DIVERSE ROLES FOR EGF RECEPTOR SIGNALING IN THE BREAST CANCER TUMOR MICROENVIRONMENT

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1 DIVERSE ROLES FOR EGF RECEPTOR SIGNALING IN THE BREAST CANCER TUMOR MICROENVIRONMENT By NIKOLAS G. BALANIS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Cathleen R. Carlin, Ph.D. Cell Biophysics Program Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY January, 2014

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the Thesis/dissertation of NIKOLAS BALANIS Candidate for the Ph.D. degree *. (chair of committee) WITOLD SUREWICZ SUSANN BRADY-KALNAY THOMAS EGELHOFF CATHLEEN CARLIN STEPHEN JONES (date) JULY 10, 2013 * We also certify the written approval has been obtained for any proprietary material contained therein 1

3 Dedication Στη Τοùλα, Γεώργιο, και θàλασσα. Σας αγαπώ πàρα πολù. 2

4 Table of Contents LIST OF FIGURES...5 LIST OF ABBREVIATIONS...6 Abstract...8 Chapter Overview The ErbB family of receptor tyrosine kinases Overview Integrin cell surface receptors Integrin/EGFR Crosstalk Rho-GTPase function and regulation Overview RhoA Regulatory Proteins Signal transducer and activator of transcription Cancer stem cells in breast cancer Triple Negative Breast Cancer Epithelial-to-Mesenchymal Transition and Triple Negative Breast Cancer Mitogen Inducible Gene Implications Figures Chapter Summary Introduction Materials and Methods Results Discussion Acknowlegements Figures Chapter Summary Allosteric Mechanism for EGFR Activation

5 3.3 Integrin-EGFR Complex Formation and p190rhogap Activation p190rhogap Activity and Focal Adhesions Perspectives Acknowledgements Figures Chapter Summary Introduction Materials and Methods Results Discussion Acknowledgements Figures Chapter Results Acknowledgements Figures Chapter Discussion Future directions EGFR and the cytoskeleton EGFR trafficking and cancer Stat3 and the cytoskeleton Jak2 : Drug Targeting EGFR and Mig6: Clinical Perspectives EGFR and Mig6: EGFR function EGFR and Mig6: Clinical Targeting Conclusions Figures BIBLIOGRAPHY

6 LIST OF FIGURES Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure

7 LIST OF ABBREVIATIONS BC EBR ECM EGFR EMT ER ErbB Erk1/2 FA FAK FN GEF Breast cancer EGFR binding region Extracellular matrix Epidermal growth factor receptor Epithelial to mesenchymal transition Estrogen receptor Avian erythroblastosis oncogene B Extracellular signal-regulated kinase ½ Focal Adhesion Focal adhesion kinase Fibronectin Guanine exchange factor HER2 Human epidermal growth factor receptor 2 IFN Interferon IL-6 Interleukin 6 JAK2 Janus kinase 2 MIG6 Mitogen inducible gene 6 NMuMG p120 p190 PR Normal mouse mammary gland p120 RasGAP p190 RhoGAP Progesterone receptor PYK2 Protein tyrosine kinase 2 RALT Receptor associated late transducer 6

8 RhoA RTK SRC Ras homology gene family member A Receptor tyrosine kinase Sarcoma kinase STAT3 Signal transducer and activator of transcription 3 TGF-β TNBC Transforming growth factor β Triple negative breast cancer 7

9 Diverse Roles for EGF Receptor Signaling in the Breast Cancer Tumor Microenvironment Abstract By NIKOLAS G. BALANIS The ligand/ cell surface receptor interaction is a paradigm for how cells utilize extracellular cues to signal to their intracellular environment. Ligand/receptor interactions are important in almost all biological processes. However, focusing solely on the ligand/receptor interaction excludes the many possible ligand-independent modes of surface receptor action, vis a vis those that occur in trans. Our studies sought to uncover the role of extracellular matrix, important molecules in the tumor microenvironment, as they function through integrin receptors to regulate the receptor tyrosine kinase, EGF Receptor (EGFR). This thesis links EGFR/integrin crosstalk to the control of cellular protrusions such as lamellipodia and filopodia, as well as the control of stress fiber formation necessary for cell contractility. We have also linked EGFR/integrin crosstalk to activation of the Signal transducer and activator of transcription 3 (Stat3). We have shown that activation of Stat3 is necessary for EGFR induced transformation of normal mammary epithelial cells. We provide mechanistic insights into how certain breast 8

10 cancers switch from EGFR/Stat3 signaling to Fibronectin/Stat3 signaling following epithelial-to-mesenchymal transition (EMT). We have shown that following EMT, breast cancers are desensitized to EGFR inhibition and become sensitized to Janus kinase 2 (Jak2) inhibitors. This finding may describe why certain cancers are resistant to EGFR directed therapies. Finally, we have identified the EGFR inhibitor protein Mitogen inducible gene 6 (Mig6) as essential to cell survival in Triple-negative breast cancer (TNBC). The observations provided in this thesis uncover the role of EGFR in the tumor microenvironment and provide insight into novel therapies for TNBC. 9

11 Chapter 1 Introduction 1.1 Overview Our research focuses on signaling from the extracellular matrix protein, fibronectin (FN), to specific integrins which form macromolecular complexes with the EGFR. We find that the EGFR is trans -activated independent of ligand upon cell adhesion to FN. We also find that the EGFR regulates a host of morphological outcomes upon adhesion to FN, characterized by two important findings. First, we report that the EGFR and β3 integrin form a physical complex which is capable of activating the RhoA GTPase activating protein (RhoGAP), p190rhogap (p190), which drives the formation of filopodial membrane protrusions on FN. A dominant inhibitory mutant of the EGFR cannot form a complex with β3 integrin and instead complexes with β1 integrin. The EGFR/β1 integrin complex does not signal through p190, and forms lamellipodial protrusions in lieu of filopodia. Second, we report that EGFR-null fibroblasts reconstituted with exogenous EGFR no longer form the elongated parallel-ordered ventral stress fibers that are a hallmark of contractile cells, that are important for their response to mechanical strain. Although the exact mechanism of this loss is unknown we find reduced recruitment of the actin polymerization protein zyxin to peripheral focal adhesions (FAs), the site of stress fiber formation. The generation of cancer cells that have altered morphological features is an important step in metastatic progression. EGFR/integrin crosstalk has the ability to both 10

12 alter cellular protrusions and the cytoskeleton, and thus may play a critical role in metastatic progression. Metastatic progression of many breast carcinomas show increased and altered expression patterns of integrins, EGFR, and FN, with the last two often linked to a poor clinical prognosis (1). EGFR regulation of both tumor formation and metastatic progression has been studied at length; however these studies are often in the context of ligand-dependent activation of EGFR. We hypothesized that FN/EGFR signaling may contribute to breast cancer progression. We focused on Triple-negative breast cancer (TNBC), the most lethal breast cancer subtype, and the only subtype that remains without an FDA-approved targeted therapy. Interestingly, even though EGFR is highly expressed in TNBC, EGFR inhibitors have done poorly in clinical trials of metastatic TNBC. We report that EGF dependent activation of Signal Transducer and Activator of Transcription Factor 3 (Stat3) is necessary for primary tumor formation in an EGFR overexpression mouse mammary gland epithelial cell model. Upon induction of EMT, an important step in metastatic progression, cells cannot activate Stat3 via a Src-dependent EGFR pathway and instead activate Stat3 via a FN:Jak2 pathway. EMT is a coordinated series of steps resulting from both genetic and epigenetic changes that results in epithelial cell losing polarity, and going various biochemical changes resulting in a more mesenchymal phenotype with higher migratory capacity, invasiveness, and in cancer the ability to metastasize. Following EMT, these cells with enhanced metastatic capacity become sensitized to Jak2 inhibitors. This is the first evidence that EMT may sensitize to any class of inhibitors and has important clinical ramifications for targeting metastatic breast cancer. Additionally, activation of Stat3 via this FN/β1-integrin/Jak2 dependent pathway is recapitulated in a 11

13 human model of metastatic TNBC in which EGFR no longer activates Stat3. Blocking EGFR function in this TNBC model with specific tyrosine kinase inhibitors has no effect on growth in a 3D-organotypic culture model that mimics the pulmonary microenvironment a frequent metastatic site in breast cancer. TNBC may have bypassed a requirement for EGFR to induce Stat3 signaling as an adaptive mechanism to maintain Stat3 signaling in its absence. We found that this breast cancer subtype overexpresses the EGFR inhibitory protein Mig6, which can squelch downstream signaling of the EGFR. Interestingly, Mig6 appears to block pro-apoptotic functions of EGFR as knockdown of Mig6 results in increased caspase activity and apoptosis. Additionally, we find that Mig6 is necessary for metastatic TNBC cell survival in the pulmonary microenvironment in nu/nu mice. These findings are in contrast to Mig6 function in primary tumors from other cancers where Mig6 is a known tumor suppressor. Hence our studies suggest that EGFR function is in a pro-apoptotic pathway in metastatic TNBC, while Mig6 functions to block this pro-apoptotic function. The FN/Jak2/Stat3 pathway is used to maintain activation of Stat3 and its growth promoting properties in the absence of EGFR function. In conclusion, the synergistic effects of Mig6 and Stat3 are essential to the cell survival and metastatic outgrowth in TNBC. Our studies indicate the critical importance of cellular microenvironment in controlling cellular fate in both normal and cancer cell physiology and the evolving role of EGFR during tumor progression. 12

14 1.2 The ErbB family of receptor tyrosine kinases Overview Members of the ErbB family such as the EGFR, ErbB2, ErbB3, and ErbB4 are receptor tyrosine kinases that serve as important signaling nexuses (Figure 1) (2-4). Each ErbB member has unique aspects; ErbB2 has an active kinase domain but no known ligand, and generally functions in heterodimers with ErbB1 and ErbB3. Although ErbB3 has a ligand binding domain its kinase is inactive. Hence, ErbB3 heterodimerizes with other ErbB members to serve as a scaffold for their downstream signaling. EGFR and ErbB4 are activated following dimerization induced by ligand. Canonically, EGFR is activated by binding of one of its many cognate ligands such as Epidermal Growth Factor (EGF), Amphiregulin, and HB-EGF among others (5). Upon activation by ligand at the cell surface EGFR dimerizes and is subsequently internalized and trafficked through the endocytic pathway (6,7). Eventually the EGFR is trafficked to the lysosome for degradation (6). Activated EGFR at both the cell surface and along the endocytic pathway can phosphorylate substrate proteins with its intrinsic kinase activity or autophosphorylate its C-terminal tail to serve as a signaling scaffold for other proteins. We are interested in the identity of the proteins that become phosphorylated and that form molecular complexes with the EGFR, as they impact numerous biological outcomes. In normal cell physiology the EGFR controls disparate physiological processes such as renal organogenesis, renal electrolyte balance, and cardiac function (8,9). Furthermore it controls processes such as cell survival, cell proliferation, and cell adhesion in a litany of cell types. When EGFR function becomes dysregulated these processes are altered 13

15 contributing to diseases such as polycystic kidney disease, and numerous forms of cancer (10). Recently the mechanism of EGFR activation was revealed, which gives us insight into both its normal and dysregulated function(11). Allosteric Model of Tyrosine Kinase Activation Each kinase domain of a monomeric EGFR is composed of two distinct lobes, the N and C lobe, and a central activation pocket. This pocket is also the site of the activation loop that contains residue Tyrosine 845 that is phosphorylated in trans by the nonreceptor tyrosine kinase Src (12). However unlike other related tyrosine kinases, phosphorylation of this residue is not necessary for activation of EGFR. It was traditionally thought that activation of the EGFR requires the kinase domains of two monomers to form a symmetric dimer, as crystallographic structures have shown this arrangement (13). However, a recent study showed that mutation of the symmetric dimer interface had very little impact on EGFR activation (11). Current evidence points to the formation of an asymmetric dimer that is quite similar to the CDK/Cyclin complex (14) (Figure 2). In this model EGFR is activated through allosteric interactions following ligand engagement (11). The EGFR is autoinhibited in its basal state; ligand engagement unmasks the EGFR kinase domain dimerization interface (15). The C-lobe domain of the activator kinase domain interfaces with the N-lobe of the activated kinase domain, resulting in its subsequent activation and autophosphorylation. In this model the juxtamembrane region of the activated kinase latches to the C-lobe of the activator kinase (14). Recent evidence suggests that the inactive monomeric EGFR kinase also resides in a third intrinsic disordered state. In this state the interface of the N-lobe EGFR kinase domain is intrinsically disordered (16). In this model EGFR oncogenic mutations remove 14

16 the disorder at the N-lobe that is a feature of EGFR in its basal state, allowing for easier N lobe/c lobe interaction and anomalous activation independent of ligand engagement (16). Traditionally, biologists have asked questions about the EGFR solely in the context of its ligand-dependent signaling. However over the last 10 years the EGFR has been shown to function outside the bounds of this simplified model of EGFR function (17). Our studies focus on the activation of EGFR due to its interaction and crosstalk with integrin receptors. We report that integrins employ Src to phosphorylate the EGFR in trans, and this phosphorylation is necessary for subsequent activation of Stat3. The allosteric activation model provides a potential explanation for the function of Tyrosine 845 in the activation loop of the EGFR. As stated earlier in other kinases the phosphorylation of the equivalent residue is necessary for activation, while in the EGFR it is not (18,19). This model predicts that Tyrosine 845 activation shifts the N-lobe interface to a more ordered state, allowing for dimerization to occur more readily. This model hypothesizes that lateral propagation and amplification of EGFR activation can thus occur through phosphorylation of Tyrosine 845 without the need for further ligand binding. Our research has uncovered a 679-LL dileucine motif residing in the C-terminal portion of the juxtamembrane domain that is necessary for activation of Tyrosine 845, and EGFR signaling and trafficking (Figure 2) (6,20,21). We have uncovered that this motif is an important regulator of complex formation between the EGFR and integrin molecules. 15

17 1.3 Integrin cell surface receptors Integrins are cell surface receptors that are composed of both an α and β subunit (22). They serve to attach cells to the extracellular matrix (ECM) via interactions with the extracellular portion of these α and β subunits (22). The eighteen α and eight β subunits in mammalian cells can form up to twenty-four distinct integrin receptors with different affinities for various matrix molecules (22). This allows for incredible plasticity and specificity, facilitating integrins binding of a diverse set of matrix molecules, including collagen, laminin, vitronectin, and most importantly FN (22). Furthermore, integrins serve as linkages to the cell cytoskeleton through various adaptor proteins such as α- actinin, vinculin, and zyxin on their internal face. This link between the extracellular and intracellular cytoskeleton provides the requisite rigidity to foster cell migration and support adhesion. Upon attachment to ECM proteins, integrins also cluster and enhance the activation and recruitment of proteins that regulate the cytoskeleton. As such integrins are signaling platforms, serving as scaffolds for many cytosolic kinases involved in cell adhesion, cell spreading, and cell migration such as the non-receptor tyrosine kinase known as focal adhesion kinase (FAK). FAK regulates turnover of constituent molecules at the FA that are important in regulation of adhesion and migration (23). The FA is the site of integrin binding to extracellular matrix, it is a cluster of integrins that extracellulary is attached to matrix molecules such as fibronectin, collagen, and laminin (24). Internally FA resident integrins are connected to the actin cytoskeleton via various adaptor proteins such as α-actinin, vinculin, and zyxin. This allows the FA to take extracellular cues from matrix and transmit this information to the the actin cytoskeleton, to regulate cell morphology, the types and number of actin protrusions, and the internal 16

18 rigidity of the cell. Integrins are essential in the formation of these various cellular membrane protrusions such as lamellipodia and filopodia. Lamellipodial protrusions are formed from a dendritic network of branched actin filaments that line the plasma membrane (20). Lamellipodia drive cell motility by generating forces through a combination of cell adhesion, actin polymerization at the periphery, and retrograde actin flow (20). While not absolutely essential for cell motility, cells use filopodia to form new adhesive contacts for motility and explore their environment (20). Two important integrins are the FN binding receptors a 5 β 1 and a v β 3. In specific cell models, both a 5 β 1 and a v β 3 are sufficient to downregulate the activation of the Rho-GTPase RhoA that is necessary in the early stages of cell spreading (25). However, upon binding to the same ECM proteins cells expressing one or the other form of integrin have dramatic differences in cellular morphology (26). These integrins have altered ability to affect recovery of RhoA activity following early inhibition after cell spreading: a 5 β 1 supports recovery of RhoA activity while a v β 3 does not. These integrins also recruit various kinases such as Src, and RhoGAP regulatory proteins by different mechanisms. Our work has highlighted the ability of EGFR to alter the constituent RhoA regulatory molecules in complex with these two integrins, specifically regulating the formation of membrane protrusions (21). 1.4 Integrin/EGFR Crosstalk Work from our laboratory has highlighted the importance of a dileucine motif (679-LL) in the juxtamembrane region of the EGFR that is required for Tyrosine

19 phosphorylation of the EGFR upon cell adhesion to FN (6,20,21,27). The EGFR (679- LL) motif is situated in the C-terminal portion of the juxtamembrane region, and is within the interface of the N and C lobe interaction domain in ligand-induced asymmetric dimers (11). When these dileucine residues are mutated to alanines (679-AA) the EGFR is no longer phosphorylated upon Tyrosine 845; however a limited number of C-terminal residues still undergo auto-phosphorylation upon both EGF stimulation, and EGFR activation following FN-dependent adhesion. Alterations in EGFR phosphorylation impact specific downstream pathways including RhoA and Stat3 signaling (21) (28). We speculate that the AA mutation restricts the conformational changes necessary for exposure of the activation loop, while still allowing for the autophosphorylation of a subset of residues in the C-terminal tail. Our first paper highlighted the fact that this mutation regulated the ability of β1 or β3 integrins to form a molecular complex with the EGFR (20). To accomplish this goal we used the NIH-3T3 derived NR6 mouse cell line, which is EGFR-null (29). In this NR6 Fibroblast (EGFR-null) reconstitution model we exogenously expressed Wild-Type Human EGFR (WT-EGFR) which binds β3 integrins or EGFR(679-AA) which binds β1 integrins (20). While WT-EGFR/β3-integrin complexes led to formation of filopodia, EGFR (679-AA)/β1-integrin complexes suppress filopodia formation and instead display lamellipodia. These effects are manifested through the function of the RhoGAP p190 which regulates Rho-GTPase RhoA. We found that EGFR is capable of directly binding to p190 and activating its RhoGAP function (Figure 3). 18

20 1.5 Rho-GTPase function and regulation Overview Rho-GTPases are important downstream effectors of integrin-mediated cell adhesion and spreading (30). They act as molecular switches that regulate the formation of membrane protrusions (31). This is accomplished by regulating the assembly and disassembly of the actin cytoskeleton which is essential to the formation of filopodia and lamellipodia, the two most well studied forms of membrane protrusions (32). Rho GTPases cycle between an active GTP bound from and inactive GDP bound form. Guanine Nucleotide Exchange Factors (GEFs) increase the dissociation of GDP and increase the association with GTP thus pushing the GTPase into the active form. RhoGAPs function in the opposite direction, increasing the speed of GTP hydrolysis to promote formation of inactive GDP-bound RhoA. Our first study focused on the interplay between the EGFR, the RhoA GTPase, and its GAP protein p190, following integrinmediated FN adhesion. RhoA Regulatory Proteins When the two known isoforms of p190, p190-a and p190-b, are active, they inhibit RhoA by inducing GTP hydrolysis. Following p190 phosphorylation on residue Y1105 by Src, FAK or Arg kinase, p190 forms a complex with the RasGAP p120rasgap (p120). To effectively inhibit RhoA p190 must be in this complex as well as undergo recruitment to the cell periphery (30). p120 has SH2-SH3-SH2 protein 19

21 interaction domains that are speculated to bind p190 at one SH2 domain and FAK at the other (24). The SH2-SH3-SH2 domain in p120 thus allows it to create a link between integrins through FAK and the RhoGAP regulatory proteins directly at the FA which is the site of actin assembly and disassembly (24). Our work has highlighted the ability of EGFR to recruit a p120/p190 complex to the cell periphery upon cell adhesion (20). In the NR6 reconstitution model EGFR-null and EGFR expressing cells display dramatic differences in morphology. EGFR-null cells form abundant ordered ventral stress fibers, in contrast to cells expressing Wild-Type EGFR showing disordered stress fibers and elongated filopodia. We speculate that the integrin/egfr interaction designates localization of RhoA inhibition at the cell periphery thus regulating morphological outcomes (Figure 3). We also speculate that the upstream molecules that phosphorylate p190 may play a role in determining the localization and function of p190. In EGFR null cells we suspect FAK is essential to p190 function as has been previously reported (24). Upon expression of the EGFR, Src (which is also present in the integrin/egfr macromolecular complex) may be ideally situated to phosphorylate p190 upon FNdependent adhesion (33). Thus, integrin/egfr and integrin/fak complexes may not only be located at different FA sites but also regulate the activation of p190 by different mechanisms (Figure 3). 1.6 Signal transducer and activator of transcription 3 The Stat3 transcription factor consists of a N-terminal domain, a coiled-coil domain, a DNA binding domain, a linker domain, an SH2 domain necessary for 20

22 formation of the Stat3 homodimer, and a transactivation domain that contains a tyrosine residue that upon phosphorylation binds to the SH2 domain of an opposing Stat3 molecule (34). Stat3 was initially thought to be activated solely through conventional immune response pathways elicited by Interferon (IFN) or by cytokines such as IL-6 (35). In this process Stat3 binds to receptors such as gp130 that lack intrinsic kinase activity, but that associate with cytosolic Janus tyrosine kinase family members (JAKs) which phosphorylate Stat3 at a specific tyrosine residue leading to its subsequent activation. It was soon found that receptor tyrosine kinases such as Platelet derived growth factor (PDGFR) and EGFR were able to activate Stat3 as well (36,37). Stat3 is also a substrate for the oncoprotein v-src, which unlike cellular Src (c-src) lacks the C-terminal autoinhibitory domain (38,39). Constitutively active Stat3 enhances cell survival, promotes proliferation, and is able to transform cells, providing evidence of its oncogenic ability (40). Stat3 regulates genes that are common to both wound healing and cancer, and is thought to play a major role in tumor formation (41). Elevated levels of Stat3 in immune cells in the tumor microenvironment negatively affect their ability to mount an immunological response (42). Stat3 also plays roles in regulating the microtubule cytoskeleton which is essential in cellular trafficking and regulating protrusions such as filopodia. Stat3 interaction with the protein Stathmin inhibits the formers ability to destabilize microtubules (43). Although it remains to be seen if Stat3 phosphorylation plays a role in this interaction, our pilot studies with the direct Stat3 inhibitor Stattic show it is able to interfere with filopodia formation (28). Importantly for our work, Stat3 activity seems to be elevated in stem-like breast cancer cells (44). 21

23 1.7 Cancer stem cells in breast cancer Single tumors often have diverse populations of cells that respond differently to targeted therapy (45). Very recent evidence points to single cancer stem cells giving rise to the various diverse daughter cells resident in the tumor (46,47). In the exceptional study by Schepers et al, their group was able to track single cancer stem cells using a fluorescent color and prove that they gave rise to differentiated daughter cells of various identities in adenomas (47). It remains to be seen whether this type of stem/daughter relationship is a general mechanism among all forms and subtypes of cancer. If true the implications for breast cancer research are paramount because the stem-like population in breast cancer represents an important clinical target, as these cells may give rise to the intra-tumor heterogeneity seen in the solid tumor (48,49). Directed therapies to this cell population may solve the problems that arise with targeting a heterogeneous tumor cell population. Stat3 appears be preferentially activated in the stem-like population in human breast tumors, and is also important for their growth in xenografts (44). It thus seems to be an ideal target for therapeutic intervention towards these stem-like cells. Inter-tumor heterogeneity of breast cancer presents a problem as well, as therapies directed to certain types of breast cancer are ineffective in others (50). The importance of Stat3 in the stem cells of the various subtypes of breast cancer is currently unknown. We have focused our study of Stat3 in TNBC the sole breast cancer subtype without a proven FDA-targeted therapy, and one whose cells share characteristics of stem-like cells of the breast (44,48,49,51). 22

24 1.8 Triple Negative Breast Cancer Breast cancer is the most commonly diagnosed form of cancer and the second leading cause of death due to cancer in US women. Approximately 15% of breast cancer patients develop TNBC. TNBC is especially prevalent in African-American and Hispanic women under the age of 40 where clinical management is often exacerbated by socioeconomic disparities. TNBC tumors are characterized by lack of progesterone receptor (PR) expression, estrogen receptor (ER) expression, and lack of amplification of human epidermal growth factor receptor 2 (HER2). Targeted therapies for metastatic breast cancer target both ER and HER2, however since these are lacking in TNBC there are no FDA approved targeted therapies for TNBC. Thus the current TNBC treatment regimen consists of surgical or chemotherapeutic options. TNBC (ER-, PR-,HER2-) is one of the 4 genotypically distinct breast cancer subtypes which include luminal A (ER+,PR+,HER2-), luminal B (ER+,PR+,HER2 +), and HER2 positive (ER-,PR-,HER2+). Interestingly, EGFR is overexpressed in TNBC, is often used as a marker for basal breast cancer, and is correlated with poor overall survival (52,53). There are two classes of EGFR inhibitors in clinical use: those that inhibit kinase activity via interactions with the kinase domain such as Gefinitib and Erlotinib which have proven effective in treating certain forms of lung cancer; and monoclonal antibodies such as cetuximab that target the extracellular domain of the EGFR that have proven effective in treating metastatic colon cancer, and squamous cell carcinomas of the head and neck. However Phase I and Phase II trials have shown that both types of EGF inhibitors are ineffective in treating TNBC (54). Our research has uncovered a mechanism whereby TNBC may circumvent EGFR which activates pro-oncogenic Stat3 23

25 in the primary tumor through a FN/integrin signaling pathway that maintains prooncogenic Stat3 activity during metastatic outgrowth. 1.9 Epithelial-to-Mesenchymal Transition and Triple Negative Breast Cancer Epithelial-to-mesenchymal transition (EMT) is a series of pre-programmed genetic and epigenetic changes that normally occur early in development resulting in loss of epithelial cell characteristics and gain of mesenchymal characteristics with enhanced migratory capacity and ability to invade. In development, EMT is an essential to giving rise to the various tissues and organs in the organism (55,56). EMT can also occur in adult tissue remodeling and wound healing (57). Via EMT, cells of epithelial origin lose epithelial cell markers such as E-cadherin and gain mesenchymal markers such as N- cadherin, FN, and integrin β1 (56,58). In cancer, EMT can be improperly co-opted at the primary tumor, leading to the formation of a population of mesenchymal-like cells with increased growth, migratory, and invasive properties that enhance their ability to disseminate to distant organs (59). In addition, cells that have undergone EMT more are prone to form tumors at the primary site. Futhermore, the post-emt population which has colonized distant organs may also be able to re-seed the primary tumor (60). EGFR overexpression is sufficient to transform normal mammary epithelial cells (61). In a normal mouse mammary gland (NMuMG) breast cancer model, Stat3 is downstream of EGFR overexpression following ligand stimulation or FN adhesion (28). EGFR overexpression in this model is necessary for primary tumor growth. Mutating 24

26 EGFR at the 679-LL motif, blocks activation of Stat3 but not other downstream signals such as Erk1/2 and Akt, and abrogates primary tumor growth (28). This model can also be induced to undergo EMT with TGF-β resulting in cells with mesenchymal properties that also become TNBC-like. In this post-emt cell population Stat3 activity is independent of EGFR activity irrespective of the signal that activates EGFR. Using a human TNBC cell line we showed that Stat3 activity is activated by a FN/β1 integrin signaling axis(28). Inhibition of Stat3 in this human TNBC cell model upon FN adhesion reduces their mesenchymal like appearance suggesting it may affect metastatic potential. We also find that in this model Stat3 activity is necessary for growth in a 3-dimensional (3D) culture model that mimics the pulmonary microenvironment, a preferred site of TNBC metastasis. We found that β1-integrin engagement of FN leads to activation of Stat3 through Proline Rich Tyrosine Kinase 2 (Pyk2)/FAK upstream of Jak2. Pyk2 is a FAK homolog that shares many functions with FAK. Furthermore its expression is upregulated following EMT (62). It has also been shown to directly associate with JAK2 in the context of immunological signaling (63). We found that that 3D organotypic outgrowth of TNBC cells line becomes sensitized to Jak2 inhibitors following EMT. Finally, we found that TNBC model cell lines more highly express the EGFR inducible inhibitor protein Mitogen Inducible Gene-6 (Mig6), which may explain the loss of EGFR dependence. 25

27 1.10 Mitogen Inducible Gene-6 Recently, a number of inducible proteins have been identified that fine-tune EGFR signaling by restraining its robust protein tyrosine kinase activity (64). Upon activation of various upstream RTKs such as EGFR, de novo mrna and protein synthesis results in the upregulated expression of these inducible feedback inhibitors. As such, these intrinsic molecules are particularly attractive candidates for unleashing cell autonomous regulation of unrestricted EGFR signaling in cancer. One such protein is the putative tumor suppressor Mig6, which prevents EGFR-mediated transformation (65). Mig6 is a cytosolic protein that inhibits EGFR by binding to an extended surface of the EGFR catalytic domain involved in allosteric activation in ligand-induced asymmetric dimers (66). Dysregulated EGFR activity and loss of Mig6 expression can induce apoptosis in both normal and malignant mammary epithelial cells (67,68). Thus, Mig6 may be endowed with both tumor promoting and suppressing functions at different stages of tumor development. Mig6 is a cytosolic protein that binds ligand activated EGFR through its ErbB binding region (EBR domain) to block kinase function. Mig6 also has a RALT endocytic domain (RED) which links it to intersectins and AP-2 which are important for clathrindependent internalization. Although the mechanism is unclear Mig6 can route the EGFR to lysosomes for degradation by a mechanism that is independent of both EGFR kinase function and ubiquitination (Figure 1.4) (64). Mig6 is thought to be a tumor suppressor as it is capable of blocking transformation induced by EGFR overexpression (65,69). Other studies however find that genetic knockdown of Mig6 can induce apoptosis in endothelial cells(67,68). The role Mig6 plays in the regulation of EGFR in TNBC is 26

28 undefined. We sought to determine what role Mig6 plays in TNBC growth and metastasis Implications Our research has uncovered the central role EGFR/integrin crosstalk plays in both normal and cancer cell physiology. Specifically we have furthered our understanding of the role of this crosstalk in formation of cellular protrusions necessary for cell migration, the formation of stress fibers necessary for cell contractility, and tumor progression in TNBC. Most exciting however are the implications our research has in the treatment of TNBC, as they provide explanation to the failure of EGFR targeted drugs. First they open the possibility that Mig6 and Jak2 represent new prognostic biomarkers, second they suggest that targeting Mig6 and Jak2 in metastatic TNBC is a therapeutic option. 27

29 1.12 Figures Figure 1.1. The four ErbB family members EGFR, ErbB2, ErbB3, and ErbB4. In every ErbB monomer but ErbB2 ligand enters a ligand binding pocket formed by Domains I and III and binds simultaneously to both domains. In ErbB2 domains I and III are in close apposition occluding the ligand binding pocket. In every ErbB monomer but ErbB2 Domains II and IV interact in the unliganded receptor and bury the dimerization arm present in Domain II. Upon ligand binding the II/IV interaction is altered and the dimerization arm is exposed. This arm can then interact with a Domain II from another liganded monomer, eventually leading to dimerization. It is not important if these are homo or heterodimers, as long as the Domain II dimerization arm can interact. ErbB2 is different in that its dimerization arm is consituitively exposed. All ErbB members contain a transmembrane domain, and C-terminal phophorylation sites. Unlike other ErbB members ErbB3 has a kinase domain that is non-functional thus requiring heterodimerzation with other ErbB members for ErbB3 to become phosphorylated and serve as a scaffold. (70). 28

30 29

31 Figure 1.2. In the EGFR symmetric dimer model (left) the two kinase domains are rotated relative to each other. Recent studies that have mutated residues in the purported symmetric dimer interface had no impact on downstream activation of residues in the C-terminal tail suggesting this model of the EGFR is not physiologically relevant. In the asymetric dimer model activation of the EGFR results from the interaction of the activator kinase C- lobe and the activated kinase N-lobe. The activated kinase can then phosphorylate residues in the C-terminus of the activator. 30

32 31

33 Figure 1.3. EGFR re-localizes the site of RhoA inhibition consequently altering morphological outcome. In the presence of EGFR RhoA antagonist p190rhogap is recruited to EGFR through p120 and tyrosine phosphorylated by Src kinase that is presumably associated with a v β 3. Tyrosine phosphorylation of p190 is necessary for its activation. These FA complexes support the formation of filopodia but cannot form parallel-ordered stress fibers. In the absence EGFR p190rhogap is recruited through FAK directly to integrins. RhoA inhibition here supports the formation of actin stress fibers but not filopodia. 32

34 33

35 Figure 1.4. In the activated EGFR (left) residues in the C-terminal tail are phosphorylated by the intrinsic kinase activity of the activator kinase domain which becomes the receiver domain in the reciprocal orientation. Mig6 binds to the receiver kinase (right) through EBR domain segment one (blue), allowing for the insertion of EBR domain segment two (green) into the kinase domain inhibiting its activity and preventing the receiver kinase from becoming the activator kinase in the reciprocal orientation. The EBR domain also inserts into the kinase domain of the active EGFR and blocks kinase activity (green). Following binding of Mig6 to EGFR, Mig6 links to endocytic machinery through its RED domain. The RED domain binds both AP-2 and intersectins to direct EGFR through the endocytic pathway for eventual degradation at the lysosome. 34

36 35

37 Chapter 2 β3 integrin-egf receptor cross-talk activates p190rhogap in mouse mammary gland epithelial cells 2.1 Summary Active RhoA localizes to plasma membrane, where it stimulates formation of FAs and stress fibers. RhoA activity is inhibited by p190rhogap following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190rhogap is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120rasgap. This complex then translocates to the cell surface, where p190rhogap down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with β3 integrin to regulate p190rhogap activity in mouse mammary gland epithelial cells. Adhesion to FN stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that FN-activated EGFR recruits p120rasgap to the cell periphery. Expression of an inactive β3 integrin subunit abolishes p190rhogap tyrosine phosphorylation, demonstrating a mechanistic link between β3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The β3 integrin/egfr pathway also has 36

38 a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since FN is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition. 2.2 Introduction Multicellular organisms rely on cell migration throughout their lifespan. Cells specified in one region of the embryo migrate over long distances to form functionally distinct tissues (71,72). Cell migration facilitates repair mechanisms in the adult notably during wound healing when fibroblasts and inflammatory cells migrate to sites of injury (73). Cell migration is also important in organs such as mammary glands that restructure epithelial tissues during morphogenesis (74). Cells become invasive and undergo metastasis when migration processes involved in epithelial morphogenesis are reactivated in solid human tumors (75,76). Despite major advances in therapies that curb tumor growth, metastatic disease has proven difficult to control (77,78). Metastasis is the leading cause of death in cancer patients underscoring the importance of understanding fundamental cell migration mechanisms. The extension of the plasma membrane is one of the first steps in cell migration (79,80). Animal cells produce membrane protrusions such as lamellipodia and filopodia through coordinated action of actin cytoskeleton, cell adhesions, and plasma membrane (81). Lamellipodia are broad, thin membrane protrusions with a dendritic network of 37

39 branched actin filaments abutting the plasma membrane that generate motile force through a combination of plus-end actin polymerization, retrograde flow, and cell adhesion (82). Filopodia are finger-like membrane projections filled with bundles of parallel actin filaments (83). While not absolutely required for cell migration, cells use filopodia to explore their environment and form new adhesive contacts for motility and spreading (84,85). Filopodia have also been implicated in conveying long distance signals important for pattern formation in developing epithelia (86). Most cultured cells produce both organelles upon initial contact with extracellular matrix (ECM). Actin cytoskeleton and ECM are physically connected via cell adhesions initiated by activation of integrin transmembrane receptors (22). Signaling receptors such as the EGF receptor (EGFR) modulate normal cell behavior by forming physical and functional complexes with integrins that bind fibronectin (FN) (33,87). Elevated EGFR expression contributes to carcinoma development and metastasis by stimulating proliferation and enhancing survival of FN-attached cells in the absence of ligand (88,89). EGFR also affects tumor angiogenesis by modifying branching behavior in endothelial cells within the tumor microenvironment (90). Previous studies indicate that elevated EGFR expression is sufficient to transform normal mammary epithelial cells similar to polyoma middle T using the NMuMG cell model (61). These same studies showed that elevated EGFR expression enhances mesenchymal characteristics induced by EGF or TGF-β, and also primes NMuMG cells for TGF-β-driven epithelial-to-mesenchymal transition (EMT). Although integrin-egfr signaling is implicated in multiple cell migration pathways, relatively little is known about its involvement in formation of motile membrane protrusions in normal and pathological settings (33,87,91). 38

40 Ligand-induced EGFR activation involves formation of asymmetric dimers between adjacent tyrosine kinase domains (see Figure 2.1A) (11). This mode of activation is regulated by residues that form a flexible interface between the C-terminal lobe of one tyrosine kinase domain and the N-terminal lobe of its dimer partner (Figure 1A). The intracellular EGFR juxtamembrane region plays a crucial role in stabilizing these asymmetric dimers (92,93). Residues in the juxtamembrane region and dimer interface modulate a number of other functions suggesting they integrate EGFR activation with cellular responses (94-97). Residues Leu679 and Leu680 (679-LL) located in the N-terminal lobe interface regulate post-endocytic sorting to lysosomes (6,27). Receptors with a 679-AA dialanine substitution [EGFR (679-AA)] undergo ligand-accelerated internalization similar to wild-type EGFR (WT-EGFR), but are routed away from lysosomes to a recycling pathway where they are coupled to novel signaling complexes (98). These findings provide new insights to signaling pathways up-regulated in breast cancer cell lines where EGFR forms dimers with related ErbB family members that divert activated EGFRs to recycling endosomes similar to EGFR (679-AA) (98-100). The role of these residues during EGFR trans-activation independent of growth factor stimulation has not been addressed. We demonstrate here that EGFR has a significant role in determining how both normal mouse mammary gland epithelial cells and those poised for EMT respond to FNenriched adhesive environments. Our data support a model that signaling through β3 integrin FN receptors via EGFR activates the RhoA antagonist p190rhogap by recruiting its binding partner p120rasgap to plasma membrane. We also provide evidence that the β3 integrin-egfr pathway is a positive regulator of filopodia formation. 39

41 EGFR with the 679-AA substitution had an instrumental role in dissecting this pathway, since it behaves as a dominant inhibitory molecule capable of interfering with β3 integrin-egfr signaling. 2.3 Materials and Methods Antibodies and Reagents These antibodies were purchased from commercial sources: actin and vinculin mouse monoclonal antibodies from Sigma (St. Louis, MO); FITC-conjugated integrin β1 and β3 antibody and matched FITC-conjugated isotype controls from EBioscience (San Diego, CA); β1 integrin rabbit antibody from Chemicon (Billerica, MA); β3 integrin, FAK, phosphotyrosine, and p130cas mouse monoclonal antibodies from BD Biosciences (San Jose, CA); mouse-specific EGFR goat and EGFR rabbit antibody from R & D Systems (Minneapolis, MN) and Fitzgerald Industries (Concord, MA), respectively; phospho-egfr (Tyr845, Tyr992, Tyr1068, Tyr1173), phospho-fak (Tyr397, Tyr526/527), phospho-p130cas (Tyr165, Tyr249, Tyr410), and Src rabbit antibodies from Cell Signaling Technology (Beverly, MA); p120rasgap and p190rhogap mouse monoclonal antibodies from Millipore (Billerica, MA); p190rhogap rabbit antibody from Bethyl Laboratories (Montgomery, TX); and Rac1 and RhoA rabbit antibodies from Cytoskeleton, Inc. (Denver, CO). Human-specific EGFR1 mouse monoclonal antibody was produced using the ascites method. All secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Rhodamine-conjugated phalloidin, Alexa594-wheat germ agglutinin (WGA), and the nuclear counter-strain DAPI were 40

42 purchased from Molecular Probes (Eugene, OR). Receptor-grade EGF, selective inhibitors for EGFR [N-(3-Chlorophenyl)-6,7-dimethoxy-4-quinazolinamine or AG1478] and Src [4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-(3,4-d)pyrimidine or PP2], extracellular matrix proteins, and FITC-conjugated phalloidin were from Sigma, and EMgrade paraformaldehyde from EM Science (Gibbstown, NJ). Cell Lines NR6 cells are an NIH-3T3 variant lacking endogenous mouse EGFR (29). NMuMG cells are epithelial cells derived from normal mouse mammary gland (101). NR6 cells expressing human WT-EGFR or receptors with 679-LL converted to 679-AA [EGFR (679-AA)], and NMuMG cells with inactive D119A β3 integrin, are described elsewhere (6,98); (102). Permanent NMuMG cell lines expressing WT-EGFR, or EGFR (679-AA) were produced using established protocols (61,103). All cells were routinely maintained at 37 o C in a humidified atmosphere of 5% CO 2 and 95% air in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% L- glutamine. Media for NMuMG cell lines were additionally supplemented with 10 μg/ml insulin. Cell lines expressing recombinant proteins were selected and maintained in media supplemented with the appropriate antibiotic [G418 (MP Biomedicals; Solon, OH) or puromycin (Invitrogen; Carlsbad, CA)]. Cell Adhesion Cells grown to approximately 80% confluence were serum-starved in media supplemented with 0.5% BSA and 1% L-glutamine for 5 h and detached from tissue culture plastic with 0.25% trypsin-edta. Trypsin was inactivated with a 2-fold volume 41

43 of serum-free media supplemented with soybean trypsin inhibitor (0.5 mg/ml) (Invitrogen). Cells were allowed to adhere to polystyrene dishes or glass coverslips coated with ECM proteins (10 µg/ml) at a density of approximately cells/mm surface area. Control cells were kept in suspension in polystyrene dishes coated with RIA-grade BSA (10 µg/ml). Analysis of Cell Spreading Cells were incubated with Alexa594-WGA and DAPI for 15 min, and then fixed with 3% paraformaldehyde-pbs. Data for mono-nucleated cells were collected using a Nikon Eclipse TE200 microscope equipped with a digital camera. NIH ImageJ software was used to quantify the mean surface area per cell based on WGA staining for 500 cells in two independent experiments for each cell line. Results are presented as mean surface area (in arbitrary units or AU)/cell ± SEM as a function of time post-adhesion. Surface area adhesion rates (AU/min) were calculated by linear regression analysis for data from the 5, 10, and 20 post-adhesion time points. Confocal and Video Microscopy Cells were perforated with 0.5% β-escin in a solution of 80 mm PIPES, ph 6.8, supplemented with 5 mm EGTA and 1 mm MgCl 2 for 5 min and fixed with 3% paraformaldehyde PBS for 15 min as described previously (104). Non-specific binding was blocked with 5% normal serum from the host animal used to generate the secondary antibody. Cells were stained overnight at 4 C or 1 h at room temperature with antibodies diluted in a solution containing 0.5% β-escin and 3% RIA-grade BSA. Confocal images 42

44 were acquired with a Zeiss LSM 510 Meta laser scanning microscope (Carl Zeiss MicroImaging, Jenna, Germany) using diode (excitation 405 nm), Argon (excitation 488 nm), and HeNe (excitation 543 and 633 nm) lasers, 40 or 100 Plan Apo NA 1.4 objectives, and Zeiss LSM software (Carl Zeiss MicroImaging, Jenna, Germany). For video microscopy, cells were placed in a temperature-controlled chamber at 37 C in an atmosphere of 5% CO 2. Data were analyzed using a Leica 6000 B inverted microscope equipped with a Retiga EXI 12 bit camera (Q imaging, Vancouver, BC, Canada) and MetaMorph image analysis software (Universal Imaging, Downington, PA, USA). Stress Fiber Alignment Stress fiber alignment was carried out using a previously described Sobel filter algorithm to analyze fixed cells stained with rhodamine-conjugated phalloidin (105). The Sobel filter algorithm determines the orientation of the pixel intensity gradient in a 3 3 pixel area. Images of individual cells were subdivided into pixel grids and grids lacking pixels with phalloidin signals were eliminated from the analysis. The average angle of pixel intensity gradient for each grid (i.e. the angle of stress fibers passing the grid) was computed and the histograms of angle distribution were generated. The kurtosis (i.e., peakedness) of the angle histogram was also determined and defined as "Alignment Index". Data are presented as mean alignment index ± SEM, n = 100 cells. Cell Morphometry Measurements Quantification of both filopodia and lamellipodia was done using fixed cells stained with rhodamine-conjugated phalloidin. Filopodia were counted using MetaMorph software. Briefly, individual cells were filtered by determining the threshold values for average 43

45 pixel intensity. The number of filopodia per cell was determined by counting only filopodia with above average fluorescent intensity following thresholding that crossed the cell edge and were longer than 2 µm. Lamellipodia were quantified using a custom program in the MetaMorph software package that traces and measures the whole-cell perimeter and cell perimeter with adjacent lamellipodial network. The fraction of cell perimeter occupied by lamellipodial network was used as a parameter for quantification. Data presented as mean number of filopodia/cell, or percent lamellipodia/cell, ± SEM, n = 60 cells from 3 independent experiments. The form factor, or 4Aπ/P 2 where A is the cell area and P is the perimeter, were calculated using the Image Morphometry Analysis feature in MetaMorph software on thresholded cell images. Cell Lysis Cells were washed 3 times with PBS supplemented with 5 mm EDTA, 5 mm EGTA, and a phosphatase inhibitor cocktail (10 mm NaF, 10 mm Na 4 P 2 O 7, and 1 mm Na 3 VO 4 ). Cells were lysed with 1% NP-40 in a solution of 50 mm Tris-HCl, ph 8.5, supplemented with 150 mm NaCl, the phosphatase inhibitor cocktail, and a cocktail of protease inhibitors (100 µm phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 4 µg/ml pepstatin). Cell lysates were clarified by high-speed centrifugation and protein concentrations were determined by Bradford assay (Bio-Rad; Hercules, CA). Metabolic Labeling Cells were pre-incubated in methionine and cysteine-free medium for 1 h. Amino acidstarved cells were pulse-labeled with 35 S-Express Protein Labeling Mix (2.5 mci/ml; 44

46 PerkinElmer Life Sciences, Boston, MA) diluted in amino acid-deficient medium supplemented with 10% dialyzed FBS and 0.2% BSA for 1 h. The radio-labeled cells were then incubated in chase medium supplemented with a 10-fold excess of nonradioactive methionine and cysteine for 3 h. Immunoprecipitation and Immunoblotting Conventional immunoprecipitations were carried out by incubating cell lysates with antibodies to specific proteins for 1 h followed by protein-a-sepharose beads (Sigma) for an additional 1 h at 4 C. Surface immunoprecipitations were carried out as described previously (106). Briefly, newly adherent cells were washed with DMEM supplemented with 1% BSA, and then incubated with EGFR1 antibody that recognizes an extracellular human EGFR epitope, or an isotype matched antibody to IL2Rα, diluted in the PBS-BSA solution (10 µl/ml) on ice for 1 h. Cells were washed 4 times with PBS-BSA and cell lysates were incubated with protein A beads for 1 h at 4 o C to collect immune complexes formed at the cell surface. Immune complexes eluted with sample buffer or equal aliquots of total cellular protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes using standard methods. Blots were blocked in TBS-T (10 mm Tris, 150 mm NaCl, and 0.1% Tween-20) supplemented with 5% non-fat dry milk or 5% BSA. Membranes were incubated with primary antibodies overnight at 4 o C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature for detection by ECL. ECL signals were quantified using ImageQuant TM LAS 4000 digital imaging system and ImageQuant TL software (GE Healthcare, Piscataway, NJ). 45

47 Flow Cytometry Cells were trypsinized and either analyzed immediately (EGFR) or following a 3-h incubation in bacterial dishes (β1 and β3 integrins). Cells were stained with primary or secondary antibodies diluted in PBS supplemented with 1% BSA for 30 min on ice, fixed with 2% paraformaldehyde, and analyzed on an icyte Reflection Flow Cytometer (Champaign, IL). Rho Family GTPase Activation Assays GTPase activity was measured using Activation Assay Kits BK036 (Cdc42 and Rac) and BK035 (Rho) from Cytoskeleton, Inc. (Denver, CO) according to the manufacturer s instructions. Briefly, cell extracts were prepared with ice-cold cell lysis buffer and immediately snap frozen in liquid nitrogen and stored at 80 o C to minimize GTP hydrolysis. An aliquot was set aside for protein concentration determination using the Precision Red Advanced Protein Assay Reagent supplied with the kit. Equal protein aliquots were added to individual wells in an 8-well strip coated with an appropriate Rho GTPase binding domain (RBD) and plates were incubated on an orbital shaker at 4 o C for 30 min. Strips were washed and incubated with Rho GTPase-specific primary antibodies followed by HRP-conjugated secondary antibodies and then an HRP detection reagent supplied with the kit. Absorbance was measured at 490-nm using a microplate spectrophotometer (SpectraMax 340 Microplate Reader; Molecular Devices, Downingtown, PA). Samples from at least two independent experiments assayed in triplicate. Data are presented as percent change relative to cells in suspension ± SEM as a function of time post-adhesion. Pulldown assays for active RhoA were carried out with a GST fusion protein containing the RBD of Rhotekin (gift of Danny Manor, Case 46

48 Western Reserve University, Cleveland, OH) using established methods (107). Briefly, cells were lysed in a solution of 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-HCl (ph 7.2), 500 mm NaCl, and 10 mm MgCl 2 supplemented with protease inhibitors. Clarified lysates were incubated with 10 µg of GST-RBD immobilized on glutathione-agarose beads for 90 min at 4 C. Bound proteins were eluted in sample buffer, and RhoA was detected by immunoblotting. Statistical Analyses Statistical analyses were performed using the Student s t test. A P value of < was considered statistically significant. Image Preparation Digital images were prepared using Adobe Photoshop CS4 and Adobe Illustrator CS4 software packages. 2.4 Results The 679-AA Mutation Selectively Blocks Tyrosine Phosphorylation on a Subset of EGFR Residues in Reconstituted EGFR-Null Cells The overall goal of these studies was to determine the role of EGFR during the early stages of cell migration when cells form initial contacts with FN. Initial studies were carried out using human EGFR expressed in NR6 mouse fibroblastic cells which lack endogenous receptor (29). This heterologous expression system has been used 47

49 previously to study effects of EGFR on cell morphology and motility ( ). NR6- derived cells are also suitable for studies with FN since they express both major forms of FN receptors (α 5 β 1 and α v β 3 ) (113). In addition to wild-type EGFR (WT-EGFR), cells reconstituted with EGFR (679-AA) were examined since this mutant had already yielded novel insights to ligand-induced EGFR activation (6,27). Reconstituted cell lines were selected with equivalent levels of total cellular and cell surface EGFR expression based on immunoblotting and flow cytometry, respectively, using human-receptor specific antibodies (Figure 2.1B-C). Cells were also incubated with FITC-conjugated β1 or β 3 integrin antibodies and subsequently analyzed by flow cytometry to determine whether ectopic EGFR expression alters basal trafficking of FN integrins. All three cell lines expressed similar cell surface levels of both FN receptors with β1 integrin expression relatively high compared to β3 integrin (Figure 2.1C). Furthermore, both EGFR proteins were biosynthetically stable up to three hours post-adhesion to FN indicating cell adhesion does not route either receptor to lysosomes (Figure 2.1D). In contrast, WT- EGFR but not EGFR (679-AA) was rapidly degraded following ligand stimulation (Figure 2.1E), confirming the original EGFR (679-AA) phenotype (6,27). We next asked whether WT-EGFR and EGFR (679-AA) were activated by FN binding. WT-EGFR was modified on a subset of tyrosine residues that also serve as major autophosphorylation sites after EGF stimulation (Tyr992, Tyr1068, and Tyr1173) (Figure 2.2A). In addition, WT-EGFR was phosphorylated on Tyr845 (Figure 2.2A), a Src-specific substrate located in the activation loop in the tyrosine kinase domain (Figure 1A) (114,115). EGFR (679-AA) however was primarily phosphorylated at Tyr992 in newly adherent cells (Figure 2.2A). Consistent with reports in the literature (33,116), 48

50 EGFR- and Src kinase inhibitors (AG1478 and PP2, respectively) interfered with FNinduced tyrosine phosphorylation of WT-EGFR and EGFR (679-AA) (Figure 2.2B). Furthermore, neither EGFR protein was activated by cell adhesion to collagen (Figure 2B) that also binds β1 integrin (22), confirming that EGFR trans-activation is FN-specific in NR6 mouse fibroblasts. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase involved in many aspects of integrin-mediated signal transduction including cell spreading, migration, and survival (117). Activation of FAK by integrin clustering leads to autophosphorylation at Tyr397. We demonstrated that FAK was phosphorylated at Tyr397 as a parallel readout of integrin activation in all three cell lines (Figure 2.2C). The multifunctional adaptor protein p130cas is a prominent Src substrate that binds EGFR during FN-induced cell adhesion (118). The p130cas protein underwent rapid tyrosine phosphorylation in all three cell lines confirming that endogenous Src is activated independent of EGFR expression (Figure 2.2D). Furthermore, p130cas was phosphorylated on the same set of tyrosine residues in all cell lines, indicating it forms similar molecular complexes (Figure 2.2D). Altogether these data suggest that 679-AA is a dominant inhibitory mutation that selectively modifies EGFR-specific cell adhesion responses without affecting other FN-induced pathways. EGFR Enhances Membrane Protrusive Activity on FN Preliminary studies indicated that all three cell lines were maximally spread on FN by 20 min (Figure 2.3A). Cells adhered for 20 min (Figure 2.4A-C) or 1 h 49

51 (Supplementary Figure 2.3B) were stained with rhodamine-phalloidan to determine the effect of FN on morphology and F-actin cytoskeleton. Cells were co-stained with an antibody to vinculin, an adapter protein which localizes to integrin-mediated cell matrix adhesions (119). The three cell lines displayed a spectrum of phenotypes that correlated with EGFR expression. EGFR-null cells exhibited a typical fibroblastic elongated shape with abundant parallel-ordered ventral actin stress fibers tethered at each end by prominent vinculin-positive focal adhesions (Figure 2.3B, Figure 2.4A,). Cells with WT-EGFR had prominent curved stress fibers, a network of cortical actin filaments abutting the plasma membrane, and multiple filopodial extensions around the entire periphery of the cell (Figure 2.3B, Figure 2.4B). Vinculin was present on filopodial actin shafts in addition to the cell periphery (Figure 2.4B). Cells with WT-EGFR were also imaged by time-lapse microscopy starting approximately 10 min after FN seeding (Supplementary Videos A, B). Figure 2.4D-E shows sequential images of two cells with prominent filopodia that grow by extending from the cell periphery over a period of 60 to 120 seconds. Cells with EGFR (679-AA) also lacked prominent ventral stress fibers. However in contrast to WT-EGFR, cells with the mutant receptor had broad lamellipodial protrusions giving them a more rounded appearance, a dense ring of branching actin filaments associated with small dot-like vinculin-enriched focal adhesions located at the lamellum-lamellipodia border, and a sparse branching actin network in the lamellum (Figure 2.3B, Figure 2.4C). Morphometric analysis showed that cells with WT-EGFR had a statistically significant four-fold increase in filopodia/cell compared to EGFR-null cells and a 2 to 3-fold increase compared to cells with EGFR (679-AA) (Figure 2.4F). 50

52 Conversely, the mutant EGFR was associated with a 2 to 3-fold increase in the percentage of lamellipodial perimeter compared to WT-EGFR (Figure 2.4G). To quantify adhesion-induced changes in stress fiber orientation we adapted a previously described Sobel filter algorithm that measures stress fiber alignment of phalloidan-stained actin filaments (105). Individual cells were divided into pixel grids and the angles of stress fibers in each grid were measured and binned to generate histograms of fiber orientation for individual cells (Figure 2.4H - J). The kurtosis (i.e. peakedness) of angle distribution plots was determined for 100 cells and averaged to derive a relative stress fiber alignment index for each cell type. These analyses confirmed that the frequency of parallel-ordered stress fibers was significantly reduced in cells expressing WT-EGFR or EGFR (679-AA) compared to EGFR-null mouse fibroblasts (Figure 2.3C). In contrast to FN, all three cell lines had similar phenotypes on collagen (Figure 2.4K). Collectively, these findings support two main conclusions. First, EGFR regulates either the type or mode of actin stress fiber formation on FN. Second, the 679-AA mutation interferes with a regulatory pathway associated with filopodia formation. The 679-AA Mutation Interferes with FN-Induced RhoA Inhibition Cell adhesion is frequently associated with an initial decline in RhoA activity (107,120). Experiments were carried out to determine whether this was also true in the heterologous EGFR expression system using ELISA-based assays to measure GTPbound Rho family members which were expressed at similar levels in all three cell lines (Figure 2.3D). Similar to EGFR-null cells, cells reconstituted with WT-EGFR exhibited 51

53 a rapid decline in relative GTP-RhoA levels during the early stages of FN adhesion (Figure 2.5A; see Supplementary Table I for unadjusted absorbance data). However, cells with EGFR (679-AA) exhibited steady RhoA activity immediately following FN adhesion (Figure 4A). In contrast, all three cell lines displayed similar patterns of Rac1 (Figure 2.5B) and Cdc42 (data not shown) activities on FN. Previous studies have shown that adhesion-induced inhibition of RhoA is mediated by Src-dependent tyrosine phosphorylation of p190rhogap necessary for its activation as a RhoA GTP-GDP exchange activating protein (GAP) ( ). p190rhogap also forms an active molecular complex with p120rasgap (122,125). In addition to tyrosine phosphorylation and p120rasgap complex formation, p190rhogap must be recruited to plasma membrane in order to be active (30). Since p120rasgap is known to bind autophosphorylated EGFR (126,127), we hypothesized it might serve as a molecular link between FN-activated EGFR and p190rhogap. Coimmunoprecipitation studies revealed that p190rhogap formed a molecular complex with p120rasgap in all three cell lines on FN (Figure 2.5C). While p120rasgap was also present in a complex with WT-EGFR after FN binding, the 679-AA mutation blocked this interaction (Figure 2.5D). Surface co-immunoprecipitation studies were carried out to determine whether these molecular complexes were present at the cell surface. Newly adhered cells were incubated with EGFR1 antibody which recognizes an extracellular human EGFR epitope, and cell lysates were then incubated with IgG-affinity purification beads to capture EGFR immune complexes specifically formed at the cell surface. Control studies were carried out using an isoform matched monoclonal antibody to the IL2 receptor alpha subunit (IL2Rα). p120rasgap was present in the same 52

54 molecular complex as WT-EGFR but not EGFR (679-AA) (Figure 2.5E). In addition, WT-EGFR formed a complex with β3 integrin, in contrast to EGFR (679-AA) which was associated with β1 integrin at the cell surface (Figure 2.5E). β1 integrin also binds collagen. However, EGFR (679-AA) was not activated by collagen ligation (Figure 2.2B) and cells expressing the mutant receptor did not produce a discernible phenotype on collagen (Figure 2.4K) suggesting the 679-AA mutation interferes with the ability of β1 integrin to activate EGFR signaling. We next tested the hypothesis that WT-EGFR causes p190rhogap to redistribute to membrane protrusions during cell adhesion using confocal microscopy. The p190rhogap protein was concentrated in puncta that were overlapping or interspersed with EGFR-positive puncta along filopodial shafts in cells expressing WT-EGFR (Figure 2.5F; Figure 2.5 G-H shows two additional examples of co-localized EGFR-p190RhoGAP on FN-induced filopodia). In contrast, lamellipodial protrusions were devoid of p190rhogap at the leading edge in cells expressing EGFR (679-AA) (Figure 2.5I). The β3 Integrin-EGFR Pathway Activates p190rhogap in Mouse Mammary Epithelial Cells with Physiological Levels of Endogenous EGFR The NR6 heterologous expression system allowed us to hypothesize a role for EGFR in FN dependent cell adhesion involving activation of the RhoA antagonist p190rhogap. This system also identified EGFR (679-AA) as a dominant inhibitory molecule in this pathway. We next sought to confirm and extend these results in normal murine mammary gland (NMuMG) epithelial cells with physiological levels of endogenous EGFR. NMuMG cells expressing recombinant β3 integrin with a D119A 53

55 mutation that abolishes FN binding and subsequent β3 integrin activation were also examined (102,128). FN binding induced a robust activation of endogenous EGFR compared to cells kept in suspension (Figure 2.6A). Endogenous EGFR was also detected in molecular complexes with β3 integrin and p120rhogap when parental cells were seeded on FN (Figure 2.6B). The D119A mutation abolished FN-induced EGFR tyrosine phosphorylation and also prevented EGFR-p120RasGAP complex formation confirming a role for β3 integrin in the FN-EGFR pathway (Figure 2.6B). It was not possible to test whether endogenous EGFR recruits p120rasgap to the plasma membrane using the cell surface co-immunoprecipitation assay due to lack of a suitable mouse-specific EGFR antibody. However, β3 integrin-egfr-p120rasgap cell surface complexes were readily detectable using a previously published NMuMG cell line expressing recombinant human EGFR at high levels (61) (Figure 2.6C). These studies were carried out using a monoclonal antibody that specifically recognizes human EGFR. We cannot rule out the possibility that the recombinant human receptor forms a heterodimer with endogenous mouse EGFR following integrin engagement. There are several different routes linking integrin ligation with Src activity (129). Both FN integrin receptors activate FAK creating a binding site for Src that subsequently phosphorylates FAK at Tyr576/577 (130). FN binding also induces formation of specific β3 integrin-src complexes that lead to Src activation (131). We therefore utilized β3 integrin (D119A) to distinguish between Src substrates modified downstream of these two alternative modes of Src activation. Src-dependent tyrosine phosphorylation of p190rhogap was greatly attenuated in cells expressing β3 integrin (D119A) compared to control NMuMG cells (Figure 2.6D). D119A also interfered with tyrosine 54

56 phosphorylation of a second FN-dependent Src substrate p130cas (Figure 5E). In contrast, FAK underwent Src-dependent phosphorylation in both cell lines suggesting this pathway is regulated in part by a β3 integrin independent mechanism (Figure 2.6F). The D119A mutation did not completely abolish formation of p120rhogapp190rasgap complexes whose assembly is regulated by both phosphotyrosinedependent and-independent mechanisms (Figure 2.6D) (122). However, our data suggest that the D119A substitution results in loss of phosphotyrosine-dependent complex formation required for p190rasgap activation as a RhoA GAP ( ). We also demonstrated that FN engagement leads to an initial reduction in GTP- RhoA levels by a mechanism blocked by β3 integrin (D119A) expression using the Rho binding domain (RBD) of the Rhotekin protein expressed as a GST-fusion protein (Figure 2.6G) (107). Thus as in the heterologous expression system there was a positive correlation between activation of the β3 integrin-egfr-p190rasgap pathway and reduced levels of GTP-RhoA in cells with physiological levels of endogenous EGFR. The β3 Integrin-EGFR Pathway Induces Filopodial Membrane Protrusions in the NMuMG Cell Model The next set of experiments tested whether β3 integrin-egfr signaling was associated with increased membrane protrusive activity in the NMuMG cell model. The cells were first imaged by time-lapse microscopy after FN seeding (Supplementary Video C). Figure 2.7A shows sequential images from one cell in the video displaying dynamic growth of a filopodial-like membrane protrusion. Parental NMuMG cells as well as cells 55

57 expressing β3 integrin (D119A) were also fixed and co-stained with phalloidin and a vinculin antibody to image actin cytoskeleton and focal adhesion complexes, respectively, on FN. Similar to NR6 cells reconstituted with human EGFR, the NMuMG cells displayed multiple filopodia with vinculin staining associated with filopodial actin shafts along with prominent curved stress fibers and a cortical actin filament network (Figure 2.7B). The D119A substitution was associated with a very different phenotype characterized by prominent dorsal stress fibers attached to vinculin-positive focal adhesions at one end (132) (Figure 2.7B). Cells with β3 integrin (D119A) were also generally devoid of filopodial membrane protrusions and differences between filopodial formation in these cells versus parental NMuMG cells was statistically significant (Figure 2.7C). Parental NMuMG cells were also seeded on FN in the presence of PP2 Src kinase inhibitor, which significantly reduced filopodia formation on FN (Figure 2.7D-E). Collectively these data support a mechanistic link between β3 integrin, Src kinase, and EGFR and filopodial membrane protrusive activity in a cell model with physiological levels of endogenous EGFR. EGFR Regulates FN-Dependent Responses in Normal Breast Epithelial Cells Sensitized for EMT Although not normally present in the adult mammary tissue, greatly increased FN levels have been found both in development as branching morphogenesis occurs, and in various types of mammary tumors (133). Given the importance of EMT in normal development and tumor progression we asked whether EGFR signaling has a significant 56

58 role in normal epithelial cells poised for EMT as they respond to new FN-enriched adhesive environments (133,134). We took advantage of the dominant inhibitory 679-AA mutation identified in the heterologous expression system to test a role for EGFR in the FN-induced phenotype in NMuMG cells. Cells were produced that expressed matched levels of recombinant human WT EGFR and EGFR (679-AA) (Figure 2.7F). These cells were then seeded on FN and stained with phalloidin to examine actin cytoskeleton by confocal microscopy. Expression of either form of the EGFR led to increased spread area on FN compared to cells with endogenous receptor (Figure 2.8A). However, filopodia formation was significantly reduced by expression of dominant inhibitory EGFR (679-AA) (Figure 2.7G-H). Cells with WT-EGFR also displayed an elongated mesenchymal-like shape compared to cells with EGFR (679-AA) that were rounded in appearance with broad lamellipodial membrane protrusions (Figure 2.8B-C). We used form factor analysis to quantify these differences in cell shape (135). This value varies from 1 to 0, from perfectly circular perimeters to less rounded perimeters, respectively (136). Elongated cells expressing WT-EGFR displayed a low form factor compared to cells with EGFR (679-AA) which had a relatively high form factor (Figure 2.8C). Altogether these data are consistent with the idea that EGFR expressed at high levels plays a role in changes in epithelial cell shape and motility that occur during EMT in FNrich microenvironments. 2.5 Discussion Prior studies have shown that integrin engagement suppresses RhoA activity (124). Src-dependent phosphorylation of p190rhogap Tyr1105 creates a binding site 57

59 for one of the two SH2 domains in p120rasgap (122,125). This complex then translocates to the cell surface where it can down-regulate RhoA via interactions mediated by pleckstrin homology (PH) and/or Ca 2+ -dependent phospholipid binding (CaLB) domains in p120rasgap (30,137,138). Our data support an alternative mode of p190rhogap membrane translocation involving recruitment of p120rasgapp190rhogap complexes to activated EGFRs (Figure 2.9). Similar to EGF stimulation, activated EGFR physically associates with p120rasgap in response to β3 integrin engagement (139). Furthermore p190rhogap co-localizes with EGFR on membrane protrusions. The association between p120rasgap and EGFR is likely mediated by p120rasgap SH2 domain binding to a phosphorylated tyrosine residue in EGFR (127). EGFR Tyr992 can be excluded as a p120rasgap docking site since it is the only tyrosine phosphorylation event induced by FN binding in EGFR (679-AA) which does not physically associate with p120rasgap. However, we cannot rule out the possibility that EGFR-p120RasGAP association is mediated by an unknown adaptor protein. EGFR may also synergize with pathways that activate p120rasgap PH and CaLB membrane docking sites to enhance p190rhogap activation. Active RhoA localizes to the plasma membrane where it stimulates formation of focal adhesions and stress fibers (140,141). Previous studies have shown that p190rasgap activation correlates with decreased RhoA activity accompanied by a reduction in stress fiber formation and a corresponding increase in membrane protrusive activity (124,142). However data obtained using the heterologous NR6 expression system suggests RhoA down-regulation is not always sufficient to reduce stress fiber formation. It was only when cells were reconstituted with human EGFR that we 58

60 observed a significant reduction in actin stress fibers suggesting EGFR may have a key role in recruiting p190rhogap to the plasma membrane where it can down-regulate RhoA. EGFR expression was also associated with a dramatic increase in filopodia membrane protrusions. Although de novo pathways have been described (143,144), filopodia usually form by gradual convergence of lamellipodial actin filaments that then elongate from their tips (145,146). The dominant inhibitory 679-AA mutation attenuated filopodia formation and was associated with a dramatic increase in lamellipodia. Our data therefore suggest EGFR regulates lamellipodia-filopodia transition and that residues Leu679 and Leu680 have a positive role in filopodia formation. EGFR (679-AA) is also tyrosine phosphorylated by FN adhesion but forms a stable complex with β1 integrin instead of β3 integrin. In addition to blocking p190rhogap activation, we hypothesize that FN-induced β1 integrin-egfr (679-AA) complexes sequester one or more components required for EGFR-dependent lamellipodia-filopodia transition accounting for the phenotype associated with the mutant receptor. However, the mutant receptor is phosphorylated on a single tyrosine residue suggesting FN-induced β1 integrin-egfr (679-AA) complexes probably have restricted signaling capacity. Thus it remains to be seen if the β1 integrin-egfr interaction unmasked by the 679-AA mutation is biologically significant. Leu679 and Leu680 reside in a region of the EGFR kinase domain with considerable conformational flexibility (11). A rare L679F mutation associated with enhanced EGFR activity in some non-small cell lung cancer patients provides further support these residues have a critical role in EGFR function (93). It is conceivable EGFR acquires a native conformation involving these 59

61 residues with a preference for β1 integrin interactions which could lead to distinct biological outcomes. Interestingly the 679-LL motif is not conserved in other EGFR family members suggesting related receptors could evoke distinct cell morphologies and signaling pathways because of preferred binding to different FN receptors (27). The pathway depicted in Figure 7 was also activated in normal mouse mammary gland epithelial cells expressing physiological levels of endogenous EGFR. NMuMG cells with inactive β3 integrin (D119A) displayed prominent dorsal stress fibers and relatively few filopodia compared to parental NMuMG cells confirming a central role for β3 integrin in the FN-EGFR pathway. Furthermore FN-activated EGFR was physically associated with p120rasgap, and p190rhogap was specifically activated downstream of β3 integrin-src signaling. Since the β1 integrin-src pathway has also been linked to p190rhogap activation (147), our data suggest there may be subtle differences in spatial or temporal p190rhogap regulation depending on the specific mode of Src activation. Stress fiber re-orientation has been linked to mechano-sensing at integrin-based focal adhesions (148,149). Our data indicate that EGFR-integrin subtype specific complexes play a role in regulating the type or mode of stress fibers upon FN-dependent adhesion, an important avenue for future study. Previous studies have shown that β3 integrin promotes activation of Src that has been primed through disruption of intra-molecular interactions between a COOH-terminal phosphotyr527 residue and the Src SH2 domain that fold Src into an inactive auto-inhibitory conformation (150). Src auto-inhibition can be alleviated by phosphotyr527 dephosphorylation or interaction with a phosphotyrosine residue in another molecule which has a higher affinity for the Src SH2 domain than phosphotyr527 (151). In addition to p190rhogap, the D119A substitution also 60

62 attenuated Src-dependent phosphorylation of the p130cas adaptor protein. Given its involvement in many aspects of adhesion-dependent cell behavior (118), we cannot exclude the possibility that p130cas also contributes to β3 integrin-egfr signaling. Interestingly p130cas has been implicated in Src priming by recruiting protein tyrosine phosphatase 1B capable of dephosphorylating phosphotyr527 to p130cas-src complexes (152). It is conceivable that EGFR also contributes to Src priming. Mammary gland is an important model of mammalian branching morphogenesis (76). Studies employing 3D Matrigel cultures of primary mammary epithelial cells indicate branching morphogenesis is driven by collective cell migration without formation of leading actin-based membrane protrusions (153). FN appears to have only a moderate effect on mammary gland branching morphogenesis, specifically retarding mammary epithelial cell proliferation (154). However, FN undergoes a threefoldincrease in expression between puberty and sexual maturity and remains high during pregnancy and lactation (155). Furthermore, FN has a critical role in alveologenesis during pregnancy (154). Thus β3 integrin-egfr may regulate formation of filopodia in certain differentiated epithelial cells during post-natal mammary gland development independent of EGFR ligand. The β3 integrin-egfr pathway may also have a critical role in human breast cancer since FN is an important component of the tumor microenvironment (156). Mouse mammary epithelial cells with elevated EGFR expression are associated with formation of β3 integrin-egfr-p120rasgap complexes at the cell surface, abundant filopodia, and a shift form epithelial towards mesenchymal cell shape downstream of FN binding. 61

63 Previous studies have shown that elevated EGFR expression transforms these cells and also sensitizes them to undergo TGF-β induced EMT (61). Mouse mammary epithelial cells with elevated expression of the mutant EGFR have reduced numbers of filopodia and lack an elongated mesenchymal appearance. TGF-β induces β3 integrin expression (102) suggesting elevated EGFR expression may have a major role in EMT-driven changes in cell shape and motility. In addition, breast cancer has a predilection to metastasize to bone matrix enriched for RGD-containing bone sialoprotein (157,158). Filopodia regulatory proteins such as Arp2/3, fascin, and Ena/VASP are also associated with increased risk of transformation or metastasis in various cancers when they are expressed at high levels, and many invasive cancer cells have abundant filopodia ( ). Triple-negative breast cancers (TNBC) have a particularly aggressive phenotype and EGFR overexpression is associated with poor prognosis in TNBC patients (163). TNBC patients do not generally respond to standard EGFR inhibition therapies that may even exacerbate the disease by selecting for EGFR resistance (164). Further investigation of the β3 integrin-egfr pathway will help identify novel molecular targets that drive metastasis in the tumor microenvironment. These future studies will also provide a rationale for development of new therapies that overcome problems associated with EGFR resistance in TNBC and possibly other cancers characterized by EGFR dysfunction. 2.6 Acknowlegements Chapter 2 resulted in the publication of a first author publication (20).All experiments were performed by Nikolas Balanis except for Figure 2.4H which was performed in 62

64 collaboration with Masaaki Yoshigi from a method adapted from his previous publication (105). Useful discussion as well as NMuMG cell lines were provided by Michael Wendt and Bill Schiemann. 63

65 2.7 Figures Figure 2.1. Reconstituted EGFR Does Not Alter FN Integrin Cell Surface Expression in EGFR-Null Fibroblasts. (A) Model depicting interactions between EGFR cytosolic tail sequences leading to allosteric kinase activation in ligand-induced asymmetric dimers [adapted from (165)]. The C-terminal lobe of one kinase domain (light grey) interacts with the N-terminal domain of a second kinase domain (dark grey) via critical residues located at the dimer interface [depicted as uuuu (unoccupied) or vvvv (occupied)]. The 679-LL lysosomal sorting signal maps to the N-terminal lobe dimer interface (27). Sequences in adjacent juxtamembrane (Jx) regions form an anti-parallel amphipathic helix that facilitates dimerization (14). Tyrosine residues that undergo auto- or Src-dependent phosphorylation (open or black circles, respectively) and activation loops (dashed lines) are also depicted. (B) Equal protein aliquots were immunoblotted with specific antibodies listed in the figure. (C) Histograms for cell surface expression of EGFR and FN integrins detected by flow cytometry (dashed lines) versus background fluorescence associated with secondary antibodies (solid lines). The y-axis represents cell number and x-axis is fluorescence intensity on a logarithmic scale. Flow experiments were repeated at least twice. (D - E) Cells were metabolically labeled with 35 S-labeled amino acids for 30 min followed by a 3-h chase. Cells were either adhered to FN (D) or stimulated with EGF (E) for times indicated. EGFR immune complexes were resolved by SDS-PAGE and radioactive proteins were detected by fluorography. 64

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67 Figure 2.2. EGFR is Trans-activated by FN Binding in Mouse Fibroblastic Cell Models. (A) EGFR immune complexes from suspended and FN-adhered cells were immunoblotted with phospho-specific EGFR antibodies listed in the figure. Bottom panels were immunoblotted with an activation-independent EGFR antibody. (B) Cells were pre-treated with vehicle or EGFR (1 µm AG1478) or Src (10 µm PP2) tyrosine kinase inhibitors for 30 min and then adhered to FN, or were adhered to collagen in the absence of kinase inhibitors. EGFR immune complexes were immunoblotted for phosphotyrosine (top panels). Blots were stripped and re-probed with an EGFR antibody to control for total protein loading (bottom). (C) Cells were adhered to FN for 20-min. Equal protein aliquots were immunoblotted with a phospho-specific antibody to activated FAK (top) or an antibody to total FAK protein (bottom). (D) p130cas immune complexes from cells kept in suspension or adhered to FN for times indicated were immunoblotted with antibodies listed in the figure. (A D) Representative of three (A, B) or two (C, D) independent experiments. 66

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69 Figure 2.3. EGFR regulates cell morphology upon fibronectin (A) Serum-starved NR6 cell lines adhered to FN for various times were stained with Alexa594-WGA and DAPI to quantify cell surface area and identify mono-nucleated cells, respectively. Cell surface area was determined with NIH ImageJ software. Data presented as mean surface area (in arbitrary units or AU)/cell ± SEM (n 100 from 3 independent experiments) as a function of time, *P < EGFR-null cells displayed a statistically significant increase in cell spread area and also achieved maximal spread area more rapidly than cells expressing recombinant EGFR proteins. Spread area rates were 4.13 arbitrary units (AU)/min for EGFR-null cells, 3.67 AU/min for cells with WT- EGFR, and 3.23 AU/min for cells with EGFR (679-AA). (B) Confocal images of NR6 cell lines adhered to FN for 1 h stained with rhodamine-conjugated phalloidin. Scale bars, 10 µm. (C) Bars represent mean kurtosis as an index of stress fiber alignment (mean ± SEM; n = 100 cells) corresponding to data in Figure 3H I. (D) Equal aliquots of total cell protein from NR6 cell lines were immunoblotted with Rho GTPase-specific antibodies. 68

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71 Figure 2.4. EGFR Modulates FN-Dependent Cell Morphology in Mouse Fibroblasts. (A - C) Confocal images of EGFR-null cells (A) and cells expressing WT-EGFR (B) or EGFR (679-AA) (C) adhered to FN for 20 min stained with phalloidin (red) to visualize actin cytoskeleton. Cells were also stained with a vinculin-specific antibody (green). Magnified images of boxed areas are shown to right. Bracket, lamellum. (D - E) Time lapse images of two cells expressing WT-EGFR starting 590 s (D) or 490 s (E) after FN seeding. Arrows point to thin structures consistent with filopodia. Images from Supplementary Videos A and B, respectively. (F G) Bars represent the average number of filopodia/cell (mean ± SEM; n 60 cells) (F), or the average percentage of cell perimeter occupied by lamellipodial network/cell (mean ± SEM; n 60 cells from 3 independent experiments) (G). All measurements were made using phalloidin-stained images obtained 20-min post-adhesion to FN. White bars, EGFR-null cells; light grey bars, cells with WT-EGFR cells; dark grey bars, cells with EGFR (679-AA). Asterisks indicate differences between cells that were statistically significant (P < 0.001) as determined by Student's t test. (H - J) Representative cell images for EGFR-null cells (H) and cells expressing WT-EGFR (I) or EGFR (679-AA) (J) divided into grids for analysis of stress fiber alignment. Stress fibers were color-coded by remapping the fiber angle to the hue angle of HSV color space. This color-coding shows vertical stress fibers in red and horizontal stress fibers in blue. Histograms of fiber orientation are shown to the right of each image. The x-axis represents stress fiber angles from 90 to + 90 degrees. Stress fiber angles in individual grids were binned based on 4 degree intervals. The y-axis represents the relative percentage of grids with a specific bin designation. Mean kurtosis as an index of stress fiber alignment (mean ± SEM; n = 100 cells) corresponding to data in H I shown in Supplementary Figure 1C. (K) Confocal images of EGFR-null, WT- EGFR, or EGFR (679-AA) adhered to collagen for 20 min stained with phalloidin (red) to visualize actin cytoskeleton. (A D, K) Scale bars, 10 µm. 70

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73 Figure 2.5. EGFR Regulates p190rhogap Activity. (A - B) Cells were kept in suspension or adhered to FN for times indicated, and GTPase activities were determined using RhoA (A) or Rac1 (B) specific ELISAs. Data for cells in suspension were set to 1, and data from adherent cells were plotted as percent change relative to 1 ± SEM (n = 3) as a function of time. See Supplementary Table I for unadjusted absorbance data. (C) The p120rasgap immune complexes from cells adhered to FN for 20 min were immunoblotted with a p190rhogap antibody (top). Filters were stripped and re-probed with a second p120rasgap to control for protein loading (bottom). Lysates were also immunoprecipitated with an irrelevant isotype matched IgG to control for non-specific binding. Input lanes are aliquots of total cell protein set aside before immunoprecipitation. (D) EGFR immune complexes from cells adhered to FN for 20 min were immunoblotted with a p120rasgap antibody (top) and re-probed with a second EGFR antibody (bottom). (E) Cells were incubated with an antibody to an extracellular human EGFR epitope or an isotype-matched negative control (IL2Rα antibody) as they were adhering to FN. Cells were harvested 20 min postadhesion and immune-complexes with membrane-exposed EGFRs were immunoblotted with an antibody to p120rasgap. The same blot was stripped and successively reprobed with antibodies to β3 integrin, β1 integrin, and EGFR. Input lanes are aliquots of total cell protein set aside before immunoprecipitation. (C E) Representative of three (C, D) or two (E) independent experiments. (F) Confocal images of cells with WT- EGFR adhered to FN for 20 min stained with phalloidan (green) and antibodies to EGFR (red) and p190rhogap (blue). Magnified images of individual and merged channels for boxed areas are shown to right. (G - H) Images show two additional examples of colocalized EGFR and p190rhogap on filopodial shafts in FN-adhered cells expressing WT-EGFR. (I) Cells with EGFR (679-AA) adhered to FN for 20 min were stained with phalloidan (green) and antibodies to EGFR (red) and p190rhogap (blue). Magnified images of individual channels for boxed areas are shown to right. Arrowheads, lamellipodial cell edge. Scale bars, 10 µm. 72

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75 Figure 2.6. β3 Integrin-EGFR Crosstalk Activates p190rhogap in Normal Mammary Gland Epithelial Cells. (A) Endogenous EGFR immune complexes from NMuMG cells expressing a control EGFP plasmid kept in suspension or adhered to FN for 1 h were immunoblotted with a phosphotyrosine antibody. The same blot was stripped and re-probed with an EGFR antibody. (B) Endogenous EGFR immune complexes from NMuMG cells expressing a control EGFP plasmid or β3 integrin with an inactivating D119A substitution adhered to FN for 1 h were immunoblotted with a phosphotyrosine antibody. The same blot was stripped and re-probed with antibodies to β3 integrin, p120rasgap, and EGFR. (C) NMuMG cells expressing human EGFR at high levels were incubated with an antibody to an extracellular human EGFR epitope or an isotype-matched negative control (IL2Rα antibody) as they were adhering to FN. Cells were harvested 1 h post-adhesion and immune complexes with membrane-exposed human EGFRs were immunoblotted with an antibody to β3 integrin. The blot was successively re-probed with antibodies to p120rasgap and EGFR. (B C) Input lanes are aliquots of total cell protein set aside before immunoprecipitation. (D) p190rhogap immune complexes from cells adhered to FN for times indicated were immunoblotted with a phosphotyrosine-specific antibody. Filters were stripped and re-probed with a p120rasgap antibody and then a p190rhogap antibody to control for protein loading. (E - F) Cells adhered to FN for 1 h were harvested to recover p130cas immune complexes (E) or collect total cellular protein (F). p130cas immune complexes were immunoblotted with a phosphotyrosine antibody (E) and equal aliquots of total cellular protein blots with a phosphotyr576/577 FAK antibody (F). Blots were stripped and re-probed with appropriate activationindependent antibodies. (G) Cells were adhered to FN for 1 h and cell lysates were incubated with glutathione beads alone or glutathione beads loaded with a GST-RBD fusion protein to capture active GTP-RhoA. Bound proteins were immunoblotted with a RhoA-specific antibody (top). Equal aliquots of total cellular proteins were immunoblotted with a RhoA-specific antibody (bottom). Representative of three independent experiments. 74

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77 Figure 2.7. β3 Integrin-EGFR Interactions Induce Filopodia Membrane Protrusions in Normal Mammary Gland Epithelial Cells. (A) Time lapse images of NMuMG cell taken from Supplementary Video C starting 600 s after FN seeding. Arrow points to thin structure consistent with filopodia. (B) Confocal images of NMuMG cells with control EGFP plasmid or β3 integrin (D119A) adhered to FN for 1 h stained with phalloidin to visualize actin cytoskeleton (red) and a vinculin-specific antibody (blue). (C) Bars represent the average number of filopodia/cell (mean ± SEM). White bar, control NMuMG cells (n = 15); grey bar, NMuMG cells with β3 integrin (D119A) (n = 23). (D) Confocal images of control NMuMG cells adhered to FN in the absence or presence of Src kinase inhibitor (10 µm PP2) stained with phalloidin (red). (E) Bars represent the average number of filopodia/cell (mean ± SEM). White bars, control NMuMG cells on FN (n = 28); light grey bars, control NMuMG cells on FN in the presence of PP2 (n = 39). (F) Equal total protein aliquots from NMuMG cells with elevated levels of WT human EGFR or EGFR (679-AA) were immunoblotted with a human-specific EGFR antibody. (G) NMuMG cells with elevated levels of human WT-EGFR or EGFR (679-AA) adhered to FN and stained with phalloidin (red). (H) Bars represent the average number of filopodia/cell (mean ± SEM). White bar, NMuMG cells with WT human EGFR (n = 54); grey bar, NMuMG cells with EGFR (679-AA) (n = 57). (C, E, H) Asterisks indicate differences between cells that were statistically significant (P < 0.001) as determined by Student's t test. (B, D, G) Magnified images of individual channels for boxed areas are shown to right. Size bars, 10 µm. (C, F, H) Representative of two independent experiments. 76

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79 Figure 2.8. EGFR expression regulates cell shape and spreading (A) Serum-starved NMuMG cells with physiological levels of endogenous mouse EGFR which had been transfected with EGFP control plasmid and cells expressing high levels of human WT-EGFR or EGFR (679-AA) were adhered to FN for 20 minutes and were stained with Alexa594-WGA and DAPI to quantify cell surface area and identify mononucleated cells, respectively. Cell surface area was determined with NIH ImageJ software. Data presented as mean surface area (in arbitrary units or AU)/cell ± SEM (n 53), *P < (B, C) NMuMG cells with elevated levels of human WT-EGFR or EGFR (679-AA) adhered to FN for 20 minutes and stained with phalloidan (red). These images are representative of images used to calculate average shape factor/cell (C). White bars, NMuMG cells transfected with an EGFP control plasmid; light grey bars, cells with WT-EGFR cells; dark grey bars, cells with EGFR (679-AA). Asterisks indicate differences between cells that were statistically significant (P < 0.001) as determined by Student's t test. 78

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81 Figure 2.9. Summary Model. EGFRs trans-activated in β3 integrin complexes are modified at multiple autophosphorylation sites as well as the Src-specific substrate Tyr845 located in the kinase activation loop (autophosphorylation and Src substrates in white and black, respectively). β3 integrin engagement is required for Src-dependent phosphorylation of p190rhogap leading to its association with a p120rasgap SH2 domain. The D119A is a mutation in the extracellular cation coordinating site of β3 integrin blocking its binding to ligand. Parenthetically, p120rasgap also has SH3, pleckstrin homology (PH), and Ca 2+ -dependent phospholipid binding (CaLB) domains in addition to a catalytic GAP domain. EGFR recruits p120rasgap-p190rhogap complexes to plasma membrane where they may negatively regulate RhoA activity. The EGFR-p120RasGAP association probably involves SH2 binding to an autophosphorylated EGFR tyrosine residue. However we cannot rule out the possibility this interaction is mediated by an unknown adaptor molecule. 80

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83 Chapter 3 Mutual Cross-Talk between Fibronectin Integrins and the EGF Receptor: Molecular Basis and Biological Significance 3.1 Summary Extension of the plasma membrane is one of the first steps in cell migration. Understanding how cells choose between various types of membrane protrusion enhances our knowledge of both normal and cancer cell physiology. The EGF receptor is a paradigm for understanding how transmembrane receptor tyrosine kinases regulate intracellular signaling following ligand stimulation. Evidence from the past decade indicates that EGF receptors also form macromolecular complexes with integrin receptors leading to EGF receptor transactivation during cell adhesion. However, relatively little is known about how these complexes form and impact cell migration. Our recent work characterized a molecular complex between EGF receptor and β3 integrin which recognizes RGD motifs in extracellular matrix proteins. Complex formation requires a dileucine motif (679-LL) in the intracellular juxtamembrane region of the EGF receptor that also controls whether or not the receptor undergoes Src kinase-dependent phosphorylation at Tyr-845. In contrast to wild-type receptors, mutant EGF receptors 82

84 defective for Tyr-845 phosphorylation form complexes with β1 integrin that also binds RGD motifs. In addition, we have discovered that EGF receptor antagonizes small GTPase RhoA by mediating membrane recruitment of its regulatory GAP p190rhogap. In this article we discuss a potential new role for Src-dependent EGF receptor transactivation in integrin/egf receptor complex formation. We also discuss how our study fits with previous observations linking p190rhogap to RhoA-dependent cytoskeletal rearrangements involved in cell migration, and provide new data that the EGF receptor is compartmentalized to relatively immature zyxin-poor focal adhesions which are the likely site of p190rhogap signaling. 3.2 Allosteric Mechanism for EGFR Activation EGF receptors (EGFR) form molecular complexes with multiple integrin extracellular matrix receptors leading to EGFR transactivation and fine-tuning of adhesion-induced cell signaling responses ( ). How EGFR activation is achieved remains an active area of investigation. Adhesion-activated integrins may sequester EGFRs leading to receptor oligomerization and activation independent of soluble ligand. Alternatively, or in concert with increased receptor density, adhesion-induced Src kinase activity may have a critical role in EGFR transactivation. Src kinase phosphorylates EGFR residue Tyr-845 which is located in the activation loop of the EGFR catalytic domain (169). The corresponding tyrosine residues in other receptor tyrosine kinases are autophosphorylated following ligand stimulation and phenylalanine substitutions significantly impair kinase signaling and downstream signaling. In contrast, EGFR Tyr- 845 phosphorylation is not required for ligand-induced EGFR activation but may instead 83

85 represent a primary mechanism for EGFR transactivation (169). Our recently published results have identified a specific dileucine motif (679-LL) located in the EGFR juxtamembrane (JM) domain that is absolutely required for Tyr-845 phosphorylation during cell adhesion (20). These results were obtained by adhering cells to the extracellular matrix protein fibronectin (FN) containing an RGD sequence (Arg Gly Asp) recognized by several integrin receptors. Our studies were focused on RGDdirected β1 integrin and β3 integrin receptors. EGFR 679-LL was originally identified as a lysosomal sorting signal following ligand-stimulated EGFR down-regulation (27). A 679-AA substitution does not affect ligand-induced internalization but promotes EGFR recycling and enhanced activation of a subset of EGFR tyrosine kinase substrates in recycling endosomes (98). Subsequently, this motif was shown to have a critical role in ligand-induced EGFR activation. An important study by Zhang et al. showed that EGFR is activated by an allosteric mechanism involving formation of an asymmetric dimer in which one kinase domain induces an active conformation in the other (11). This occurs through intra-molecular interactions between the C-terminal lobe of a activator kinase domain and the N- terminal lobe of a acceptor kinase domain leading to its catalytic activation (11). In this model 679-LL resides in the asymmetric dimer interface adjacent to a JM segment that latches the acceptor domain to the activator domain to achieve full activation of EGFR (14,93,170). We speculate that integrin/egfr complex formation triggers conformational changes in the activation loop that make Tyr-845 accessible to Src kinase. 679-AA does not block integrin complex formation per se, but interferes with Tyr-845 exposure preventing EGFR transactivation by Src kinase. 84

86 Our data are consistent with a model in which the 679-LL motif is necessary to form a stable β3 integrin-egfr complex. However, EGFRs form complexes with different integrin receptors in other cell models (33). How then is integrin-egfr complex formation regulated? Based on the resistance of EGFR (679-AA) to Srcdependent Tyr-845 phosphorylation, we hypothesize that Src kinase has a critical role in assembling and/or stabilizing β3 integrin-egfr complexes. β1 and β3 integrins both activate focal adhesion kinase (FAK) leading to activation of Src kinase (171). However, β3 integrins also promote Src activation via direct interactions between Src and β3 integrin cytoplasmic tails (172). Our data therefore raise two interesting possibilities that are not mutually exclusive. First, Tyr-845 phosphorylation may be preferentially mediated by Src kinase activated via direct interaction with β3 integrin cytoplasmic tail. Second, β3 integrin-egfr complexes may be stabilized by interactions between Src and its EGFR substrate Tyr-845. Thus alternative integrin-src activation events may determine the integrin constituents of the integrin-egfr complex. 3.3 Integrin-EGFR Complex Formation and p190rhogap Activation The small GTPase RhoA is a well-known regulator of actin cytoskeletal rearrangements associated with formation of focal adhesions (FAs), actin stress fibers, and filopodia and lamellipodia membrane protrusions (31,142,173). Previous reports have shown that RhoA is antagonized by p190rhogap, the most abundant GAP for RhoA in mammalian cells, during the early stages of cell adhesion (142). To become fully active p190rhogap must be tyrosine phosphorylated by Src kinase, form a complex with p120rasgap, and undergo recruitment to the cell periphery (30,142,174). 85

87 Different membrane targeting mechanisms have an important role in determining the effect of local changes in GTP Rho activity. Our recent study showed that EGFR mediates p190rhogap activation by promoting its membrane recruitment (20). In this context, p120rasgap serves as a bridge, physically linking EGFR and p190rhogap. We found that adhesion-activated EGFRs form a complex with p120rasgap, EGFRp120RasGAP complex formation is associated with a reduction in RhoA activity, and p190rhogap is recruited to the cell periphery where it co-localizes with EGFR (20). Two lines of evidence indicate the β3 integrin/egfr axis is primarily responsible for p190rhogap activation. First, a D119A inhibitory mutation in β3 integrin that blocks RGD binding inhibits EGFR-p120RasGAP complex formation (20). Second, EGFR (679-AA) which forms a complex with β1 integrin does not recruit p120rasgap (20). We also provide new data that wild-type EGFR promotes p190rhogap tyrosine phosphorylation necessary for p120rasgap-p190rhogap complex formation (Figure 3.1A). Similar to cells reconstituted with wild-type EGFR, p190rhogap is tyrosine phosphorylated (Figure 3.1A) and also antagonizes RhoA activity in newly adherent EGFR-null cells (20). Although the mechanism for p190rhogap activation in the EGFR-null cells is not known, previous studies indicate that p120rasgap-p190rhogap membrane recruitment is mediated by FAK following FN engagement (24). Our data suggest the mechanism of p190rhogap membrane recruitment has a dramatic effect on cell morphology (20). The EGFR-null cells form prominent ventral stress fibers (Figure 1B), compared to cells reconstituted with wild-type EGFR that form abundant filopodial extensions (Figure 3.1C). Remarkably, EGFR (679-AA) expression abolishes adhesioninduced p190rhogap tyrosine phosphorylation (Figure 3.1A). Furthermore, RhoA 86

88 inhibition which usually accompanies cell adhesion, is compromised in cells expressing EGFR (679-AA) (20), and this dominant-inhibitory phenotype is associated with formation of broad lamellipodial membrane protrusions (Figure 3.1D). We cannot assess whether complex formation between β1 integrin and EGFR (679-AA) represents a physiological signaling unit or an experimental artifact due to the introduction of the dialanine substitution. However, these results raise the possibility that EGFR may inhibit certain adhesion-induced responses. Overall our results have uncovered an unexpected role for EGFR in p190rhogap activation during formation of membrane protrusions involved in cell migration (20). They also underscore the critical role p190rhogap membrane recruitment has on allowing cells to switch between different types of cytoskeletal rearrangements. 3.4 p190rhogap Activity and Focal Adhesions Upon engagement of extracellular matrix, integrins cluster and recruit various anchor/adapter and signaling proteins to help form FAs (175). Adapter proteins such as p130cas, vinculin, α-actinin, and zyxin provide links to the actin cytoskeleton, allowing for the formation of tension necessary to change cell morphology (175). The RhoA antagonist p190rhogap is also localized to FAs during cell adhesion (24). FAs are formed by sequential recruitment of different anchor proteins that are involved in binding the actin cytoskeleton to the membrane (176,177). Our recent studies showed that EGFR expression is associated with altered distribution of vinculin, one of the first proteins recruited to newly formed FAs, when cells are adhered to FN (20,31). Here we extend 87

89 these studies by examining additional FA proteins, the actin binding protein α-actinin and the anchor protein zyxin, which serve as markers for mid and late stage FAs, respectively [17]. α-actinin is concentrated in elongated FAs at stress fiber tips in EGFR-null mouse fibroblasts (Figure 3.1B). Similar to vinculin, α-actinin is enriched at the base of filopodial actin filaments in cells with wild-type EGFR (Figure 3.1C) (20). Cells with EGFR (679-AA) display a distinct α-actinin staining pattern characterized by small puncta at the cell periphery along with periodic staining in the dense cortical actin region and the sparse actin network in the lamellum. The late stage FA marker zyxin is recruited to FAs at stress fiber tips in EGFR-null mouse fibroblasts (Figure 3.2A). In contrast, cells expressing either wild-type or mutant EGFR were characterized by a statistically significant reduction in zyxin incorporation at peripheral adhesion complexes compared to EGFR-null cells (Figure 3.2B - D). Altogether our data suggest EGFR is compartmentalized to relatively immature FAs during cell adhesion (Figure 3.3A). As FAs mature they grow in size and incorporate additional constituent molecules (178). Immature FAs are smaller in size and relatively short-lived. This may explain why EGFRs form complexes with β3 integrin since α v β 3 is relatively enriched in zyxin-negative FAs compared to other RGD directed integrin receptors (177) (Figure 3.3A). β3 integrin/egfr compartmentalization could serve two purposes. First, it may confine EGFR-dependent p190rhogap activation to relatively immature FAs. Second, compartmentalization may contribute to the reduction in stress fiber formation. Zyxin is recruited to FAs under increased tension (149). Zyxin also senses mechanical load and enhances actin polymerization at FAs contributing to stress fiber growth (149,179,180). Membrane p190rhogap recruitment to mature FAs is 88

90 probably mediated by FAK in EGFR-null cells [11] (Figure 3.3B). Thus EGFR may reduce stress fiber formation by driving a switch in how p190rhogap is activated. 3.5 Perspectives Our work raises several interesting questions for future investigation. The first question relates to how EGFR chooses to interact with different RGD directed integrin binding partners. Our data suggest integrin/src cross-talk may have a significant role in this selection process. It has also been shown that the p130cas signaling adaptor is required for EGFR complex formation with integrins recognizing RGD motifs (33), and we suspect additional adaptor proteins have yet to be identified. Integrin/EGFR complex formation and its impact on adhesion-induced signaling have traditionally been viewed as distinct processes. We propose that these processes are intimately linked and that regulation is bidirectional. The ability of EGFR to switch integrin binding partners is probably important in allowing cells to parse and adjust to extracellular cues in a physiological context. Data from this article suggest that EGFR/integrin complexes are resident in immature FAs which have a higher rate of turnover compared to mature FAs (175). Because of their constant turnover immature FAs can rapidly sample and adjust to the extracellular environment. Rapid turnover is also necessary for cell migration. Thus immature FAs represent an ideal platform to integrate information from extracellular cues to regulate formation of membrane protrusions. We also suspect interactions with different RGD-containing extracellular matrix proteins may influence formation of distinct integrin/egfr complexes. As in vivo environments are seldom homogeneous in matrix composition, cells undoubtedly integrate signals from many distinct EGFR/integrin complexes simultaneously. How these signals are interpreted and their 89

91 hierarchy in relation to membrane protrusion outcomes is an interesting future direction. We believe these questions will further establish a pivotal role for the EGFR in allowing cells to switch between different mechanisms of membrane protrusion and hence regulate cell migration in response to a dynamic microenvironment. 3.6 Acknowledgements Chapter 3 resulted in a first author publication (21). All experiments were performed by Nikolas Balanis. Both Nikolas Balanis and Cathleen Carlin wrote the manuscript. 90

92 3.7 Figures Figure 3.1. A) EGFR Modulates FN-Dependent Phosphorylation of p190rhogap. FN-dependent adhesion assays were performed as previously described (20). Briefly, cells were adhered to FN for 1 h and p190rhogap immune complexes were analyzed by SDS-PAGE followed by immunoblotting with phosphotyrosine or p190rhogap antibodies. p190rhogap undergoes tyrosine phosphorylation in EGFR-null fibroblasts (left), and cells reconstituted with wild-type EGFR (middle), but not in cells expressing EGFR (679-AA) (right). (B - D) EGFR Modulates α-actinin Distribution. Confocal projections of EGFR-null cells (B) and cells with WT-EGFR (C) or EGFR (679-AA) (D) adhered to FN for 20 min co-stained with phalloidan to visualize actin (red) and α-actinin antibody (green). Magnified images of individual and merged channels for boxed areas are shown to right. Scale bars, 10 µm. 91

93 92

94 Figure 3.2 (A - C) EGFR is Compartmentalized to Zyxin-Poor Focal Adhesions. Confocal imaging was performed as previously described (20). EGFR-null cells (A) and cells with wildtype EGFR (B) or EGFR (679-AA) (C) were adhered to FN for 20 min and co-stained with phalloidan (red) and zyxin antibody (green). Magnified images of individual and merged channels for boxed areas are shown to right. Asterisks indicate zyxin positive FAs. Scale bars, 10 µm. (D) Bars represent the average nuµber of zyxin-positive adhesion complexes 20-min post-adhesion to FN. White bar, EGFR-null cells; light grey bark cells with WT-EGFR; dark grey bar, cells with EGFR (679-AA). Asterisks indicate differences between cells that were statistically significant (P < 0.001) as determined by Student s t test. These results show a significant reduction in zyxin-positive FAs in cells expressing either form of EGFR. 93

95 94

96 Figure 3.3 EGFR Regulates RhoA-Mediated Cytoskeletal Rearrangements by Reconfiguring p190rhogap Activation. (A) β3 intergin/egfr complexes localize to relatively immature FAs where EGFRs recruit p120rasgap/p190rasgap complexes to the cell periphery. (B) FAK binds p120rasgap which facilitates a bridge between FAK and p190rasgap in EGFR-null cells with zyxin-positive mature FAs [11]. This model predicts EGFR modulates cell migration by switching modes of p190rhogap activation and antagonizing GTPase RhoA in relatively immature FAs. 95

97 96

98 Box 3.1 Glossary 679-LL: Dileucine motif in EGFR juxtamembrane domain required for Src kinase-mediated Tyr-845 phosphorylation and β3 integrin complex formation; 679-AA substitutions block these activities. α-actinin: Actin filament cross-linking and bundling protein; commonly used marker for mid-stage focal adhesions. Fibronectin (FN): High-molecular weight glycoprotein of the extracellular matrix with an RGD motif that binds to membrane-spanning integrin receptors. Focal adhesions (FA): Large, dynamic protein complexes at cell surface connecting actin cytoskeleton to extracellular matrix. Focal adhesion kinase (FAK): Cytosolic protein tyrosine kinase recruited to early-stage cell-matrix adhesions which mediates many downstream responses. p120rasgap: A negative regulator of Ras signaling that also forms a complex with p190rhogap contributing to GTPase RhoA inhibition during cells adhesion; may bind directly to activated growth factor receptors via an SH2 domain. p190rhogap: The most prominent GAP for GTPase RhoA in mammalian cells; probably linked to activated growth factor receptors through p120rasgap; antagonizes RhoA during cell adhesion. RGD (Arg Gly Asp): Conserved motif found in a variety of adhesive proteins present in extracellular matrices including fibronectin, vitronectin, and osteopontin; β1 and β3 integrins are RGD-directed adhesion receptors. RhoA: Small GTPase known to regulate actin cytoskeletal rearrangements associated with formation of focal adhesions, actin stress fibers, and filopodia and lamellipodia membrane protrusions. Tyrosine-845: Tyrosine residue residing in the EGFR activation loop which is phosphorylated by Src kinase; not required for ligand-mediated EGFR activation. Src kinase: Non-receptor tyrosine kinase responsible for phosphorylating Tyrosine 845 on the EGFR; present in integrin/egfr complex. Vinculin: Cytosolic protein involved in attaching actin cytoskeleton to plasma membrane; commonly used marker for earlystage focal adhesions. Zyxin: Cytosolic protein involved in actin polymerization at focal adhesions; commonly used marker for mature focal adhesions. 97

99 Chapter 4 Epithelial-to-Mesenchymal Transition Promotes Breast Cancer Progression via a Fibronectin- Dependent Stat3 Signaling Pathway 4.1 Summary It has been previously established that overexpression of the EGF receptor (EGFR) is sufficient to induce tumor formation by otherwise non-transformed mammary epithelial cells, and that the initiation of epithelial-mesenchymal transition (EMT) is capable of increasing the invasion and metastasis of these cells. Using this breast cancer (BC) model, we find that in addition to EGF, adhesion to fibronectin (FN) activates Signal Transducer and Activator of Transcription 3 (Stat3) through EGFR-dependent and -independent mechanisms. Importantly, EMT facilitated a signaling switch from Srcdependent EGFR:Stat3 signaling in pre-emt cells to EGFR-independent FN:Jak2:Stat3 signaling in their post-emt counterparts, thereby sensitizing these cells to Jak2 inhibition. Accordingly, human metastatic BC cells that failed to activate Stat3 downstream of EGFR did display robust Stat3 activity upon adhesion to FN. Furthermore, FN enhanced outgrowth in three-dimensional organotypic cultures via a mechanism that is dependent upon β1 integrin, Janus kinase 2 (Jak2), and Stat3 but not EGFR. Collectively, our data 98

100 demonstrate that matrix-initiated signaling is sufficient to drive Stat3 activation, a reaction that is facilitated by EMT during BC metastatic progression. 4.2 Introduction The extracellular matrix (ECM) plays an integral role in the development and homeostatic maintenance of different organ systems (181). However, the signaling mechanisms that empower cancer cells to aberrantly utilize the ECM remain poorly defined (156). Recent evidence suggests that cooperation between integrins and growth factor receptors alters downstream signaling and may contribute to pathological responses of cancer cells to soluble ligands (166,167). Indeed, it has been shown that integrins form physical complexes with EGFR and Transforming Growth Factor Beta (TGF-β) receptors giving rise to oncogenic signaling patterns and pathological responses to ligands (20,61,102). High levels of EGFR expression within primary mammary tumor are strongly linked to the poor prognosis of human breast cancer (BC) (53,182). Our previous studies indicate that EGFR is capable of transforming normal mammary epithelial cells (61). These findings and others (183) suggest that paracrine EGF signaling functions in concert with the process of epithelial-mesenchymal transition (EMT) to facilitate tumor cell invasion and dissemination (61). Collectively, these studies have led to the clinical evaluation of EGFR inhibitors as potential targeted molecular therapies for BC (53,184). Unfortunately, the results of these trials strongly indicate that targeted inhibition of EGFR does not offer any clinical benefit to BC patients (54). Consistent with this inherent resistance of BC to EGFR inhibition, it was recently established that EGFR 99

101 expression is actually diminished in the later stages of metastasis (185). Interestingly, metastatic breast carcinomas also display enhanced expression and altered distribution of fibronectin (FN) compared to normal breast (1). Along these lines, enhanced expression of FN is a well-established marker of the EMT process (186), and survival analysis of BC patients with high levels of FN expression is predictive for an increased risk of mortality (1). Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that regulates cell proliferation and survival; it also functions as a major player that drives the growth of stem-like BC cells (44,187). STAT3 can be activated by several signaling systems including those stimulated by interleukin-6 (IL-6) (188), Oncostatin M (189), interferons (190), and EGF (191). In light of our recent study that demonstrated the ability of the matrix to directly activate EGFR (20), we hypothesized that FN:EGFR signaling could play a pivotal role in the activation of STAT3 during BC progression. We show herein that FN is readily capable of activating STAT3 in the absence of any soluble ligands, a reaction that is markedly increased in metastatic BC cells. Mechanistically, we demonstrate that EMT leads to pathway switching of STAT3 activation, such that non-metastatic BC cells employ a SRC-dependent EGF:EGFR axis to stimulated STAT3, while their metastatic counterparts switch to a pathway activated by β1 integrin activation via binding to FN, leading to activation of FAK and Pyk2 which phosphorylate JAK2 which subsequently phosphorylates and activates STAT3 (FN:β1 integrin:fak/pyk2:jak2:stat3 pathway). Importantly, interdiction of FN:STAT3 signaling, but not that of EGFR:STAT3, readily prevents the three-dimensional (3D) outgrowth of metastatic BC cells in a culture that mimics the pulmonary 100

102 microenvironment (pulmonary organotypic culture). In summary, our data suggest a mechanism by which metastatic BC cells exploit EMT to circumvent growth factor signaling, and directly engage the ECM to activate critical downstream signaling pathways. 4.3 Materials and Methods Cell lines, cell culture, and reagents Normal mouse mammary gland (NMuMG) epithelial cells and human MDA-MB-231 and MDA-MB-468 BC cells were purchased from ATCC (Mannassas, VA) and cultured as described previously (61,62,192). Mouse NR6 fibroblasts are an NIH-3T3 variant lacking endogenous EGFR expression (29). Construction of NMuMG and NR6 cell lines expressing human WT-EGFR or EGFR with 679,680-LL converted to AA (EGFR-AA) were described previously (20,98). Cells were maintained at 37 C in a humidified atmosphere of 5% CO 2 and 95% air in Dulbecco s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cell lines expressing recombinant proteins were selected and maintained in media supplemented with the appropriate antibiotic [G418 (MP Biomedicals, Solon, OH) or puromycin (Invitrogen, Carlsbad, CA)]. NMuMG cells were stimulated with TGF-β1 (5 ng/ml) to induce EMT as described previously (185). The human MCF10A parental cell line and its increasingly tumorigenic variants T1k and Ca1h were cultured according to published methods (193). Pharmacological inhibitors were as follows (AG1478, target: EGFR, Cayman Pharmaceuticals),(Erlotinib, target: EGFR, Cayman Pharmaceuticals), (PF573228, target: FAK, Pfizer Inc), (PF562271, target: FAK/PYK2, Pfizer Inc), 101

103 (WP1066, target: JAK2, EMD Millipore), (Stattic, target: STAT3, EMD Millipore), (STAT3 inhibitor VII, target: upstream activators of STAT3, EMD Millipore). Cell harvesting and immunoblotting Cells were washed 3 times with PBS supplemented with 5 mm EDTA, 5 mm EGTA, and a phosphatase inhibitor cocktail (10 mm NaF, 10 mm Na 4 P 2 O 7, and 1 mm Na 3 VO 4 ). Cells were lysed with 1% NP-40 in a solution of 50 mm Tris-HCl, ph 7.4, supplemented with 150 mm NaCl, the phosphatase inhibitor cocktail, and a protease inhibitor cocktail (100 µm phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 4 µg/ml pepstatin). Cell lysates were clarified by high-speed centrifugation and protein concentrations were determined by Bradford assay (Bio-Rad; Hercules, CA). Equal aliquots of total cellular protein were resolved by SDS-PAGE and transferred to nitrocellulose filters for immunoblotting using standard methods. Antibody concentrations and suppliers are as follows Cell Signaling Technologies [EGFR(Tyr845),1:1000, #2231; EGFR(Tyr992), 1:1000, #2235; EGFR(Tyr1086), 1:1000, #2220; EGFR, 1:1000, #4267; β3 Integrin, 1:1000, #4702; β1 Integrin, 1:2000, #4706; perk1/2, 1:2000, #4377; Erk1/2, 1:1000, #9102; pstat3, 1:1000, #9145; STAT3, 1:1000, #9132; psmad2, 1:1000, #3108; SMAD2/3, 1:1000, #8685], Harlan Labrotories [EGFR1, 1:50 FACS, custom], BD Bioscience [E-Cadherin, 1:5000, #610181], BD Pharmigen [β1 Integrin Neutralizing, 10 µg/ml, #562219; Fibronectin, 1:5000, #610077], Santa Cruz Biotechnologies [β-actin, 1:1000, sc47778], Sigma Aldrich [β-actin,1:2500, #A5441]. Cell morphometry measurements Cells were permeabilized with 0.5% β-escin in a solution of 80 mm PIPES, ph 6.8, supplemented with 5 mm EGTA and 1 mm MgCl 2 for 5 min and fixed with 3% paraformaldehyde PBS for 15 min as described previously 102

104 (104). Cells were stained with rhodamine-conjugated phalloidin and confocal images acquired with a Zeiss LSM 510 Meta laser scanning microscope (Carl Zeiss MicroImaging, Jenna, Germany) using a 40 Plan Apo NA 1.4 objective and Zeiss LSM software (Carl Zeiss MicroImaging, Jenna, Germany). Filopodia were counted using MetaMorph software (Molecular Devices, Sunnydale, CA). Briefly, individual cells were filtered by determining threshold values for average pixel intensity. The number of filopodia per cell was determined by counting only filopodia with fluorescent intensity above the average intensity following thresholding that crossed the cell edge and were 2 µm. Cell shapes were determined by computing shape factor parameters which measure cell area as a function of cell perimeter (equation 4Aπ/P 2 where A is the cell area and P is the perimeter) as described previously (20,135). This value varies from 0 to 1 for elongated to more rounded shapes respectively (136). Cell biological assays For cell adhesion experiments, cells grown to 80% confluency were serum-starved for 5 h in media supplemented with 0.5% bovine serum albumin (BSA). NMuMG cell populations were serum-starved in DMEM that was also supplemented with insulin (10 µg/ml) and if applicable TGF-β1 (5 ng/ml). Cells were detached from tissue culture plastic with 0.25% trypsin-edta which was inactivated with a two-fold volume of serum-free media supplemented with soybean trypsin inhibitor (0.5 mg/ml; Invitrogen). Cells were allowed to adhere to polystyrene dishes or glass coverslips coated with ECM proteins (10 µg/ml) at a density of cells/mm surface area. Control cells were kept in suspension in polystyrene dishes coated with BSA (10 mg/ml). DNA synthesis was measured by [ 3 H]-thymidine incorporation as previously described (193). Caspase 3/7 activity was quantified using the Caspase 3/7 Glo reagent 103

105 (Promega, Madison, WI), and cell fractionation was performed using a Nuclear/Cytosol Fractionation Kit (Biovision, Milpitas, CA), according to the manufacturers instructions. 3D organotypic growth assays Ninety-six well plates were coated with Cultrex (50 µl/well; Trevigen Inc., Gaithersburyg, MD) and cells were resuspended in DMEM supplemented with 10% FBS and 4% Cultrex (150 μl/well). To assess FN specific growth effects, ninety-six well plates were similarly coated with Cultrex or a 2:1 mixture of Cultrex:FN using a 1 mg/ml FN stock. Luciferase expressing MDA-MB-231 or NMuMG-EGFR cells were resuspended in DMEM supplemented with 2% FBS and 2% Cultrex, or with a 2% solution of a 1:3 Cultrex:FN mixture. Cells were seeded at a density of cells/well. Media was replaced every 4 days and organoid outgrowth was detected by adding D-luciferin potassium salt (Caliper Life Sciences, Hopkinton, MA) to induce bioluminescence which was quantified using a GloMax-Multi detection system (Promega, Madison, WI). Longitudinal cell growth was normalized to an initial reading taken 30 min after seeding as a baseline. Organotypic cultures were also examined by phase-contrast microscopy to assess their morphology. Tumor growth NMuMG cell lines were resuspended in sterile PBS supplemented with 5% Matrigel ( cells/50 µl) and subsequently injected directly into the nipple of 6-week old female nu/nu mice (Charles River, Wilmington, MA) to allow seeding within the mammary ducts. Tumor growth was monitored by digital caliper measurements at the indicated time points using the following equation: volume = (length 2 ) (width) (0.5). In-silico analyses The Cancer Cell Line Encyclopedia contains a repository of log 2 expression data derived from Affymetrix U133A Arrays for 947 unique human 104

106 cancer cell lines. Human BC cell lines were annotated based on literature search for their basal versus luminal BC status (50,194). Expression data for FN was extracted for each cell line using a robust microarray (RMA) algorithm and reconverted from a log 2 to a linear scale as described in (195). GEO Dataset GSE36953 contains expression data using the Affymetrix U133A for MDA-MB-231 cells under various culture conditions. The dataset contained MAS5.0 normalized expression data which was used to determine fold changes between groups. Fold-change in transcript expression was determined by comparing the levels observed in MDA-MB-231 tumors versus those measured in their respective 2D cultured counterparts. Kaplan-Meier plots The Kaplan-Meier plot is an on-line biomarker validation tool that compares the proportional survival of patient groups based on relative biomarker expression using microarray data. This tool was employed to estimate survival probabilities for BC patients split into two groups based on FN gene expression. This analysis was carried out by extracting microarray data for 2878 BC patients and overall survival data for 1027 patients from a database described in (196) using the sole _at probe (214702_at). Statistical analyses Statistical analyses were carried out using an unpaired Student s T-test where P values < 0.05 were considered statistically significant. 4.4 Results FN activates an EGFR:STAT3 signaling axis We previously established that FN and EGFR form a signaling complex coupled to the activation of the RhoA antagonist p190rhogap in newly adherent cells (20). However, the extent to which FN:EGFR 105

107 cross-talk induces other signaling pathways remains unexplored. To address this question, we engineered NR6 cells which lack endogenous EGFR expression to stably express equivalent levels of either wild-type EGFR (WT-EGFR) or an EGFR mutant with a dialanine substitution for Leu679 and Leu680 (EGFR-AA) (6,20,27). Importantly, the EGFR-AA mutant is defective for Tyr845 phosphorylation by SRC, an event that is necessary for STAT3 activation (197). Accordingly, EGF stimulation of WT-EGFR expressing cells elicited robust phosphorylation of EGFR at Tyr845 and STAT3, a reaction that failed to occur in EGFR-AA expressing cells (Figure 4.1A). Importantly, adhesion to FN induced the phosphorylation of Tyr845 in cells expressing WT-EGFR, but not in those expressing EGFR-AA (Figure 4.1B). Furthermore FN-mediated Tyr845 phosphorylation of EGFR was dependent on the kinase activities of EGFR and SRC and was not triggered by the alternative ECM protein laminin (Figure 4.1C). Along these lines, EGFR expression was specifically required for FN-induced activation of STAT3, which contrasts with that of Erk1/2 that was similarly stimulated in control and EGFR expressing cells (Figure 4.1D). Similar to ligand-mediated activation of STAT3, FNmediated STAT3 signaling was sensitive to EGFR and SRC kinase inhibitors (Figure 4.1E). Consistent with its activation and role as a transcription factor, phosphorylated STAT3 underwent nuclear translocation in response to FN exposure, opening the possibility of unique FN-induced STAT3 gene expression profiles (Figure 4.1F). In addition to its nuclear function, cytoplasmic STAT3 also plays a role in regulation of cytoskeleton (43). Accordingly, FN-induced formation of filopodial membrane protrusions was potently blocked by treating NR6-EGFR cells with Stattic, an inhibitor of STAT3 dimerization (198) (Figure 4.1G and 1H). Collectively, these findings show that 106

108 FN is capable of activating the STAT3 signaling system independent of ligand stimulation. STAT3 activation is critical for EGFR-mediated transformation of mammary epithelial cells We recently established that Normal Murine Mammary Gland (NMuMG) cells are transformed by overexpression of EGFR (hereafter referred as NME cells) (61). Interestingly, we show here that overexpression of EGFR-AA failed to transform NMuMG cells (hereafter referred to as NME-AA cells; Figure 4.2A-D). Consistent with these in vivo findings, parental and NME-AA cells formed small differentiated acinar structures when propagated in 3D-organotypic cultures (Figure 4.2E). In stark contrast, NME cells produced much larger filled organoids that are characteristic of transformed cells (199) (Figure 4.2E). This aberrant growth in 3D-culture was normalized by administration of the EGFR kinase inhibitors AG1478 and Erlotinib, as well as by that of two different STAT3 inhibitors: (i) Inhibitor VII, which targets STAT3 pathway members, and (ii) Stattic, which blocks STAT3 dimerization and activation. In contrast, the aberrant growth of the NME cells was not affected by WP1006 (Figure 4.2F), which inhibits JAK2-mediated activation of STAT3. Unlike parental NR6(EGFR-null) fibroblasts (20), the parental NMuMG cells express endogenous EGFR. They readily activate Erk1/2 and Akt in response to EGF (Figure 4.2G and 2H left panels) (185). However, only NME cells displayed robust STAT3 phosphorylation when stimulated with EGF (Figure 4.2G and 2H). Moreover, EGF-stimulated STAT3 activation was dependent on SRC kinase, while Erk1/2 activation was not (Figure 4.2H). Collectively, these data establish that STAT3 is aberrantly activated downstream of Src-dependent EGFR signaling, and suggest that this 107

109 pathway likely plays a critical role in the transformed phenotype of mammary epithelial cells that overexpress this growth factor receptor. Autocrine expression of FN is associated with aberrant STAT3 phosphorylation To further examine the potential role of STAT3 in BC progression, we analyzed the HRASdriven human MCF10A BC progression series, consisting of normal (10A), indolent (T24-HRAS-transformed T1K), and malignant (Ca1h) cell lines (200). Figure 4.3A shows that malignant Calh cells acquire a mesenchymal phenotype characterized by downregulation of E-cadherin and upregulation of FN and β1 integrin as compared to their nonmalignant counterparts. Furthermore, autocrine FN expression in Ca1h cells was highly correlated with constitutive STAT3 activation by an EGFR-independent mechanism (Figure 4.3A and 3B). These findings suggest that EMT-mediated upregulation of FN and β1 integrin may facilitate the activation of the STAT3 in more aggressive BC. EMT selectively drives a pathway switch upstream of STAT3 activation To further examine the role of EMT in EGFR:STAT3 and FN:STAT3 signaling we utilized TGF-β1 to induce a mesenchymal state in the NME cells, a measure we previously observed to be capable of increasing the invasion and metastasis of these cells (61). IL-6 stimulation of STAT3 was unaffected by induction of EMT (Figure 4.4A). In contrast, short- (6 h, 48 h) or long- (4 wk) term exposure of NME cells to TGF-β1 prior to EGF stimulation led to a dramatic reduction in the ability of these cells to activate STAT3 via this pathway (Figure 4.4B-D). Furthermore, ex vivo NME cells harvested from post-emt tumors selectively displayed reduced EGF-mediated STAT3 signaling as compared to their pre-emt counterparts despite the fact that ligand-induced Erk1/2 activation was maintained in both 108

110 cell populations (Figure 4.4E). In sharp contrast, TGF-β1-induced EMT potently enhanced the ability of FN to activate STAT3 (Figure 4.5A). Importantly, EGFR and Src inhibitors that abrogated FN:STAT3 signaling in pre-emt cells had essentially no effect on FN:STAT3 signaling in their post-emt counterparts (Figure 4.5B). Similar to Figure 4.4E, the activation of Erk1/2 by FN was unaffected by the EMT-status of the cells (Figure 4.5A), and was similarly impaired by EGFR kinase inhibition in both cell populations (Figure 4.5B). Concomitant with this enhanced response to FN, STAT3 phosphorylation switched from a pathway regulated by Src and EGFR (Figure 4.5B) to one dependent upon JAK2 (Figure 4.5C). Importantly, this pathway switching sensitized post-emt NME cells to pharmacologic inhibition of JAK2 (Figure 4.5D and 5E). Taken together, these data strongly suggest that EMT programs mediate a pathway switch in STAT3 activation away from EGFR-dependent signaling in pre-emt cells to an EGFRindependent pathway regulated by FN and JAK2 during EMT and the metastatic progression of BCs. FN facilitates STAT3 signaling in metastatic BC cells independent of EGFR Thus far we have shown that FN is capable of activating STAT3 independent of EGFR following induction of EMT (Figure 4.5A and 5B). Therefore, we hypothesized that FN adhesion maintains STAT3 signaling independent of growth factor receptor signaling during metastatic outgrowth. This hypothesis was tested in human MDA-MB-231 BC cells, which were originally derived from a pleural effusion of a metastatic BC patient (201). Interestingly, Src-dependent EGFR signaling failed to elicit STAT3 phosphorylation in MDA-MB-231 cells as compared to the MDA-MB-468 cells, in which EGFR is genomically amplified and therefore highly expressed (Figure 4.6A) 109

111 (202,203). In contrast, IL-6 activated STAT3 via a JAK2-dependent manner in the MDA- MB-231 cells (Figure 4.6B). Consistent with the lack of EGFR:STAT3 signaling, FN adhesion elicited robust STAT3 phosphorylation in MDA-MB-231 cells (Figure 4.6C) by a mechanism that was independent of EGFR and SRC kinase activity (Figure 4.6D). Treatment of the MDA-MB-231 cells with TGF-β1 can enhance their pulmonary metastasis (204). Along these lines, treatment of MDA-MB-231 cells with TGF-β1 led to potent induction of β1 integrin and FN expression (Figure 4.6E). Interestingly, gene microarray data from in vitro basal-like BC cell lines, including MDA-MB-231 cells, exhibit high levels of FN expression as compared to luminal BC (Figure 4.6F). Furthermore, expression of FN is enhanced in MDA-MB-231 primary tumor xenografts versus in vitro cultured cells (Figure 4.6G). Thus, enhanced autocrine FN expression by cells at the primary tumor may provide a local oncogenic niche that not only maintains STAT3 signaling independent of other extracellular factors, but enhances STAT3 signaling in surrounding cells. Accordingly, we found that high FN expression in the primary tumor was linked to poor overall survival in a cohort of 1027 BC patients (Figure 6H) (196). FN:STAT3 signaling is regulated by a focal adhesion kinase-dependent:jak2 pathway in MDA-MB-231 cells We next sought to elucidate the mechanism whereby FN activates STAT3 independent of EGFR. Integrins are transmembrane receptors that sense the ECM and are linked to intracellular signaling modules via the focal adhesion complex (205). Indeed, we previously established that several integrins and focal adhesion complex proteins are upregulated during TGF-β-induced EMT (62,102,193). These findings raise the possibility that an integrin-mediated axis could facilitate 110

112 FN:STAT3 signaling during EMT and metastasis. Accordingly, adhesion to FN elicited robust activation of two related focal adhesion kinases, FAK and PYK2, as well as that of JAK2 (Figure 4.7A). FN-induced STAT3 activation was blocked by treating cells with a small molecule inhibitor PF (PF228) that specifically targets FAK (Figure 4.7B). Use of a related compound, PF (PF271), that targets both FAK and PYK2, was more effective at lower concentrations than PF228, suggesting a preferential role for PYK2 in mediating FN:STAT3 signaling in the MDA-MB-231 metastatic BC cell line (Figure 4.7B). Moreover, FN-mediated JAK2 phosphorylation was also dependent on FAK/PYK2, but independent of EGFR kinase activity (Figure 4.7C). Similar to post- EMT NME cells (Figure 4.5E), FN-induced STAT3 activation was also blocked by the JAK2 inhibitor WP1066 in MDA-MB-231 cells (Figure 4.7D). Consistent with a potential role for acute STAT3 activation in modulating the cytoskeleton (Figure 4.1), administration of JAK2 (WP1066) or STAT3 (Stattic) inhibitors prevented MDA-MB-231 cells from acquiring an elongated mesenchymal morphology when cultured on FN (Figure 4.7E and 7F). In contrast, targeting EGFR kinase activity under these same conditions had little-to-no effect on cell morphology (Figure 4.7E and 7F). Collectively, these findings have delineated a novel FN:FAK/PYK2:JAK2 signaling axis that drives the sustained activation of STAT3 in metastatic BC cells. The FN:STAT3 signaling axis regulates 3D outgrowth of metastatic BC cells The importance of cytoskeletal dynamics (206) and β1 integrin (156,207) in regulating the metastatic outgrowth of BC has been previously established. Therefore, we next sought to identify the impact of inactivating FN:STAT3 signaling in preventing 3D-outgrowth. 111

113 Using a 3D-organotypic culture system to recapitulate the compliance of the pulmonary microenvironment, we found that the outgrowth of MDA-MB-231 cells was significantly enhanced by FN supplementation (Figure 4.8A and 8B). Moreover, supplemental FN enabled MDA-MB-231 cells to transition from branched morphologies to cell-dense 3Dstructures (Figure 4.8A), a phenotype that we previously been linked to enhanced metastatic capacities of BC cells (61,185). Underscoring the importance of β1 integrin in mediating these events, we found that administration of neutralizing β1 integrin antibodies prevented FN-induced 3D outgrowth as compared to an isotype control antibody (Figure 4.8C). In contrast, MDA-MB-231 cells were resistant to EGFR kinase inhibition in the presence of supplemental FN (Figure 4.8A and 8D). Pharmacological inhibition of STAT3 using two mechanistically distinct compounds (STAT3 inhibitor VII and Stattic) completely abrogated 3D outgrowth of MDA-MB-231 cells (Figure 4.8A and 8D). In contrast to NME cells, which are non-metastatic (Figure 4.4H), outgrowth of MDA-MB-231 cells was also blocked by administration of WP1066 to inhibit JAK2 (Figure 4.8A and 8D). Collectively, these data establish a role for a FN:STAT3 signaling module in driving the 3D outgrowth of metastatic BC. Furthermore, our data demonstrate a role for β1 integrin in a complementary STAT3 pathway that employs FN linked to a JAK2:STAT3 signaling network in place of EGFR in late-stage BC. 4.5 Discussion BC can be divided into several genetically distinct subgroups. Clinically, those BCs belonging to the triple-negative (TNBC) subtype comprise ~10-20% of all BCs and are 112

114 unique in their metastatic aggressiveness, increased rate of relapse, and poor overall prognosis (50,208). Unfortunately, TNBC remains a diagnosis of exclusion defined only by their lack of estrogen receptor (ER-α) and progesterone receptor (PR), and by their absence of human epidermal growth factor receptor 2 (HER2) amplification (208). As such, effective targeted molecular therapies against TNBC do not exist. Interestingly, TNBCs often exhibit elevated levels of EGFR expression, which is associated with decreased overall survival (53,209). These findings suggest that administration of EGFR targeted therapies would alleviate TNBC disease progression, a supposition that has not been born out in clinical trials (210). Unlike other carcinomas where EGFR inhibitors are initially highly effective and resistance is acquired through tumor evolution, TNBCs are inherently resistant to EGFR inhibition. The mechanisms that drive this disconnect between the diagnostic and therapeutic efficacy of EGFR in BC remained poorly defined. Our studies demonstrate the ability of FN to activate STAT3, thus contributing to aberrant cell growth within a physiologically relevant tumor microenvironment. These finding are supported by other recent studies demonstrating the ability of the ECM and cytoskeletal dynamics to directly initiate critical signaling pathways (211) and metastatic outgrowth (206). Furthermore, FN is a secreted matrix protein whose production is potently increased upon the induction of EMT. Therefore, our data establish a mechanism by which EMT may act in trans to activate STAT3 and influence the growth and/or metastasis of surrounding tumor cells. Moreover, disseminated tumor cells that have undergone EMT have the ability to establish a metastatic niche through EMT-induced autocrine FN production that supports STAT3 activation and metastatic outgrowth. Indeed, using a BC model in which transformation is initiated by EGFR overexpression, 113

115 we show that EGF-mediated STAT3 signaling is actually diminished following the induction of EMT. Importantly, loss of growth factor responsiveness is associated with a concomitant increase in the ability of FN to stimulate this critical oncogenic pathway. Mechanistically, our data suggest that BCs switch away from SRC:EGFR-dependent STAT3 signaling to a compensatory pathway whereby β1 integrin receptors signal to PYK2/FAK during metastatic progression. Of course the new FN:JAK2:STAT3 signaling pathway that we have elucidated in this study is only one facet of the unique molecular phenotype of metastatic BC cells that is acquired through both genomic and non-genomic means. Other potentially critical factors that may be relevant in TNBC include the interplay between EGFR and insulinlike growth factor receptor (IGFR) signaling systems and production of cell surface effectors such as the subclass of heparin sulfate proteoglycans known as syndecans ( ). Understanding how these different factors are integrated to drive metastatic progression will provide the basis for improved prognostic screening, guide individualized therapeutic choice, and further illuminate clinical resistance to EGFRtargeted therapies in TNBC patients. Somewhat surprisingly, our data also appear to exclude a role for autocrine IL-6 since EMT programs fail to diminish the ability of NME cells to activate STAT3 in response to exogenous IL-6. Other investigators have shown that PYK2 forms a specific molecular complex with JAK2 responsible for activating selective JAK2 signaling responses (63,216). Thus, it is reasonable to assume that FN-activated PYK2/FAK may fulfill a similar role in a JAK2:STAT3 signaling network, which is governed in an EMTdependent manner. Underscoring the importance of this switch in upstream STAT3 114

116 signaling, we show that inhibition of JAK2 had no effect on BC organoid growth prior to induction of EMT. However, once these same cells have undergone EMT pharmacologic inhibition of JAK2 prevents BC outgrowth. These findings are quite remarkable as several recent studies suggest that the process of EMT is strongly linked to the acquisition of a stem-like and drug-resistant state (217). Indeed, our data clearly shows that BC cells acquire the ability to engage the matrix to facilitate cell signaling in the face of EGFR inhibition following EMT. Therefore, our JAK2 findings are quite unique in that they represent the first known example whereby EMT actually sensitizes BC cells to a targeted chemotherapy. These findings support the notion that JAK2 inhibition may be a viable option for treatment of late-stage BC. Accordingly, inhibition of JAK2 is effective in abrogating 3D growth of the human metastatic MDA-MB-231 cells that are resistant to inhibition of EGFR. Interestingly there are already phase II clinical trials addressing the effectiveness of JAK2 inhibition in BC (218). Human MDA-MB-231 cells harbor activating Ras and BRAF mutations that are associated with constitutive MEK- Erk1/2 activation, which could account for EGFR-independent metastatic outgrowth (219). However, the effectiveness of β1 integrin, JAK2, and STAT3 inhibition in decreasing the outgrowth potential of these cells strongly implicates STAT3 as a critical signaling node mediating tumor outgrowth in FN-rich microenvironments even in cells harboring an alternative oncogenic pathway. Collectively, our findings establish a novel form of EMT-mediated pathway switching that engages the tumor microenvironment to facilitate activation of critical signaling pathways. Our data illustrate a switch away from SRC-dependent EGFR:STAT3 signaling downstream of ligand stimulation or FN engagement as tumor 115

117 cells undergo EMT. This is accompanied by a concomitant activation of an alternative pathway whereby FN activates β1 integrin receptors to elicit a FAK/PYK2:JAK2:STAT3 signaling pathway (Figure 4.9). In addition to supporting the use of JAK2 inhibitors to specifically target post-emt and metastatic BCs, our findings also indicate that development of novel strategies to prevent the switch in upstream STAT3 signaling may represent an important first step in re-sensitizing late-stage BCs to EGFR inhibitor therapies. 4.6 Acknowledgements Chapter 4 resulted in a first author publication for Nikolas Balanis of which Michael Wendt was co-author (28). Experimental design was conceived by Nikolas Balanis, Michael Wendt, and Cathleen Carlin. Experiments were performed by Nikolas Balanis, Michael Wendt, and Barbara Schiemann. Mouse and 3D-culture experiments were performed primarily by Michael Wendt and Barbara Schieman in the lab of Bill Schiemann with some assistance from Nikolas Balanis. Experiments involving fibronectin were primarily performed by Nikolas Balanis, those with EGF primarily by Michael Wendt with some exceptions. Writing was performed by Nikolas Balanis, Michael Wendt, Cathleen Carlin, and Bill Schiemann. 116

118 4.7 Figures Figure 4.1. EGFR-dependent STAT3 signaling in a heterologous reconstitution system. A, EGFR-null NR6 cells reconstituted with wild-type human EGFR (NR6-EGFR) or EGFR with a 679-LL dialanine substitution (NR6-EGFR-AA) were harvested under basal conditions ( ) or following a 30-min EGF stimulation (+). Equal protein aliquots were immunoblotted with phospho-specific and total EGFR and STAT3 antibodies. B, NR6- EGFR or NR6-EGFR-AA cells were left in suspension ( ) or adhered to FN for 20 min (+). Equal protein aliquots were immunoblotted with phospho-specific and total EGFR antibodies. C, NR6-EGFR cells pretreated with vehicle, EGFR (1 µm AG1478) or SRC (10 µm PP2) kinase-specific inhibitors and subsequently adhered to FN as in panel A or laminin. Equal protein aliquots were immunoblotted with phospho-specific and total EGFR antibodies. D, Parental and EGFR-expressing NR6 cells were adhered to FN for the indicated amounts of time and equal protein aliquots were immunoblotted with phospho-specific (p) and total (t) STAT3 and Erk1/2 antibodies. E, NR6-EGFR cells were pretreated with the indicated kinase inhibitors and subsequently adhered to FN. Equal protein aliquots were analyzed by immunoblotting with phospho-specific and total protein antibodies for STAT3. F, NR6-EGFR cells were kept in suspension or adhered to FN for the indicated amounts. Cells were subjected to cell fractionation prior to immunoblotting with phospho-specific and total STAT3 antibodies. Cytosolic (Cyt) and nuclear (Nu) fractions were confirmed by immunoblot for GAPDH and Lamin B1, respectively. G, Representative confocal images of NR6-EGFR cells adhered to FN for 20 min in the presence of vehicle or the STAT3 inhibitor Stattic (5 µm) and then stained with phalloidin to visualize the actin cytoskeleton. H, Data for NR6-EGFR cells in G quantified as described in Experimental Procedures. Data are the mean number of filopodia per cell 2 mm in length/cell ± SE. 117

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120 Figure 4.2. STAT3 activation is required for EGFR-dependent transformation of NMuMG cells. A, NMuMG cells were stably transfected to express either wild-type human EGFR (EGFR-WT) or a 679-LL dialanine substitution (EGFR-AA), whose cell surface expression in stable polyclonal NMuMG cell populations was verified flow-cytometry and cells were sorted for equivalently elevated levels of EGFR in indicated (denoted as sorted ). B, Control (GFP), EGFR-WT (NME), or EGFR-AA (NME-AA) expressing NMuMG cells ( ) isolated in Panel A were engrafted onto the mammary fat pad of female nu/nu mice. Shown are all mice from each group 54 days after fat pad inoculation (yellow line denotes outline of mammary lesions). C and D, Control NMuMG cells ( ) expressing GFP, WT-EGFR (NME), or EGFR-AA (NME-AA) were injected into mammary fat pads of female nu/nu mice. Mean tumor size over a 54 day period (C) and mean tumor weight at day 54 (D). Data are mean (± SE) of 5 mice per group. E, Representative photomicrographs of cells propagated in 3D organotypic culture for 10 days. Bar, 200 magnification. Insets, high magnification images of hollowed acinar structures formed by control and NME-AA cells. F, Representative photomicrographs of NME propagated in 3D-cultures in absence (NS) or presence of indicated inhibitors (1 µm each) for 10 days. Bar, 100 magnification. C D, representative data from at least three independent experiments. G, NMuMG-derived cells were stimulated with EGF up to 30 min. H, NMuMG cells were pretreated with SRC (10 µm PP2) or EGFR (1 µm AG1478) kinase-specific inhibitors 2 h prior to EGF stimulation. G H, Equal protein aliquots were analyzed by immunoblotting with indicated antibodies. 119

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122 Figure 4.3. Autocrine FN production correlates with STAT3 activation in a human BC progression model. A, Human MCF10A (10A) cells and tumorigenic variants T1k and Ca1h serum deprived for 24 hours and subsequently stimulated with EGF for 30 m. Equal protein aliquots were analyzed by immunoblot with the indicated phospho (p) and total (t) antibodies. B, Malignant Ca1h cells were treated with the EGFR inhibitor (1 µm AG1478) and analyzed by immunoblot with phospho-specific and total EGFR and STAT3 antibodies. Data are representative of at least 2 independent experiments. 121

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124 Figure 4.4. EMT inhibits EGFR-dependent STAT3 signaling. A, NME cells were serum deprived for 6 h in the presence or absence of TGF-β1 (5 ng/ml) and subsequently stimulated with IL-6 (50 ng/ml) for 30 m. B, NME cells were serum deprived in the presence or absence of TGF-β1 as in panel A and subsequently stimulated with EGF (50 ng/ml) for 30 min. C and D, NME cells were cultured in the presence of TGF-β1 for 48 h (C) or 4 wk (D), to elicit an EMT. These pre- and post-emt cell populations serum deprived for 6 h and stimulated with EGF as in panel B. E, NME cells were cultured ex vivo from mammary fat pad tumors generated by pre- and post-emt cell populations. These cells were then serum deprived and treated with EGF as in panel B. A E, Equal protein aliquots were analyzed by immunoblotting with indicated antibodies and data are representative of at least 2 independent experiments. 123

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126 Figure 4.5. EMT enhances FN:STAT3 signaling via a JAK2-dependent mechanism. A - C, Pre- and Post-EMT NME cell populations (48 h pretreatment with TGF-β1) were seeded on FN up to 4 h (A), for 2 h following a 30-min pretreatment with SRC (10 µm PP2), EGFR (1 µm AG1478), or STAT3 (5 µm Stattic) inhibitors (B), or a 30-min pretreatment with the JAK2 (5 µm WP1066) or STAT3 (10 µm STAT3i VII) inhibitors prior to FN adhesion. Α C, Equal aliquots were immunoblotted with phospho-specific and total protein antibodies as indicated. D, Representative photomicrographs of preand post-emt NME cell populations generated as in Panel A, propagated in 3D organotypic culture for 4 days the absence (NS) or presence of inhibitors to JAK2 (WP1066) or STAT3 (VII). Bar, 400 magnification. E, 3D cell growth in (D) quantified at 4 days post plating by bioluminescence. Data are the mean of two independent experiments completed in triplicate (± SE). 125

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128 Figure 4.6. FN activates STAT3 in human metastatic MDA-MB-231 cells. A, MDA-MB- 231 and MDA-MB-468 cells were serum deprived for 24 h and subsequently stimulated with EGF (50 ng/ml) for 30 min. B, MDA-MB-231 cells were serum deprived for 5 h and either left unstimulated (NS) or stimulated with IL-6 (20 ng/ml) for 30 min in the absence or presence of EGFR (1 µm Erlotinib; 1 µm AG1478), JAK2 (5 µm WP1066), and STAT3 (5 µm Stattic; 5 µm STAT3i VII) inhibitors for 30 min. C and D, MDA-MB-231 cells were left in suspension for 20 min or adhered to FN for up to 4 h (B), or adhered to FN for 2 h in the absence ( ) or presence (+) of EGFR (1 µm AG1478), SRC (10 µm PP2) or STAT3 (10 µm Stattic) inhibitor (C). A D, Equal aliquots were analyzed by immunoblotting with the indicated phospho-specific and total protein antibodies. E, MDA-MB-231 cells were treated with TGF-β1 (5 ng/ml) for 48 h and analyzed by immunoblotting for expression of fibronectin (FN) and β1-integrin (β1-int). Actin served as a loading control. F, Log2 mrna Expression data for FN was collected for 58 human breast cancer cell lines housed in the Cancer Cell Line Encyclopedia and is shown here on a linear scale following Robust Muli-Array Averaging (RMA). The indicated cell lines were annotated as basal or luminal based on review of the literature resulting in 48 classified cell lines. FN is expressed at higher levels in basal-like versus non-basal-like counterparts. G, Fibronectin (FN), and β1 integrin expression are increased in MDA-MB- 231 tumors compared to their in vitro cultured counterparts, while the expression of E- cadherin shows an inverse relationship between xenografts and cultured cells. H, Kaplan-Meier plot correlating FN expression and the probability of survival over a 25-yr period in a cohort of 1027 BC patients split into low (black) and high (red) FN expression groups. The number of patients at risk in the low and high FN expression group is indicated below the x-axis. 127

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130 Figure 4.7. A FN:FAK/PYK2:JAK2 pathway activates STAT3 in human metastatic MDA-MB-231 cells. A, MDA-MB-231 cells were left in suspension ( ) or adhered to FN for indicated times. B, MDA-MB-231 cells were adhered to FN for 2 h in the presence of increasing concentrations of the FAK/PYK2 inhibitor PF (PF271) or FAK-specific inhibitor PF (PF228). C, MDA-MB-231 cells were adhered to FN in the absence ( ) or presence (+) of EGFR (1 µm Erlotinib or 1 µm AG1478) or FAK/PYK2 (1 µm PF271) inhibitor. D, MDA-MB-231 cells were adhered to FN in the absence ( ) or presence (+) of JAK2 inhibitor (1 µm WP1066). A D, Equal aliquots analyzed by immunoblot with the indicated phospho-specific and total protein antibodies. E, Representative confocal images of MDA-MB-231 cells adhered to FN for 2 h in the presence of vehicle or EGFR (1 µm Erlotinib or 1 µm AG1478), JAK2 (3 µm WP1066), or STAT3 (3 µm Stattic) inhibitors. Cells were fixed and stained with phalloidin to visualize the actin cytoskeleton. Size bars, 5 µm. F, Average shape factor/cell calculated from confocal images with values ranging from 0 to 1 for elongated to more rounded shapes respectively (136). *All P-values <

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132 Figure 4.8. FN:STAT3 signaling is required for 3D organotypic outgrowth of MDA-MB- 231 cells. A, Representative photomicrographs of cells propagated in 3D-organotypic cultures for 11 days under control [basement membrane extract (BME)] or FNsupplemented conditions in the absence or presence of EGFR (1 µm Erlotinib or 1 µm AG1478), JAK2 (1 µm WP1066), or STAT3 (1 µm Stattic or 1 µm STAT3i VII) inhibitors. Bars, 100 magnification. B, MDA-MB-231 cells were propagated in 3Dorganotypic cultures with or without supplemental FN and longitudinal outgrowth was quantified by bioluminescence at the indicated time points. C, MDA-MB-231 cells were propagated under FN-supplemented 3D conditions as in Panel B in the absence (IgM) or presence of β1 integrin neutralizing antibody (α-β1-int). 3D cellular outgrowth was quantified using a bioluminescence 11 days post plating. D, 3D cellular outgrowth of the MDA-MB-231 cells cultured in the absence (NS) or presence of EGFR (1 µm Erlotinib or 1 µm AG1478), JAK2 (1 µm WP1066) or STAT3 (1 µm STAT3 Inhibitor VII or 1 µm Stattic) inhibitors. Data in Panels B - D are the mean (± SE) of two independent experiments completed in triplicate. 131

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134 Figure 4.9. STAT3 pathway switching is induced by EMT during breast cancer progression. Early-stage BCs utilize SRC-dependent EGFR signaling to drive STAT3 activation and primary tumor growth. Although EGFR is capable of activating STAT3 downstream of FN engagement, the growth factor-stimulated pathway appears to be the prominent mode in pre-emt cells. Late-stage BCs that have acquired EMT phenotypes utilize β1 integrin receptors to engage the ECM containing aberrant levels of FN thereby activating a FAK/PYK2:JAK2:STAT3 signaling cascade. This pathway may be clinically relevant at the metastatic tumor site and likely contributes to the resistance of BC to EGFR targeted therapies. Targets for pharmacological and immunological inhibitors used in this study are also highlighted. 133

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136 Chapter 5 Mig6 prevents apoptosis and is necessary for pulmonary tumor outgrowth in TNBC 5.1. Results Given its key role in breast cancer progression, we hypothesized that FN adhesion maintains Stat3 signaling by a complementary mechanism that bypasses the FN-EGFR axis to account for inherent resistance to EGFR inhibition in TNBCs. We speculated that TNBC cells may circumvent EGFR activity by up-regulating Mig6 expression. We analyzed a comprehensive array of in-silico derived expression data of human breast cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia (220) to determine if any of the known intrinsic EGFR feedback inhibitors could potentially empower metastatic TNBCs to disable EGFR-dependent Stat3 signaling. The EGFR inhibitory molecule Mig6 was consistently expressed at higher levels in a basal-like TNBC cell lines, including MDA-MB-231 cells, as compared to those classified as luminal breast cancer cell lines (Figure 1.5A). Furthermore, Mig6 expression was elevated in patients diagnosed with basal-like breast cancer relative to patients with a non-basal subtype diagnosis (Figure 1.5B). Therefore, Mig6 expression was depleted in MDA-MB-231 cells using a shrna approach to test its role in EGFR-dependent signaling (Figure 1.5C). As shown in Figure 1.5D, Mig6 knockdown alleviated the block to Tyr845 phosphorylation which is necessary for EGF-dependent activation of Stat3. Furthermore, Mig6 knockdown resulted in a robust increase in Akt activation (Figure 1.5D). 135

137 Unexpectedly, Mig6 depletion did not have a discernible effect on ligand-induced Erk1/2 phosphorylation, suggesting that Mig6 fine-tunes EGFR function by selectively inhibiting downstream pathways that either require Src-dependent Tyr845 phosphorylation or activation of Akt during tumor metastasis. Mig6 has tumor suppressor activity in a number of primary human cancers (65,69). To address its role in metastatic TNBC, we depleted Mig6 expression in luminescent MDA-MB-231 cells whose in vivo growth and metastasis can be tracked by bioluminescent imaging (Figure 1.6A). Surprisingly, the outgrowth of MDA-MB-231 cells in 3D culture was dramatically reduced when Mig6 was depleted using two independent shrnas as compared to cells expressing a control scrambled shrna (Figure 1.6B-C). Morphologic examination of the 3D cultures also revealed the appearance of multiple apoptotic bodies (arrows in Figure 1.6B). Accordingly, Mig6- depleted MDA-MB-231 cells had increased levels of Caspase3/7 activity as compared to control cells, supporting the hypothesis that Mig6 inhibits apoptotic cell death during metastatic progression (Figure 1.6D). Along these lines, we also observed enhanced MDA-MB-231 cell death within the pulmonary microenvironment in nu/nu mice 48 h after cells were injected into the lateral tail vein (Figure 1.6E). Importantly, this enhanced cell death dramatically inhibited pulmonary tumor formation by Mig6-depleted cells (Figure 1.6F and 1.6G). Thus Mig6 is essential in mediating the survival and outgrowth of pulmonary TNBC metastases. Furthermore patients with primary tumors that were classified as basal-like showed almost three fold increase in expression of Mig6 as compared to non-basal like tumors. These exciting findings implicate Mig6 in the control 136

138 of cell survival at the secondary metastatic site, opening the possibility of a new therapeutic target for TNBC Acknowledgements The data presented in Chapter 5 here have not yet resulted in a primary publication. Mouse experiments were performed by Micahel Wendt, western blotting and gene array database searches and manipulations were performed by Nikolas Balanis 137

139 5.3. Figures Figure 5.1. Mig6 is highly expressed in human TNBC cell models. (A) Mig6 mrna expression levels in 48 breast cancer lines Broad-Novartis Cancer Cell Line Encyclopedia that were annotated as basal (red) or luminal (blue) based on literature search (50,194). Green arrows highlight MDA-MB-231 cells. RMA, Robust Multi-array Average. (B) Mig6 mrna expression values in log 2 format for human tumor samples from patients with basal or non-basal breast cancer obtained from GeoData Set GSE (C) Equal aliquots from MDA-MB-231 cells expressing scrambled shrna control (scram) versus two independent Mig6 shrnas immunoblotted with antibodies to Mig6 and actin. (D) Protein aliquots from control and Mig6-depleted cells under basal conditions or following 30 min incubation with EGF immunoblotted with antibodies listed in figure. 138

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141 Figure 5.2. Mig6 depletion induces apoptosis and prevents pulmonary metastasis of MDA-MB-231 TNBC cells. (A) Luciferase-expressing MDA-MB-231 cells stably expressing scrambled (sc) shrna and three independent Mig6 shrnas screened for Mig6 and actin levels by immunoblotting. (B & C) Outgrowth of MDA-MB-231 cells grown under 3D culture conditions and quantified by bioluminescence. Photomicrographs in (B) are representative structures formed by control (scram) and Mig6-depleted (shmig6) cells. Arrows, apoptotic cell morphologies. Data in (C) are average bioluminescent values normalized to values measured immediately after plating (T0) ± SE from two independent experiments carried out in triplicate. (D) Floating cells assayed for Caspase 3/7 activity with luciferase reporter. RLU, relative light units. (E) Control (scram) and Mig6-depleted (shmig6) MDA-MB-231 cells injected into lateral tail veins of nu/nu mice. Longitudinal bioluminescent images from the same mice shown immediately following injection (T0) and indicated time points thereafter. (F) Data are mean pulmonary bioluminescent values ± SE normalized to the injected value (T0) (n = 5 mice/group). (G) Representative images of lungs from mice sacrificed 49 days post tail vein injection confirming that Mig6 depletion blocks pulmonary metastases. 140

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143 Chapter 6 Discussion and Future Directions 6.1 Discussion There are 58 Receptor Tyrosine Kinases (RTKs) in humans, which are subdivided into 20 subfamilies (221). Like many other receptor tyrosine kinases, binding of ligands to the extracellular face activates EGFR by inducing dimerization (221). There are numerous paradigms for how ligand induces RTKs to dimerize (221). In other RTK members such as TrkA, KIT, and FGFR, extracellular ligands either make the sole direct contribution to the dimerization interface (TrkA), or dimerization is induced by some combination of ligand and receptor contacts between the dimers (KIT, FGFR). ErbB1 receptors are unique in that their dimerization interface is mediated solely by interactions between receptors (Figure 5.1). Recent evidence demonstrates that RTKs can be activated independent of ligand. Based on how they associate on their extracellular face ErbB1 members are uniquely poised to mediate ligand-independent modes of RTK activation, as they do not require ligand in their dimerization interface. Integrin-mediated adhesion to ECM molecules such as FN, has been shown to activate the EGFR independent of ligand (17,166). These studies showed that EGFR and integrins form a complex and impact downstream signaling through molecules such a Erk1/2 and Akt (17,33,222). However little was known about the role of EGFR/integrin crosstalk in regulation of other downstream 142

144 signaling molecules such as Stat3. Integrins have previously been shown to regulate cell morphology. The impact of EGFR/integrin crosstalk on cell morphology which had yet to be studied, became the basis for my first two papers. Importantly, EGFR has been shown to be both an important regulator and molecular signature of various cancers such as forms of glioblastoma, lung, colon, and breast cancer. One problem with treatment of many of these cancers with targeted drugs is the tumor s ability to resist drug treatment, either through acquired or inherent resistance. My thesis has provided important information on how inherent resistance to EGFR targeted therapies may be occurring in a subset of breast cancer known as TNBC. The process of EMT/metastatic progression turns off the ability of EGFR to activate Stat3, which is now activated through an integrin pathway. Furthermore, EMT sensitizes cancer cells to Jak2 inhibitors thus providing insight that may inform drug discovery. My research has furthered our understanding of EGFR and integrin crosstalk in both normal and cancer cell physiology, as well as elucidating mechanisms of inherent resistance that may be important in many cancers. We showed first that distinct EGFR/integrin complexes impact the activation of the morphology regulator RhoA through p190rhogap and directly influence the types of membrane protrusions cells form (20). Second we implicated a role for the EGFR in restricting the formation of ventral stress fibers, and in blocking the ability of the actin polymerization molecule zyxin to localize to FAs (20,21). Lastly, we identified a FN mediated EGFR/integrin pathway that activates the Stat3 transcription factor (28). We showed that both EGF-dependent activation and EGFR/integrin crosstalk can activate Stat3. Both these pathways are operant in breast cancers that have not undergone EMT, 143

145 and Stat3 activation is necessary for survival of pre-emt cells. However following metastatic progression, ligand-dependent activation of Stat3 is lost, as is Stat3 activation via EGFR/integrin crosstalk, both due presumably to the upregulation of the EGFR inducible inhibitor protein Mig6. Instead a β1-integrin/pyk2/jak2 pathway activates Stat3 in these cells. Additionally, following EMT these cells become sensitized to Jak2 inhibition in 3D organotypic growth assays. We believe that both Jak2 and Mig6 may represent new therapeutic targets in the treatment of metastatic TNBC. The work of my thesis has provided new information on the importance of ligand-independent mediated activation of the EGFR in both normal and cancer cell physiology and hinted at the possibility of new therapeutic targets. As stated earlier, in normal cell physiology the impact of EGFR/integrin crosstalk on cellular morphology had not been elucidated. To address this question we used the EGFR-null NR6 mouse fibroblast reconstitution model. Using flow cytometry we determined that both β1 and β3 integrins are present in these cells which previously had been implicated in EGFR/integrin crosstalk (166). Following EGF mediated activation the EGFR is internalized via a clathrin-dependent process, trafficked through the endocytic pathway into multi-vesicular bodies (MVBs) and eventually routed to lysosomes where it is degraded (223). Using radiolabeled EGFR we determined that fibronectin-mediated activation of EGFR does not result in EGFR degradation. One possibility is that the EGFR remains at the cell surface following activation, and hence never engages the internalization machinery. Alternatively, the EGFR may internalize but route through recycling endosomes back to the cell surface. This is an important avenue 144

146 of future study as how EGFR is trafficked through endosomes can determine its downstream signals (224). We also found that WT-EGFR is activated following FN-dependent adhesion and is dependent on both the kinase activity of the EGFR and Src. As stated earlier, leucine residues 679 and 680 in the EGFR are in the N-lobe interface necessary for asymmetric dimer formation. Using phospho-specific antibodies we showed that mutation of these residues to alanines blocks autophosphorylation of EGFR on a subset of residues such as Tyr845, while allowing others such as Tyr992 to be phosphorylated. We speculate that this mutation interferes with transfer of conformational change from the receiver kinase to the donor kinase that is necessary for full activation; however it does not completely abrogate this transfer of conformational information. Interestingly the WT-EGFR only binds β3-integrin while the 679-AA mutant strictly binds β1-integrin. WT-EGFR/β3- integrin complexes bind to p120rasgap, a p190rhogap binding partner at the cell surface. It has been known that p190rhogap needs to not only be phosphorylated, but also recruited to the cell surface to become active (24,30). Hence our work presented a novel mechanism of recruitment to the cell periphery via EGFR, other than the already established recruitment methods through FAK (24). We showed that as a result cells expressing the two forms of EGFR display either filopodia or lamellipodia, respectively. We believe that 679-AA mutant may mimic a transient native conformational state of the EGFR, and hence both WT-EGFR/β1-integrin and WT-EGFR/β3-integrin complexes may be possible physiologically. Our work further delineated that only EGFR/β3- integrin complexes could bind to p190rhogap which were then able to inhibit RhoA activation, leading to the formation of filopodia. Our first paper demonstrated that the 145

147 EGFR has an important role in regulation of not only intracellular signaling events following adhesion, but also the types of morphological structures involved in cell migration. The EGFR has been implicated in the regulation of numerous types of cancer. We extended studies using the 679-AA mutant to uncover the role of the EGFR specifically in breast cancer. Using the tumorigenic NMuMG-E cell line, we found that EGFR activation of Stat3 is necessary for primary tumor growth. Following metastatic progression induced by EMT in these cells, or in a human model of metastatic TNBC, EGFR no longer functioned in activating Stat3. We asked how metastasis bypasses the requirement for EGFR activation of Stat3. We found that the Mig6 protein, a EGFR inducible inhibitor, was overexpressed in serial lung metastasis of NMuMG-E cells compared to pre-metastatic cells. Furthermore human TNBC cell lines more highly expressed Mig6 compared to the many other cancer cell line models, and Mig6 is essential for survival following dissemination to the lung. These data provide an explanation for how EGFR function may be blunted, but not how Stat3 is activated in the absence of EGFR. We delineated a β1-integrin/pyk2/jak2 pathway that activates Stat3 in the absence of EGFR function in the human TNBC model. Interestingly this pathway is not active in pre-metastatic NMuMG-E cells, as inhibiting Jak2 in these cells does not block Stat3 or inhibit cell survival. Does Mig6 overexpression following EMT/metastatic progression drive the cell to switch to this alternative Stat3 pathway? Or is Mig6 overexpression an adaption by these metastatic cells to blunt EGFR signaling that is detrimental to their survival. Numerous questions remain as to how Mig6 functions in TNBC. 146

148 My thesis has answered many important questions concerning the role of EGFR/integrin crosstalk in both normal and cancer cell physiology, however it has also brought to light many new questions as well. How does EGFR alter stress fiber formation? Although we have evidence that it regulates the identity of FAs, we do not know how the EGFR performs this function. Is the EGFR directly localized to FAs in the newly adhering cell, and if so by what mechanism does it regulate them? With respect to Mig6 very little is known clinically. Is it a clinically relevant target in TNBC? Does Mig6 restrict EGFR function, or does it have independent pro-survival or anti-apoptotic functions? How does EMT/metastatic progression alter EGFR localization/function? Do the acute functions of Stat3 contribute to cancer progression? These and many other important questions remain. Our work has opened whole new avenues that can contribute to the understanding of normal and cancer cell physiology. Most importantly however are the potential insights into the etiology and progression of TNBC, which is the breast cancer subtype with the poorest prognosis, and the sole breast cancer subtype that has no FDA approved therapies 6.2 Future directions EGFR and the cytoskeleton As we have shown EGFR has a role in regulation of the formation of membrane protrusions and in the formation of actin stress fibers. But, from what compartment does EGFR execute these functions? We hypothesize that the EGFR may be present in FAs to block the recruitment of molecules essential for the formation of ventral stress fibers, such as zyxin. Our current studies have been able to place the EGFR in association with 147

149 FA proteins such as integrins; however, we have not been able to see these molecules colocalized using microscopy. It is possible that EGFR association with the FA is only transient, or is associated with integrins only prior to integrin clustering. To determine if EGFR is localized to FAs, we propose using FRET based methods of localization with EGFR and FA resident proteins β1/β3 integrins; both molecules have been studied using FRET previously (225,226). Another possibility is that EGFR does not need to be in the same complex to regulate stress fiber formation, but regulates FAs from a distance via downstream signaling. If this is the case it may be interesting to determine either through site directed mutagenesis, deletion mutants, or via sirna of EGFR pathway members which are readily available in our lab to determine what EGFR motifs and EGFR pathway members are necessary for these functions. However, multiple phosphorylation sites on the EGFR have redundant function which may complicate both deletion and site directed mutagenesis approaches. EGFR trafficking and cancer It has been shown that EGFR is routed to lysosomes for degradation following ligand stimulation (6). In contrast, we find that EGFR receptor is not degraded following activation via integrins suggesting either a functional difference in the endocytic pathway of the EGFR, or rerouting through non-degradative internal compartments (20). Interestingly, in many cancers defective or altered endocytosis of RTKs is linked to the development of cancer (227). We speculate that EGFR is either maintained at the cell surface or routed through recycling endosomes following activation via integrin crosstalk. 148

150 We propose performing simple staining experiments of newly adhered cells with already established markers of the endocytic pathway to determine if and where the EGFR is routed following adhesion. We hypothesize that is routed through recycling endosomes, however the identity and function of these endosomes is not clear. Alternatively FNinduced activation of the EGFR may not result in internalization; it is possible that receptor is in a different microdomain of the plasma membrane, or is not recruited to sites of internalization such as clathrin-coated pits. As stated earlier these questions are important as in many cancer cells defective endocytosis of RTKs is linked to cancer development. As we have previously shown integrin mediated activation of EGFR is relevant to tumor progression, EMT, and metastasis. We suspect that the specifics of the endocytic pathway following integrin mediated activation may be relevant in developing new targets in cancer therapy. Stat3 and the cytoskeleton One unexplored avenue in our studies is the direct role that Stat3 transcription factor may have on the cytoskeleton. Stat3 blocks the microtubule destabilizing ability of the protein Stathmin (228). As stated earlier we have shown that the EGFR is upstream of Stat3 activity upon adhesion to FN (28). We believe that Stat3 may influence morphological outcomes and migratory capacity in newly adhering cells independent of its transcriptional activity. One future direction to pursue is the role of acute Stat3 function in the adhering cell. Equally important is to understand the contribution of this acute function in disease states such as cancer where cells are characterized by their altered morphologies, and increased migratory capacity. We have already shown that 149

151 Stat3 is an important regulator in breast cancer and our initial studies have also shown that inhibiting Stat3 activity in cells adhering to FN inhibits filopodia formation acutely (28). We hypothesize that Stat3 contributes to cancer progression via both non-genetic and genetic means. To properly address this question we must uncouple genetic and nongenetic functions of Stat3. The Stathmin binding site on Stat3 is not currently known, hence we propose mapping this site using a series of truncation mutants with myc tagged Stat3 as described previously (228). Depending on the site of Stathmin binding it may be possible to create point mutations that block the acute functions of Stat3 while leaving its transcriptional activity intact. With this tool we can address questions directed solely at the acute functions of Stat3 on microtubules and the cytoskeleton, and determine how the acute functions of Stat3 affect both tumorigenicity and the ability of cancer cells to metastasize. Jak2 : Drug Targeting Our research has shown Jak2 inhibition is sensitized following EMT. Whereas Jak2 inhibitors have no effect Pre-EMT, following the EMT process Jak2 inhibits cell growth. Studying Jak2 inhibitors in human metastatic breast cancer patients is an important future direction. There are already Phase II trials in place studying the Jak2 inhibitor Ruxolitinib specifically in breast cancer (218). 150

152 EGFR and Mig6: Clinical Perspectives As stated earlier Mig6 is an inducible inhibitor protein of the EGFR that we find is necessary for cell survival at the secondary metastatic site. However in other forms of cancer it has been implicated as a tumor suppressor. One of the first steps in uncovering the role of Mig6 in TNBC is determining the clinical characteristics of Mig6 in TNBC patients. Currently little is known about Mig6 expression in human tumors as only the very newest gene arrays have Mig6 probesets. There are currently no large repositories of its expression in human TNBC biopsies, thus we propose assaying a large enough data set of both primary and metastatic tumor biopsies in both TNBC and the other 3 subtypes of breast cancer (as a control) for Mig6 expression. It may also be interesting to determine if there are subtypes within that the TNBC cohort which overexpress Mig6 (50). We hypothesize that Mig6 will be overexpressed in TNBC biopsies compared to controls. We have observed that Mig6 is overexpressed in human basal like vs. nonbasal-like breast cancers samples, however this is from a smaller data set with an n=40 (28). Furthermore, our evidence indicates that Mig6 may function primarily in a subtype of TNBC known as the Mesenchymal-Stem-Like subtype (50). Classification of the TNBC biopsies into the various TNBC subtypes would also be informative as to which breast cancer patients Mig6 is most likely operant. Finally we propose studying the regulation of Mig6 at the mrna level to determine what factors are important in inducing its overexpression. 151

153 EGFR and Mig6: EGFR function Another question that remains is why and how does Mig6 restrict EGFR function in TNBC. Although we do not have evidence that Mig6 and EGFR interact directly, Mig6 blocks ligand-induced Tyr845 phosphorylation in MDA-MB-231 cells in the presence of ligand. One possibility is that EGFR signals apoptotically in TNBC. Recent papers have uncovered that when activated EGFRs reside on the limiting membranes of endosomes versus becoming sequestered in intraluminal vesicles they generate apoptotic signals (224). As Mig6 binds active EGFR, it may simply restrict activated EGFRs involved in apoptotic signaling in endosomes. It is known that EGFR signaling is compartmentalized at multiple levels that are highly dependent on cellular context. Receptors are in constant flux between different membrane sub-domains and sub-cellular compartments such as endosomes, each with unique accessibility to signaling substrates ( ). In addition, EGFR is constantly switching between different inactive dimeric conformations that may represent intermediates in the transition between inactive monomers and ligand-bound asymmetric dimers that are catalytically active (232). Thus the simplest explanation for our data is that EMT triggers changes in cellular compartmentalization that trap EGFR in one of these intermediate conformations leading to sustained signaling by a mechanism that is resistant to EGFR therapies (Figure 5.2). Furthermore unique conformations of inactive and active tyrosine kinases are increasingly attractive targets for drug design (233). One possible future direction is to test this hypothesis and try to uncover the conformation of activated EGFR in TNBC. 152

154 Another possibility is that EGFR is compartmentalized differently due to interactions with Sodium-Glucose Transport Receptors (SGLTs). EGFR interacts with SGLT1 in cancer cells, and this interaction contributes to the maintenance of internal glucose level and consequently cell survival even if EGFR kinase activity is blocked (234). One possibility is that Mig6 overexpression maintains a kinase inactive EGFR in these complexes, and hence conserves cells ability to uptake this basic substrate necessary for their survival. Future studies directed at this pathway would focus on the role of Mig6 in the interaction between EGFR and SGLT in TNBC. We would study the impact of either depleting Mig6 or blocking its ability to bind EGFR and determine how these manipulations affect glucose metabolism through SGLTs. EGFR and Mig6: Clinical Targeting Of course the main goal of research into cancers such as TNBC is to find cures or effective treatments. As such Mig6 may represent a new therapeutic target in metastatic TNBC. We propose targeting Mig6 via inhibitory antibodies, small molecule inhibitors, or peptide inhibitors that mimic the Mig6 binding region of EGFR to block Mig6 function. Theoretically to abrogate Mig6 function the most direct design of these molecules would be to interfere with the EGFR binding region (EBR) of Mig6. However our experiments have not ruled out the ability of some secondary function of Mig6 that regulates metastatic cell survival that is unrelated to EGFR. Hence an alternative design would be to use clinically targeted sirna constructs, although various problems with delivery to tumorigenic cells could be raised. However designs are moving forward in Phase II trials with RNAi constructs for other diseases, so it is a possibility. Another 153

155 potential problem is that Mig6 has been shown to be a tumor suppressor in other cancers. As such this may represent a very targeted patient group and hence may not be an economically feasible target. Regardless the correct identification of Mig6 function in the tumor prior to treatment will need to be established before the development of any treatment targeting it. Mig6 may instead serve better as a prognostic biomarker in TNBC. 6.3 Conclusions In normal cell physiology we have shown that the EGFR/integrin crosstalk is an important regulator of membrane protrusion and actin stress fiber formation. Furthermore we have shown that this crosstalk may have a key role in recruiting molecules involved in actin polymerization to the FA. In cancer cell physiology we have delineated a Stat3 pathway necessary for tumorigenesis, and for survival at the secondary metastatic site. Stat3 activation is primarily EGFR dependent before metastatic progression, but following metastatic progression it no longer requires EGFR instead activating through β1integrin/pyk2/jak2 pathway. We have found that following EMT, cells are sensitized to Jak2 function. The EGFR inhibitor protein Mig6 is required for survival in these postmetastatic cells. We believe that Mig6 may also represent a new therapeutic target for TNBC. Our results suggest that blocking Mig6 function in TNBC may result in cancer cell apoptosis. Our research also indicates that Mig6 function in TNBC is different than has been previously reported in other cancers, where Mig6 functions as a tumor suppressor in the primary tumor. As such Mig6 has an evolving role throughout cancer progression, switching between both tumor-suppressor and tumor promoting functions, 154

156 depending on the stage of progression and extrinsic cues from the microenvironment. Thus, it is critically important that the correct therapeutic population is targeted, as this represents a targeted patient group. We believe many EGFR/integrin complexes have yet to be elucidated. Although our studies focused on the roles of β1 and β3 integrins in EGFR crosstalk, numerous other integrin/egfr complexes may form (235,236). Furthermore other ErbB receptors may crosstalk with integrins and each pair may have unique signaling outcomes. Whatever the outcome it has become clear that crosstalk between matrix receptors and ErbB receptors could influence a host of cellular outcomes. This crosstalk is perfectly situated to fine tune extracellular signals based on both cellular microenvironment and intrinsic cellular cues. 155

157 6.4 Figures Figure 6.1 ErbB receptor dimerization interaction is mediated without growth factor. Ligand binds simultaneously to two sites (DI and DII) in the EGFR and through a series of conformational changes exposes a previously buried dimerization arm in Domain II. Before ligand binding, this arm is completely buried within the membrane proximal domain IV. Domain IV orients the dimers in a configuration for maximal activation. Adapted from (221) 156

158 . 157

159 Figure 6.2 (top) EGF ligand activates the receptor by inducing dimerization arms in the extracellular domain followed by the formation of an asymmetric dimer. (bottom) Following TNBC metastatic progression the EGFR is trapped in an altered compartment which signals apoptotically. Mig6 functions to block this receptor from functioning hence abrogated apoptotic signaling. Lateral propagation between receptors is required for activation of Stat3. 158

160 159