Sensitization to death receptor stimuli and anchoragedependent cell death through induction of endoplasmic reticulum stress

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1 Sensitization to death receptor stimuli and anchoragedependent cell death through induction of endoplasmic reticulum stress by Kikanwa Brenda Lydia Hope Anyiwe A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Medical Biophysics University of Toronto Copyright by Kikanwa Anyiwe 211

2 ii Sensitization to death receptor stimuli and anchorage-dependent cell death through induction of endoplasmic reticulum stress Abstract Kika Anyiwe Master of Science Department of Medical Biophysics University of Toronto 211 Activation of the unfolded protein response follows induction of endoplasmic reticulum (ER) stress, resulting in widespread inhibition of protein expression. FLIP protein is particularly sensitive to stresses that perturb protein translation; as such, a reduction in FLIP is likely an early outcome of ER stress. Due to the anti-apoptotic role of FLIP, it is anticipated that potential decreases in FLIP would bring about an increase in sensitivity to death receptor stimuli and anoikis, a form of anchorage-dependent cell death. It was hypothesized that induction of ER stress results in downregulation of FLIP expression, resulting in sensitization of resistant tumour cells to death receptor stimuli and anoikis. From this hypothesis, it was determined that induction of ER stress through treatment of cells with brefeldin sensitized tumour cells to Fas-mediated cell death and anoikis. Moreover, over-expression of FLIP appeared to protect against ER stressinduced sensitization to cell death.

3 iii Acknowledgements The advice, help, and encouragement of many people formed an integral part of the completion of this thesis. I am especially grateful for the tireless support of Dr. Aaron Schimmer, my supervisor, whose wise guidance helped me to become a more skilled and thoughtful student. I greatly appreciate the astute counsel of Dr. Robert Bristow and Dr. Eldad Zacksenhaus, within my thesis committee, who prompted me to consider the work I was doing in new and useful ways. I would like to thank Marcela Gronda, Rose Hurren, Craig Simpson, Reza Beheshti-Zavareh, Dr. Wei Xu, Dr. Mahadeo Sukhai, and all the members of the Schimmer lab, whose proficiency and experience in the lab helped me immeasurably. I appreciate the funding support from the CIHR Banting and Best Canada Graduate Scholarship Master s award. Most importantly, thank you to my family my mom, dad, and brother who made everything possible.

4 iv Table of Contents Introduction 1 Apoptotic cell death 1 Extrinsic apoptosis 1 Tumour necrosis factor receptor superfamily 1 Intracellular transduction of death stimuli 3 FLIP 5 Cross-talk between extrinsic and intrinsic apoptotic pathways 6 Anoikis anchorage-dependent cell death 9 Anoikis proceeds through extrinsic and intrinsic apoptosis 1 Extrinsic pathway and anoikis 1 Bcl-2 family proteins and anoikis 11 Integrins 12 Cadherins 14 Mechanisms of anoikis resistance 15 Oncogenic signalling 15 Epithelial to mesenchymal transition 17 Reactive oxygen species and hypoxia 18 Endoplasmic reticulum stress 19 Unfolded protein response 2 PERK 21 ATF6 22 IRE1 22 Divergent outcomes depend on severity of stress 23 ER stress and cell death 24 Rationale and Hypothesis (I) Endoplasmic reticulum stress and cell death 27 Materials and Methods 3 Results 34 Effects of treatment with brefeldin on Fas-mediated cell death 34 Effects of treatment with brefeldin A on anchorage-independent cell death 36 Expression of markers associated with increased endoplasmic reticulum 38 stress following treatment with brefeldin A Effects of treatment with thapsigargin on Fas-mediated and anchorage- 43 independent cell death Effects of brefeldin A and thapsigargin on clonogenic growth 46 The involvement of FLIP in Fas-mediated cell death and anchorage- 48 dependent cell death Conclusions and discussion 52 Opposing outcomes and complexity of the unfolded protein response 52 Other possible unfolded protein response-related factors 53 Future directions 55 Dose response effects 55 Understanding mechanisms of resistance 55 Role of eif2 in PERK-deficient cells 56

5 Possible involvement of eukaryotic translation factor eif4e 57 Pro-survival function of BI-1 58 Breflate and geldanamycin clinically-relevant compounds 59 Mouse models in vivo effects Rationale and Hypothesis (II) Screen for anoikis sensitizers 61 Materials and Methods 63 Results 65 Screen Results 65 Conclusions and discussion 116 Future directions 116 Works Cited 118 v

6 vi List of Figures Figure A Schematic representation of Fas-mediated apoptosis. Figure 1 Treatment with brefeldin A sensitizes and OVCAR3 cells, but not or HT-29 cells, to Fas-mediated cell death. Figure 2 Treatment with brefeldin A sensitizes and OVCAR3 cells, but not or HT-29 cells, to Fas-mediated cell death as measured by trypan blue exclusion assay. Figure 3 Treatment with brefeldin A sensitizes cells, but not cells, to anoikis. Figure 4 Activation of the PERK pathway of the endoplasmic reticulum stress-induced unfolded protein response following treatment with brefeldin A. Figure 5 Activation of endoplasmic reticulum stress in cells. Figure 6 Treatment with thapsigargin sensitizes cells, but not cells, to Fas-mediated cell death and anoikis. Figure 7 Treatment with thapsigargin sensitizes cells, but not cells, to Fas-mediated cell death and anoikis as measured by trypan blue exclusion assay. Figure 8 Treatment with brefeldin A and thapsigargin sensitizes cells to Fasmediated cell death as measured by colony formation assay. Figure 9 Involvement of Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP) in Fas-mediated cell death and anoikis. Figure 1 Overexpression of FLIP protects against sensitization to Fas-mediated cell death. Figure 11 Screen Results

7 1 Introduction Apoptotic cell death Apoptosis is a process of programmed cell death initiated by cellular damage or the receipt of extracellular anti-survival signals. Apoptosis is characterized by a series of morphological changes including cell shrinkage and blebbing, chromatin condensation, endonuclease-mediated fragmentation of DNA, eventually terminating in cell death (1, 2). Apoptosis can proceed through either the extrinsic death receptor-mediated or the intrinsic/mitochondrial pathway (3, 4). Extrinsic apoptosis is triggered by ligation of death receptor ligands to their cognate receptors, while intrinsic cell death occurs largely due to mitochondrial damage resulting from cytotoxic compounds, cellular stress, or radiation. Deficiencies in apoptosis can lead to inappropriate growth, survival and proliferation of cells, thereby contributing to tumorigenic transformation (1-5). Extrinsic apoptosis Tumour necrosis factor receptor superfamily Extrinsic apoptosis is initiated upon the binding of death-inducing ligands to receptors within the tumour necrosis factor (TNF) receptor superfamily (6-8). Currently, eight members of the TNF receptor family have been identified, including tumour necrosis factor receptor 1 (TNFR1/DR1), Fas (CD95/DR2), tumour necrosis factor receptor-related apoptosis-mediating protein (TRAMP/DR3), tumour-related apoptosisinducing ligand receptor 1 (TRAIL-R1/DR4), tumour-related apoptosis-inducing ligand

8 2 receptor 2 (TRAIL-R2/DR5), tumour necrosis factor superfamily member 21 (TNFRSF- 21/DR6), ectodysplasin A receptor (EDAR/DR7), and nerve growth factor receptor (NGFR/DR8) (7, 9). Of these, the best studied are the TNF receptor, the Fas receptor, and the TRAIL receptors 1 and 2, activated by binding of tumour necrosis factor (TNF), Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL), respectively (8, 9). Apoptotic signalling initiated by both the Fas and TRAIL receptors necessitates formation of the death-inducing signalling complex (DISC), followed by cleavage and activation of downstream caspases (6, 8). Fas receptor-mediated apoptotic signalling has been demonstrated to participate in cell death of tumour cells treated with anti-cancer compounds. An upregulation of both Fas receptor and Fas ligand expression has been observed following treatment of cancer cells with canonical chemotherapeutic agents (1, 11). Moreover, treatment of colon carcinoma cells with cytotoxic compounds induced enhanced assembly of the proapoptotic death-inducing signalling complex, through an increase in expression of Fasassociated death domain protein (FADD) and procaspase 8 (11). In addition to apoptosis induced through Fas signalling, the association of TRAIL with its receptors has been found to yield increased apoptosis of tumour cells. Cancer cells more readily express TRAIL receptors TRAIL-R1 and TRAIL-R2 relative to normal untransformed cells, which tend to express decoy receptors (TRAIL-R3 and R4) that lack functional intracellular death domains, and are incapable of transducing extracellular death-promoting stimuli (8, 12). As such, TRAIL preferentially kills cancer cells rather than normal cells; the comparative insensitivity of normal cells to TRAIL makes this ligand an attractive candidate for therapeutic use. Humanized monoclonal

9 3 agonist antibodies to the TRAIL receptor including mapatumumab and lexatumumab are currently in phase I and II clinical trials for anti-cancer treatment as a single agent, as well as in combination with conventional chemotherapeutic drugs (13, 14). Intracellular transduction of death stimuli death effector domain-containing proteins The transmission of a pro-apoptotic signal through the extrinsic or death receptormediated pathway of caspase activation requires the involvement of proteins within the death domain superfamily. These proteins facilitate assembly of the death-inducing signalling complex following binding of Fas ligand or TRAIL to either the Fas or TRAIL receptor. Proteins within the death domain (DD) family contain death effector domains (DEDs) regions that function in protein-protein interaction. Recognized among the death domain family are seven main proteins Fas-associated death domain protein (FADD), caspase 8, caspase 1, cellular Fas-associated death domain-like interleukin-1- converting enzyme-like inhibitory protein (FLIP) death effector domain containing DNA binding (DEDD), DEDD2, and phosphoprotein enriched in astrocytes 15-kDa (PEA-15) (15, 16). Recruitment of these proteins to the DISC is accomplished through the formation of homotypic death effector domain interactions between adjacent molecules; each domain consists of a conserved six helices arranged in an amphipathic antiparallel conformation (16). These helices form hydrophobic regions the association between DED domains of neighbouring proteins precipitates increased protein aggregation within the DISC, thereby facilitating activation of procaspases (16, 17).

10 4 Binding of Fas ligand to its receptor induces receptor oligomerization; the presence of a death domain within the cytoplasmic portion of the Fas receptor facilitates recruitment of the FADD adaptor protein, which carries both an N-terminal DD and C- terminal DED domain, and is the apical regulator of all TNF-mediated apoptosis. Procaspase 8 then binds with FADD through homotypic DED interactions (15, 16). As procaspase 8 molecules aggregate within the death-inducing signalling complex, autocatalytic cleavage results in self-activation of this initiator caspase, followed by cleavage and activation of downstream executioner caspases, including caspases 3, 6 and 7 (16). Stimulation of the caspase cascade ultimately results in widespread cellular proteolysis, resulting in cell death (17). In contrast to the autocatalytic cleavage model of caspase 8 function, more recent work has demonstrated that activation of this initiator caspase is dependent on dimerization of subunits. The formation of a dimer composed of two identical procaspase 8 molecules is necessary to generate an active complex, while catalytic cleavage not in and of itself an activating step helps to stabilize this active conformation (18). Typically, apoptosis follows formation of the death-inducing signalling complex and activation of caspases. However, two proteins within the DED superfamily c-flip and PEA-15 can bind to FADD in place of procaspase 8, thereby preventing further caspase cleavage and activation, blocking apoptosis (11, 16, 17).

11 5 Fas ligand Fas receptor FADD DISC procaspase 8 caspase 3 Apoptosis Figure A Schematic representation of Fas-mediated apoptosis Fas-associated death domain-like interlekin-1-converting enzyme-like inhibitory protein (FLIP) Cellular FLIP is an endogenously expressed protein that antagonizes apoptosis. FLIP shares homology with caspase 8 due to the presence of tandem death effector domains at its amino terminus; the protein is expressed as both short and long isoforms (FLIP L and FLIP S ) (19). While FLIP S contains two death effector domains, FLIP L contains both DEDs along with a pseudo-caspase domain at its carboxy terminus that is catalytically inactive (2). This similarity in homology facilitates competitive binding of FLIP to the adaptor FADD protein in place of procaspase 8, thereby preventing cleavage and activation of proximal effector caspases. The anti-apoptotic effect of FLIP is largely dependent on the cellular FLIP-to-caspase 8 ratio FLIP most effectively suppresses

12 6 apoptosis where it is overexpressed. Inappropriate expression of FLIP has been associated with disorders characterized by insufficient apoptosis namely cancer (2, 21). PEA-15, although containing a single death effector domain, functions in a manner similar to FLIP through association with FADD and blockage of further caspase activation (15). However PEA-15 mediated caspase-inhibition involves a further phosphorylation step; phosphorylation of PEA-15 at serines 14 and 116 has been observed following TNF induction, and is thought to be carried out by protein kinase C (PKC ), protein kinase B (Akt), or calcium/calmodulin kinase II (CamKII) (22, 23). Cross-talk between extrinsic and intrinsic apoptotic pathways With respect to Fas-mediated apoptotic stimuli, there are two major types of death signalling that can result. Type I signalling proceeds through the canonical death receptor-mediated pathway, with caspase 8 alone functioning as the apical initiating caspase (18). This class of signalling demonstrates robust assembly of death-inducing signalling complexes along with high levels of activated caspase 8. In this pathway, activation of downstream effectors results solely through caspase 8-mediated cleavage of effector caspases, and apoptosis proceeds without involvement of the intrinsic/mitochondrial cell death pathway (18, 19). However, type II signalling predominates in the vast majority of cells, characterized by greater interaction between extrinsic death-receptor and intrinsic/mitochondrial pathways (24). Type II signalling involves less observed DISC formation and caspase 8 cleavage, along with greater

13 7 activation of caspase 9 and various other components typically associated mitochondrialmediated apoptosis (24). Regulation of the BH3-only protein Bid has been observed in response to both death receptor-mediated and mitochondrial signalling, making Bid an additional putative link facilitating communication between these two distinct cell death pathways (25). Traditionally Bid a BH3-only protein functions by binding and inhibiting antiapoptotic Bcl-2 proteins Bcl-2, Bcl-XL, and Mcl-1 through interactions with the BH3 domain of these proteins (26, 27). In this way, Bid helps to sequester anti-apoptotic proteins, and suppress their inhibition of pro-apoptotic Bax and Bak. However, Bid has also been demonstrated to stimulate mitochondrial membrane permeabilization in a more direct way following TNF- receptor activation (28). Activated caspase 8 can cleave fulllength Bid into truncated Bid (t-bid), which translocates to the mitochondria where it, along with Bax and Bak, directly participates in outer membrane pore formation (28, 29). Increased release of cytochrome c, followed by apoptosome formation and caspase 9 activation leads to enhanced cleavage of downstream caspases. The generation of t-bid represents activation of mitochondrial apoptosis secondary to TNF-mediated signalling, whereby typically intrinsic pathways can reinforce apoptosis initiated by death-receptor signalling (29). Intrinsic/mitochondrial apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins (3). Members of this family are both pro- and anti-apoptotic in function; it is the balance in expression between the two opposing groups of Bcl-2 proteins that influences the net survival or death promoting effect (31). Bcl-2 proteins can be classified according to the presence of Bcl-2 homology domains 1-4 short

14 8 motifs of less than twenty residues (32, 33). Anti-apoptotic Bcl-2 proteins Bcl-2, Bcl- XL, Mcl-1, Bcl-w, Bfl1/A-1, and Bcl-B contain homology domains 1-4 and function to maintain cell survival (34, 35). Multidomain proteins Bax, Bak, and Bok have homology domains 1-3 and are pro-apoptotic. Bim, Bad, Bid, Bik, Bmf, Noxa, Puma, and Hrk are BH3-only proteins having only the BH3 domain and function to promote cell death (36, 37). During apoptosis, Bax and Bak translocate to the mitochondria and oligomerize, prompting permeabilization of the outer mitochondrial membrane. Pore-formation within the mitochondrial membrane permits release of apoptogenic factors into the cytoplasm, including cytochrome c and Smac/DIABLO (31, 32). The apoptosome is formed through association of cytochrome c with apoptosis protease-activating factor 1 (Apaf1), followed by recruitment of procaspase 9. Localization to the apoptosome complex causes activation of caspase 9 the main initiator caspase in mitochondrial cell death followed by cleavage and activation of caspase 3 and downstream effectors (38). Activation of caspase 3 represents the convergence of both extrinsic and intrinsic apoptosis in commitment to downstream proteolysis and cell degradation. The integration between the death receptor and mitochondrial cell death pathways has an influence on potential therapeutic approaches concerning death receptor agonists in the treatment of cancer cells. Fas or TRAIL receptor agonists may demonstrate increased apoptotic activity when used in combination with agents that induce cellular stress, due to the capacity of death receptor activation to also trigger mitochondrial-mediated cell death.

15 9 Anoikis anchorage-dependent cell death Normal cells grow within the context of contact with neighbouring cells and the extracellular matrix, which provide scaffolding for correct cell growth and cues regulating survival and proliferation. When cells lose contact with neighbouring cells or the extracellular matrix, they undergo a form of anchorage-dependent apoptosis known as anoikis (39). Anoikis is death resulting from a state of cell homelessness, where apoptosis is induced following a withdrawal of cell-to-cell contacts and adhesion (). Anchorage-dependent cell death is an important feature regulating normal cell growth. Physiologically, anoikis is important for the regulation of limb formation during embryonic development, and for homeostasis of tissues, particularly those with high turnover, such as the intestinal epithelium (41, 42). Anoikis prevents cells from continuing to grow, survive and proliferate in the absence of external cues, thereby preventing tumourigenic transformation (39). Induction of anoikis upon loss of adhesion also precludes the growth of cells within inappropriate extracellular matrices; a cell is unable to proliferate when in an improper extracellular matrix context, which inhibits dysplastic growth (43, 44). Indeed, the ability of a cell to maintain survival in an anchorage-independent fashion is a necessary step in oncogenic transformation during cancer progression (45). It is thought that anoikis resistance permits processes of metastatic spread cells become impervious to the withdrawal of signals reinforcing growth and survival that accompany loss of adhesion (46). Neoplastic cells that are anoikis resistant have the capacity to detach from a primary tumour, migrate through lymphatic and blood vessels, and grow in a distant site without undergoing apoptosis due to an absence of cell-to-cell contacts (47). Since

16 1 anchorage-independent tumour cells can maintain proliferation even within an inappropriate extracellular microenvironment, such cells are more likely to seed a secondary metastatic tumour far from the primary site, facilitating the spread of distant metastatic lesions (45-47). Anoikis proceeds through extrinsic and intrinsic apoptosis Ultimately, as with both death receptor and mitochondrial-mediated apoptosis, anoikis results in the activation of a terminal proteolytic caspase cascade. Factors that regulate both extrinsic and intrinsic cell death have been associated with the progression of anoikis (45). Extrinsic pathway and anoikis Components of the death receptor cell death pathway participate in anchoragedependent apoptosis. Within non-transformed human umbilical vein endothelial cells (HUVEC) and Madin Darby canine kidney cells, anoikis induction through detachment was mediated through cleavage and activation of caspase 8 (48, 49). Detachment of cells from extracellular contacts has been reported to induce an upregulation in the expression of the Fas receptor and FADD proteins, along with downregulation of both FLIP protein and mrna (48). The FADD adaptor protein, which is necessary in extrinsic apoptosis to potentiate tumour necrosis factor-mediated signals, functions in a similar manner to promote anoikis. Inhibiting FADD through dominant-negative overexpression of a nonfunctional mutant isoform yields both reduced caspase 8 activity, and reduction in anoikis following cell detachment (49).

17 11 The involvement of Fas-mediated signalling during anoikis is thought to be a result of changes in morphology following cell detachment (). Loss of adhesion results in rounding of cells; it has been suggested that this change of shape could bring TNF receptors within the cell membrane into closer proximity, triggering downstream activation of Fas signalling. Moreover, anti-apoptotic factors that prevent TNF-mediated death also reduce susceptibility to anchorage-dependent death; cells that overexpress FLIP protein are more resistant to anoikis (48). Bcl-2 family proteins and anoikis Detachment-induced apoptosis has been associated with a reduction in the expression and activity of anti-apoptotic Bcl-2 family proteins, including Bcl-2, Bcl-XL, and Mcl-1 (51, 52). Moreover, loss of adhesion yields increased accumulation of proapoptotic BH3-only proteins, which help facilitate anoikis progression. Loss of outer mitochondrial membrane integrity is prompted by increased activation of Bid, Bad, and Bim, which facilitate Bax and Bak oligomerization, leading to greater pore formation and membrane permeabilization, resulting in the enhanced release of apoptogenic factors from the mitochondria (53). In particular, loss of contact prompts enhanced Bim activity. Bim remains quiescent within cytoplasmic dynein complexes prior to detachment, whereupon Bim is released and translocates to the mitochondria, where it opposes the influence of anti-apoptotic Bcl-2 (54). Bim translocation to the mitochondria is also aided by a cell-contact dependent reduction in proteasomal degradation; loss of anchorage abrogates kinase phosphorylation of Bim, thereby preventing its digestion in the proteasome. Other BH3-only factors, including Noxa and Puma, have also been

18 12 associated with anoikis progression. Bad overexpression in MDCK cells resulted in increased anchorage-dependent cell death (55). Conversely, reduced anoikis following detachment of cells is correlated with stabilization of anti-apoptotic Bcl-2 and Mcl-1 (52). Down-regulation of BH3-only factors relative to Bcl-2 proteins has been linked with anoikis resistance. sirna-targeted downregulation of Bim and Bid was carried out in MCF1A cells and FSK-7 mammary epithelial cells respectively, and yielded a reduction in detachment-induced apoptosis (56). Anoikis resistance has also been hypothesized to occur due to upregulation of factors within the inhibitor of apoptosis (IAP) family of proteins. The family includes X- linked inhibitor of apoptosis protein (XIAP), as well as inhibitor of apoptosis proteins 1 and 2 (IAP1, IAP2), and function to oppose mitochondrially-mediated cell death through inhibition of downstream caspase activity (57, 58). IAP1 and 2 also trigger pro-survival nuclear factor-kb (NF- B) signalling (58). Integrins Integrins help mediate the attachment of a cell to its extracellular matrix through binding to fibronectin and laminin glycoproteins within the extracellular matrix that facilitate adhesion (59). Integrin function is sensitive to activation of growth factor receptors, including epidermal growth factor receptor (EGFR), hepatocyte growth factor (HGF), insulin receptor, platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR) (59). In normal cells, integrin-ecm attachment is necessary in order to maintain cellular survival; within neoplastic cells, integrin-mediated survival signalling becomes dysregulated (). Uncoupling of integrin

19 13 attachment from receptor activation in cancerous cells means that these receptors can be stimulated independently of their cognate ligands (). Loss of integrin attachment has been associated with oncogenic signalling and increased apoptosis in cells that lose anchorage. One of the ways in which cancer cells prevent anoikis through regulation of attachment to the extracellular matrix occurs through a change in the profile of integrin expression, which becomes apparent with more advanced disease progression (59). Various different integrins ( 1 1, 2 1, 3 1, 5 1, 6 1, 6 4, and V 3) affect cell survival, and alterations in patterns of typical expression can be correlated with cancer progression (61). For example, melanoma cancer cells express both V 1 and V 3. V 1 integrin is expressed only in melanocyte precursor cells and early melanoma cancer cells, while V 3 is expressed in later stage melanoma (62). Appearance of the 3 integrin subunit is indicative of more advanced disease. In addition, while V 1 mainly interacts with fibronectin, V 3 has the capacity to bind to a wider range of ECM glycoproteins, facilitating the attachment of melanoma cells to collagen within the dermis, and inhibiting the anoikis which would normally be induced from growth of epidermal melanocyte cells within the dermis (63). As such, integrin modulation helps trigger anoikis resistance and local invasiveness. Association of integrins with extracellular matrix proteins stimulates activity of various oncogenic signalling pathways, including MAPK/ERK, PI3K-Akt, JNK, thereby increasing pro-survival gene expression in part through the fos and Jun transcription factors (64). Integrins also induce growth signalling through the focal adhesion and integrin-linked kinases (FAK and ILK) (65, 66). In normal cells that have undergone detachment, FAK is cleaved by effector caspases, producing a C-terminal focal adhesion

20 14 targeting domain, which functions to negatively regulate FAK (65). However, attachment of integrins prompts phosphorylation of FAK, inhibiting its cleavage and inactivation, thereby allowing FAK to positively reinforce its downstream targets that maintain cell survival, including Akt and JNK. Similarly, ILK helps activated PI3K signalling through phosphorylation of Akt (66). Activity of these kinases is upregulated in anoikis-resistant cells; overexpression of FAK and ILK can rescue cells from apoptosis resulting from loss of adhesion (66). Integrin-mediated adhesion also triggers NF- B signalling which helps upregulate Bcl-XL expression, a process that is maintained in anoikis-resistant cells, characterized by increased induction of NF- B (67). Cadherins While integrins mediate cell-extracellular matrix adhesion, cadherins facilitate cell-to-cell contacts these membrane proteins regulate attachment to homotypic or heterotypic cell types in a calcium-dependent manner, thereby preventing anoikis induction (68). Cadherins are necessary to control anoikis, particularly in tissue with high cell turnover like the intestinal epithelium, where increased cell death results from dysregulated cadherin expression. Increased expression of -catenin, a downstream substrate modulated by cadherin function, confers resistance to anchorage-dependent apoptosis (69). Cadherin expression contributes to PI3K-Akt signalling; where cell-tocell contacts are formed, Akt activity is increased, prompting inhibition of pro-apoptotic factors within the Bcl-2 family (7).

21 15 Mechanisms of anoikis resistance FLIP, an endogenous factor that confers protection from apoptotic stimuli, also mediates cellular responses to anoikis; where FLIP is overexpressed, cells are resistant to detachment-induced cell death. sirna-mediated downregulation of FLIP expression was found to sensitize previously resistant tumour cells to death receptor stimuli. Upon triggering of death receptor signalling through treatment with Fas ligand, FLIP-depleted cells experienced greater reductions in cell growth and viability relative to controls (71). Moreover, abrogating FLIP expression also influences the in vivo capacity for metastasis and invasion. Inhibition of FLIP expression led to a substantial reduction in the ability of tumour cells to form distant metastatic lesions in the lungs, liver and bone following injection into mice (71). Oncogenic signalling In addition to overexpression and dysregulation of FLIP, other mechanisms have been reported to be associated with anoikis resistance. Two pro-survival pathways that are strongly activated in anchorage-independent cells are the Raf-MAPK mitogen activated protein kinase)/erk (extracellular regulated kinase) kinase (MEK) pathway, and the phophatidylinositol-3-kinase signalling pathway (72, 73). The Raf-MAPK/ERK pathway is responsive to extracellular matrix contacts and growth signals regulating cell survival, and affect transcription of factors controlling growth and cell cycle regulation (72). Upon receipt of growth-inducing stimuli, Srcmediated signalling prompts activation of Ras, a GTPase, that prompts recruitment of Raf, a serine-threonine kinase, to the cell membrane where it is phosphorylated and

22 16 activated (74). Raf participates in the phosphorylation of downstream MAP kinase kinase substrates, leading to the phosphorylation of ERK, along with other terminal MAP kinases such as JNK (c-jun-nh2-kinase) and p38. Once activated, ERK moves to the nucleus where it alters transcription of pro-survival genes (74, 75). Phosphatidylinositol-3-kinases (PI3Ks), required for phosphorylation of membrane lipids, respond to external growth signals by catalyzing the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to form phosphatidylinositol (3-5)- triphosphate (73). PIP3 is a second messenger that facilitates recruitment of Akt kinase, a pleckstrin homology (PH) domain-containing protein. At the cell membrane, Akt is phosphorylated at threonine 38 and serine 473 by phosphoinositide-dependent kinase 1 and 2 (PDK1, 2) (76). Once activated Akt moves to the nucleus, facilitating transcription of genes promoting proliferation and growth (77). Cells that are anoikis resistant maintain constitutive MAPK/ERK and PI3K signalling even in the absence of appropriate growth signals. Both Raf-MAPK/ERK and PI3K signalling pathways function to alter the expression and regulation of Bcl-2 proteins, enhancing the activity of anti-apoptotic factors while simultaneously suppressing the function of pro-apoptotic proteins (78). In particular, the function of two BH3-only proteins Bim and Bad has been observed to be negatively regulated through direct phosphorylation by both ERK and Akt (79). Alternatively, their expression can also be reduced through ERK and Akt-mediated downregulation of transcription (). The presence of fewer Bim and Bad molecules or inactive phosphorylated Bim and Bad interferes with their inhibition of anti-apoptotic Bcl-2 and Mcl-1 at the mitochondria, resulting in the preservation of mitochondrial membrane

23 17 integrity, and the prevention of cytochrome c release (54). Moreover, these oncogenic signalling pathways inhibit the transcription of other pro-apoptotic BH3-only proteins such as Puma and Noxa by affecting their regulation by p53 and FOXO transcription factors (81). Activated Akt also works to decrease cleavage and activation of caspase 9 (). PI3K activation can also be triggered in an autocrine signalling fashion. For example, melanoma cells that are highly proliferative with a propensity to metastasize demonstrate increased expression of TrkB a tyrosine kinase receptor that is upregulated in cells resistant to anoikis. Upregulation of TrkB greatly stimulates PI3K activity, and functions to inhibit caspase-mediated anchorage-dependent cell death (82). Epithelial to mesenchymal transition The epithelial-mesenchymal transition, wherein cells gain the capacity for greater motility through resistance to anchorage-dependent cell death, also functions during processes of metastasis and invasion in epithelially-derived cancers (83). Anoikisresistant tumour cells become capable of growth in foreign extracellular matrix microenvironments, facilitating their survival in distant secondary tumour sites. Cells that are anoikis resistant demonstrate increased expression of factors traditionally linked with epithelial-mesenchymal transition (84). PI3K-Akt signalling induces expression of these mesenchymal factors that help confer resistance to cell death during loss of adhesion (84). Snail, a zinc finger transcription factor, functions to alter cell-to-matrix and cell-to-cell adhesion, through inhibition of E-cadherin expression, and upregulation of the production of ECM glycoproteins, including fibronectin and vimentin (85). Snail

24 18 also positively reinforces PI3K-Akt activity, and inhibits cleavage of downstream effectors within the apoptotic caspase cascade (86). Twist is a helix-loop-helix transcription factor that impinges upon the ratio of Bcl-2 family proteins by upregulating expression of anti-apoptotic factors (87). With greater expression indicative of worse disease prognosis, Twist upregulation has been observed in cancer progression (88). Reactive oxygen species and hypoxia The production of reactive oxygen species (ROS) is typically increased within cancer cells relative to untransformed cells; increased generation of ROS has been associated with resistance to adhesion-dependent apoptosis (89). ROS production contributes to constitutive activity of the oncogene Src kinase by triggering its oxidation (9). As a result, Src continually phosphorylates growth factor receptors, such as epidermal growth factor receptor (EGFR), prompting growth factor-mediated survival signalling even in the absence of growth factors (9). This ligand-independent activity functions to substantially enhance resistance to anchorage-dependent cell death. Accordingly, ligand-independent EGFR signalling and accompanying adhesionindependence is abrogated through treatment with antioxidants, which limit ROS generation (9). ROS production is thought to be enhanced due to intratumoural hypoxia, characteristic of highly malignant and invasive solid tumours, and associated with poor prognosis (91, 92). Hypoxia functions to bring about a cadherin switch, where E- cadherin is downregulated and replaced with N-cadherin, a typical feature of the epithelial-mesenchymal transition (92). This helps confer resistance to anoikis; N-

25 19 cadherin cell contact prompts Akt survival signalling and functions in maintaining expression of -catenin, an anti-apoptotic factor (93). Taken together, hypoxic conditions enhance processes of anoikis resistance. Endoplasmic reticulum stress Within the cell, the endoplasmic reticulum is the organelle principally responsible for protein synthesis. The endoplasmic reticulum is also necessary for maintenance of proper three-dimensional folding of proteins, a process that is highly sensitive to alterations within the microenvironment of the ER (94). Changes in glucose and nutrient availability, Ca 2+ ion concentration, and reduction-oxidation status of cells result in detrimental stress to the endoplasmic reticulum; this limits its capacity for N-linked protein glycosylation, calcium-dependent chaperone protein function, and disulfide-bond formation, respectively (95). Dysfunction of these post-translational protein modifications compromises the ability of the endoplasmic reticulum to successfully fold proteins and maintain conformational integrity (96). As a result, stress to the ER causes the accumulation of misfolded proteins within the ER lumen. Increased quantities of unfolded proteins within the ER are cytotoxic; ER stress has been implicated in disorders such as cell death following ischaemia and in diabetes (97).

26 2 Unfolded protein response Collection of misfolded proteins within the ER results from stress the ER exceeds its capacity for ensuring conformational folding fidelity (98). Misfolded protein accumulation within the ER prompts activation of the unfolded protein response (UPR) an evolutionarily conserved mechanism to deal with an excess load of misfolded proteins in an attempt to regulate and normalize ER activity (94). The response is primarily initiated as a cytoprotective measure to limit excess protein formation until the ER has regained function at an optimal level. The UPR is initiated by three main ER transmembrane receptors pancreatic ER kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) (99). In addition to stresses relating to energy availability, Ca 2+ ion regulation and oxidation, the unfolded protein response can also be induced by stimuli that limit proteasome function; inhibition of retrograde transport of proteins from the ER to the cytoplasm for proteasomal degradation contributes to an excess load of improperly folded proteins within the ER (). UPR upregulation also occurs following viral infection, a defense mechanism to limit the assembly and release of competent virus particles (11). When the ER is functioning normally, the calcium-dependent ER chaperone protein Grp78 interacts with the N-terminal portions of each of the three receptors (PERK, ATF6, IRE1) within the ER lumen (95). However, the aggregation of improperly folded proteins, induced by ER stress, prompts dissociation of Grp78 from PERK, ATF6, and IRE1, as increased levels of Grp78 are required to associate with nascent proteins to govern correct folding (96). Due to the removal of repression, the

27 21 cytosolic C-terminal portions of the three mediators transduce UPR signalling. The unfolded protein response initially attempts to resolve stress to the endoplasmic reticulum as a means of promoting survival. Therefore, in early stages of ER stress, the UPR is cytoprotective. However if stress is prolonged or severe, the UPR functions instead in promoting apoptosis (96-98). PERK Upon release of Grp78, pancreatic ER kinase (PKR)-like kinase, a serinethreonine kinase, forms a dimer, and undergoes activation through autophosphorylation. The kinase domain shares structural homology with its proximal substrate the eukaryotic initiation factor 2 (eif2 ) (12). eif2 is the regulatory subunit of eukaryotic translation initiation factor 2, which mediates the association of the ribosome with the initial methionine-carrying trna (12). Phosphorylation of eif2 by PERK inhibits the initiation of ribosomal translation, leading to widespread suppression of capdependent translation (13). Though the majority of protein translation is arrested, as a means of relieving an excess protein load within the ER, expression of select genes is permitted (14). Where internal ribosomal entry sites (IRES) or other regulatory sequences are present within 5 promoters, translation continues (15). One of the pivotal genes expressed during PERK-mediated inhibition of general translation is activating transcription factor 4 (ATF4). ATF4 upregulates expression of C/EBP homologous protein (CHOP), a pro-apoptotic factor that induces cell death (16).

28 22 ATF6 Initiating activating transcription factor 6-mediated UPR signalling involves translocation of ATF6 from the ER to the Golgi complex following release of Grp78 (94). At the Golgi, ATF6 is activated through cleavage by site 1 and 2 proteases; ATF6 then translocates to the nucleus where it functions to upregulate transcription of prosurvival genes (17). ATF6 increases expression of ER chaperone proteins, such as Grp78, Grp94, calreticulin, and protein disulfide isomerase, as well as factors involved in amino acid regulation and peptide translocation; its targets typically possess ER stress response elements (ERSEs) within their 5 untranslated regions (18). ATF6 also induces increased expression of X box-binding protein 1 (XBP1), which functions as a substrate within IRE1 signalling (17). IRE1 IRE1, the third UPR transmembrane regulator, functions as both a serinethreonine kinase as well as an endoribonuclease upon stress to the ER. With its endonuclease domain, IRE1 activates XBP1 through splicing and removal of an intron sequence; spliced XBP1 (sxbp1) then functions as a transcription factor (96). Binding to target genes possessing unfolded protein response element (UPRE) and ER stress enhancer (ERSE) promoter sequences, sxbp1 triggers expression of factors that regulate retrograde transport of proteins for proteasomal degradation, thereby reducing the load of excess misfolded proteins within the ER (19). sxbp1 also upregulates expression of ER-resident chaperone and heatshock proteins, that facilitate protein folding (19).

29 23 While the IRE1 pathway has pro-survival effects mediated by activation of the sxbp1 transcription factor, IRE1 also induces pro-apoptotic signalling; overexpression of IRE1 induces apoptosis. The E3 ligase TNF-receptor-associated factor 2 (TRAF2) associates with IRE1 following UPR activation, and activates apoptosis signal regulating kinase (ASK1) a MAPK kinase kinase that functions in phosphorylation and activation of downstream MAPK substrates, JNK and p38 (11). Apoptotic JNK regulates the balance of pro- and anti-apoptotic proteins at the mitochondria through phosphorylation; anti-apoptotic Bcl-2 is inhibited, while the activity of pro-apoptotic BH3-only proteins is upregulated (111). Divergent outcomes depend on severity of stress There are two directly opposing outcomes of unfolded protein response initiation UPR activation can lead to reduced ER stress and continued cell survival; conversely, if stress is severe or maintained, cell death will result. The divergent cytoprotective and apoptotic functions of the unfolded protein response are facilitated by the sequential activation of each of the three pathways (94-96). The PERK pathway is triggered first, with an accompanying suppression of protein translation as the principal response. This is followed by activation of the ATF6 arm, which generally functions to upregulate cytoprotective and pro-survival pathways (94, 112). IRE1 upregulation follows with an initial anti-apoptotic programme intended to confer cell survival through induction of proteins that facilitate correct folding (113). However, following continued stress to the ER and UPR activation, IRE1 begins to stimulate expression of P58 IPK, a heatshock protein that negatively regulates PERK. P58 IPK inhibits PERK signalling by binding

30 24 PERK and preventing its phosphorylation of eif2 ; in this way, P58 IPK allows protein translation to resume (94, 114). P58 IPK is thought to represent cessation of UPR activity, as it is one of the later factors to appear following stress to the ER. It is possible that P58 IPK signals the end of the survival phase of the UPR its activation facilitates continued translation. If by this point ER stress has been improved, the cell resumes usual activity. If, however, ER stress is prolonged, the end of translation inhibition permits the IRE1 pathway to induce expression of pro-apoptotic JNK and p38 (94, 115). JNK signalling might then function to reinforce cell death stimulation initiated by earlier PERK-mediated expression of pro-apoptotic CHOP (15). ER stress and cell death Participation of various factors has been associated with a cell death response to ER stress and unfolded protein response activation, including CHOP, Gadd34, and caspase cascade effectors. CHOP is also known as growth-arrest-and-dna-damage-inducible gene 153 (Gadd153); despite its increased activity in stimulating apoptosis as a result of DNA damage, during the unfolded protein response activation of the PERK pathway is necessary to induce its expression (116). There are a number of different ways CHOP is hypothesized to bring about apoptosis following endoplasmic reticulum stress. As a transcription factor, CHOP reduces expression of members within the anti-apoptotic Bcl- 2 protein family, thereby allowing enhanced activity of pro-apoptotic factors at the mitochondria (117, 118). Increased expression of several BH3-only proteins, including Noxa and Puma, has been observed following induction of ER stress, as demonstrated

31 25 through microarray expression analysis (119, ). CHOP is also thought to promote apoptosis through upregulating expression of Tribbles-related protein 3 (Trb3), a factor associated with ER stress-induced cell death (121). sirna downregulation of Trb3 has been observed to protect against apoptosis following chemical induction of ER stress. Trb3 is thought to bind to Akt, inhibiting phosphorylation of downstream substrates and oncogenic activity (122). In addition, CHOP function is also subject to post-translational phosphorylation on serines 78 and 81 due to p38 MAPK activity, a factor activated through IRE1 signalling. This interaction suggests that the IRE1 and PERK pathways might reinforce each other s pro-apoptotic activity in the event of severe stress, with CHOP functioning as a point of interaction (123). Gadd34 functions in concert with protein phosphatase 1 (PP1), in order to facilitate PP1-mediated dephosphorylation of eif2. This inhibition of PERK signalling permits protein translation to resume (124). Gadd34, in inhibiting PERK, functions in a manner similar to P58 IPK ; however, its effect on ultimate cell fate is opposite to the prosurvival effect of P58 IPK (125). Upregulation of Gadd34 has been observed to accompany greater ER stress-induced cell death (125). Activation of the proteolytic caspase cascade occurs during ER stress-mediated apoptosis; caspases 12, 3, 6, 7, 8 and 9 have all demonstrated activation (126). Yet, which caspase functions as the primary initiating activator of the cascade remains unknown. Caspase 12, in particular, has been considered to function as the apical regulator of ER stress-induced apoptosis (126). However, as it is only expressed in rodents due to mutational silencing in humans, its involvement is called into question due to the conservation of the unfolded protein response across species (127). Caspase 4 is

32 26 closely related to caspase 12 and is functional in humans; however, its activation following ER stress has not been categorically demonstrated (128).

33 27 Rationale and Hypothesis (I) The extrinsic death receptor-mediated pathway of apoptosis involves binding of a death-inducing stimulus, such as Fas ligand, to its corresponding TNF receptor at the extracellular surface membrane, prompting the assembly of the death-inducing signalling complex (DISC) (6, 8). Recruitment of procaspase 8 results in its autocatalytic cleavage, leading to activation of downstream effector caspases, and ultimately, cell death (16, 17). However, various endogenous factors resist this process of Fas-mediated apoptosis; one such protein is Fas-associated death domain-like interleukin-1 converting enzyme (FLIP) protein (2). As a result of shared homology with caspase 8, FLIP inhibits caspase 8 activation by competitively binding within the DISC, functioning to inhibit downstream apoptosis (18). Where FLIP is overexpressed, it is preferentially recruited to the DISC in place of caspase 8, conferring enhanced apoptosis resistance (2). FLIP overexpression is relevant to malignancy and neoplastic progression, since upregulation of FLIP and concomitant insensitivity to death receptor-mediated stimuli is characteristic of tumour cell apoptosis evasion (21). Previously, the Schimmer lab has identified FLIP as an important target for sensitizing resistant cells to death receptor stimuli, in particular, treatment with Fas ligand. FLIP protein expression levels were reduced in tumour cells through transfection of a FLIP-specific sirna. sirna-targeted downregulation of FLIP sensitized tumour cells to death receptor stimuli; these cells experienced a reduction in viability upon treatment with Fas ligand, indicating sensitization of previously resistant cells to Fasmediated cell death (71).

34 28 The unfolded protein response is activated in response to ER stress, and as part of its programme induces widespread global inhibition of protein translation (98, 99). As discussed in greater detail below, FLIP is a short half-life protein. Given the role of FLIP in resistance to death receptor stimuli, it was hypothesized that ER stress would sensitize resistant cells to death stimuli through a mechanism at least partly related to reductions in FLIP protein. In order to activate the unfolded protein response, brefeldin A a known ER stress inducer was selected as a model. Brefeldin A, typically used in the study of protein secretion, inhibits the movement of proteins from the endoplasmic reticulum to the Golgi, causing an accumulation of proteins within the ER (129). Brefeldin A inhibits the activity of ADP ribosylation factor 1 (ARF1), a GTPase that functions in the recruitment and assembly of coat proteins during GTP-dependent transport vesicle formation. By binding to the guanine exchange factor that regulates GTP-GDP cycling, brefeldin A maintains ARF1 in an inactive conformation, resulting in inhibition of transport vesicle formation, which is required for protein movement between organelles (13, 131). Within the unfolded protein response, the PERK pathway is of particular relevance due to its inhibition of protein expression. As a protein previously established to be tied to the resistance of cells to Fas-mediated apoptosis, FLIP expression is sensitive to stresses that perturb protein translation. FLIP is a short half-life protein, as demonstrated by prior studies reporting that FLIP expression within cells was abolished within 1 to 2 hours of treatment with cyclohexamide (132). Accordingly, a reduction in FLIP is likely an early outcome of ER stress. Due to the anti-apoptotic role of FLIP, it is anticipated that potential decreases in FLIP would bring about an increase in sensitivity

35 29 to death receptor stimuli and anchorage-dependent cell death a type of detachmentinduced apoptosis that proceeds through some of the same Fas-mediated apoptotic pathways. Specific aims Therefore, it is hypothesized that induction of ER stress results in downregulation of FLIP expression, resulting in a sensitization of resistant tumour cells to death receptor stimuli and anoikis. This hypothesis generated the following specific aims: 1) Determine cell growth and viability following induction of ER stress with respect to death receptor stimuli and anchorage-dependent cell death. 2) Assess activation of ER stress through expression of factors within the PERK unfolded protein response pathway. 3) Investigate the role of FLIP in mediating response to ER stress induction.

36 3 Materials and Methods Reagents Brefeldin A and thapsigargin were obtained as powders (Sigma-Aldrich, St. Louis, MO) and stored at 4 C. Stock solutions of these compounds were prepared in dimethyl sulfoxide (DMSO) to final concentrations of 1 and 5 mm, respectively, and stored at -2 C. FAS receptor-activating monoclonal antibody (CH-11) was obtained from MBL (Nagoya, Japan). Cell culture and prostate, HT-29 colon, and OVCAR3 ovarian cancer cells were cultured in RPMI 16 media supplemented with 1% fetal calf serum (FCS) (Hyclone, Logan, UT), along with penicillin ( IU/mL) and streptomycin ( µg/ml). cells stably expressing FLIP were cultured in complete RPMI 16 media as described above, along with.8 mg/ml G418 to select for plasmid-containing cells. Cells were incubated at 37 C with 5% CO 2. To evaluate growth and viability under anchorage-dependent conditions, cells were cultured under adherent conditions in 96-well polystyrene plates (Greiner Bio-one Cellstar, Monroe, NC). To evaluate growth and viability under anchorage-independent conditions, cells were cultured under non-adherent (suspension) conditions in hydrogelcoated ultralow binding 96-well plates (Corning, Acton, MA).

37 31 Cell growth and viability The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4- sulfophenyl)-2h-tetrazolium inner salt (MTS) reduction assay (CellTiter 96 AQ ueous One Solution Cell Proliferation Assay) (Promega, Madison, WI) was used to assess cell growth and viability according to the manufacturer s instructions and as previously described (71). A trypan blue exclusion assay was also used to confirm cell viability as previously described (133). A colony formation assay was used to assess clonogenic growth. Cells were treated with either brefeldin A or thapsigargin for 24 hours. After treatment, cells were harvested and washed in phosphate-buffered saline. Cells () were re-plated into 6- well plates, and permitted to grow under adherent conditions for 6 days. Colonies, defined as containing a minimum of cells, were then fixed and stained with.3% methylene blue, and counted. Real-time reverse transcription-polymerase chain reaction Following treatment with brefeldin A, SuperScript II reverse transcriptase along with random primers were used to generate complementary DNA (cdna) from total cellular RNA isolated from collected and cells, in accordance with manufacturer s protocols (Qiagen, Mississauga, ON). Using resultant cdna, SYBR Green PCR Master mix, and gene-specific primers, real-time reverse transcriptionpolymerase chain reaction assays were carried out and analyzed using an ABI77 Sequence Detection System (Applied Biosystems, Foster City, CA). Forward and

38 32 reverse PCR primer pairs for human cdnas were as follows: FLIP-L (GenBank NM- 3879) (5 - CCT AGG AAT CTG CCT GAT AAT CGA 3 / 5 TGG ATA ACC TGC TAC GAG TG 3 ), FLIP-S (GenBank U9775) (5 GCA GCA ATC CAA AAG AGT CTC A 3 / 5 TTT TCC AAG AAT TTT CAG ATC AGG A 3 ), 18S (GenBank U13369) (5 AGG AAT TGA CGG AAG GGC AC 3 / 5 GGA CAT CTA AGG GCA TCA Ca 3 ). Expression of Grp78 and FLIP mrna was quantified relative to the expression of 18S mrna. Immunoblot analysis and cells treated with brefeldin A, and cells transfected with both empty and FLIP-expressing plasmids were collected, washed with cold PBS, and lysed using Laemmli sample buffer ( mm Tris-Cl (ph 6.8), 2% sodium dodecylsulfate (SDS), and 1% glycerol). The concentration of protein lysates was quantified (DC Protein Assay, Bio-Rad, Mississauga, ON) prior to electrophoresis in 12% SDS-polyacrylamide gel electrophoresis gels and transference to nitrocellulose membranes. The following primary antibodies were used to probe membranes: rabbit anti-human Grp78 (1:3 vol/vol dilution), rabbit anti-human phosphorylated eif2 (1: vol/vol dilution), rabbit anti-human ATF4 (1:1 vol/vol dilution), mouse antihuman CHOP (1: vol/vol dilution), rabbit anti-human eif2 (1:2 vol/vol dilution), mouse anti-human FLIP (1: vol/vol dilution), and rabbit anti-actin (1:1 vol/vol dilution). Membranes were incubated in 5% skim-milk powder in PBS with.5% Tween (PBS-T) containing the corresponding primary antibody. Membranes were then washed in PBS-T. Horseradish peroxidase-conjugated anti-rabbit and anti-

39 33 mouse secondary antibodies were applied to membranes at concentrations of 1:, 1:, 1:, 1:, 1:, 1:, and 1:2 to detect anti-grp78, antiphosphorylated eif2, anti-atf4, anti-chop, anti-flip, anti- eif2, and anti-actin respectively, in 5% milk PBS-T for 1 hour at room temperature. Visualization was done using enhanced chemiluminescence reagents (Immobilon, Etobicoke, ON) according to manufacturer s protocols.

40 34 Results Effects of treatment with brefeldin A on Fas-mediated cell death Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP) inhibits Fas-mediated apoptosis by competitively binding to the Fasassociated death domain protein (FADD) in place of procaspase 8, thereby preventing downstream caspase cleavage and activation (71). With its short half-life and high turnover, FLIP is sensitive to stresses that alter protein translation (132). Stress to the endoplasmic reticulum results in shutdown of protein translation affecting the production of short half-life proteins, such as FLIP (96). Brefeldin A is a known ER stress inducer. The response of resistant cells when treated with brefeldin A in the presence of CH-11 an agonistic antibody that targets the FAS death receptor would aid in the characterization of brefeldin A in the sensitization of resistance tumour cells to death receptor stimuli. In order to investigate the effect of brefeldin A on Fas-mediated cell death, PPC- 1,, HT-29 and OVCAR3 cells were treated overnight with increasing concentrations of brefeldin A either in the presence or absence of Fas ligand ( ng/ml) CH-11 (Figure 1). After incubation, cell growth and viability was measured by the MTS assay.,, HT-29, and OVCAR3 cells were all resistant to Fas ligand. Cotreatment of and OVCAR3 cells with brefeldin sensitized and OVCAR3 cells to CH-11 compared to cells treated with brefeldin or CH-11 alone (Figure 1A, 1C). In contrast to the effects in and OVCAR3 cells, and HT-29 cells that are resistant to Fas were not sensitized to this death ligand by brefeldin (Figure 1B, 1D). The

41 35 sensitization to brefeldin mediated by CH-11 in but not cells was confirmed with a trypan blue exclusion assay (Figure 2). Figure 1 A) B) DU CH-11 + CH-11 - CH-11 + CH Brefeldin ( M) Brefeldin ( M) C) D) OVCAR3 - CH-11 + CH-11 2 HT-29 - CH-11 + CH Brefeldin ( M) Brefeldin ( M) Figure 1 Treatment with brefeldin A sensitizes and OVCAR3 cells, but not or HT-29 cells, to Fas-mediated cell death. A) prostate, B) DU-145 prostate, C) OVCAR3 ovarian, and D) HT-29 colon cancer cells were cultured under adherent conditions. Cells were then treated with increasing concentrations of brefeldin A overnight in the absence (closed squares ) or presence (open squares ) of ng/ml CH-11. Resultant cell growth and viability was assessed using MTS. Points, mean viability of cells post-treatment; bars, standard deviation.

42 36 Figure 2 A) B) Brefeldin ( M) - CH-11 + CH-11 2 DU Brefeldin ( M) - CH-11 + CH-11 C) D) OVCAR3 - CH-11 + CH HT-29 - CH-11 + CH Brefeldin ( M) Brefeldin ( M) Figure 2 Treatment with brefeldin A sensitizes and OVCAR3 cells, but not or HT-29 cells, to Fas-mediated cell death as measured by trypan blue exclusion assay. A) prostate, B) DU-145 prostate, C) OVCAR3 ovarian, and D) HT-29 colon cancer cells were cultured under adherent conditions. Cells were then treated with increasing concentrations of brefeldin A overnight in the absence (closed squares ) or presence (open squares ) of ng/ml CH-11. Resultant cell growth and viability was assessed using a trypan blue exclusion staining assay. Points, mean viability of cells post-treatment; bars, standard deviation. Effects of treatment with brefeldin A on anchorage-independent cell death Anchorage-independent cell death is also dependent on FLIP and activation of the death receptor pathway of caspase activation. Therefore, the ability of brefeldin to sensitize resistant cells to anoikis was examined. and anoikis-resistant

43 37 cells were treated overnight under adherent and suspension conditions, with increasing concentrations of brefeldin (Figure 3). After incubation, cell growth and viability was measured by the MTS assay. Brefeldin reduced the growth and viability of cells under suspension conditions that promote anoikis, but had minimal effects on the cells cultured under adherent conditions (Figure 3A). Brefeldin-mediated sensitization to anoikis in cells was confirmed by a trypan blue exclusion assay (Figure 3B). In contrast, brefeldin did not sensitize to anoikis, consistent with the inability of brefeldin to sensitize these cells to CH-11 (Figure 3C, 3D).

44 38 Figure 3 A) B) Brefeldin ( M) Brefeldin ( M) C) D) Brefeldin ( M) Brefeldin ( M) Figure 3 Treatment with brefeldin A sensitizes cells, but not cells, to anoikis. A), B) prostate and C), D) prostate cancer cells were cultured under both adherent conditions (closed squares ) and suspension conditions (open squares ). Cells were then treated with increasing concentrations of brefeldin A overnight. Resultant cell growth and viability was assessed using A), C) MTS and B), D) trypan blue exclusion staining. Points, mean viability of cells post-treatment; bars, standard deviation. Expression of markers associated with increased endoplasmic reticulum stress following treatment with brefeldin A Brefeldin A was used in the above assays to stimulate stress to the endoplasmic reticulum in keeping with its known function of inhibiting vesicle formation and protein

45 39 transport (129). Therefore, to confirm the intended effects of brefeldin on the tested cells, the effects of brefeldin on expression of ER stress markers was examined. cells were cultured under adherent and suspension conditions with brefeldin ( µm) for, 6, 18, 24 or 36 hours. As a control, cells were also treated with vehicle under adherent and suspension conditions for and 36 hours. After these times of incubation, cells were harvested and expression levels of markers associated with the PERK pathway of the unfolded protein response Grp78, phosphorylated eif2, ATF4, and CHOP were assessed by immunoblotting (Figure 4A). In cells, brefeldin increased expression of ER stress markers Grp78, phosphorylated eif2, ATF4, and CHOP at times that preceded and concentrations associated with sensitization to CH-11 and anoikis. In contrast, no change in ER stress markers was observed after culturing vehicle-treated cells under suspension conditions. Consistent with the ability to induce ER stress, brefeldin also increased mrna levels of Grp78 (Figure 4B).

46 Figure 4 A) Brefeldin (hrs) GRP A S A S A S A S A S Control (hrs) 36 A S A S phospho-eif2 ATF4 CHOP total eif2 B) Relative Grp78 mrna expression p <.1 p <.1 Control Brefeldin ( M) Control Brefeldin ( M) Figure 4 Activation of the PERK pathway of the endoplasmic reticulum stress-induced unfolded protein response following treatment with brefeldin A. A) cells were cultured in adherent and suspension conditions and treated with vehicle alone for or 36 hours, or with brefeldin A for 6, 18, 24, or 36 hours. Immunoblotting was performed to assess expression levels of Grp78, phospho-eif2α, ATF4, and CHOP relative to levels of total eif2α. B) cells were cultured in adherent and suspension conditions and treated with brefeldin A, or with vehicle alone as a control for 24 hours. Real-time reverse transcription-polymerase chain reaction was used to assess expression of Grp78.

47 41 The effects of brefeldin in cells, that were not sensitized to CH-11 or anoikis by brefeldin, were also examined. Here, cells were treated overnight under adherent conditions with increasing concentrations of brefeldin A. After incubation, cells were harvested, and expression of Grp78 was assessed by immunoblotting (Figure 5A). Brefeldin increased expression of Grp78 protein. Likewise, brefeldin increased expression of Grp78 mrna in these cells (Figure 5B). Figure 5 A) Brefeldin (µm) GRP78 β-actin Figure 5 Activation of endoplasmic reticulum stress in cells. A) cells were cultured in adherent conditions and then treated with increasing concentrations of brefeldin A for 24 hours. Immunoblotting was performed to assess expression levels of Grp78.

48 42 Figure 5 cont. B) 1. Relative Grp78 mrna expression Control Brefeldin ( M) Control Brefeldin ( M) Figure 5 cont. Activation of endoplasmic reticulum stress in cells. B) cells were cultured in adherent and suspension conditions and treated with brefeldin A, or with vehicle alone as a control for 24 hours. Real-time reverse transcription-polymerase chain reaction was used to assess expression of Grp78.

49 43 Effects of treatment with thapsigargin on Fas-mediated and anchorage-independent cell death To begin to understand whether induction of ER stress was functionally important for brefeldin A s ability to sensitize resistant cells to CH-11 or anoikis, cells were treated with thapsigargin, a compound that interferes with the function of sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatases (SERCAs), thereby inducing ER stress (134). cells were treated with increasing concentrations of thapsigargin with and without CH-11 or under adherent and suspension conditions (Figure 6). After incubation, cell growth and viability was measured by the MTS assay. Similar to the effects of brefeldin, thapsigargin also sensitized cells to CH- 11 and anoikis (Figure 6A, 6B). Cell death was confirmed by trypan blue staining (Figure 7A, 7B). In contrast, thapsigargin did not sensitize cells to CH-11 or anoikis (Figure 6C).

50 44 Figure 6 A) B) C) Thapsigargin ( M) Thapsigargin ( M) - CH-11 + CH-11 - CH-11 + CH Figure 6 Treatment with thapsigargin sensitizes cells, but not cells, to Fas-mediated cell death and anoikis. A), B) prostate and C) prostate cancer cells were cultured under adherent conditions alone (A, C) or under both adherent and suspension conditions (B). For cells cultured under adherent conditions alone, cells were then treated with increasing concentrations of thapsigargin overnight in the absence (closed squares ) or presence (open squares ) of ng/ml CH-11. For cells cultured under both adherent (closed squares ) and suspension (open squares ) conditions, cells were treated with increasing concentrations of thapsigargin overnight. Resultant cell growth and viability was assessed using MTS. Points, mean viability of cells posttreatment; bars, standard deviation. Thapsigargin ( M)

51 45 Figure 7 A) B) Thapsigargin ( M) - CH-11 + CH Thapsigargin ( M) C) CH-11 + CH Thapsigargin ( M) Figure 7 Treatment with thapsigargin sensitizes cells, but not cells, to Fas-mediated cell death and anoikis as measured by trypan blue exclusion assay. A), B) prostate and C) prostate cancer cells were cultured under adherent conditions alone (A, C) or under both adherent and suspension conditions (B). For cells cultured under adherent conditions alone, cells were then treated with increasing concentrations of thapsigargin overnight in the absence (closed squares ) or presence (open squares ) of ng/ml CH-11. For cells cultured under both adherent (closed squares ) and suspension (open squares ) conditions, cells were treated with increasing concentrations of thapsigargin overnight. Resultant cell growth and viability was assessed using trypan blue exclusion staining. Points, mean viability of cells posttreatment; bars, standard deviation.

52 46 Effects of brefeldin A and thapsigargin on clonogenic growth To further characterize the ability of brefeldin and thapsigargin to sensitize cells to CH-11, these compounds were evaluated in a clonogenic growth assay. Here, cells were treated overnight with increasing concentrations of brefeldin or thapsigargin under adherent conditions with and without CH-11 (Figure 8). After incubation, cells were plated into clonogenic growth assays; six days later, colony formation was assessed. By the clonogenic growth assay, both brefeldin and thapsigargin sensitized cells to CH-11 (Figure 8A, 8B). Taken together, these results suggest that ER stress induced by brefeldin and thapsigargin sensitize cells to CH- 11 and anoikis.

53 47 Figure 8 A) Number of colonies Brefeldin ( M) - CH-11 + CH-11 B) Number of colonies CH-11 + CH Thapsigargin ( M) Figure 8 Treatment with brefeldin A and thapsigargin sensitizes cells to Fasmediated cell death as measured by colony formation assay. cells were cultured under adherent conditions. Cells were then treated with increasing concentrations of A) brefeldin A or B) thapsigargin overnight in the absence (closed squares ) or presence (open squares ) of ng/ml CH-11. Cells were collected and then re-plated under adherent conditions; colonies were allowed to grow for five to six days before being stained with methylene blue and counted. Columns, mean number of colonies; bars, standard deviation.

54 48 The involvement of Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP) in Fas-mediated cell death and anchoragedependent cell death Cellular Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP) is an endogenously expressed protein that antagonizes Fasmediated apoptosis and anoikis (132). FLIP is a short half life protein, so it was hypothesized that induction of ER stress and subsequent inhibition of protein translation would preferentially decrease levels of FLIP protein that would be functionally important for the observed effects of brefeldin CH-11 and anoikis sensitization. To test this hypothesis, and cells were treated overnight with brefeldin ( µm). After incubation, levels of FLIP protein and mrna were measured by immunoblotting and quantitative RT-PCR, respectively. Brefeldin A reduced levels of FLIP protein but did not reduce levels of FLIP mrna in cells (Figure 9A, 9B). Likewise, in cells FLIP protein levels were reduced following overnight treatment with brefeldin A (Figure 9C). Therefore, these results indicate that brefeldin decreases levels of FLIP, consistent with its known mechanism as an inducer of ER stress. Moreover, brefeldin decreased FLIP in cells, indicating that resistance to CH-11 and anoikis in these cells is due to mechanisms beyond expression of FLIP.

55 49 Figure 9 A) Treatment Control Brefeldin A S A S FLIP -actin B) Relative FLIP mrna expression p =.21 p =.17 Control Brefeldin ( M) Control Brefeldin ( M) Figure 9 Involvement of Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP) in Fas-mediated cell death and anoikis. A) cells were cultured in adherent and suspension conditions and treated either with vehicle alone, or with brefeldin A for 24 hours. Immunoblotting was performed to assess expression levels of FLIP. Fold change in expression quantified through densitometry as 1., 1.,.7,.4, relative to control. B) cells were cultured in adherent and suspension conditions and treated with brefeldin A, or with vehicle alone as a control for 24 hours. Real-time reverse transcription-polymerase chain reaction was used to assess expression of FLIP.

56 Figure 9 cont. C) Treatment Control Brefeldin A S A S FLIP -actin Figure 9 cont. Involvement of Fas-associated death domain-like interleukin-1- converting enzyme-like inhibitory protein (FLIP) in Fas-mediated cell death and anoikis. C) cells were cultured in adherent and suspension conditions and treated either with vehicle alone, or with brefeldin A for 24 hours. Immunoblotting was performed to assess expression levels of FLIP. Fold change in expression quantified through densitometry as 1., 1.,.2,.1, relative to control. To determine whether reductions in FLIP protein were functionally important for brefeldin sensitization to CH-11, cells were stably transfected with FLIP cdna. Over-expression of FLIP was confirmed by immunoblotting (Figure 1A). Both FLIPover-expressing cells and control cells transfected with an empty vector were cultured in the presence of CH-11. Compared to control cells, over-expression of FLIP abrogated the ability of brefeldin to sensitize cells to CH-11, as measured by both MTS and trypan blue exclusion (Figure 1B, 1C). Therefore, FLIP appears functionally important for brefeldin-mediated CH-11 sensitization.

57 51 Figure 1 A) Empty vector FLIP overexpressing FLIP -actin B) C) 2 FLIP Empty vector 2 FLIP Empty vector Brefeldin ( M) Brefeldin ( M) Figure 1 Overexpression of FLIP protects against sensitization to Fas-mediated cell death. A) cells were transfected with either a FLIP-overexpressing vector. Immunoblotting was performed to compare levels of FLIP protein in cells transfected with an empty vector, and cells transfected with a FLIPoverexpressing vector. B) cells that were transfected with either a FLIPoverexpressing (closed squares ) or an empty vector (open squares ) were cultured under adherent conditions. Cells were then treated with increasing concentrations of brefeldin A in the presence of ng/ml CH-11. Resultant cell growth and viability was assessed using B) MTS and C) trypan blue exclusion staining. Points, mean viability of cells post-treatment; bars, standard deviation.