THE ROLES OF A LIM DOMAIN PROTEIN, HIC-5/ARA55, IN TGF-β SIGNALING IN PROSTATE CANCER CELLS HUI WANG

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1 THE ROLES OF A LIM DOMAIN PROTEIN, HIC-5/ARA55, IN TGF-β SIGNALING IN PROSTATE CANCER CELLS by HUI WANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. David Danielpour Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January 2009

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of candidate for the degree *. (signed) (chair of the committee) (date) *We also certify that written approval has been obtained for any proprietary material contained therein.

3 Table of Contents List of Tables... 5 List of Figures... 6 Acknowledgements... 8 Abbreviations Abstract Chapter 1 Introduction Overview of prostate and prostate cancer Two important signaling pathways in prostate Androgen signaling in prostate TGF-β signaling in prostate Hic-5 and its biological function Hic-5 and the LIM protein superfamily Hic-5 at focal adhesions Hic-5 in the nucleus Hic-5 and TGF-β signaling Hic-5 and prostate

4 1.4 Purpose of this research Chapter 2 Hic-5 functions as an inhibitor for the regulatory Smad Results Hic-5 blocks transcriptional activation by Smad Hic-5 can block Gal4-CA-Smad3 MH2-ML activity Hic-5 physically interacts with Smad The binding of Hic-5 and Smad3 can occur in a cell-free system Smad3 MH2 domain is responsible for the interaction of Hic-5 with Smad Hic-5 C-terminus is critical for the inhibition of Smad3 activity Hic-5 can reverse TGF-β-induced cell cycle arrest Summary Materials and Methods Chapter 3 Hic-5 can upregulate Smad2-dependent TGF-β signal transduction through downregulating the inhibitory Smad7 protein level Results

5 3.1.1 Hic-5 can bind to Smad Hic-5 can induce Smad7 protein loss Effects of Hic-5-Smad7 interaction on TGF-β Signaling Functional Inactivation of Smad7 by Hic Effect of endogenous Hic-5 on Smad2-dependent TGF-β responses Silencing Hic-5 represses TGF-β-induced EMT in prostate cancer cells The proteasomal and the lysosomal pathways may not be responsible for Hic-5-induced Smad7 protein loss Summary Materials and Methods Chapter 4 Discussion and future directions Hic-5, AR and TGF-β signaling Hic-5 could function as an adaptor for Smad Smad3 and Smad7 may not compete with each other for the sake of binding to Hic

6 4.4 Smad2, Smad3 and the biological significance The mechanisms leading to Smad7 protein loss by Hic Hic-5, focal adhesions and EMT Hic-5 could be a clinical marker indicating prostate cancer progression Summary Bibliography

7 List of Tables Table 1. Primers for PCR amplification of Hic-5 and its truncations 75 Table 2. Primers for PCR amplification of Smad3 and its truncations.75 5

8 List of Figures Figure 1-1. Structure of Hic-5 39 Figure 1-2. Endogenous Hic-5 protein levels in different prostatic cell types 41 Figure 2-1. Hic-5 can selectively block Smad3 activity.61 Figure 2-2. Hic-5 can block 3TP-lux induction 64 Figure 2-3. Hic-5 can block Gal4-CA-Smad3 activity 66 Figure 2-4. Hic-5 can bind to Smad Figure 2-5. Smad2 MH2 domain is critical for the binding to Hic-5.70 Figure 2-6. Hic-5 LIM3 domain is required for the binding to Smad3..72 Figure 2-7. Hic-5 can block the cytostatic effect of TGF-β 74 Figure 3-1. Hic-5 can bind to Smad7 directly 101 Figure 3-2. Hic-5 can selectively induce Smad7 protein loss Figure 3-3. Hic-5-Smad7 interaction can enhance TGF-β signal transduction Figure 3-4. Hic-5 can reverse the inhibitory effect of Smad7 on the reporter induction by TGF-β in a cell line-dependent manner.109 Figure 3-5. Silencing Hic-5 can downregulate TGF-β signal transduction

9 Figure 3-6. Hic-5 is required for TGF-β-induced EMT..113 Figure 3-7. The proteasomal and the lysosomal pathways are not likely to be involved in Smad7 protein downregulation by Hic Figure 4-1. Hic-5 has dual roles in TGF-β signaling

10 Acknowledgements First of all, I thank my advisor Dr. David Danielpour for his continuous support in my Ph.D. study. Dr. Danielpour is undoubtedly a great mentor, always being there to listen and to give advice. He taught me all the skills essential for a successful scientist and encouraged me to think actively and formulate my own ideas. In the past years, what I learned in his laboratory includes not only the lab-techniques, the knowledge and the ability to work independently, but also the efficient way to balance the work and the personal life. All in all, Dr. Danielpour opened the door of science for me and led me on the right track for my future career. I thank the rest of my dissertation committee, Dr. Paul N. MacDonald, Dr. Charles L. Hoppel and Dr. Bingcheng Wang, for their patience, time and advice over the years. Their critical questions and insightful comments helped me understand how a good scientist thinks about and accomplishes his project. 8

11 I thank my colleagues in Danielpour lab: Dr. Kyung Song, Tracy L. Krebs, Jiayi Yang, Reema Wahdan-Alaswad and Dorjee Tamang. Without their help, cooperation and encouragement, my project would definitely have been harder complete. I thank the Department of Pharmacology, where I learned the fundamentals of drug action and where I was inspired to pursue my future goal for a career in drug design and development. I also thank the department for providing so many opportunities to improve my presentation skill. I am indebted a lot to my wife and my parents for their unconditional support throughout the years of hardship. I truly thank you all. 9

12 Abbreviations 3TP-lux, a TGF-β-responsive reporter containing the promoter region of plasminogen activator inhibitor-1; Ad, adenovirus vector; AR, androgen receptor; AD, NF-κB transcription activation domain; ARA55, androgen receptor-associated protein 55; ARE-lux, a Smad2-respomsive reporter containing activin response element; BD, Gal4 DNA binding domain; CA, constitutively active; co-ip, co-immunoprecipitation; DHT, dihydrotestosterone; DMEM/F-12, Dulbecco s modified Eagle s medium/ham s F-12; EMT, epithelial mesenchymal transition; FAK, focal adhesion kinase; FAST-1, forkhead activin signal transducer-1; FBS, fetal bovine serum; HEK293, human embryonic kidney cell line 293; GST, Glutathione-S-transferase; Hic-5, hydrogen peroxide-inducible clone-5; MH1, Mad Homology 1, MH2, Mad Homology 2; ML, middle linker; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate buffered saline; PYK2, proline-rich tyrosine kinase 2; R. L. A., relative luciferase activity; RT-PCR, reverse transcription-polymerase chain reaction; SBE, Smad-binding element; shrna, small hairpin RNA; Smurf, Smad ubiquitination regulatory factor; 10

13 TGF-β, transforming growth factor-β; TβRI, TGF-β type I receptor; TβRII, TGF-β type II receptor; WT, wild-type. 11

14 The Roles of a LIM Domain Protein, Hic-5/ARA55, in TGF-β Signaling in Prostate Cancer Cells Abstract by HUI WANG Findings from our laboratory as well as from others support that transforming growth factor beta (TGF-β) functions as a critical tumor suppressor of the prostate. During the progression of a variety of cancers, the function of TGF-β switches to that of an oncogene. The underlying mechanism behind this switch is poorly understood. Data provided here suggest that hydrogen peroxide-inducible clone-5 (Hic-5, also known as androgen receptor-associated protein 55, ARA55), a LIM domain protein, plays a role in this oncogenic switch. We observed that enforced expression of Hic-5 inhibits transforming growth factor (TGF)-β-induced Smad binding element (SBE)-luciferase reporter activity and the cytostatic effects of TGF-β in prostate cancer cells. Co-immunoprecipitation studies show a physical interaction between Hic-5 and Smad3, which is dependent on the MH2 12

15 domain of Smad3 and the LIM3 domain of Hic-5. In addition, we also found that Hic-5 can instead enhance other TGF-β responses through an alternative mechanism involving Smad7, a key negative regulator of TGF-β signaling. Hic-5 interacts directly with Smad7, enhancing Smad2-dependent TGF-β signaling through inducing loss Smad7 protein. Overexpression of Hic-5 reverses the ability of Smad7 to suppress TGF-β-induced phosphorylation of Smad2 and activation of the Smad2-dependent promoter construct FAST-1/ARE-lux. Correspondingly, silencing of endogenous Hic-5 reduces ARE-lux reporter response to TGF-β in PC3 human prostate carcinoma cell line, consistent with the elevation of endogenous Smad7. Further work suggests that Smad7 protein loss is caused by its physical interaction with Hic-5; however, the proteasomal and the lysosomal pathways are not likely involved in this protein downregulation. Taken together, our findings support that Hic-5, which is overexpressed during prostate cancer progression, interacts with both Smad3 and Smad7; these interactions lead to suppression of Smad3 but induction of Smad2 activity and transcriptional responses. We suggest that this impact of Hic-5 on TGF-β signaling causes tumor progression by inducing epithelial-mesenchymal transition 13

16 and by suppressing the cytostatic responses of TGF-β in prostate cancer cells. Thus, strategies that suppress the expression of Hic-5 in prostate cancer cells or intercept Hic-5 s impact on TGF-β signaling in tumor epithelium are likely to impact on the therapeutic intervention of prostate cancer. 14

17 Chapter 1 Introduction Prostate cancer is the most common life-threatening malignancy in elderly men, particularly in the Western hemisphere. Unless caught early, prostate cancer is an incurable disease with limited therapeutic options for long-term survival. This is largely attributed to our relatively poor understanding of the fundamental events that cause this disease and drive its progression. Elucidating the etiology and pathobiology of this malignancy, particularly at the molecular level, is thus tantamount to the development of new therapeutics. Towards our effort of uncovering the inner secrets of prostate cancer cells, we have focused our attention on the role of a protein named Hic-5, which is a focal adhesion-related protein. In our studies here, we have observed that Hic-5 can regulate prostate tumor cell behavior through physiological interactions with transforming growth factor-β (TGF-β) signaling mediators/regulators, Smad3 and Smad7. 15

18 1.1 Overview of prostate and prostate cancer Human prostate is the male reproductive organ surrounding urethra and below bladder, mainly responsible for producing proteins and ions that constitute the seminal fluid. As a tubuloalveolar gland, prostate is composed of ducts lined with the secretory epithelium, which, in turn, is enclosed by the stroma. Prostate cancer is adenocarcinoma, arising from mutations in normal secretory cells of the prostate gland. It is the most common cancer type in men and is the second leading cause of cancer-related death in men in industrialized countries [1]. The primary risk factors increasing the burden of this disease include diet, race and age. The incidence of prostate cancer in Asian males living in Asia is radically lower than in men in Western countries [2, 3]. One of remarkable features of this cancer is that it develops quite slowly in most cases. Many patients may not be aware that they have it and often die of unrelated causes. Nevertheless, prostate tumor could metastasize to such sites as lymph node and bone, causing severe conditions. The major treatments for prostate cancer are surgery, radiation and hormone 16

19 therapy. The optimal choice greatly depends on the stage of the disease. Surgical resection is typically performed in patient presenting with the early stage, while radiation therapy is an option available at all stages of this disease. In hormonal therapy, the main strategy is surgical or chemical castration to prevent cancer cells from being stimulated by androgens, the steroid hormones required for prostate cell growth. However, prostate cancer is rarely cued through this therapy, as tumor cells acquire resistance to androgen deprivation after a few years of therapy [4]. 1.2 Two important signaling pathways in prostate Prostate epithelial cells are controlled by numerous signaling pathways. Our laboratory has been particularly interested in the role of two of these pathways, namely the androgen and TGF-β pathways. It is well known that androgen supply is critical for the growth of prostate [5], whereas TGF-β is a major negative regulator that induces cell cycle arrest and apoptosis in the normal prostate epithelium [6]. We suggest that androgen and TGF-β cooperate to achieve a 17

20 homeostatic balance of cell growth and cell death in the normal prostate; this balance is disregulated in prostate cancer Androgen signaling in prostate The steroid hormone androgen, particularly in the form of testosterone or dihydrotestosterone (DHT), plays essential roles in growth, differentiation and maintenance of prostate [5, 7, 8]. After androgen ablation, prostate tissue undergoes significant apoptosis and atrophy [9]. Androgen receptor (AR) is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. Without ligand, AR distributes in both the nucleus and the cytoplasm. Following hormone binding, AR molecules dimerize with each other and translocate into the nucleus. The DNA-binding domain of AR contains two zinc fingers and enables AR dimers to bind to specific canonical palindromic DNA sequence, the androgen-responsive element [10]. The resistance of prostate cancer to androgen ablation therapy is often associated with amplification of AR gene [11]. Analyses of AR targets have shown that AR primarily modulates the G1/S phase 18

21 in cell cycle [12]. For example, ligand-bound AR can induce D-type cyclin expression through mammalian target of rapamycin (mtor)-dependent pathway in prostate cancer cells [13], leading to activation of cyclin-dependent kinase (CDK)/cyclin D1 complex, retinoblastoma protein (Rb) phosphorylation/degradation and thereby S-phase progression. Consistently, removal of androgen leads to early G1 arrest in prostate cancer cells [14]. In live cells, both DNA and ligand binding capacities of AR are influenced by a number of coregulators, which further control AR s biological actions [15]. Hic-5, which we will discuss below, is one of them TGF-β signaling in prostate TGF-β, for which there are three mammalian isoforms, is a multifunctional cytokine involved in regulation of proliferation, cell cycle arrest, differentiation, apoptosis, angiogenesis, wound healing and immunosuppression [16-18]. Two transmembrane serine/threonine kinase receptors, TβRI and TβRII, initiate TGF-β signaling [19-21]; and the major downstream effectors are called Smad proteins. 19

22 These Smad proteins are evolutionarily conserved from invertebrate to human, particularly in the N- and C- termini, which are named as MH1 and MH2 domain, respectively [22]. They are divided into three groups: (a) the receptor-regulated Smads (R-Smads), including Smad 2 and 3; (b) the common Smads (co-smad) Smad4, which binds to R-Smads to form heteromeric dimers; (c) the inhibitory Smad7, which antagonizes Smad-mediated signaling. Within the signal transduction, binding of ligand to TβRII promotes recruitment of TβRI to TβRII to form a heteromeric receptor/ligand complex. A constitutively active kinase domain of TβRII then phosphorylates the glycine-serine domain of TβRI, causing activation of the latter [23]. With the assistance of Smad anchor for receptor activation (SARA), Smad2, and possibly Smad3, is recruited to TGF-β receptor complex [24]. The activated TβRI kinase then phosphorylates the two-terminal serines of the receptor-associated Smads [25], thereby promoting their open conformation. Receptor-activated Smads dimerize with co-smad4. and then translocate into the nucleus, where they bind to Smad-binding element (SBE) and interact with other transcription factors to regulate target gene expression [26-29]. The stability of phosphorylated Smads is controlled by 20

23 phosphatase such as PPM1A, which induces dephosphorylation and further nuclear export of R-Smads [30]. Besides Smads, TGF-β receptors can activate other pathways, including Erk, p38 and JNK, involving activation of TGF-β-activated kinase 1 (TAK1), small GTPase Ras and so on [31, 32]. On the other hand, Smad7 also binds to TβRI; but as a negative regulator, it blocks TGF-β signaling mainly through recruiting the E3 ubiquitin ligases to induce proteosomal degradation of TβRI [33, 34]. Its function will be discussed further in Chapter 3. The past decade has seen an accumulation of evidence demonstrating both tumor suppressor and oncogenic activities of TGF-β. In the normal prostate tissues or early-stage cancer, the suppressor activity of TGF-β is dominant. Expression and activation of TGF-β and Smads 2 and 3 are suppressed by physiological levels of androgens in mature prostate [35-37]. Following castration-induced androgen withdrawal, a robust induction of TGF-βs and activation of Smads 2 and 3 occur in the prostate, concomitant with the onset of apoptosis and involution [35-39]. Both in vivo and in vitro studies show that TGF-β is a potent inducer of apoptosis, growth arrest, differentiation of prostate 21

24 epithelia [40-45], and thus implicate a critical role of TGF-β signaling on the dependence of androgen for cell survival. Upon malignant transformation, prostate epithelial cells acquire resistance to TGF-β-induced apoptosis and growth arrest, correlating with downregulation of TGF-β receptor levels and escape from androgen dependence [16, 46-48]. A number of recent in vivo studies demonstrate that loss of TGF-β signaling may play a causal role in carcinogenesis of the prostate [43, 49-52]. However, during tumor progression, TGF-β pathways appear to favor the oncogenic activity, probably associated with hyperactivation of Smads-independent TGF-β signals, such as ras/mapk [17]. It has been reported that secretion of TGF-β by tumor cells, particularly in the late-stage cancer, contributes to invasion and metastasis and reduces host-tumor immune responses [16]. One of such mechanisms is epithelial-mesenchymal transition (EMT). EMT is a biological process necessary for tissue remodeling during embryonic development as well as for tumor progression [53]. During EMT, the highly organized epithelial cells reorganize their actin cytoskeleton, leading to loss of cell-cell adhesions and remodeling of cell-extracellular matrix (ECM) interaction. 22

25 The polarized epithelia then transdifferentiate into separate single fibroblast-like cells with relatively high mobility [53]. TGF-β was the first identified EMT inducer in normal mammary epithelial cells [54]. Later evidence indicated that TGF-β stimulates the expression of Snail and Slug, which are strong inducers of EMT and transcriptional repressors of cell-cell contact molecules, such as E-cadherin [55, 56]. These findings implicate TGF-β signaling as a potential therapeutic target. The crosstalk of androgen and TGF-β pathways has been discussed by many publications. Briefly, AR can associate with Smad3 [57]. Ligand-bound AR inhibits Smad3 activity through blocking Smad3-DNA interaction [57]. Alternatively, Smad3 could upregulate or downregulate AR-mediated transactivation in a cell line-specific manner [58, 59]. 1.3 Hic-5 and its biological function Hic-5 and the LIM protein superfamily Hic-5 (hydrogen peroxide-inducible clone-5) was initially identified as a 23

26 hydrogen peroxide or TGF-β-inducible protein in mouse osteoblast MC3T3-E1, and was shown to prompt cellular senescence [60, 61]. Another research group later found that this protein is able to interact with AR in a ligand-dependent manner and thus renamed it as ARA55 (AR-associated protein 55) [62]. Recent publications have revealed that Hic-5 plays roles in a broad range of biological processes including integrin signaling, steroid hormone action, cell differentiation and tumorigenesis [62-66]. In the human genome, Hic-5 gene has been localized to chromosome 16p11 [67]. Alternate splicing results in a number of Hic-5 isoforms [68]. Among them, there are two typical ones, designated as Hic-5α (longer) and Hic-5β (shorter) (Figure 1-1), respectively. They differ by 17 extra amino acid residues at the N-terminus [69]. Further evidence showed that Hic-5α and Hic-5β may have different functions during the differentiation of mouse myoblast cell line C2C12. Myogenesis is controlled by Hic-5α, but not Hic-5β [70]. At the protein level, immunohistochemistry showed that expression of Hic-5 is essentially restricted to smooth muscle and myoepithelial cells in human tissues, in contrast to its homolog paxillin, whose expression is more widespread [71]. 24

27 Hic-5 belongs to the LIM protein superfamily, according to its four conserved tandem LIM domains at the C-terminus (Figure 1-1). The LIM domain is a cysteine-rich structure with two repeated zinc fingers, which was originally identified in three transcription factors: lin-11 (C. elegans), isl-1 (rat) and mec-3 (C. elegans) [72]. These LIM domains might possess specific DNA sequence-binding ability [73], although no such a sequence has been identified. LIM domains are found in numerous proteins from yeast to human. Depending on the sequence similarity and the overall protein structure, LIM proteins could belong to one of three groups [74]. Members of group I are named as homeodomain proteins, such as LHX proteins and LMO protein (Figure 1-1). They primarily localize in the nucleus and function as transcription factors. Group II LIM domain proteins are cytoplasmic proteins mainly composed of LIM domains, including cysteine-rich proteins known as CRP or CRIP, which could regulate cell differentiation and cytoskeletal remodeling (Figure 1-1) [75]. Compared with the former two, group III LIM domain proteins are more heterogeneous, containing a variable number of LIM domains at their C-terminus. According to their individual N-terminal sequences, this group is further divided 25

28 into two subfamilies. One of them is the zyxin subfamily including zyxin, thyroid hormone interacting protein 6 (Trip6) and lipoma preferred partner (LPP). All zyxin subfamily proteins have three LIM domains at their C-termini, while their N-terminal halves are rich in proline. Typical members of the other subfamily, known as the paxillin subfamily, are paxillin, Hic-5 and leupaxin. They are featured by four C-terminal LIM domains and several N-terminal conserved leucine-abundant regions called LD motifs. Although group III members predominantly reside in cytosol, particularly at focal adhesions, they usually harbor N-terminal nuclear export signals, allowing them to shuttle between the cytoplasm and the nucleus and thus could function as signal transducers [76-79]. Their C-terminal LIM domains mainly serve as protein-protein interaction interfaces for localization at focal adhesions or the nuclear matrix [80, 81]. Additionally, members in this group have been found to bind to the nuclear receptors through their LIM domains and therefore act as the nuclear receptor co-regulators [62]. For example, Trip6 was first identified in a two-hybrid screen as a protein interacting with thyroid hormone receptor in a hormone-dependent manner [82]. Both paxillin and Hic-5 can associate with AR and GR. They have 26

29 the potential to enhance the transactivation mediated by these nuclear receptors [62, 83] Hic-5 at focal adhesions Focal adhesions are dynamic macromolecular structures forming around the heterodimeric transmembrane receptor integrin, through which cells contact with ECM proteins including fibronectin, laminin, collagen and vitronectin. A large number of structural proteins and signaling regulators, such as vinculin, talin, focal adhesion kinase (FAK) and Crk-associated substrate (CAS), are components of focal adhesions. Therefore, focal adhesions can not only relay extracellular mechanical force to cell skeleton for cell anchorage or mobility, but also serve as hubs to transmit signals from ECM to the nucleus and thus affect cell growth, survival or apoptosis. Group III LIM proteins are scaffolding parts of focal adhesions, providing binding sites for tyrosine kinases and structural proteins. In the case of paxillin, LD motifs are responsible for the binding to vinculin or FAK [84]. Although none 27

30 of LIM domain-binding partners has been found at focal adhesions, LIM domains are required to target paxillin to focal adhesions [85]. Engagement of integrin with ECM proteins can cause N-terminal phosphorylation of paxillin possibly by FAK, creating binding sites for Srk homology 2 (SH2) domain and thus resulting in recruitment of the adaptor proteins Crk and CrkL [86, 87]. This tyrosine-phosphorylated paxillin-based complex further binds to tyrosine kinases such as Src and then positively impacts downstream signal transduction [88, 89]. Hic-5 has a similar binding profile to paxillin at focal adhesions with respect to interaction with FAK, vinculin [63] and GIT1, an Arf GTPase-activating protein [90], in spite of using different regions for binding. However, Hic-5 seems to play an interceptive role in integrin-mediated signaling. It can interact with the negative regulator, C-terminal Src kinase (Csk), and thus cause inactivation of Src [63]. Another mechanism for this negative effect could be mediated through FAK, which interacts with Hic-5 but does not phosphorylate it [61]. The resultant competition between paxillin and Hic-5 to bind to FAK could decrease activation of Src and Rho-family small GTPases [91]. Accordingly, the binding of Hic-5 to FAK accounts for Hic-5-induced cell senescence, since Hic-5 level was 28

31 diminished correspondingly during immortalization in FAK-positive, but not FAK-negative, mouse embryonic fibroblast [92]. Through a similar mechanism, overexpression of Hic-5 reduced spreading of mouse fibroblast NIH 3T3 on fibronectin by Hic-5 [91]. FAK-like kinase proline-rich tyrosine kinase-2 (PYK2) can also associate with Hic-5 at focal adhesions [93] or cytoskeleton [94]. Most importantly, it phosphorylates Hic-5 tyrosine residues 47 and 60 at Hic-5 N-terminus (Figure 1-1, Hic-5α numbering, [93, 95]). Although PYK2 is structurally similar to FAK, this kinase is a negative regulator of cell proliferation and survival [96, 97]. The phosphorylation of Hic-5 by PYK2 suggests that Hic-5 is a transducer downstream of PYK2, confirming the disruptive role of Hic-5 in integrin signaling. Conforming to this negative function, the tyrosine-phosphorylated Hic-5 can block epidermal growth factor (EGF)-induced lamellipodia formation through inhibition of Rac activation [98]. Interestingly, Hic-5 was also found to bind to protein-tyrosine phosphatase PEST at focal adhesions as well [99], but the physiological implication of this interaction is not defined yet. Besides focal adhesion-related proteins, another typical cytoplasmic 29

32 Hic-5-binding partner is the heat shock protein 27 (hsp27). This interaction has been reported to repress the cellular protective effect of hsp27 [100] Hic-5 in the nucleus While the majority of Hic-5 has a cytoplasmic distribution, this protein is present in the nucleus as well. It was reported that Hic-5 can form a nuclear-cytoplasmic shuttling complex with PINCH (a LIM-only protein) and integrin-linked kinase [101]. Hic-5 nuclear export signal is composed of LD3 motif and two nearby cysteine residues [81, 102]. Its nuclear localization signal resides at Hic-5 LIM4 domain, since deletion of LIM4 domain significantly decreases binding of Hic-5 to nuclear matrix [80]. Recent findings strongly support the involvement of Hic-5 in gene regulation. So far, two Hic-5-responsive genes have been well demonstrated: p21 and c-fos [103, 104]. As an adaptor protein, the regulatory effect of Hic-5 on gene expression could depend on its binding partners, including some transcription factors, in the nucleus. For example, in Wnt signaling, Hic-5 interacts with 30

33 Lef/Tef transcriptional complex and functions as a repressor [105]. Our laboratory and another research group reported that Hic-5 can bind to the transcription factor Smad3 and further suppress or enhance their activity, respectively [103, 106]. Recent reports support the role of Hic-5 as co-activator for such nuclear receptors as AR, glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor γ (PPARγ) in a ligand-dependent manner [62, 80, 102, 107]. These interactions are mediated by Hic-5 LIM domains, but not LD motifs. Hic-5 binds to the ligand-binding domain of AR and dramatically enhances the androgen-responsive promoter activity in prostate cancer cell lines [62]. Based on this positive effect, Hic-5 is known as ARA55 (AR receptor-associated 55). In some circumstances, disruption of Hic-5-AR interaction is inhibitory for AR activity. For example, PYK2 blocks AR transaction via interaction and further phosphorylation of Hic-5, thus sequestering Hic-5 from AR in prostate carcinoma cells [108]. Hic-5 A413T (alanine to threonine mutation) mutant, which loses AR-binding ability but still dimerizes with wild-type Hic-5, can suppress AR activity [109]. 31

34 Hic-5 also controls GR activity, as extracellular signal-regulated kinase-8 (ERK-8) blocks GR activity through sequestering Hic-5 in a kinase-independent manner [110]. Hic-5-binding region in GR is the tau2 motif containing about 30 amino acid residues, which possesses transcriptional activation and nuclear matrix targeting activity [102]. LIM3 and LIM4 domains of Hic-5 are necessary, but not sufficient, for strikingly promoting effects on GR transactivation [80]. In breast cancer cells, sequential chromatin immunoprecipitation assays indicated that the Hic-5-GR complex associates with many general transcription factors at the glucocorticoid-responsive mouse mammary tumor virus (MMTV) and c-fos promoters in hormone-treated cells, including transcriptional intermediary factor2, receptor-associated coactivator 3, camp response element binding protein and p300 [111]. Silencing Hic-5 could reduce the recruitment of these transcription factors to these promoters. Interestingly, without hormone treatment, Hic-5 was also detected at the glucocorticoid-responsive MMTV promoter region. However, it binds to the nuclear receptor corepressor (NCoR), implicating a scaffolding function of Hic-5 in steroid hormone receptor signaling [111]. PPARγ is another Hic-5-binding nuclear receptor, known to be both necessary 32

35 and sufficient to induce the differentiation of adipocytes [112]. In pre-adipocytes, enforced expression of Hic-5 was found to induce epithelial markers in the presence of PPARγ ligand, while silencing Hic-5 inhibited this differentiation [107] Hic-5 and TGF-β signaling Hic-5 mrna and protein levels are induced by TGF-β in various cell lines [60, 113]. Through such induction, Hic-5 may mediate cell proliferation induced by TGF-β. Recent findings nicely support such a role of TGF-β in hypertrophic scar fibroblasts (HTSF) [114]. HTSF have myofibroblast properties, expressing a relatively higher level of Hic-5 than normal dermal fibroblast cells. After severe injury to dermis, these cells become hyperactive and accumulate in hypertrophic scars. TGF-β slows the HTSF cell growth through inducing cell cycle inhibitors p21 and p15 in a Hic-5-dependent manner [114]. Silencing Hic-5 abates cellular response to either autocrine or exogenous TGF-β-induced p21 and p15 expressions, while overexpression of Hic-5 enhances the protein levels of these two molecules [114]. 33

36 Additionally, Hic-5 is essential for the maintenance of a myofibroblast phenotype by TGF-β in HTSF. Silencing Hic-5 in HTSF dramatically lowers autocrine TGF-β1 production, leading to decreased expression of ECM proteins and the myofibroblast marker, smooth muscle α-actin [115]. This result confirms the role of Hic-5 in cell differentiation. Although TGF-β normally functions a negative regulator of cell growth, this function of TGF-β may be switched to that of a tumor promoter enhancing tumor progression through multiple mechanisms including EMT [17]. Recent reports support the involvement of Hic-5 and its homolog, paxillin, in TGF-β-regulated EMT. Paxillin δ isoform was first found to be downregulated during TGF-β-induced EMT in normal murine mammary gland (NMuMG) epithelial cells, while its overexpression disrupts this biological process [113]. Correspondingly, Hic-5 and paxillin δ was shown to be expressed in a reciprocal manner, suggesting that Hic-5 could facilitate EMT by TGF-β. Later on, the same research group confirmed that Hic-5 contributes to EMT in rat proximal tubule epithelial (MCT) cells and MCF10A human normal mammary gland cells after TGF-β treatment [66]. This function of Hic-5 apparently involves activation of the 34

37 small GTPase RhoA pathway, which regulates actin stress fiber formation [116]. Consistently, silencing Hic-5 suppresses TGF-β-dependent RhoA activation and cell migration. Our laboratory also substantiated this function of Hic-5 in DU145 human prostate carcinoma cells, in which silencing Hic-5 represses the mesenchymal marker vimentin induction and the epithelial marker E-cadherin translocation by TGF-β (Figure 3-6A and 6B). The possible mechanism underlying this observation will be discussed below Hic-5 and prostate Immunohistochemical staining of the rat prostate reveals that Hic-5 localizes primarily in the stroma, with no detection in the epithelial layers [65, 117]. Our laboratory confirmed this result in different prostatic cell lines and cell strains. Prostatic fibroblast, myofibroblast or metastatic cancer cells express markedly higher levels of Hic-5 than prostatic epithelial cells (Figure 1-2). A recent publication proposed that Hic-5 may regulate tumorigenesis through epithelial-stromal interaction [65]. In WPMY-1 prostatic myofibroblast cell line, 35

38 silencing of Hic-5 abolishes the response of keratinocyte growth factor (KGF) to androgen [65, 118]. Moreover, Hic-5 has the potential to affect autocrine TGF-β levels in HTSF cells, which possess myofibroblast phenotype [115]. Both KGF and TGF-β represent important factors by which myofibroblasts communicate with tumor epithelium within tumor microenvironment [119], and therefore Hic-5 may modulate epithelial tumorigenesis through controlling stromal secretion of paracrine growth factors. Furthermore, clinical analysis indicated that higher Hic-5 protein levels may be associated with shorter survival time of patients with hormone-refractory prostate cancer [120]. Nevertheless, further effort is definitely required to confirm this epithelial-stromal model for Hic Purpose of this research In summary, Hic-5 plays multiple physiological roles in integrin signaling, nuclear receptor action, cell senescence, cell differentiation and tumorigenesis [60, 62, 64, 66, 101, 107, 121]. In our laboratory, we are interested in the functions of Hic-5 in prostatic cancer cells. 36

39 The homeostasis of the prostate could depend on crosstalk of androgen and TGF-β signalings. Our laboratory previously showed that ligand-bound AR blocks Smad3 activity in NRP-154 and LNCaP prostate adenocarcinoma cell lines, occurring through a direct binding of AR with Smad3 [57]. However, ligand-bound AR failed to suppress Smad3 in a non-tumorigenic prostatic epithelial cell line, NRP-152 (data not shown). Since AR activity is controlled by many AR co-regulators [15], we initially hypothesized that NRP-152 cells express certain AR binding proteins that intercept the ability of AR to antagonize Smad3 responses. To confirm this hypothesis, we screened a series of AR co-regulators including ARA24, ARA54, Hic-5/ARA55, ARA70 and ARA160. Our preliminary data suggested that Hic-5 may actually enhance the inhibitory effect of ligand-bound AR on Smad3 in NRP-154 cells. However, further work revealed an intriguing finding that Hic-5 inhibited Smad3 independent of either androgen or AR. Considering the differential protein levels of Hic-5 in normal prostatic epithelial cells and metastatic prostatic cancer cells, our finding suggested that Hic-5 may play a role in tumorigenesis by interfering with TGF-β responses [66]. We 37

40 therefore hypothesized that abnormal expression of Hic-5 may be a trigger for tumor progression in the prostate. In this dissertation, we will show that Hic-5 can communicate with both Smad3 and Smad7 through direct protein-protein interactions, resulting in enhanced activation of Smad2 activity while blocking the overall activity of Smad3 down-stream of its activation. Therefore, the induction of Hic-5 by TGF-β in prostate cancer may facilitate the progression of this malignancy. 38

41 Figure 1-1 Group I N LIM1 LIM2 C LMO Group II N LIM Gly-rich C CRIP Group III Hic-5β start aa18 NES LD1 C47&74 N Y43&60 LD2 LD3 LD4 LIM1 LIM2 LIM3 LIM4 Hic-5α start aa1 C Hic-5 39

42 Figure 1-1. Schematic structures of LIM domain proteins. Typical proteins [122]: LMO (Group I) CRIP (Group II), Hic-5 (Group III). Hic-5α starts from amino acid (aa) residue No.1, while Hic-5β starts from amino acid residue No. 18 [69]. NES: the nuclear export signal is composed of the cysteine residues 47 and 74 and the LD3 motif [81]. Y43&60: the tyrosine residues 43 and 60 are the phosphorylation sites of proline-rich tyrosine kinase 2 (PYK2) [98, 108]. 40

43 Figure 1-2 NRP-154 NRP-152 DU145 PC3 CWR22V1 LNCaP IB: Hic-5 rugm WPMY-1 rdpf rvpf NRP-154 PC3 IB:Hic-5 41

44 Figure 1-2. Endogenous Hic-5 protein levels in different prostatic cell types. NRP-154, rat prostatic tumorigenic cell line; NRP-152, rat prostatic non-tumorigenic cell line; DU145, human prostate carcinoma cell line from brain metastasis; PC3, human prostate carcinoma cell line from bone metastasis; CWR22V1, human prostate carcinoma cell line; LNCaP, human prostate carcinoma cell line; rugm, rat urogenital mesenchymal cell; WPMY-1, human prostate myofibroblast; rdpf, rat dorsal prostate fibroblast; rvpf, rat ventral prostate fibroblast. Prostatic malignant carcinoma cells or stromal cells express high levels of Hic-5. 42

45 Chapter 2 Hic-5 functions as an inhibitor of the regulatory Smad3 As mentioned above, previous work from our laboratory showed that ligand-bound AR blocks TGF-β signaling in the prostate carcinoma cells lines, NRP-154 and LNCaP, through the direct association of Smad3 to the ligand binding domain of AR [57]. However, this inhibition appears to be cell line-specific, as it does not happen in NRP-152 rat prostate non-tumorigenic cell line (unpublished data). These data led us to hypothesize that AR co-regulators may play a role in such differential inhibition, since AR activity is controlled by many coregulators [15]. Therefore, we tested a series of AR-binding proteins including ARA24, ARA54, Hic-5 (ARA55), ARA70 and ARA160 with respect to their ability to affect Smad3 inhibition by ligand-bound AR. We observed that Hic-5 blocks Smad3 activity, however, in an androgen and AR-independent manner. In addition, Hic-5 protein level was found to be higher in stromal or carcinoma cells than in epithelial cells in prostate, suggesting that Hic-5 may be involved in tumorigenesis. These observations intrigued us to explore the physiological role of Hic-5 in TGF-β signal transduction in prostate cancer cells, 43

46 although it was not what we planed to pursue in the first place. In this chapter, we report that Hic-5 effectively inhibits Smad3 activity. Experimental support is provided that such suppression occurs through a direct interaction of the LIM3 domain of Hic-5 with the MH2 domain of Smad3 in the nucleus. It is likely that Hic-5, which is induced by TGF-β in certain cells types, may function in a negative feedback loop to impact TGF-β responses. 2.1 Results Hic-5 blocks transcriptional activation by Smad3 As mentioned above, we originally hypothesized that AR-coregulators could control the ability of AR to suppress Smad3-mediated transcription. Subsequent screening with the luciferase reporter assays showed that Hic-5 might be a candidate. To further test the effect of Hic-5 on AR-Smad3 interaction, NRP-154 cells, which are highly responsive to TGF-β, were co-transfected with the reporter SBE4 BV -luciferase and various combinations of the expression vectors for constitutively active Smad3 (CA-Smad3), AR, Hic-5 or the corresponding empty 44

47 vectors as control. The next day, cells were supplemented with 10 nm DHT or vehicle, and luciferase activity was measured 24 h later. Transfection of CA-Smad3 caused SBE4 BV -luciferase reporter induction, and as before [57] this induction was blocked by ligand-bound AR (Figure 2-1A). Expression of Hic-5 further reduced CA-Smad3 activity to the basal level. Unexpectedly, this inhibitory effect by Hic-5 was not lost in the absence of AR or DHT (Figure 2-1A, bar 6 and 7), suggesting such suppression was independent of both DHT and AR. In a similar assay, TGF-β-induced SBE4 BV -luciferase reporter activity was also repressed by Hic-5 in NRP-154 cells, suggesting the inhibition of endogenous Smad3 (Figure 2-1B). In addition, co-transfection with Hic-5 repressed SBE4 BV -luciferase activity-induced by CA-Smad3 (Figure 2-1C) and TGF-β (data not shown) in NRP-152 cells. To examine if this inhibition is a common feature of LIM proteins, we developed pcdna3-based Myc-paxillin and Myc-zyxin expression constructs and examined their effects as above. In contrast to Hic-5, enforced expression of neither paxillin nor zyxin altered activation of SBE4 BV -luciferase by CA-Smad3 (Figure 2-1D). 45

48 We further characterized the ability of Hic-5 to suppress Smad3-dependent TGF-β responses using a plasminogen activator inhibitor-1 (PAI-1) promoter reporter, 3TP-lux, which is widely used to test response to TGF-β [20]. In both NRP-154 and NRP-152 cells, enforced expression of Hic-5 by transient transfection of FLAG-Hic-5-pcDNA3 effectively blocked 3TP-lux induction by CA-Smad3 (Figure 2-2A and 2B). We extended this experiment to the human prostatic androgen-responsive carcinoma cell line, LNCaP. Because these cells respond to TGF-β very weakly due to low levels of TβRII [123], they were co-transfected with pcmv5-tβrii expression plasmid to enhance response to TGF-β. Similar to NRP-154 and NRP-152 cells, transfection of FLAG-Hic-5-pcDNA3 expression substantially suppressed either TGF-β or CA-Smad3-induced 3TP-lux activity in LNCaP cells (Figure 2-2C). We then explored the effect of overexpressing Hic-5 in LNCaP cells on the levels of TGF-β-regulated proteins by Western blot analysis. For this, LNCaP cells were efficiently transfected with TβRII-pCMV5 and either FLAG-Hic-5-pcDNA3 or empty vector alone. The next day, cells were treated with 10 ng/ml TGF-β1 or vehicle, and 48 h later cells were lysed in RIPA buffer. Western blot analysis of 46

49 total cell lysates from the above revealed that Hic-5 blocks the ability of TGF-β to induce PAI-1 expression (Figure 2-2D) but not the loss of cyclin D2 expression (data not shown). These results suggest that Hic-5 selectively suppresses some TGF-β-induced responses while not affecting others Hic-5 can block Gal4-CA-Smad3 MH2-ML activity Hic-5 could bind to specific DNA sequences [73], suggesting that this protein may interact directly with SBE and then block interaction of Smad3 to SBE. To test this possibility, we developed a DNA construct encoding a fusion protein, Gal4 binding domain (BD)-CA-Smad3. Transfection of NRP-154 cells with this construct resulted in the induction of the reporter Gal4-luciferase activity. Co-transfection of FLAG-Hic-5-pcDNA reduced this induction relative to the empty vector control (Figure 2-3), consistent with the results shown above. Thus, Smad3 or another Smad protein, but not SBE, is the target of Hic-5-mediated inhibition. 47

50 2.1.3 Hic-5 physically interacts with Smad3 Many proteins have been found to bind to Hic-5 through either N-terminal LD motifs or C-terminal LIM domains [51, 62, 90, 92, 93, 99, 100, 103, 124]. We speculated that the inhibitory effect of Hic-5 on Smad3 results from a direct interaction between Hic-5 and Smad3. Using co-immunoprecipitation (co-ip) experiments, we tested the possible binding of Hic-5 to Smad2, Smad3, Smad4 or CA-Smad3. HEK293 cells were co-transfected with pcdna3-based expression plasmids for each of the FLAG-tagged Smad proteins together with Myc-tagged Hic-5 or paxillin. Twenty-four h later, the FLAG-tagged proteins were immunoprecipitated with anti-flag IgG and Myc-tagged Hic-5 was detected by immunoblotting. This experiment revealed a physical interaction between Hic-5 and wild-type Smad3 (WT-Smad3), CA-Smad3 or Smad4 (Figure 2-4A), although the latter two interactions appeared weaker. Alternatively, when FLAG-Hic-5 was immunoprecipitated with anti-flag IgG, Myc-WT Smad3 was co-immunoprecipitated (Figure 2-4B). 48

51 2.1.4 The binding of Hic-5 and Smad3 can occur in a cell-free system A GST pull-down assay was done to test if Hic-5 can bind to Smad3 in vitro. Lysates prepared from HEK293 cells transfected with either Myc-Hic-5 or empty vector were incubated overnight at 4 with either GST or purified GST-Smad3 fusion protein in the presence of Glutathione-Sepharose resin. GST-Smad3 was then pulled down, and the material eluted from the washed resin was immunoblotted with antibody against Myc. The above experiment showed that the binding of Hic-5 to Smad3 can occur under a cell-free condition (Figure 2-4C) Smad3 MH2 domain is responsible for the interaction of Hic-5 with Smad3 Smad3 has a N-terminal MH1 domain and a C-terminal MH2 domain, separated by a middle linker region (ML) (Figure 2-5A, [18]). The MH1 domain is responsible for recognizing and binding to SBE [125], while the MH2 domain recruits other transcription factors that initiate transcription [126]. To define the individual domains of Smad3 binding to Hic-5, we developed a series of 49

52 pcdna3-based constructs encoding FLAG-tagged Smad3 truncations (Figure 2-5A). HEK293 cells were co-transfected with Myc-tagged Hic-5 and each of the FLAG-tagged Smad3 truncations, and 24 h after transfection cell lysates were subjected to immunoprecipitation analysis as in described above. The M2 mouse monoclonal antibody against FLAG captured the complex of Hic-5 with either Smad3 MH2 alone or Smad3 MH2-ML, while the latter seemed to have a stronger affinity for Hic-5 (Figure 2-5B). This suggests that MH2 domain is required for the Hic-5-Smad3 interaction, while ML domain may enhance this binding. We used a mammalian two-hybrid system to test if the physical interaction between Hic-5 and MH2-ML region of Smad3 occurs in intact cells. In this experiment, NRP-154 cells were co-transfected with the construct encoding BD-Hic-5 fusion protein, the construct encoding NF-κB activation domain (AD) fused to either the MH2-ML or MH1-ML of Smad3, and the Gal4-luciferase reporter. Co-transfection of the BD-Hic-5 construct with AD-MH2-ML induced more luciferase activity relative to negative controls or the AD-MH1-ML construct (Figure 2-5C), consistent with our co-ip data. These data further suggest that a physical interaction is formed between Hic-5 and the MH2-ML region of Smad3 in 50

53 live cells rather than after cells are lysed Hic-5 C-terminus is critical for the inhibition of Smad3 activity Binding of Smad3 and Hic-5 could occur through Hic-5 LD motifs or LIM domains, since both parts were reported to serve as protein-protein interaction interfaces. To define the region of Hic-5 essential for the inhibition of Smad3 activity, we prepared Myc-tagged Hic-5 truncations, named as A2 to A8 (Figure 2-6A). In co-ip experiments using HEK293 cells, all Hic-5 truncations except A6, A7 and A8 were pulled down by WT-Smad3 (Figure 2-6B). The results from the luciferase promoter-reporter experiments were consistent with these observations (Figure 2-6C). It shows that A2, A3 and A4, which contain the intact C-terminus of Hic-5, promote similar inhibition of CA-Smad3-induced SBE activity, compared with the full-length Hic-5. These results strongly support our model that Hic-5 suppresses transcriptional effects of Smad3 through forming a physical interaction with this transcription factor. However, A6, A7 and A8 did not appear to interact with Smad3 (Figure 2-6B,) yet they partially inhibited transcription by 51

54 Smad3. Thus, other mechanisms that do not involve the physical interaction of Hic-5 with Smad3 may also be involved in inhibition of Smad3 activity by Hic Hic-5 can reverse TGF-β-induced cell cycle arrest Since Smad3 is a major factor determining the cytostatic effect of TGF-β in NRP-154 cells (data not shown), Hic-5 was expected to reverse TGF-β-induced cell cycle arrest and even apoptosis. To test this hypothesis, we inspected the DNA content in individual cells by flow cytometry. NRP-154 cells infected with Hic-5-adenovirus (Ad) or control Ad were treated with TGF-β or vehicle for 2 d. Afterwards, cells were stained with propidium iodide and then analyzed by flow cytometry. In this way, essentially 100% of these cells can be transduced, assessed by β-galactosidase-ad infection and the following conversion of the substrate X-gal to blue product. The level of Hic-5 in NRP-154 cells expressed with this adenovirus construct was comparable to that expressed at endogenous level in normal rat prostate fibroblasts (data not shown), and it was thereby within a physiological range. 52