A ROLE FOR MP1 SCAFFOLD PROTEIN IN BREAST CANCER CELLS By Mihaela Marina A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Physiology 2011 ABSTRACT A ROLE FOR MP1 SCAFFOLD PROTEIN IN BREAST CANCER CELLS By Mihaela Marina Breast cancer cells are characterized by their uncontrolled proliferation, high motility and ability to escape cell death. These pathophysiological processes are driven by aberrant expression and activity of various intracellular pathways, including ER, PI3K/AKT/mTOR, MAPK and integrins. Activated by estrogen, growth factors, nutrients, and other extra- and intracellular stimuli, these signaling pathways can cooperate or compensate for each other in order to promote tumor growth, metastasis and drug resistance. In normal cells, signaling can be tightly regulated by scaffold proteins that can turn on or off pathways, according to the cellular context. One such scaffold protein is MP1, which was initially discovered within the MAPK pathway. Subsequent studies revealed its association with additional pathways and its implication in regulating spreading, migration, proliferation, and differentiation. Since the pathways MP1 interacts with regulate breast tumorigenesis, we hypothesized and MP1 can directly participate in regulating the proliferation and survival of breast cancer cells. A panel of human mammary epithelial cell lines, consisting of ER-positive and ERnegative tumorigenic and non-tumorigenic cells was used. MCF-7 ER-positive and MDA-MB-231 ER-negative cells represented the main models for our studies. We confirmed that MP1 was expressed in all the mammary cell lines investigated, with collectively higher levels in ER-positive breast cancer cells compared to ER-negative ones. We then used siRNA duplexes to investigate the role of MP1 in breast cancer cells. First, we observed that MP1 silencing specifically induced cell death of ERpositive cells, whereas ER-negative cell lines examined were largely unaffected. Next, we established that the observed cell death effect was apoptosis, as indicated by PARP cleavage and downregulation of Bcl-2 pro-survival protein. These data suggested a novel pro-survival role of MP1 protein that is specific to ER-positive breast cancer cells. After establishing the biological relevance of MP1 silencing, we undertook a mechanistic approach and investigated the effects of MP1 siRNA on several signaling pathways. Partial depletion of MP1 was correlated with significant decrease in AKT activity and ER levels and activity. In contrast, MAPK activity was not affected, whereas reduced β1 integrin expression did not reach statistical significance. In addition, the pro-apoptotic effects triggered by MP1 silencing were not dependent on ER signaling, which is also not required for survival of MCF-7 cells. Finally, we re-confirmed that in contrast to MDA-MB-231 cells, MCF-7 cells require PI3K for survival and active AKT was able to partly rescue the cell death phenotype induced by MP1 silencing. In summary, the studies presented here indicate a novel role for MP1 scaffold protein in ER-positive breast cancer cells. MP1 silencing resulted in cell death of ER-positive breast cancer cells only, therefore, expression of MP1 appears to be a requirement for promoting survival of this subset of breast cancer cells. They further suggest that the mechanism of cell death that occurred is apoptosis and this was mediated via PI3K/AKT signaling with possible cooperation from ER and β1 integrin. ACKNOWLEDGEMENTS Graduate school, especially the PhD research, is often regarded as a tedious and solitary journey and my experience was no exception to this belief. While most of the time I was alone trying to unveil the secrets of breast cancer cells, there were many people I could share my excitement and frustrations with. I would like to thank Dr. Susan Conrad for her support and patience and for modeling my scientific thinking. The lessons I learnt from her will be very valuable during my research career. My guidance committee was extremely helpful therefore, I express my gratitude to Drs. Kathy Gallo, Richard Miksicek, Hua Xiao and Vilma Yuzbasiyan-Gürkan. Another supportive group was the Breast Cancer Signaling Network Consortium that informally guided me especially at the beginning of my project. Throughout my studies, I have always counted on the support provided by the Department of Physiology, especially Dr. Arthur Weber and Ms. Kim Crain. My journey would not have been complete without the help of my student fellows and friends here, at Michigan State University. Last but not least, I am grateful for the unconditional love and support of my family and friends from Romania.     iv TABLE OF CONTENTS LIST OF FIGURES..........................................................................................................vi LIST OF ABBREVIATIONS...........................................................................................viii CHAPTER 1.....................................................................................................................1 Literature review I.Breast cancer incidence, risk factors, and the importance of targeted therapies...........2 II.Breast cancer subtypes and therapies..........................................................................3 A.Estrogen receptor-positive breast tumors.....................................................................4 B.HER2-overexpressing breast tumors............................................................................5 C.Triple negative breast tumors........................................................................................6 III.Signaling pathways in breast cancer and the importance of scaffold proteins.............8 A.Estrogen receptor..........................................................................................................8 B.PI3K/AKT/mTOR.........................................................................................................10 C.MAPK..........................................................................................................................13 D.PAK.............................................................................................................................14 E.Integrins.......................................................................................................................15 F.Scaffold proteins..........................................................................................................18 G.MP1 scaffold protein...................................................................................................19 IV.Apoptosis....................................................................................................................21 References ....................................................................................................................27 CHAPTER 2...................................................................................................................46 The Role of MP1 Scaffold Protein in Survival of Breast Cancer Cells Abstract...........................................................................................................................47 Introduction.....................................................................................................................47 Materials and methods...................................................................................................49 Results............................................................................................................................54 Discussion......................................................................................................................73 References ....................................................................................................................79 APPENDIX.....................................................................................................................83 The Role of MP1 Scaffold Protein in Estrogen-regulated Functions of Breast Cancer Cells Abstract...........................................................................................................................84 Introduction.....................................................................................................................85 Materials and methods...................................................................................................87 Results............................................................................................................................91 Discussion......................................................................................................................97 References.....................................................................................................................99 CONCLUDING REMARKS..........................................................................................103 v LIST OF FIGURES Figure 1: Signaling pathways in breast cancer...............................................................25 Figure 2: MP1 expression in breast cancer cell lines.....................................................55 Figure 3: MP1 silencing using two independent siRNA sequences................................57 Figure 4: MP1 expression is required for attachment and survival of ER-positive breast cancer cells.....................................................................................................................59 Figure 5: Quantitation of detachment and death induced by silencing of MP1 in ER positive breast cancer cells.............................................................................................60 Figure 6: MP1 expression is not required for attachment or survival of ER-negative mammary epithelial cells................................................................................................61 Figure 7: Quantitation of detachment and death induced by silencing of MP1 in ERnegative mammary epithelial cells..................................................................................63 Figure 8: MP1 knockdown induces apoptosis of MCF-7 but not of MDA-MB-231 cells.................................................................................................................................65 Figure 9: MP1 silencing decreases ER mRNA levels and transcriptional activity..........66 Figure 10: ER silencing does not rescue the apoptosis induced by MP1 siRNA in MCF-7 cells.................................................................................................................................67 Figure 11: Effect of MP1 knockdown on cellular signaling pathways.............................69 Figure 12: The PI3K/AKT pathway is required for survival of MCF-7 but not MDA-MB231 cells.........................................................................................................................71 Figure 13: Constitutively active AKT1 partially rescues MCF-7 cells from the apoptosis induced by MP1 siRNA...................................................................................................72 Figure 14: Proposed model of MP1 siRNA-induced apoptosis of ER-positive breast cancer cells.....................................................................................................................75 Figure 15: Cell cycle arrest rescues the apoptosis induced by MP1 siRNA in MCF-7 cells.................................................................................................................................92 Figure 16: Effect of MP1 knockdown on DNA synthesis................................................94 vi Figure 17: Effect of MP1 knockdown on estrogen-stimulated migration of MCF-7 cells.................................................................................................................................95 vii LIST OF ABBREVIATIONS ADH atypical ductal hyperplasia AI aromatase inhbitor ALH atypical lobular hyperplasia AMP adenosine monophosphate AP-1 activating protein-1 Apaf-1 apoptotic protease activating factor 1 Bcl-2 B cell lymphoma 2 BrdU 5-bromo-2-deoxyuridine BSA bovine serum albumin Ccd42 cell division control protein 42 CDK cyclin-dependent kinase CSS charcoal stripped serum DAPI 4',6-diamidino-2-phenylindole DCIS ductal carcinoma in situ DISC death-inducing signaling complex E2 estrogen, 17-β estradiol ECM extracellular matrix EGF epidermal growth factor EGFR epidermal growth factor receptor ER estrogen receptor alpha ERE estrogen response element viii ERK extracellular signal-regulated kinase FADD Fas-associated death domain FAK focal adhesion kinase Fas fatty acid synthase FBS fetal bovine serum FLIP FLICE inhibitory protein Gab2 GRB2-associated-binding protein 2 GIP1 GBF interacting protein 1 GSK-3 glycogen synthase kinase-3 GTP guanosine triphosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HER2 human epidermal growth factor receptor 2 IAP inhibitor of apoptosis ICI ICI 182,780 IGF insulin-like growth factor IFG-IR insulin-like growth factor 1 receptor ILK integrin-linked kinase JNK Jun N-terminal kinase LCIS lobular carcinoma in situ MAPK mitogen-activated protein kinase MAPKSP1 mitogen-activated protein kinase scaffold protein 1 MEK MAPK/ERK kinase MMTV mouse mammary tumor virus ix MNAR modulator of non-genomic action of estrogen receptor MP1 MEK binding partner 1 mTOR mammalian target of rapamycin mTORC mammalian target of rapamycin complex NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells PAK1 p-21-activated kinase-1 PARP poly(ADP-ribose) polymerase PBS phosphate buffer saline PDK1 phopshoinositide dependent kinase PI3K phopshoinositide 3-kinase PI3KCA phopshoinositide 3-kinase constitutively active PIP3 phosphatidylinositol-3,4,5-trisphosphate POSH plenty of SH3 domains PR progesterone receptor PTEN phosphatase and tensing homolog Raptor regulatory associated protein of mTOR Rho Ras homologous Rictor rapamycin-insensitive companion of mTOR RTK receptor tyrosine kinase SERM selective estrogen receptor modulator siRNA small interfering ribonucleic acid TNBC triple negative breast cancer TNF tumor necrosis factor x TNF-R1 tumor necrosis factor receptor 1 TRAIL TNF-related apoptosis-inducing ligand VEGF vascular endothelial growth factor xi CHAPTER 1 LITERATURE REVIEW 1 LITERATURE REVIEW I. Breast cancer incidence, risk factors, and the importance of targeted therapies Over the past decade, both the incidence and mortality rate of breast cancer have decreased, reflecting improvements in screening, diagnosis and treatment methods. In spite of this encouraging progress, breast cancer remains the most common form of cancer detected globally and the second leading cause of cancer-related deaths in women. In 2011, over 39,000 women are expected to lose their lives to this disease in the United States alone (1, 2). It is estimated than one in eight women will develop breast cancer at some point in her lifetime. The chance that a woman will be diagnosed with breast cancer can be estimated based on reproductive factors including age at onset of menarche, pregnancy status, use of oral contraceptives and hormone replacement therapies. In addition, age, family history, BRCA1 and BRCA2 mutations and lifestyle factors such as physical activity, obesity, and alcohol consumption affect breast cancer risk. Fatality from breast cancer is mainly due to metastasis and the risk of metastasis and relapse is correlated with a series of parameters including tumor size and grade, lymph node involvement, hormone receptor status, HER2 overexpression, and gene expression signatures (3). A decrease in incidence and mortality rates might be made possible by a more in depth understanding of the aforementioned clinical and biological determinants. Biomedical research continues to have a tremendous impact on the management of this disease through analysis and characterization of genetic, molecular and cellular risk factors. 2 In addition to providing reliable data about the probability of breast cancer occurrence and mortality, basic and translational research is the driving force behind improving treatment methods. Effective treatments include single use or combinations of radiation, surgery, chemotherapy and endocrine therapy. However, there are subsets of patients who do not benefit from current therapies, and breast tumors often undergo recurrence due to chemotherapy failure and acquired resistance to all therapies. The following section reviews the major subtypes of breast cancer in terms of general mechanisms of progression, incidence, prognosis, current treatment and resistance and future directions of biomedical research. II. Breast cancer subtypes and therapies The uncontrolled proliferation of mammary epithelial cells leads to the formation of premalignant lesions, called atypical ductal or lobular hyperplasia (ADH or ALH), which carry a high risk of developing breast cancer. This condition precedes the non-invasive stages called ductal or lobular carcinoma in situ (DCIS or LCIS). Since tumors progressing from DCIS have invasive potential (4), their treatment usually requires surgery, whereas patients with LCIS are subjected to careful screening or endocrine therapy (5). From the molecular standpoint, there are four main subtypes of breast tumors that are treated with current therapies: ER-positive, ER-negative, HER2-positive and triple negative (ER-/PR-/HER2-) breast tumors. Another classification scheme for breast tumors separates them into five subtypes: Luminal A (ER+ and/or PR+, HER2-) with 4259% incidence; Luminal B (ER+ and/or PR+, HER2+) with 6-12% incidence; HER2+ 3 (ER-/HER2+) with 7-12% incidence; Basal (ER-/PR-/HER2-) with 15-20% incidence; and unclassified (ER-/PR-/HER2-/CK5/6-/HER-) breast cancers with 6-10% incidence (6-13). Generally, ER-negative tumors fall into the HER2-overexpressing and triple negative breast cancers groups. Although poorly differentiated, these tumors display distinct features, including high grade, pushing margins and high p53 expression (14). Unfortunately, ER-negative tumors are more aggressive and less amenable to therapy than ER-positive tumors, and often have a poor clinical outcome. A. Estrogen receptor-positive breast tumors Estrogen receptor (ERα, for simplicity, ER) is a main regulator of mammary gland development and a potent stimulator of breast cancer proliferation. Expressed in over 70% of breast tumors (defined by >1% positive staining), ER is a therapeutic and prognostic marker, since its status is a good predictor of response to endocrine therapies and is correlated with low risk of metastasis. The estrogen hormone, its natural ligand, is synthesized by ovaries in premenopausal women and by peripheral tissue in postmenopausal women. Upon binding to ER, estrogen is a powerful growth stimulant that controls the early stages of breast tumor proliferation (15). Aiming to inhibit the proliferative effects of estrogens, endocrine therapy blocks further growth of breast tumors and is usually well tolerated by both pre- and postmenopausal women. In general, anti-estrogen therapy is adjuvant to combinations of surgery, radiation and chemotherapy, but can be also administered pre-surgery. Current endocrine therapies involve the administration of selective estrogen receptor modulators (SERMS) such as tamoxifen and raloxifene, which block binding of estrogen 4 to ER, aromatase inhibitors (AI) such as anastrozole, letrozole or exemestane, which inhibit synthesis of estradiol, and pure antiestrogens such as fulvestrant, which induce degradation of ER (16-19). After initially responding to ER inhibition, 30% of breast tumors can become tamoxifen insensitive, and many progress to a non-responsive state to all endocrine therapies (16, 20, 21). Therefore, innate and acquired endocrine resistance remains the greatest challenge in the management of ER-positive breast tumors. Evidence from cell-based studies, pre-clinical animal models, genome-wide arrays and proteomic analyses of breast tumors led to several proposed mechanisms for endocrine resistance. This phenomenon can be due to deregulation of the ER signaling pathway, including the receptor itself (22-26) or the switch of tamoxifen action from antagonist to agonist (27), impaired cell cycle regulation (28-30), impaired survival signals (31), or activation of compensatory cytosolic pathways that will promote tumor proliferation and survival in the presence of antiestrogens (32). B. HER2-overexpressing breast tumors The human epidermal growth factor (HER) family of receptors is another example of molecules that regulate cell growth in normal cells but have a potent oncogenic role in breast cancer (33, 34). Approximately 20% of breast cancer patients have tumors that overexpress HER2, an event that was historically associated with an aggressive phenotype and poor survival rate (35). HER2 signals via homo- or heterodimerization with the other HER receptors, with the strongest oncogenic unit being HER2:HER3 (36). 5 Current therapies targeting HER2-overexpressing breast tumors include the monoclonal antibody trastuzumab, which blocks receptor downstream signaling and the small molecule tyrosine kinase inhibitor, lapatinib, which inhibits the kinase activity of HER2. Both compounds were shown to block cell proliferation and promote cytotoxicity and were used as monotherapy or in combination with chemotherapeutic agents. Unfortunately, HER2-overexpressing breast cancers can acquire resistance to antiHER2 therapies and this may be attributed to the expression of truncated forms of the receptor or to misregulated signaling downstream of it, which can circumvent the inhibition of proliferative and survival signals. As evidence of their genetic and molecular heterogeneity, a number of HER2overexpressing breast tumors are also ER-positive, thus several trials have addressed the combination of these targeted therapies with promising results in terms of progression free survival (37-40). C. Triple negative breast tumors Around 10-15% of breast tumors do not express estrogen receptor, progesterone receptor, or HER2 and are classified as triple negative (TNBC) (41-43). Being nonresponsive to both endocrine and anti-HER2 therapies, these tumors have a more aggressive phenotype and a poor prognosis. Patients with TNBC have the highest risk of developing metastasis (44, 45) and a high risk of recurrence. Treatment of TNBC is based on standard cytotoxic polychemotherapy (46). Cytotoxic agents target mitotic events, including microtubule assembly, blocked by the use of taxanes, and the synthesis, replication and transcription or DNA, which can be inhibited 6 by platinum, anthracycline antibiotics or fluorouracil. Since these compounds unselectively target all proliferating cells, some common side effects include immunosuppression, alopecia, and gastrointestinal bleeding and vomiting. In spite of the systemic off-target effects, chemotherapy has been effective in the management of TNBC as neoadjuvant, adjuvant and anti-metastatic strategy (47). TNBCs are very heterogeneous and several biomarkers with therapeutic and prognostic relevance have been identified. For example, patients harboring BRCA1 and BRCA2 germ-line mutations responded positively to poly(ADP-ribose) polymerase (PARP) inhibitors. In this setting, inhibition of PARP (DNA-repairing enzyme) induced cell death, by blocking the homologous recombination DNA repair step, which is critical to the cells in the absence of BRCA1 (48). In summary, the genetic and molecular heterogeneity of breast tumors confer on epithelial cancer cells the ability to become poorly or non-responsive, to current therapies. An important goal of biomedical research is to unravel the mechanisms that cause resistance to various therapies, and to develop new drugs that will be effective for specific breast cancer subtypes. In this era of novel targeted therapies, the identification and validation of tumor biomarkers has also become an essential component of breast cancer research. Single or panels of biomarkers can potentially indicate the onset and progression of breast tumors, and/or predict the response to therapeutic agents. Tracking changes in appropriate markers in biopsies or circulating tumor cells in response to drug administration could provide correlations between the biomarker status and the efficacy of a particular drug. Ideally, this might indicate patients who are 7 less likely to benefit from newly designed compounds, and these patients could be excluded from long-term clinical trials with these drugs. III. Signaling pathways in breast cancer and the importance of scaffold proteins At the cellular level, breast cancer is not a single disease but rather a collection of conditions that vary in their biology and response to treatment. As with other tumor cells, mammary cancer cells are characterized by high uptake of nutrients from their media, abnormal growth, uncontrolled proliferation, impaired DNA replication and repair, evasion of cell death, and the acquisition of new abilities that allow them to invade the surrounding tissues, survive in the bloodstream, migrate and colonize distant organs (49). This section reviews some of the signaling pathways that are responsible for aberrant cellular processes that contribute to malignant transformation, and emphasize the biological and clinical relevance of signal transduction form these pathways and their complex interactions. A. Estrogen receptor As previously stated, ER promotes proliferation of breast cancer cells. ER is a type I ligand-activated nuclear receptor that functions as a transcription factor. Ligandactivated ER dimerizes, translocates into the nucleus where it recruits coregulators and binds promoter regions of DNA that contain estrogen response elements (ERE). Direct binding to an ERE is referred to as the classical genomic pathway (50). In addition, ER 8 can regulate gene transcription indirectly, via protein-protein interactions with other transcription factors such as c-Fos/c-Jun, which bind activating protein 1-(AP-1) containing promoters (non-classical genomic pathway) (51). By turning on genes encoding proteins implicated in tumor proliferation, such as cyclin D1 (52), IGF-IR (53) and c-Myc (54, 55), ER promotes autocrine regulation of cell proliferation (56). Emerging data point out the bidirectional cross-talk between ER and other signaling molecules. First, ER can be activated by phosphorylation on a number of conserved residues, and this activation does not necessarily require the presence of estrogen (57). The ligand-independent receptor activation occurs in response to signals from growth factor receptors, such as HER2, epidermal growth factor receptor (EGFR), and insulinlike growth factor receptor (IGF-IR) (58, 59). Downstream kinases, including MAPK, AKT, JNK, PAK1 and ILK phosphorylate and activate ER on specific sites (60-67) and phosphorylation of ER promotes ligand independent growth of breast cancer cells and has been linked to endocrine resistance (61, 68-70). The clinical relevance of ER phosphorylation at Ser167 includes the predictive of response to endocrine therapy and a good prognosis for overall survival in breast cancer patients (71-73). In addition to its genomic effects, ER has non-genomic functions. By modulating the activity of several cellular kinases, ER signaling controls a subset of genes that are usually regulated by growth factor signaling (62, 63, 74, 75). These non-genomic effects are due to a small subpopulation of extranuclear ER that may be homologue to the nuclear ER or an atypical G-coupled protein receptor (76). ERs localize to the plasma membrane, activate cyclic AMP second messenger or receptor tyrosine kinases HER2, 9 IGFR and EGFR (77-80). Additionally, ER has functional interactions with adaptor protein Shc and with Src and PI3K kinases (59, 81-85). In addition to its well-established proliferative role, several studies indicate that ER may be implicated in breast cancer cell survival. ER promotes survival of breast cancer cells via cross-talk with the PI3K/AKT pathway (61), or by regulating the activity of NF-κB (86, 87), Bcl-2 (88, 89), and inhibitor of apoptosis (IAP) family members (90). In summary, ER is a crucial regulator of breast cancer cell proliferation and survival. Its activation and signal transduction are part of intricate bidirectional interactions with other signaling pathways with known roles in malignant transformation and drug resistance. B. PI3K/AKT/mTOR The phopshoinositide 3-kinase (PI3K) pathway is an important signaling node in breast cancer biology. Activated by signals received from various sources, such as IGF-IR, integrins or ER (91), PI3K lipid kinases phosphorylate the 3’-hydroxyl group of phosphoinositides like phosphatidylinositol-3,4,5-trisphosphate (PIP3). This second messenger recruits phopshoinositide dependent kinase (PDK1) and the serine/threonine kinase AKT to the plasma membrane (92, 93). In turn, PDK1 phosphorylates AKT on Th308, and this particular phosphorylation is essential for kinase activity (94). Complete activation of AKT is achieved by phosphorylation of Ser473 by a different kinase. The lipid phosphatase and tensin homolog PTEN is the physiological inhibitor of PI3K, since it dephosphorylates PIP3. Therefore, the levels of PIP3 are important for maintaining normal cellular functions. 10 Increased PI3K signaling is associated with breast tumorigenesis and can be attributed to a number of genetic and molecular alterations including high levels of constitutive active AKT1, mutations and amplifications of PI3KCA and mutations of PTEN tumor suppressor in 30% of breast cancers (95-97). AKT is the primary target of PI3K signaling. This kinase plays a pro-survival role in breast cancer cells, where it relays signals from upstream molecules such as integrins, growth factor receptors, PI3K, and mTORC1, to downstream molecules such as FLIP, Bcl-2, Bad, and NF-κB (98-101). Data from murine models of breast cancer indicate that AKT1 cooperates with other oncogenes to promote mammary tumorigenesis (102). Both cell-based and animal models of breast tumors report the cross-talk between AKT and other oncogenic pathways, including Raf/MAPK, ER, and integrin-linked kinase (ILK). Phosphorylation of Raf by AKT inhibited the Raf-MEK-ERK pathway and induced proliferation of breast cancer cells (103). This controversial effect is supported by previous findings showing that prolonged activation of the Raf/MAPK pathway results in growth inhibition (104). Several interactions between AKT and ER signaling have been demonstrated in breast cancer cells. For example, AKT phosphorylates ER and promotes survival of breast cancer cells (61), estrogen activates AKT (105, 106) and in turn, the AKT kinase activity modulates the effects of estrogen on ER activity (107, 108). Finally, in mouse mammary glands, high levels of AKT phosphorylation are correlated with overexpression of ILK, hyperplasia, and subsequent tumor formation (109). In addition to promoting breast tumor growth and survival, activation of PI3K/AKT signaling pathway has also been linked to resistance to antiestrogens (110-114) and trastuzumab (115-117). Thus, compounds targeting the PI3K/AKT signaling pathway 11 represent a potential therapeutic tool for ER-positive and HER-2 overexpressing breast tumors with primary or acquired resistance to current therapies (118). Another downstream target of PI3K is the mammalian target of rapamycin (mTOR), which is activated in response to nutrients and growth factors in proliferating cells and is an essential regulator of nutrient uptake, protein synthesis, growth, cell cycle, and survival/apoptosis (119). Two mTOR complexes have been identified, and they differ in their protein composition and response to rapamycin, which inhibits mTORC1 but not mTORC2. Both complexes include the mTOR serine/threonine kinase and one of the associated proteins raptor and rictor, which are found in mTORC1 and mTORC2, respectively. Importantly, mTORC1 is activated by AKT (120), whereas mTORC2 signals upstream of and can phosphorylate AKT at Ser473 (121, 122). Due to its demonstrated functions in mammalian cells, it is not surprising that hyperactive mTOR signaling drives breast tumor growth by stimulating translation of mRNA and protein synthesis, cycle progression and survival of breast cancer cells (123). In conclusion, the PI3K/AKT/mTOR pathway integrates signals from growth factor receptors, integrins and ER to regulate growth, proliferation and survival of breast cancer cells, and is also implicated in drug resistance. Therefore, pre-clinical and early clinical studies have been carried out using inhibitors of PI3K, mTOR, dual PI3K/mTOR inhibitors, and compounds that selectively target the AKT kinase (118, 123-126). Ongoing trials include blockade of PI3K/AKT combined with aromatase inhibitors or trastuzumab. 12 C. MAPK The MAPK signal transduction requires sequential activation of three kinases (127, 128) and the Raf-MEK-ERK module is the most studied MAPK cascade in the context of breast cancer biology (129). The extracellular regulated kinase (ERK) receives amplified signals from its upstream partners and phosphorylates its cytosolic and nuclear targets to mediated cell proliferation (130, 131). The role of mitogen activated protein kinases (MAPKs) in breast tumorigenesis significantly overlaps with that of growth factor receptor tyrosine kinases, since the MAPK linear cascade is activated downstream of HER2, EFGR and IGF-IR. In addition to growth factors, membrane-localized ER, G-coupled protein receptors and several ligands, including insulin, prolactin, estrogen, and progesterone can initiate responses through MAPK signaling to regulate proliferation, growth, differentiation, migration, and survival of breast cancer cells (130). Moreover, activation of MAPK was observed in human tumors and was correlated with decreased disease free survival (132). In breast cancer cells, estrogen rapidly activates ERK, which translocates to the nucleus (133). Additional studies confirmed that proliferation of ER-positive breast cancer cells is partly due to activation of ERK by estrogen (134-137). Estrogen-induced activation of MAPK involves IGF-IR and EFGR to promote growth and survival of breast cancer cells (75, 138). Conversely, MAPK stimulates ER signaling, via phosphorylation of ER (139). A notable aspect of MAPK function is the compensatory activation after estrogen deprivation (140, 141). Both cell culture and in vitro models have demonstrated that ERpositive cells adapt to long term deprivation of estradiol by activating the MPAL pathway, 13 which promotes cell proliferation via ER transactivation and also independently from ER. Moreover, high levels of tyrosine kinase activity are correlated with estrogen independence and anti-estrogen resistance, resulting in increased ERK activity (142, 143). In summary, the MAPK signaling pathway is a crucial regulator of breast tumorigenesis, particularly via complex interactions with ER, in response to estrogen and growth factordriven stimulation. In clinical practice, MEK blockers are relatively new targets and ongoing trials are underway to investigate the effects of specific MEK inhibitors in solid tumors. D. PAK p21-activated kinase (PAK) is another serine/threonine kinase with well-established roles in the biology of breast cancer. In normal cells, PAK regulates actin reorganization and cell motility via binding to the small GTP-ases Rac and Cdc42, or through its scaffolding function between PDK1 and AKT (144, 145). Subsequent studies performed in fibroblasts reported a cross-talk between PAK1 and MAPK, as PAK1 activates a population of MEK1 and ERK in focal complexes during adhesion to fibronectin in the presence of MP1 scaffold protein (146, 147). PAK1 has increased expression and activity in breast tumors (148-150) and in many breast cancer cells (151), where it promotes cell migration (152, 153). PAK1 can be activated by known mitogens such as heregulin (154). In addition, high expression of PAK1 promotes malignant transformation by inducing chromosome instability in tumorigenic epithelial cells (152) and aberrant survival and transformation of non14 tumorigenic mammary epithelial cells (65, 69, 148, 155, 156). MMTV-PAK1 transgenic mice developed malignant tumors that also expressed active (phosphorylated) MEK1 (157). Several reports have noted the interaction of PAK1 with ER signaling, since ER is a direct phosphorylation target of PAK1, and the kinase was linked to tamoxifen resistance (65, 69). In conclusion, data emerging from human tumor samples together with functional studies provided the rationale for designing small molecule inhibitors of PAK1. Future pre-clinical investigations will evaluate the therapeutic potential of PAK1 inhibition in the context of breast cancer (158) . E. Integrins Proliferation and survival of breast cancer cells is highly dependent of their attachment to the extracellular matrix and this attachment is mediated by integrins. Mammalian cells have 24 distinct integrin receptors, which are dimers formed by combinations of α and β subunits. Their ligands are the extracellular matrix proteins collagen, laminin, fibronectin, and vitronectin (159, 160). While helping to maintain cell adhesion to the matrix and three-dimensional tissue structure, integrins connect extra- to intracellular molecules to regulate proliferation, survival, gene transcription and migration (161). Ligand-bound integrins function at focal adhesions, where they interact with paxillin, vinculin, talin, or alpha-actinin, intracellular proteins that regulate actin polymerization and formation of motile structures. Focal adhesions are also active signaling nodes, via activation of focal adhesion kinase (FAK), ILK, s-Src, Rho and Cdc42 as well as MAPK and PI3K (162). 15 Several integrin dimers are highly expressed in breast cancer cells and have been shown to contribute to malignant transformation. For example, α6β4 integrin activates PI3K through increasing HER2 activation (163), and the interaction of this integrin with another growth factor receptor was required for PI3K-mediated migration (164). In a 3D model of mammary acini, α6β4 integrin activates PAK1 to promote resistance to apoptosis (165) while β4 subunit was required to promote HER2-mediated tumorigenesis in vivo (166). Another laminin receptor, αvβ3 integrin, has higher expression in tumorigenic compared with nontumorigenic mammary epithelial cells (167), and its expression is correlated with the metastatic potential of breast cancer cells (168). Furthermore, inhibition of αvβ3 integrin blocks VEGF-induced angiogenesis and decreases cell proliferation (169). Finally, several studies have noted that expression and activity of another dimer, the αvβ1 integrin, is correlated with adriamycin resistance (170) and induces migration of breast cancer cells expressing Shc (171). Individual integrin subunits have been characterized in terms of expression, activity and their roles in breast cancer cells. Thus, α2 integrin inhibits metastasis in cell culture and in mouse models and is positively correlated with ER status in breast tumors (172). Significant research investigated the role of β1 integrin in breast tumorigenesis. This subunit displays aberrant expression in 30-50% of breast tumors (173) and is associated with low disease free survival (174). β1 integrin has been linked to chemotherapy resistance (175, 176). In 3D culture, β1 integrin inhibitory antibodies induce apoptosis of several breast cancer cell lines and sensitize cells to radiotherapy in xenograft models (177, 178). Moreover, β1 integrin is required for tumor formation and growth in a mouse model of human breast cancer (179). 16 Integrins can indirectly regulate the biology of breast cancer cells through signals emerging from their downstream targets such as ILK and FAK kinases. FAK is a cytosolic kinase activated upon integrin ligation and is responsible for controlling motility, proliferation and survival (180-182). In vitro studies have shown that FAK is activated by PI3K and can in turn activate Ras/MAPK pathway (146, 183, 184). Analysis of metastatic breast tumors and DCIS revealed high levels of FAK, suggesting its role in tumor initiation and metastasis (185, 186). Subsequent studies reported that its expression in breast tumor tissue was correlated with invasiveness (186, 187). Blockade of FAK expression inhibited lung metastasis in vivo and the kinase also cooperated with ERK to induce angiogenesis in breast cancer cells (188, 189). ILK is a serine/threonine kinase that binds to β1 and β3 integrin at focal adhesions (190) and requires PI3K and GSK3 to phosphorylates AKT on Ser473 (191, 192). While normal cells have low levels of ILK activity, this kinase is highly expressed in breast tumors, and its overexpression was correlated with p-AKT at Ser473 and elevated levels of IGF1 (193). Functional assays in breast cancer cells indicate that ILK suppresses anoikis, promotes cell survival through the PI3K/AKT/mTOR pathway (194196), and phosphorylates ER to facilitate estrogen-mediated migration (66). In addition, interaction of ILK with rictor protein (of mTORC2) stimulates phosphorylation of AKT1 and promotes survival (197). Finally, overexpression of ILK causes malignant transformation in vivo (109). In summary, a great body of evidence demonstrates the role of integrin signaling in breast cancer formation and progression and offers new possibilities of therapeutic interventions (198). 17 F. Scaffold proteins Cells integrate information from a large number of extracellular and intracellular signals in order to elicit specific responses. As part of this integration, the stoichiometry, stability, and compartmentalization of signaling complexes are regulated by scaffold proteins. Located most often at branch points within signaling networks, scaffolds mediate crosstalk between key molecules and can potentiate or inhibit the strength of signals. Models of pathway connections through scaffold proteins vary from simple to complex. For example, a scaffold protein can function to facilitate interactions in a linear manner or it can mediate pathway branching and amplification of output to multiple downstream partners. Finally, scaffolds can be part of more elaborate interaction networks involving feedback regulation, thus directly activating or blocking signaling pathways (199). In turn, scaffold proteins can be regulated based on a cell’s needs and the presence or absence of scaffold proteins therefore contributes to the diversity of possible cellular responses. Functional studies indicate a role for several scaffold proteins in breast cancer. For instance, modulator of non-genomic action of estrogen receptor (MNAR) mediates the interactions of ER with PI3K and Src, and is required for the rapid effects of estrogen in breast cancer cells (200). POSH is another scaffold protein the proapoptotic function of which is suppressed by AKT in breast cancer cells (201). The Gab2 adaptor/scaffold protein is required to promote mammary tumor metastasis in neu-expressing mice (202). A recent study investigated the GIP1 scaffold protein, which was previously linked to IGF-IR and other receptors (203). GIP1 gene silencing inhibited proliferation and induced apoptosis of breast cancer cells (204). 18 In conclusion, scaffold proteins are essential for complex signaling networks that impact various cellular functions. Changes in the levels of scaffold protein genes or their mislocalization can potentially alter signal transduction and lead to faulty cell decisions that may ultimately contribute to breast tumorigenesis. The following section addresses some of the currently known facts about the scaffold protein MP1 and its potential relevance in the context of breast cancer. G. MP1 scaffold protein MP1 (MEK partner 1) is a 14 KDa (124 amino acid) scaffold protein that is present in several species with various degrees of homology to humans: mouse (124 aa, 97% homology), rat (124 aa, 96%), frog (Xenopus laevis; 123 aa, 87%), fruit fly (Drosophila; 124 aa, 45%), and worm (Caenohrabditis elegans; 145 aa, 20%) (205). The 16.2 kb MP1 gene is found on chromosome 4 and has seven exons and six introns. Alternative splicing results in three transcripts, with variant 1 being the longest transcript that encodes the 124 amino acid protein. According to NCBI RefSeq, transcript 3 encodes a shorter isoform (117 aa) but neither the CCDS (Consensus CoDing Sequence) database nor the UniProt database provide public data for this second isoform. MP1 was originally identified as a scaffold protein that specifically binds MEK1 and ERK1, and its ability to regulate MAPK signaling (206) and its localization to late endosomes are the most well characterized functions of this protein. The scaffolding role of MP1 has been demonstrated in several in vitro studies that report its association with large signaling complexes located to different intracellular compartments (207). For example, in late endosomes, MP1 interacts with adaptor 19 proteins p14, p18 and KRas to induce ERK activation (208-211). p14 is required to localize MP1 to late endosomes, and although they have different primary sequences the two small proteins have similar structures and form a high affinity heterodimer (205, 212). Biochemical and crystallographic analyses found that both proteins consist of a five-stranded β-sheet flanked by three α-helices (205, 213). No specific protein interaction domains were identified. However, the structures of MP1, p14 and their heterodimer indicate the presence of several surface-exposed residues, suggesting the possibility of interaction with cytoplasmic proteins (205, 212, 213). A more recent study reports the presence of MP1 in the mTOR pathway (214). In this study, the MP1/p14/p18 trimeric, coined Ragulator, interacts with and localizes Rag GTP-ases to lysosomes. Moreover, MP1 knockdown in HEK392T cells prevents the lysosomal recruitment of mTORC1 in the presence of amino acids and inhibits the amino acid-induced stimulation of dTORC1 in Drosophila, suggesting that MP1 is required for the recruitment of mTORC1 to lysosomes and is essential for the amino acid-dependent activation of the complex. MP1 is also required for PAK1 to activate a population of MEK1 in focal complexes during adhesion of rat fibroblasts to fibronectin (215) and this interaction is sufficient to activate MEK1 in the absence of Raf, but to a lesser extent than observed with active Raf or EGF (147). Coimmunoprecipitations carried out in CCL39 mice fibroblasts shown an interaction between MP1 and RACK1, a scaffold protein that is required for the presence of active ERK to focal adhesions (216). In a family (humans) with an immunodeficiency syndrome, a mutation in 3' UTR for exon 4 lead to reduced expression of p14 gene and the affected individuals had stunted growth (217). 20 Several cell-based and in vivo studies have addressed the functional role of MP1. Silencing of MP1 delayed spreading of fibroblasts and decreased migration of prostate cancer cells on fibronectin (215, 218). Furthermore, deletion of p14 gene in mice causes defects in embryo development and embryonic lethality before gastrulation (10.5 days). Epidermis of the p14-/- mice displayed fewer cell layers and had a lower mitotic index which prompted the authors of this study to conclude that p14/MP1 complex regulates cellular proliferation during development (209). Finally, transgenic lines of Drosophila, with either silenced or over-expressing dMP1, develop ectopic veins during wing development, suggesting that MP1 ortholog is required to regulate the vein cell differentiation (219). Taken together, these findings combined with published literature in breast cancer cell biology provide evidence for a potential role of MP1 scaffold protein in regulating survival, proliferation and motility of breast cancer cells. IV. Apoptosis Apoptosis is a normal cellular process in the development of breast tissue as it takes place during the formation of ducts and lumens, at the end of menstrual cycle and during involution post lactation. In breast tumors, apoptosis is the final effect of many drugs, in particular chemotherapeutic agents that are known to induce cytotoxicity. Morphologically, apoptotic cells shrink, display membrane blebbing, chromatin condensation and eventually disintegrate into apoptotic bodies which are subjected to phagocytosis (220). 21 At a cellular level, apoptosis is defined as the programmed cell death that involves two distinct processes, in terms of their localization and activation. The intrinsic pathway of apoptosis is activated mainly in response to DNA damage and centers on the release of cytochrome c from mitochondria. The decision of cell to undergo apoptosis is dependent on the functional balance between the pro-apoptotic and pro-survival members of the Bcl-2 protein family, which control the mitochondrial channels and release of cytochrome c (221). Once released, the cytochrome c binds Apaf-1 scaffold protein and facilitates the assembly of apoptosome and activation of initiator caspases 2, 8, 9 and 10. At the same time, released mitochondrial protein Smac binds to and inactivates the inhibitor of apoptosis proteins (IAP). As a result, effector caspases are activated by proteolytic cleavage and can further cleave their downstream targets, including intermediate filament proteins, nuclear lamin, and the DNA-repair enzyme, PARP. The extrinsic pathway of apoptosis is triggered upon activation of a death receptor at the plasma membrane. TRAIL and FAS receptors bind their cognate ligands and undergo conformational changes to recruit Fas-associated death domain (FADD) protein and form the death-inducing signaling complex (DISC). FADDs recruit and mediate cleavage of caspase 8, thus activating the caspase cascade. In addition, by cleaving the pro-apoptotic Bid member of the Bcl-2 family, caspase 8 integrates the intrinsic and extrinsic apoptotic pathways. A distinct apoptotic extrinsic mechanism is represented by TNF signaling. Secreted by tumor cells or by their neighboring cells, this cytokine binds and activates TNF-R1. In turn, the receptor associates with TRADD and signals to activate caspases via interaction with FADD or to regulate gene transcription though NF-κB signaling. 22 Additional proteins have been shown to regulate apoptosis. Cellular FLICE inhibitory protein (c-Flip) prevents cleavage and activation of caspase 8 (222) and several cellbased studies revealed its requirement for cell survival. Activated AKT can repress apoptosis by inactivating caspase 9 and Bad, FLIP or by upregulating Bcl-2 (99, 101, 222). The Myc oncoprotein and NF-κB seem to have both pro-apoptotic and antiapoptotic roles. A notable regulator of apoptosis is the p53 tumor suppressor, which targets many components of the apoptotic machinery, triggers apoptosis under DNA damage events, thus contributing to the tissue homeostasis. In turn, in breast cancer cells, AKT was shown to mediate degradation of p53 protein (223). Breast cancer cells have developed strategies to evade apoptosis in order to overcome two main obstacles: to survive in the hostile environment found in the bloodstream during migration and to resist the cytotoxic effects of therapeutic agents or gamma irradiation. One way of achieving this requirement is by altering the levels of molecules directly implicated in apoptosis. For example, the Bcl-2 pro-survival protein is overexpressed in breast tumors, and this is correlated with poor prognosis (224). In addition, mutation of TP53 and loss of p53 functional protein impedes the ability of breast cancer cells to induce pro-apoptotic genes (225). An alternative mechanism of escape occurs through constitutive activation of growth factor signaling pathways and their downstream MAPK and PI3K/AKT cascades, and this strategy accounts for drug resistance in many types of breast tumors. Although challenging, understanding the mechanisms regulating apoptotic events in subtypes of breast cancer may provide valuable clues about potential novel molecules with prognostic and therapeutic 23 relevance. This dissertation research has focused on one such protein, MP1, which we identify as an important pro-survival molecule in ER-positive breast cancer cells. 24 Figure 1. Signaling pathways in breast cancer. 1. Loss of integrin expression triggers defects in cell adhesion and increased tumor cell invasiveness and metastasis. 2. Activation of ER signaling by ligands or phosphorylation leads to synthesis of cell cycle proteins and promotes cell proliferation. 3. Activation of receptor tyrosine kinases by growth factors stimulates proliferation and growth of tumor cells and also renders ER ligand-independent. 4. Activation of the PI3K/AKT pathway and of Bcl-2 pro-survival protein leads to escape from apoptosis and promotes tumor cell survival. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation. 25 Figure 1. 3 Integrin 1 2 PI3K ER ILK Akt Ras PAK1 PI3K FAK RTK PIP3 PDK1 Raf 4 PTEN PAK1 p14 MEK Rac MP1 Rho ER Fos/ Jun ERK Akt mTOR/ rictor p18 mTOR/ raptor ERK Cyt c Bcl-2 Casp 9 ER p53 PARP NF-κB Smac Bcl-2 IAP Akt 26 DR Bad Casp 3 Casp 8 REFERENCES 27 REFERENCES 1. 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Several of these pathways influence the biology of breast cancer, but the functional significance of MP1 in breast cancer cells has not been investigated. In this report, we demonstrate a specific requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of ER-positive, but not ER-negative, breast cancer cells. Inhibition of MP1 expression in ER-positive cells resulted in reduced AKT1 activity, and expression of a constitutively active form of AKT1 partially rescued the cell death phenotype observed when the MP1 gene was silenced. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/AKT pathway specifically in ER-positive breast cancer cells. INTRODUCTION The small protein MEK Partner 1 (MP1, also known as Map Kinase Scaffold Protein 1 and LAMTOR3) was originally identified as a scaffold protein that potentiates MAPK signaling by binding to MEK1 and ERK1 (1). MP1 interacts with another small protein p14, and together these two proteins are localized to endomembrane compartments as part of larger signaling complexes. For example, an MP1-p14-MEK1 complex is localized to late endosomes, and this localization is required for EGF-induced ERK1/2 signaling (2-4). A second MP1-p14-p18 Ragulator complex is required for the 47   recruitment of mTORC1 to the lysosomal surface, and is essential for its amino aciddependent signaling (5). In addition to these trimeric complexes, MP1 has been reported to bind PAK1 at the plasma membrane, and the MP1-PAK1 interaction is required for MEK phosphorylation by PAK1 in the absence of Raf (6, 7). Thus, the scaffold protein MP1 can regulate the function of several intracellular kinases in different subcellular locations. Both in vitro and in vivo approaches have been taken to investigate the biological functions of MP1. Transient inhibition of MP1 expression using RNA interference in fibroblasts resulted in decreased Rho activity and delayed cell spreading on fibronectin (7). Similar knockdown experiments in DU145 prostate cancer cells resulted in decreased migration on fibronectin (6). This effect on migration was independent of the ability of MP1 to activate ERK and PAK1, since the levels of phosphorylated ERK and PAK1 were unchanged upon MP1 knockdown. However, inhibition of MP1 expression was associated with both decreased expression of paxillin and decreased number and turnover of focal adhesions at the migratory edge. Together, these data indicate that one function of MP1 in cell culture is related to cell adhesion and migration. Studies performed in conditional p14 knockout mice and in Drosophila have addressed the in vivo functions of MP1. The endosomal p14-MP1-MEK1 complex is required for cell proliferation in the epidermis during mouse embryogenesis (3). In Drosophila, the MP1/ERK complex regulates cell differentiation during development of the wing, since both down-regulation and overexpression of dMAPKSP1 lead to an ectopic wing vein phenotype (8). In summary, MP1 is a widely expressed protein that interacts with multiple protein kinases and may impact various cellular processes including 48   proliferation, spreading, migration, and differentiation. Since many of these processes play important roles in cancer biology, we used a loss of function approach to evaluate the role of MP1 in regulating the proliferation and/or survival of breast cancer cells. Analysis of publicly available gene expression datasets indicates that MP1 RNA is expressed in both normal mammary epithelial cells and in breast cancer cells. In this report, MP1 protein expression was investigated in a panel of human mammary epithelial cell lines. The data indicate that MP1 is expressed in both estrogen receptor alpha (ER)-positive and ER-negative breast cancer cell lines, as well as in nontransformed cells. However, the effects of inhibiting MP1 expression by transient transfection with siRNA duplexes differed between the cell lines. MP1 knockdown induced apoptosis of the ER-positive breast cancer cells, but not ER-negative breast cancer or non-tumorigenic cell lines. The apoptosis observed in ER-positive cells was associated with cell detachment, and with decreased ER expression and AKT activation. The phenotype could be partially reversed by overexpressing a constitutively active form of AKT1, suggesting that MP1 plays a novel role in promoting survival of ERpositive breast cancer cells via the AKT pathway. MATERIALS AND METHODS Cell lines and culture conditions MCF-7 and LCC9 cells were obtained from the Lombardi Cancer Center. T47D, ZR-751, MDA-MB-231, BT-549, and Sk-Br-3 cells were purchased from the American Type Culture Collection. Cells were maintained in Improved Modified Eagle’s Medium (IMEM) 49   containing phenol red (GIBCO-Invitrogen-Applied Biosystems), supplemented with 5% fetal bovine serum (HyClone), and 100 Units/ml Penicillin/100 µg/ml Streptomycin (Invitrogen) and incubated at 37 ºC with 5% CO2. siRNA transfections All siRNA transfection reagents were purchased from Dharmacon-Thermo Scientific. Two independent MP1 siRNA duplexes (ON-TARGETplus), ER siRNA duplexes (ONTARGETplus siRNA Human ESR1) and a non-targeting siRNA (ON-TARGETplus siCONTROL) were used. Cells were plated in six-well plates at 105-3 x 105 cells per well in FBS containing medium. After 24 h, cells were transfected with 30-150 nM of either control or MP1 siRNA using DharmaFECT 1 transfection reagent. After 48 h, cells were harvested by scraping on ice cold PBS and prepared for protein extraction. Determination of cell death Cell death was assessed at 48 h post transfection using Trypan blue exclusion assays. Briefly, floating cells were collected, centrifuged, and resuspended in PBS, while attached cells were trypsinized, centrifuged, and resuspended in PBS. For each cell suspension, 18 µl were incubated with 2 µl trypan blue for 15 min and both total number and the number of dead cells were counted with a hemacytometer. The remaining harvested cells were processed for protein determination and immunoblotting. Immunoblotting Cell pellets were lysed in CelLytic M lysis buffer (Sigma), supplemented with cocktail tablets of protease (Roche - Complete Mini EDTA-free) and phosphatase inhibitors 50   (Roche – PhosSTOP). Protein concentrations were determined using the Bradford protein assay (Bio-Rad). Total protein (10-20 µg) was subjected to 4-20% Tris-HCl SDS-PAGE (Bio-Rad), transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore), blocked with Odyssey Blocking Buffer and then incubated with the appropriate primary antibodies. Alexa Fluor 680 anti-goat, anti-rabbit, and anti-rabbit (Invitrogen) and IRDye 800CW anti-mouse and anti-rabbit (LI-COR) secondary antibodies were used for two-color detection of proteins. Membranes were scanned and analyzed using the LI-COR Odyssey system. Luciferase assay MCF-7 cells were cultured in six-well plates at 3 x 105 cells per well. The following day they were co-transfected with 0.5 µg of ERE2-tk109-luc and 0.06 µg of pβgal-Basic, using Superfect transfection reagent (Qiagen). After 3 h, the medium was changed to transfection mixes of either control or MP1 siRNA and cells were incubated overnight. The transfection medium was then replaced with phenol red-free IMEM supplemented with 5% CSS for 24 h, then cells were stimulated with 10 nM 17β-estradiol (Sigma) for 8 h. Cells were lysed and assayed for luciferase (Promega) and β-galactosidase (Clonetech) activity as suggested by each manufacturer. RNA isolation and quantity assessment Total RNA was isolated using the Trizol/Chloroform method. Briefly, cells were lysed and homogenized with Trizol, followed by a phase separation using chloroform. Next, the samples were precipitated with isopropanol, washed and redissolved in RNA 51   storage solution (Ambion). After a new precipitation step using sodium citrate and ethanol, RNA pellets were subjected to DNase digestion (Ambion). Total RNA was quantified at 260 nm using a Nanodrop ND-1000 spectrophotometer. The purity of RNA was verified by a 260/280 ratio of 2.0 ± 0.25 and a 260/230 ratio of 1.8 ± 0.15.   Reverse transcription, semi-quantitative PCR and DNA gel electrophoresis A hundred ng (100 ng) total RNA were reverse-transcribed using Superscript One Step kit according to the manufacturer’s protocol (Invitrogen). To perform a semi-quantitative PCR analysis, 25 µl of cDNA-containing reaction solutions were subjected to various PCR conditions using a Bio-Rad Thermal cycler. The PCR products were loaded on 1.5% agarose gel containing 100 ng/ml ethidium bromide (Sigma) and bands were visualized using a thermal gel imaging system (FUJIFILM). Primers and PCR conditions The following human primers were used for PCR reactions: • MP1 forward 5’-AACGGATCCATGGCGGATGACCTAAA-3’; reverse 5’- GCCGAATTCCAGAAACTTCCACAACTTG-3’. • ER forward 5’-CATTATGGAGTCTGGTCCTGTGA-3’; reverse 5’- reverse 5’- GTTTCAACATTCTCCCTCCTCTT-3’. • Actin forward 5’-CTGGGACGACATGGAGAAAA-3’; AAGGAAGGCTGGAAGAGTGC -3’. The following PCR conditions were used: 52   • MP1 and actin primers: 56 ºC annealing temperature (30 seconds) and 30 cycles • ER primers: 54 ºC annealing temperature (30 seconds) and 36 cycles The PCR products were 391 bp for MP1, 214 bp for ER, and 563 bp for actin. Antibodies and reagents The following primary antibodies were used for Western blotting: MP1 (A-19, Santa Cruz), actin (AC-40, SIGMA), estrogen receptor alpha (AB-17, Lab Vision-Thermo Scientific, or F-10, Santa Cruz), PARP (Cell Signaling), p-AKT (T308, Cell Signaling), AKT1 (BDI111, Santa Cruz), ERK (C-16, Santa Cruz), p-ERK (Cell Signaling), Flag M2 (Sigma), β1 integrin (N-20, Santa Cruz) or Bcl-2 (BD Biosciences). Pan caspase inhibitor z-VAD-FMK was obtained from BD Biosciences, Trizol from Invitrogen and PI3K inhibitor LY294002 was purchased from Sigma. Retroviral infection of MCF-7 cells pBabe-puro (Addgene plasmid 1764) or pBabe-puro-Myr-Flag-AKT1 (Addgene plasmid 15294, (9)) were transfected into 293GPG packaging cells and retroviral stocks were prepared as previously described (10). These virus stocks were used to infect MCF-7 cells (1 ml per 10 cm dish), in the presence of polybrene (8 µg/ml), and stable colonies were selected with 0.5 µg/ml puromycin. Both single colonies and pools of 50-100 colonies were selected and propagated. Stable cell lines/pools were routinely maintained in medium supplemented with 0.25 µg/ml puromycin and plated in puromycin-free conditions for siRNA transfections. 53   Statistical Analysis Data are expressed as the mean ± S.D. Most experiments were performed three times. Paired evaluations were made for experimental and control conditions within each experiment and for comparing groups of cell lines an unpaired two-tailed evaluation was done. Significance was determined by Student’s t test. Significance level was set at p< 0.05. RESULTS MP1 expression profiling in human mammary epithelial cells Expression of MP1 protein was assessed by immunoblotting in the following human mammary epithelial cell lines: MCF10A and 184B5 (nontumorigenic), MCF-7, LCC9, T47D, and ZR-75-1 (tumorigenic, ER-positive), and MDA-MB-231, BT-547, Hs579T, and Sk-Br-3 (tumorigenic, ER-negative) (Figure 2). MP1 was present in all cell lines, although the level was variable. Actin expression also varied between cell lines, but was consistent between experiments. A comparison between ER-positive (mean=3.8±0.4) and ER-negative (mean=2±1.1) breast cancer cell lines indicated a small but significantly (p=0.03) higher ratio of MP1/Actin protein levels in the ER-positive group. Since the number of samples investigated here is small, we also queried publicly available databases for MP1 mRNA expression that were downloaded from GEO including: GSE2034, GSE3494, GSE6532, GSE4922, GSE11121, GSE7390, GSE2603 and GSE14020. Data was normalized using RMA in Affymetrix Expression console and batch effects were removed. In agreement with our protein results, MP1 was widely expressed, but showed a small but statistically significant elevation in both ER and PR 54   Figure 2. MP1 expression in breast cancer cell lines. Human mammary epithelial cell lines were grown in exponential culture and whole-cell lysates were prepared. Top panel: Immunoblot from a representative experiment. Lower panel: Quantitation of MP1/Actin ratios in three independent experiments (mean ± SD, *p<0.05). 55   positive breast cancer samples (p<0.0001 by t-test for both). One clinical study identified MP1 as a gene associated with poor prognosis in sporadic lymph-node negative breast cancer patients (11), suggesting a putative role in the context of breast tumors. However, our analysis did not reveal a correlation between high MP1 expression and disease outcome (time to distant metastasis or disease free survival). Inhibition of MP1 expression induces cell death and detachment of ER-positive breast cancer cells To study the effect of inhibiting MP1 expression in breast cancer cells, short interfering RNA (siRNA) duplexes were used. Initial experiments were carried out in ER-positive MCF-7 cells. By 48 h post-transfection, cells treated with two independent MP1 siRNAs displayed a dramatic phenotype involving cell rounding and detachment (Figure 3A). As shown in Figure 3B, MP1 protein levels were reduced more than 50% by 48 h with these two MP1 siRNAs relative to control siRNA. To determine if this response to MP1 knockdown was a general feature of ER-positive breast cancer cells, two additional ER-positive cell lines were examined: LCC9 and T47D. The LCC9 cell line is an estrogen independent and antiestrogen resistant derivative of MCF-7 cells (12), and T47D is an independently derived ER-positive cell line. The siRNA sequence that consistently yielded better knockdown (MP1 siRNA #1) was chosen for these experiments and MP1 was silenced in all of the cell lines investigated (Figure 4A). As shown in Figure 4B, LCC9 and T47D cells exhibited a similar phenotype to MCF-7. To quantitate the effect of MP1 knockdown, attached and 56   A MP1 siRNA #2 MP1 #2 siRNA CON B MP1 siRNA #1 MP1 #1 CON siRNA MP1 Actin MP1/Actin 100 35 31 Figure 3. MP1 silencing using two independent siRNA sequences. MCF-7 cells were transfected with 40 nM control or MP1 siRNAs. At 48 h cells were photographed, then harvested for counting and extract preparation. A) Photographs of MCF-7 cells transfected with two different MP1 siRNA and control siRNA sequences. B) Immunoblot of transfected samples. Numbers represent the relative MP1/Actin ratios. 57   detached cells were collected at 48 h following siRNA transfection, stained with trypan blue, and counted (Figure 5). As determined by trypan blue staining, MCF-7 cells were the most sensitive to MP1 knockdown, with more than 70% of cells detached by 48 h, and the majority of these were dead. In contrast, only 10% of cells were detached in the control siRNA transfections. Although LCC9 and T47D cells were less sensitive than MCF-7, both showed a significant increase in dead/floating cells upon MP1 knockdown, with the average percentage of dead cells being 70% for MCF-7, 42% for LCC9 and 49% for T47D. Inhibition of MP1 expression does not induce death of ER-negative breast cancer cells or non-tumorigenic cells Since MP1 is expressed in ER-negative breast cancer cells and in non-tumorigenic mammary epithelial cells (Figure 2), the effects of MP1 knockdown in representatives of these cell types were also examined. Three ER-negative breast cancer cell lines (MDAMB-231, BT-549, and Sk-Br-3) and one non-tumorigenic mammary epithelial cell line (184B5) were transfected with either control or MP1 siRNA and examined at 48 h. Although MP1 levels were decreased to the same or greater extent as that obtained in the ER-positive lines (Figure 6), no obvious changes in cell morphology were seen, and cell counting/trypan blue exclusion indicated that there was no significant increase in cell detachment or death in MP1 siRNA transfected cells compared with control samples (Figure 7). Thus, the requirement for MP1 expression for cell attachment and survival may be specific to ER-positive breast cancer cells. 58   A MCF-7 siRNA CON LCC9 T47D MP1 CON MP1 CON MP1 MP1 Actin MP1/Actin B 100 25 CON siRNA 100 21 100 40 MP1 siRNA MCF-7 LCC9 T47D Figure 4. MP1 expression is required for attachment and survival of ER-positive breast cancer cells. A) Immunoblots of MCF-7, LCC9 and T47D cells transfected with 40 nM control or MP1 siRNA. B) Representative photographs of transfected cells. Scale bar = 100 µm. 59   dead cells 100 live cells % Cells 80 60 40 20 0 Det. Att. Det. Att. Det. Att. Det. Att. Det. Att. Det. Att. CON siRNA MP1 siRNA MCF-7 CON siRNA MP1 siRNA LCC9 CON siRNA MP1 siRNA T47D Figure 5. Quantitation of detachment and death induced by silencing of MP1 in ER-positive mammary epithelial cells. Attached and floating cells were collected and analyzed. The percentage of dead cells (black bars) and live cells (white bars) in each population was determined by trypan blue exclusion assays. Error bars represent the mean ± SD of three independent experiments. 60   Figure 6. MP1 expression is not required for attachment or survival of ERnegative mammary epithelial cells. MDA-MB-231, BT-549, Sk-Br-3, and 184B5 cells were transfected with 40 nM control or MP1 siRNAs for all cell lines except 184B5, where 150 nM siRNAs were used. At 48 h cells were photographed, then harvested for counting and extract preparation. A) Immunoblots of transfected samples. Numbers represent the MP1/Actin ratios expressed as percentage of control samples. B) Representative photographs of transfected cells. Scale bar = 100 µm. 61   Figure 6. A MB-231 BT-549 Sk-Br-3 184B5 siRNA CON MP1 CON MP1 CON MP1 CON MP1 MP1 Actin MP1/Actin B 100 19 CON siRNA 100 40 100 MP1 siRNA MDAMB-231 BT-549 Sk-Br-3 184B5 62   39 100 18 dead cells 100 live cells % Cells 80 60 40 20 Att. Det. Att. Det. Att. Det. Att. Det. Att. Det. Att. Det. Att. Det. Att. Det. 0 CON MP1 CON MP1 CON MP1 CON MP1 siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA MDA-MB-231 BT-549 Sk-Br-3 184B5 Figure 7. Quantitation of detachment and death induced by silencing of MP1 in ER-negative mammary epithelial cells. Attached and floating cells were collected and analyzed. The percentage of dead cells (black bars) and live cells (white bars) in each population was determined by trypan blue exclusion assays. Error bars represent the mean ± SD of three independent experiments for all samples except 184B5 cells. For this cell line the numbers shown represent the average of two independent experiments. 63   Inhibition of MP1 expression results in apoptosis of MCF-7 cells To examine the mechanism of the cell death observed in MCF-7 cells upon MP1 silencing, expression of the anti-apoptotic protein Bcl-2 was examined. As shown in Figure 8A, Bcl-2 levels decreased approximately two fold in MP1 siRNA treated cells. In addition, cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, occurred in MCF-7 cells but not in MDA-MB-231 cells (Figure 8B). To further confirm that death was via apoptosis, cells were treated with the pan-caspase inhibitor z-VAD-FMK concurrently with siRNA transfection. As shown in Figures 8C and 8D, this treatment prevented PARP cleavage and cell rounding/detachment in MCF-7 cells. MP1 knockdown reduces ER protein and mRNA levels and transcriptional activity Because the most robust effect of MP1 silencing after prolonged treatment with siRNA is apoptosis and since ER was shown to mediate survival of breast cancer cells, the effect of MP1 knockdown on ER expression and activity was investigated. MP1 silencing reduces the protein and mRNA levels of ER (Figure 9A and B) at 48 and 30 hours, respectively. In addition, relative luciferase activity in the presence of estrogen was significantly decreased following siRNA treatment (Figure 9C). These data suggest that MP1 is required to maintain ER levels and facilitate ER-driven transcription on ERE-containing promoters. MP1 siRNA induced apoptosis does not require ER Given the results on ER levels and activity upon silencing of MP1, a double knockdown approach was undertaken in order to test whether ER is required for MP1 siRNA 64   A siRNA CON MP1 B MCF-7 siRNA CON #1 #2 Bcl-2 MP1 PARP Actin MP1 Bcl-2/Actin 100 42 C MDA-MB-231 CON #1 #2 siRNA Actin CON MP1 MP1 PARP MP1 Actin z-VAD-FMK - - + % PARP cleavage 32 86 34 D CON siRNA MP1 siRNA MP1 siRNA + 50 µM z-VAD-FMK Figure 8. MP1 knockdown induces apoptosis of MCF-7 but not of MDA-MB-231 cells. MCF-7 and/or MDA-MB-231 cells were transfected for 48 h with 30 nM control or MP1 siRNA, and cell extracts were prepared. A) Immunoblot of Bcl-2 protein in MCF-7 cells. Numbers represent the average MP1/Actin ratios expressed as percentage of control samples (n=3). B) Immunoblot of PARP in MCF-7 and MDA-MB-231 cells. C) Immunoblot and quantification of PARP cleavage in MCF-7 cells transfected with MP1 siRNA in the absence or presence of 50 µM z-VAD-FMK. D) Representative photographs of the samples analyzed in panel (C). Scale bar = 100 µm. 65   B siRNA A siRNA CON MP1 CON MP1 Actin MP1 CON MP1 ERα MP1 Actin Actin ER ER/Actin 100 23±20 C B * 9 Avg. fold change 8 7 6 5 4 3 2 1 0 CSS E2 ERE-Luc/βgal+CONsiRNA CSS E2 ERE-Luc/βgal+MP1siRNA   Figure 9. MP1 silencing decreases ER mRNA levels and transcriptional activity. A) Immunoblots of ER, MP1 and actin in MCF-7 cells transfected with MP1 siRNA for 48 hours. B) Semi-quantitative PCR indicating mRNA levels of MP1, ER and actin in MCF7 cells transfected with siRNA for 30 h. C) Quantification of luciferase/β galactosidase activity in MCF-7 cells cotransfected with plasmids expressing ERE2-tk109-luc and pβgal-Basic then treated with siRNA and stimulated with estrogen. Bars represent normalized luciferase activity in estrogen-stimulated cells relative to CSS (n=4 ± SD, *p<0.5).  66   siRNA CON MP1 ER MP1+ER PARP ER MP1 Actin Figure 10. ER silencing does not rescue the apoptosis induced by MP1 siRNA in MCF-7 cells. Cells were transfected with control, MP1, ER or combination of MP1 and ER siRNAs for 48 h then harvested and probed for PARP cleavage, ER, MP1, and actin by immunoblotting.  67   induced apoptosis of MCF-7 cells. If this process was dependent on ER, then the siRNA inhibition of ER expression concurrently with MP1 silencing, would prevent it. As illustrated by the immunoblots in figure 10, both MP1 and ER proteins were decreased with siRNA. Inhibition of MP1 expression induced PARP cleavage, which is indicative of apoptosis. Silencing of ER did not result in PARP cleavage and apoptosis, suggesting that MCF-7 cells do not depend on ER signals alone for survival. Interestingly, double silencing of MP1 and ER did not rescue cells from the MP1 siRNA-induced PARP cleavage and apoptosis, indicating that the ER is not required for apoptosis induced by MP1 silencing. MP1 knockdown reduces β1 integrin expression and decreases AKT activity but does not impact ERK expression or activity in MCF-7 cells To identify pathways affected by MP1 knockdown, expression of total and phosphorylated ERK and AKT1 and β1 integrin were examined (Figure 11). AKT1 is a pro-survival protein with a well-established role in the biology of cancer. ERK signaling is typically associated with proliferation, but may also be involved in regulating cell survival. The level of phospho-ERK was unaffected by MP1 knockdown (Figure 11A), suggesting that a loss of ERK signaling is not responsible for the observed detachment and cell death . In contrast, both phospho-AKT1 and β1 integrin levels decreased within 48 h of MP1 knockdown (Figure 11B and C). 68   A siRNA CON MP1 B ERK p-ERK MP1 Actin p-ERK/ERK 100 104±15 C MCF-7 siRNA siRNA CON MP1 Akt p-Akt (T308) Actin p-Akt/Akt 100 42 ±16 MDA-MB-231 CON MP1 CON MP1 IGNβ1 MP1 Actin IGNβ1/Actin 100 60±26 100 120±25 Figure 11. Effect of MP1 knockdown on cellular signaling pathways. MCF-7 and/or MDA-MB-231 cells were transfected with 30 nM MP1 siRNA for 48 h. A) Immunoblot of total and phospho-ERK in MCF-7 cells. The average p-ERK/total ERK ratios are expressed as percentage of control samples (n=3). B) Immunoblot of total and phosphoAKT in MCF-7 cells (n=4). C) Immunoblot of β1 integrin in MCF-7 and MDA-MB231cells (n=3, p=0.1). 69   Differential requirement for PI3K/AKT pathway for survival of MCF-7 and MDAMB-231 cells Inhibition of MP1 expression resulted in cell death in MCF-7 cells, and this was correlated with decreased phosphorylated (active) AKT1. In contrast, MDA-MB-231 cells showed no such response to MP1 knockdown. If decreased AKT activity is responsible for the cell death observed after MP1 knockdown, the lack of death in MDAMB-231 cells could be due to the fact that AKT activity is not dependent on MP1 in MDA-MB-231 cells, or that survival of these cells is not dependent upon active AKT. To test the latter possibility, MCF-7 and MDA-MB-231 cells were treated with various concentrations of the PI3K inhibitor LY294002, and the effects on AKT1 phosphorylation and cell viability were examined. As shown in Figure 12A, a concentration of 20 µM was sufficient to partially inhibit PI3K activity in both cell lines, as indicated by decreased pAKT1 levels. MCF-7 cell viability declined upon LY294002 treatment, and this was the result of apoptosis as indicated by increased PARP cleavage (Figure 12B and C). In contrast, MDA-MB-231 cells were unaffected by LY294002 treatment. These data indicate that MCF-7 cells are more dependent on PI3K/AKT1 pro-survival signaling than MDA-MB-231 cells, and are in agreement with previous reports showing a differential requirement for PI3K signaling in these two cell lines (13, 14) . Constitutively active AKT1 partially rescues MP1 siRNA induced apoptosis of MCF-7 cells MP1 knockdown was correlated with decreased activation of AKT1 in MCF-7 cells, which are highly dependent on pro-survival signals from the PI3K/AKT pathway. To 70   Figure 12. The PI3K/AKT pathway is required for survival of MCF-7 but not MDAMB-231 cells. MCF-7 and MDA-MB-231 cells were treated with various concentrations of LY294002 for 48 h. A) Immunoblot of p-AKT in MCF-7 and MDA-MB-231 cells treated LY294002. B) Effects of LY294002 treatment on viability as determined by trypan blue exclusion assays. C) Immunoblot of PARP cleavage in MCF-7 and MDA-MB-231 cells treated with LY294002. 71   Figure 13. Constitutively active AKT1 partially rescues MCF-7 cells from the apoptosis induced by MP1 siRNA. A) Immunoblot of p-AKT and Flag in stable pools of MCF-7 cells infected with control (pBabe-puro) or Myr-Flag-AKT1 expression vector as described in Materials and Methods. B) The stable pools of cells described in (A) were transfected with 30 nM control siRNA or MP1 siRNA for 48 h, and cell viability was determined by trypan blue exclusion assay. Bars represent the percentage of trypan blue-positive cells. Error bars represent the mean ± SD for three independent experiments, *p<0.05. C) Immunoblot of PARP and MP1 in a representative experiment described in (B). 72   examine whether active AKT1 is sufficient to maintain cell viability in the absence of MP1 we generated MCF-7 cells expressing constitutively active AKT1 (MCF-7/pBabepuro/Myr-Flag-AKT1). p-AKT1 was highly expressed in a pool of MCF-7/pBabepuro/Myr-Flag-AKT1 cells compared to a pool of cells containing the control pBabe-puro vector (Figure 13A). These pools of cells were transfected with MP1 siRNA or control siRNA, and the effects on cell survival were examined. As shown in Figure 13B, 64% of pBabe-puro containing cells were dead in the MP1 siRNA treated sample, but this decreased to 41% in cells expressing constitutively active AKT1. In addition, the extent of PARP cleavage was decreased in Myr-Flag-AKT1 expressing cells (Figure 13C). Together, these findings indicate that expression of active AKT1 partially overcomes the requirement for MP1 expression for survival of MCF-7 cells. They also suggest that AKT1 signals, at least in part, downstream of MP1. DISCUSSION The results presented here reveal a novel role for the small scaffold protein MP1 in breast cancer cells. Although MP1 is expressed in both ER-positive and ER-negative breast cancer cells, its depletion using RNAi-mediated suppression led to detachment and death of several ER-positive cell lines, but not three ER-negative breast cancer or a non-tumorigenic mammary epithelial cell line. Although this is a limited sample, MP1 has also been depleted in rat fibroblasts and human prostate cancer cells, and cell detachment/death was not reported in either case (6, 7). Therefore, MP1 expression seems to be required for survival in a subset of cell types, including ER-positive breast cancer cells. The observed cell death that occurred as a result of inhibiting MP1 73   expression in MCF-7 cells was shown to be due to an intrinsic apoptotic mechanism, as demonstrated by decreased Bcl-2 expression, increased PARP cleavage, and rescue of the death phenotype by treatment with the pan-caspase inhibitor z-VAD-FMK. Several interesting questions are raised by these results. One is what pro-survival pathways are affected by loss of MP1 expression in MCF-7 cells. Depletion of MP1 did not result in decreased ERK activation, indicating that its pro-survival functions are not mediated by the ERK pathway. The lack of an effect on ERK activation was somewhat surprising, since MP1 was originally identified as a scaffold protein that increased ERK signaling, but is consistent with results obtained in prostate cancer cells (6). In addition, since the expression of MP1 protein was evaluated in whole cell extracts it is possible that impaired ERK activity was localized to endosomes only, as reported by previous studies. Immunoblotting data demonstrated that inhibition of MP1 expression resulted in a greater than two fold decrease in AKT phosphorylation, suggesting that AKT1 signals, at least in part, downstream of MP1. The extent of AKT inhibition may be an underestimate, since by 48 h a majority of cells were dead, and the remaining live cells might represent ones with the lowest extent of MP1 knockdown. AKT plays a known pro-survival role in breast cancer cells, where it functions to relay signals from integrins or growth factor receptors, PI3K, or mTORC1, to downstream molecules such as Bcl-2 and NF-κB (15-20). The fact that AKT may also play a role in MP1 mediated survival is supported by the fact that expression of a constitutively active AKT1 partially rescued the cell death phenotype observed upon MP1 knockdown. There are two possible explanations for the lack of a full rescue: first, it is possible that not all of the MCF-7 cells in the pool selected here express the constitutively active AKT1 and second, the 74   Figure 14. Proposed model of MP1 siRNA-induced apoptosis of ER-positive breast cancer cells. Although not directly proven, inhibition of MP1 expression may decrease the expression of IGNβ1 and possibly other molecules (GFR, RTK) at the plasma membrane. This in turn decreases signaling from the PI3K/AKT and/or other downstream pro-survival pathways that ER-positive breast cancer cells in particular are highly dependent on, leading to Bcl-2 downregulation and apoptosis. 75   PI3K/AKT may not be the only pro-survival pathway in MCF-7 cells and silencing MP1 may trigger the activity of parallel AKT-independent apoptotic signals. Depletion of MP1 in MCF-7 cells also resulted in decreased levels of ERα protein, mRNA and reduced the transcriptional activity of the receptor, suggesting that MP1 may promote ER expression and activity. However, the apoptosis observed is unlikely to be due solely to a loss of ER signaling, since we and others have found that inhibition of ER expression using siRNA does not result in MCF-7 cell death (21). Furthermore, concurrent silencing of MP1 and ER did not rescue the cell death observed, indicating that the apoptosis induced by MP1 silencing does not depend on ER. Several studies indicate that ER may be implicated in breast cancer cell survival via cross-talk with the PI3K/AKT pathway (22), or by regulating the activity of NF-κB (23, 24), Bcl-2 (25, 26), or IAP family members (27). We therefore cannot rule out the possibility that decreased ER expression may contribute to the apoptosis observed. A second question raised by these results is the molecular basis for the differential requirement for MP1 for survival of ER-positive vs. ER-negative breast cancer cells. One possibility is that activation of pro-survival proteins such as AKT is not dependent on MP1 in ER-negative cells, and a second is that the ER-negative cells are less dependent on these pro-survival signaling pathways. Unfortunately we were not able to test the impact of MP1 silencing on AKT activity in MDA-MB-231 cells. The fact that LY294002 caused a concentration-dependent apoptotic response in MCF-7 cells, but did not affect MDA-MB-231 cells supports the latter hypothesis. This is in agreement with previous reports describing a differential sensitivity to this compound between the 76   two breast cancer cell lines (13, 14). Our findings suggest that MP1 promotes survival in part via PI3K/AKT1 signaling in MCF-7 cells. A final question is whether the cell death that we have observed is related to the previously identified roles of MP1 in cell spreading and cell motility. Since the apoptotic phenotype involves cell rounding and detachment, inhibition of MP1expression may disrupt cell adhesion signals, which could then trigger cell death. MP1 silencing in MCF-7 cells was correlated with decreased levels of β1 integrin protein expression. This receptor is of a particular interest, since 30-50% of breast tumors display aberrant expression of β1 integrin and αvβ1 heterodimer is the primary receptor for fibronectin. Data from literature address the interactions between β1 integrin and molecules that regulate cell survival. However, the direct effect of β1 integrin on breast cancer cell viability, is far from being elucidated and may be ligand- and cell-type dependent, and involve only a subpopulation of the receptor. Although not statistically significant, the decrease in β1 integrin suggests that inhibition of MP1 expression in MCF-7 cells may decrease the availability of pro-survival β1 integrin at the plasma membrane, which in turn, may trigger the activation of downstream apoptotic signals. This data partly fit a more recent study in which β1 integrin inhibitory antibodies induce apoptosis of several breast cancer cells in a 3D culture system, regardless of ER status (28). In summary, this is the first report investigating the role of the small scaffold protein MP1 in mammary epithelial cells. We have identified a novel functional interaction between MP1 and AKT1, and demonstrated that a loss of MP1 expression results in 77   apoptosis in ER-positive cells that are highly dependent upon the AKT pathway for survival. Future studies will further examine the molecular mechanism(s) by which MP1 promotes survival of ER-positive breast cancer cells, and evaluate its potential as a therapeutic target for ER-positive breast tumors. 78   REFERENCES 79   REFERENCES 1. Schaeffer HJ, Catling AD, Eblen ST, Collier LS, Krauss A, Weber MJ. MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade. Science. 1998;281:1668-71. 2. Pullikuth AK, Catling AD. Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: a perspective. Cell Signal. 2007;19:1621-32. 3. Teis D, Taub N, Kurzbauer R, Hilber D, de Araujo ME, Erlacher M, et al. p14MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis. J Cell Biol. 2006;175:861-8. 4. Teis D, Wunderlich W, Huber LA. Localization of the MP1-MAPK scaffold complex to endosomes is mediated by p14 and required for signal transduction. Dev Cell. 2002;3:803-14. 5. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. RagulatorRag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell. 2010;141:290-303. 6. Park ER, Pullikuth AK, Bailey EM, Mercante DE, Catling AD. Differential requirement for MEK Partner 1 in DU145 prostate cancer cell migration. Cell Commun Signal. 2009;7:26. 7. Pullikuth A, McKinnon E, Schaeffer HJ, Catling AD. The MEK1 scaffolding protein MP1 regulates cell spreading by integrating PAK1 and Rho signals. Mol Cell Biol. 2005;25:5119-33. 8. Mouchel-Vielh E, Bloyer S, Salvaing J, Randsholt NB, Peronnet F. Involvement of the MP1 scaffold protein in ERK signaling regulation during Drosophila wing development. Genes Cells. 2008;13:1099-111. 9. Boehm JS, Zhao JJ, Yao J, Kim SY, Firestein R, Dunn IF, et al. Integrative genomic approaches identify IKBKE as a breast cancer oncogene. Cell. 2007;129:106579. 10. Ory DS, Neugeboren BA, Mulligan RC. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci U S A. 1996;93:11400-6. 11. van 't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002;415:530-6. 12. Brunner N, Boysen B, Jirus S, Skaar TC, Holst-Hansen C, Lippman J, et al. MCF7/LCC9: an antiestrogen-resistant MCF-7 variant in which acquired resistance to 80   the steroidal antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal antiestrogen tamoxifen. Cancer Res. 1997;57:3486-93. 13. Bartucci M, Morelli C, Mauro L, Ando S, Surmacz E. Differential insulin-like growth factor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Cancer Res. 2001;61:6747-54. 14. Clark AS, West K, Streicher S, Dennis PA. Constitutive and inducible AKT activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther. 2002;1:707-17. 15. Bratton MR, Duong BN, Elliott S, Weldon CB, Beckman BS, McLachlan JA, et al. Regulation of ERalpha-mediated transcription of Bcl-2 by PI3K-AKT crosstalk: implications for breast cancer cell survival. Int J Oncol. 2010;37:541-50. 16. Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, et al. Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest. 2008;118:3065-74. 17. Kim AH, Khursigara G, Sun X, Franke TF, Chao MV. AKT phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol. 2001;21:893901. 18. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129:1261-74. 19. Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol Cell. 2006;21:749-60. 20. Nam JM, Onodera Y, Bissell MJ, Park CC. Breast cancer cells in threedimensional culture display an enhanced radioresponse after coordinate targeting of integrin alpha5beta1 and fibronectin. Cancer Res. 2010;70:5238-48. 21. Wang X, Belguise K, Kersual N, Kirsch KH, Mineva ND, Galtier F, et al. Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2. Nat Cell Biol. 2007;9:470-8. 22. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem. 2001;276:9817-24. 23. Frasor J, Weaver A, Pradhan M, Dai Y, Miller LD, Lin CY, et al. Positive crosstalk between estrogen receptor and NF-kappaB in breast cancer. Cancer Res. 2009;69:8918-25. 81   24. Riggins RB, Zwart A, Nehra R, Clarke R. The nuclear factor kappa B inhibitor parthenolide restores ICI 182,780 (Faslodex; fulvestrant)-induced apoptosis in antiestrogen-resistant breast cancer cells. Mol Cancer Ther. 2005;4:33-41. 25. Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M, et al. Mechanisms of transcriptional activation of bcl-2 gene expression by 17beta-estradiol in breast cancer cells. J Biol Chem. 1999;274:32099-107. 26. Teixeira C, Reed JC, Pratt MA. Estrogen promotes chemotherapeutic drug resistance by a mechanism involving Bcl-2 proto-oncogene expression in human breast cancer cells. Cancer Res. 1995;55:3902-7. 27. Stanculescu A, Bembinster LA, Borgen K, Bergamaschi A, Wiley E, Frasor J. Estrogen Promotes Breast Cancer Cell Survival in an Inhibitor of Apoptosis (IAP)Dependent Manner. Horm Cancer. 2010;1:127-35. 28. Park CC, Zhang H, Pallavicini M, Gray JW, Baehner F, Park CJ, et al. Beta1 integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo. Cancer Res. 2006;66:1526-35. 82   APPENDIX The Role of MP1 Scaffold Protein in Estrogen-regulated Functions of Breast Cancer Cells 83   APPENDIX ABSTRACT Proliferation and migration of breast cancer cells are regulated by various cytosolic kinases, including the members of the MAPK pathway and PAK1. In turn, as shown by studies carried out in fibroblasts, the activity of these molecules requires the participation of MP1 scaffold protein. Moreover, in ER-positive breast tumors, intracellular kinases potentiate the function of ER, even in the absence of ligands, and trigger various cellular responses. Because MP1 interacts with signaling molecules that are known to promote breast tumorigenesis, the initial aim was to investigate the role that MP1 plays in regulating the proliferation and migration of breast cancer cells. The effects of inhibiting MP1 expression by siRNA-mediated silencing were investigated. First, we observed that inhibition of cell cycle progression seemed to have a protective effect against apoptosis. Under these experimental conditions, we observed that neither short-term proliferation nor migration of MCF-7 cells requires MP1. Taken together, these studies indicate that although it is required for ER activity, MP1 scaffold protein is not essential for estrogen-mediated proliferation and migration after release from cell cycle arrest. 84   INTRODUCTION Uncontrolled proliferation is one of the acquired attributes of cancer cells and this tumoral process is promoted by mitogens such as estrogens and growth factors. Their mechanism of action involves increased transcription of molecular components of the cell-cycle machinery. For over 70% of breast tumors, ER is a major regulator of cell proliferation. Activated by estrogen, ER stimulates the expression of genes encoding the cyclins that are involved in the transition from G1 to S phase of the cell cycle Therefore, antiestrogens like fulvestrant (ICI) block cell proliferation by arresting the cells in G0/G1 phase (1). Regardless of ER status, growth factors and their receptors as well as integrins can regulate cell cycle progression, hence stimulate proliferation, mainly through the MAPK and PI3K pathways. Another hallmark of epithelial cancers is the acquisition of motility. In many breast cancers, including some ER-positive tumors, this feature is a prerequisite for the switching of neoplastic cells to a metastatic phenotype. Compelling data reveal numerous mechanisms accounting for cell migration. In addition to its proliferative function, ER also influences motility. In MCF-7 cells, estrogen-stimulated ERα positively regulates cell migration (2). In MDA-MB-231 cells transfected with ERα and ERβ, migration and invasion are inhibited in a ligand-independent manner (3). Notably, Raf, MEK and ERK, members of the mitogen activated protein kinase (MAPK) cascade, and the p-21 activated kinase-1 (PAK1) regulate cell motility in normal cells and are implicated in breast cancer signaling. Several studies have reported the direct involvement of MEK and ERK in the motility of tumor cells (4-7). PAK1 is downstream 85   of Rac and Cdc42, and its activation induces the formation of membrane protrusions (8, 9). PAK1 may also play a role in breast cancer by causing tumorigenesis and hyperplasia of the mammary epithelium (10, 11), and by regulating spreading and migration (12, 13). In addition, MAPK and PAK1 are known to trigger ligand independent effects of ER-signaling by directly activating ER (10, 14). MEK binding partner 1 (MP1) is a small scaffold protein that promotes MEK1 activation by B-Raf (15). The overexpression of MP1 enhances ERK activation in cultured fibroblasts (15). In HeLa cells and mouse fibroblasts, the adaptor protein p14 recruits MP1 to late endosomes where the MP1/p14 heterodimer is required for ERK activation (16, 17). Recently, crosstalk between PAK1 and MEK1 was demonstrated in rat fibroblasts, where PAK1 activates a population of MEK1 and ERK in focal complexes during adhesion to fibronectin. This interaction requires MP1 (18) and is sufficient to activate MEK1 in the absence of Raf, but to a lesser extent than observed with active Raf or EGF (19). Of particular interest is the involvement of MP1 in regulating attachment of fibroblasts. Knocking down MP1 inhibited their acute spreading on fibronectin, thus indicating that MP1 allows the rapid cytoskeletal reorganization necessary for membrane protrusions and cell spreading to occur. The existence of this new interaction between MP1 and PAK1, which was required for subsequent MEK1 activation, suggested a role for MP1 in coupling MAPK signaling to Rho (18). The pathways that MP1 interacts with control the proliferation and motility of breast cancer cells, therefore we reasoned that MP1 might have similar functions in mammary tumor epithelial cells and might directly contribute to the proliferation and motility of 86   breast cancer cells. To test this hypothesis, the effects of MP1 silencing on MCF-7 cell short-term proliferation and migration were examined. Our results indicate that inhibition of cell cycle appears to have a protective effect against the apoptosis induced by MP1 silencing. Consequently, when cells are released from arrest using estrogen, MP1 is not required to promote the entry in the S phase or migration. Taken together, these studies indicate that MP1 scaffold protein is not essential for estrogen-mediated proliferation and migration of breast cancer cells following arrest. MATERIALS AND METHODS Cell lines and culture conditions MCF-7 cells were obtained from the Lombardi Cancer. Cells were maintained in Improved Modified Eagle’s Medium (IMEM) containing phenol red (GIBCO-InvitrogenApplied Biosystems), supplemented with 5% fetal bovine serum (HyClone), and 100 Units/ml Penicillin/100 µg/ml Streptomycin (Invitrogen) and incubated at 37 ºC with 5 % CO2. siRNA transfections All siRNA transfection reagents were purchased from Dharmacon-Thermo Scientific. MP1 (ON-TARGETplus siRNA Human MAP2K1IP1) and a non-targeting siRNA (ONTARGETplus siCONTROL) were used. Cells were plated in six-well plates at 105-3 x 105 cells per well in FBS containing medium. After 24 h, cells were transfected or cotransfected with 30 nM siRNA using DharmaFECT 1 transfection reagent. After 48 h, 87   cells were harvested by scraping on ice cold PBS or trypsinization and prepared for protein extraction. Cell cycle arrest After 5-6 h, siRNA transfection mixes were replaced with charcoal-stripped serum (CSS) medium supplemented with 10 nM ICI182,780 for 24 hours, which induced their growtharrest in the G0/G1 phase of the cell cycle and abolished ER signaling. BrdU incorporation MCF-7 cells were plated on glass coverslips in 6-well plates, then transfected and prearrested as previously described. Following ICI pre-arrest for 24 hours, cells were treated with 10 nM 17-β estradiol or vehicle in CSS-containing medium for 19 hours. During the last five hours of treatment, cells were labeled with 25 µM BrdU, then fixed for 15 minutes with 3% methanol-free formaldehyde (Polysciences) and subjected to immunofluorescence. In parallel, cells were plated, treated and harvested at the same time point for protein extraction. Immunofluorescence Formaldehyde-fixed cells were washed with PBS, then fixed for 10 minutes with ice-cold pure methanol at -20ºC. DNA was denatured using 2N HCl for 30 min at 37ºC, then slides were neutralized by 0.1 M borax solution pH 8.5 (Sigma) for 30 min. After a brief wash with PBS, cells were blocked in 2% BSA, then incubated with anti-BrdU/Alexa Fluor conjugated antibody (Invitrogen). A DAPI nuclear counterstain was performed. 88   Eight random fields were visualized and the percentage of BrdU positive cells was calculated using the Metamorph program. Immunoblotting Cell pellets were lysed in 1% Triton XC-100/HEPES or CelLytic M lysis buffer (Sigma), supplemented with cocktail tablets of protease (Roche - Complete Mini EDTA-free) and phosphatase inhibitors (Roche – PhosSTOP). Protein concentrations were determined using the Bradford protein assay (Bio-Rad). Total protein (10-20 µg) was subjected to 420% Tris-HCl SDS-PAGE (Bio-Rad), transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore), blocked with Odyssey Blocking Buffer and then incubated with the appropriate primary antibodies overnight. Alexa Fluor 680 anti-goat, anti-rabbit, and anti-rabbit (Invitrogen) and IRDye 800CW anti-mouse and anti-rabbit (LI-COR) secondary antibodies were used for two-color detection of proteins. Membranes were scanned and analyzed using the LI-COR Odyssey system. Antibodies and reagents The following primary antibodies were used for Western blotting: MP1 (A-19, Santa Cruz), actin (AC-40, SIGMA) and estrogen receptor alpha (AB-17, Lab Vision-Thermo Scientific, or F-10, Santa Cruz) CDK2 inhibitor SU9516 was obtained from Calbiochem, ICI from Tocris, 17-β estradiol, DAPI and BrdU were purchased from Sigma. 89   Migration assay Cell migration or invasion was analyzed using a Boyden chamber transwell assay (Corning). Cells were plated, transfected, and synchronized in G0/G1 phase as previously described. Pre-arrested cells were then incubated overnight in 10 nM estrogen/CSS. Untransfected cells treated with estrogen were used as a positive control for migration, while cells in steroid-depleted media (CSS) represented the negative migration control. To prepare them for migration, cells were trypsinized, quenched with soybean trypsin inhibitor, counted and resuspended in serum-starvation media. Uncoated polycarbonate transwell permeable inserts were pre-treated with BSA in order to block the attachment to the membrane. Fifty thousand cells were seeded in the upper chambers and allowed to migrate for 24 h. The chemoattractant in the lower chamber was medium supplemented with 10% FBS. The permeable membranes were then fixed, stained with crystal violet and the migrating cells were visually scored under a phasecontrast microscope equipped with a grid-containing eyepiece, at 200X. The results are the average number of cells invading through the membrane as counted per five random grid areas, in duplicate inserts Statistical Analysis Data are expressed as the mean ± S.D. Most experiments were performed three times. Paired evaluations were made for experimental and control conditions within each experiment. Significance was determined by Student’s t test and set at p<0.05. 90   RESULTS The initial transfection protocol resulted in cell detachment and death, preventing the performance of additional functional assays under these conditions. We therefore modified the procedure, so that transfection mixes containing control or MP1siRNA were replaced with ICI-containing medium after five-six hours. This change partially rescued the observed phenotype, causing fewer cells to detach. Under these new conditions, we have investigated the requirement for MP1 in two estrogen-mediated cellular processes in MCF-7 cells: S phase entry and migration. MP1 siRNA induced apoptosis is rescued by cell cycle arrest Our observations indicate that the replacement of transfection media with ICI/CSS reduced the cell death phenotype in MCF-7 cells. ICI anti-estrogen decreases the levels of ER and also induces cell cycle arrest in G0. The same effect can be achieved by treating cells with SU9516, that specifically blocks CDK2, which is required from the transition of G1 to S phase. This latter experiment lacks a negative control, which would be represented by MCF-7 cells transfected with MP1 siRNA for 48 hours, in the absence of SU9516. Such experiment would have resulted in PARP cleavage in the presence of MP1 siRNA but not in control samples. Both ICI and SU9516 prevented PARP cleavage upon MP1 silencing, as demonstrated in figure 15, suggesting that cell cycle arrest counteracts the apoptotic signals mediated by loss of MP1 expression. 91   A FBS siRNA CON MP1 ICI CON MP1 PARP ER Actin B siRNA CON MP1 PARP MP1 Actin Figure 15. Cell cycle arrest rescues the apoptosis induced by MP1 siRNA in MCF7 cells. A) Immunoblot of PARP, ER and actin in cells transfected with siRNA mixes in FBS or ICI. B) Immunoblot of PARP, MP1 and actin in cells transfected with MP1 siRNA concurrently with 5 µM SU9516, specific CDK2 inhibitor, for 48 h.  92   Silencing of MP1 does not affect estrogen-induced S phase entry following arrest in MCF-7 cells Since the best described role of MP1 involves the scaffolding between MEK1 and ERK1 and because these kinases have been linked to ER signaling, we asked whether endogenous MP1 was necessary for MCF-7 cells to proliferate in the presence of estrogen. To better delineate the role of estrogen in S phase entry, cells were synchronized in the G0 quiescent phase of their cell cycle, then released with estrogen. The results from three independent experiments revealed that transfection with MP1 siRNA did not alter estrogen-stimulated DNA synthesis (Figure 16). Under estrogen stimulation, there were 34.5% BrdU-positive cells in control siRNA-treated wells and 30.5% with MP1 siRNA transfection. In the absence of estrogen, there were only 12% BrdU-positive cells in control wells and 10.8% in MP1siRNA transfected wells. These data suggest that basal expression of MP1 scaffold protein is not required for the estrogen-dependent S phase entry of the cell cycle. Effects of MP1 silencing on estrogen-stimulated migration after cell cycle arrest Estrogen promotes migration of MCF-7 breast cancer cells independently from its effects on proliferation (20). As shown in figure 17, our results suggest that high levels of MP1 may not be required for migration of MCF-7 cells following release from arrest. Although chemoattractants were present in the bottom wells, the migratory potential of untransfected cells increased four-fold when treated with estrogen compared to CSS alone, whereas control and MP1 siRNA transfected cells migrated at comparable levels. A specific mitosis inhibitor, such as mitomycin D, was not used in order to clearly 93   A CSS Estrogen CON siRNA MP1 siRNA C CSS E2 40 B siRNA CON MP1 MP1 Actin E2 - + - %BrdU Positive Cells 35 30 25 20 15 10 5 0 + CON siRNA MP1 siRNA Figure 16. Effect of MP1 knockdown on DNA synthesis. MCF-7 cells were plated, pre-arrested and stimulated with estrogen. A) Representative overlaid immunofluorescent images of pre-arrested MCF-7 cells transfected with MP1 siRNA (lower panels) or control siRNA (upper panels) in the absence (left) or presence of estrogen (right). Green=anti BrdU Alexa Fluor 480; blue=DAPI. B) Immunoblots of MP1 and actin in samples plated, treated and harvested under similar conditions as the BrdU labeled cells. C) Quantification of BrdU incorporation. Bars represent percentage of BrdU-positive cells (mean ±SD, n=3). 94   Figure 17. Effect of MP1 knockdown on estrogen-stimulated migration of MCF-7 cells. A) Representative images of migrating MCF-7 cells at 200X magnification. Untransfected cells (upper panels) were treated with CSS alone (left) or 10 nM estrogen (right). Cells transfected with control siRNA (lower left) or MP1 siRNA (lower right) were stimulated with estrogen. B) Immunoblots of MP1 and actin of samples shown in (A). C) Quantification of cell migration. Bars represent number of migrating cells/area. Error bars are the mean standard error of replicate wells. 95   Figure 17. A Untransfected cells CSS CON siRNA/Estrogen Estrogen MP1 siRNA/Estrogen B siRNA - - CON MP1 MP1 Actin E2 C 300 250 Cells/Area 200 150 100 50 0 CSS E2 Untransfected cells CON MP1 siRNA/E2 siRNA/E2 96   - + + + distinguish between proliferation and migration. However, even if the cells had completed a new cell cycle after their 24 h-long movement through the permeable inserts, the migration would have been masked by proliferation in both MP1 siRNA and control siRNA treated samples, since the knockdown of MP1 resulted in similar numbers of BrdU-positive cells. DISCUSSION The additional findings reported here help delineate the functions of MP1 protein in breast cancer cells. Our data indicate that its partial inhibition using siRNA transfection for two days results in apoptosis. This effect prevented us from examining additional physiologically relevant processes, such as proliferation and migration. Previous studies have clearly established the role of estrogen in promoting proliferation and migration of MCF-7 cells. Therefore, in order to investigate these functions we undertook a wellestablished strategy of pre-arresting the cells in the G0/G1 phase of the cell cycle, then releasing them with estrogen to further stimulate their proliferation or migration. Since cell transfection requires active proliferation, the ICI-mediated cell cycle arrest was done at 5-6 hours post siRNA transfection with MP1 siRNA. Interestingly, replacement of transfection media with ICI/CSS diminished the observed cell death effect and prevented the cleavage of PARP. Similar effects were noted when MCF-7 cells were transfected with MP1 siRNA in the presence of a specific CDK2 inhibitor. Cell cycle arrest is not always followed by apoptosis, since these are two distinct processes that share some regulatory factors (21). However, the molecular mechanisms that determine cell decision between proliferation and apoptosis are still 97   elusive and may be cell type and context dependent. In our experiments, is appears that inhibition of cell cycle progression has a protective effect against pro-apoptotic signals induced by MP1 silencing. Although we do not provide direct evidence to support this, it is possible that inhibition of apoptosis occurred via transcriptional regulation of proapoptotic molecules by highly active inhibitors of cell cycle, such as p21. Such mechanisms have been noted in different cell types (22, 23). Alternatively, exit of G1 phase by estrogen may activate cytosolic pro-survival pathways, such as PI3K/AKT, that can override the pro-apoptotic signals initiated by MP1 silencing (24). Under these conditions of partial MP1 silencing and reduced cell death, we carried out functional studies to establish the role of MP1 in short-term proliferation and migration of estrogen-stimulated MCF-7 cells. BrdU labeling during release from arrest revealed that MP1 siRNA transfection did not significantly decreased the percentage of proliferating cells compared to control, suggesting that sustained levels of MP1 are not required for S phase entry. MP1 was reported to regulate Rho function and its inhibition delayed spreading of fibroblasts (18) and decreased migration of prostate cancer cells (25). Generally, a suppression of tumor cell spreading is believed to be correlated with increased migration. However, there are conflicting reports on the significance of spreading relative to the metastatic potential (12, 20, 26).  Our data indicate that inhibition of MP1 expression using siRNA duplexes does not influence the migration of breast cancer cells in Boyden chamber assays. Thus, MP1 may not be required for estrogen-stimulated proliferation or migration of MCF-7 cells, following cell cycle arrest.   98   REFERENCES 99   REFERENCES 1. Clarke R, Leonessa F, Welch JN, Skaar TC. 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Biochem Biophys Res Commun. 2003;307:839-46. 102   CONCLUDING REMARKS 103   CONCLUDING REMARKS The main goal of this dissertation was to investigate how MP1 protein affects tumoral processes in breast cancer cells. Given its scaffolding function, and therefore its contribution to potentiating signals that regulate proliferation, spreading, migration, and differentiation, we reasoned that MP1 might play similar roles in breast cancer cells. We propose a novel pro-survival role for MP1 that is specific to some ER-positive cell lines. Our results indicate that MP1 silencing results in detachment and death of ERpositive breast cancer cells. In contrast, in three independent ER-negative breast cancer cells, the same approach had no significant effect on survival. More importantly, non-tumorigenic mammary epithelial cells were unaffected by MP1 silencing. In MCF-7 cells, a widely used model of estrogen-stimulated, antiestrogen sensitive human breast cancer, apoptosis induced by inhibition of MP1 expression was correlated with decreased activity of AKT and decreased levels of the Bcl-2 pro-survival protein. Interestingly, we demonstrate that MCF-7 cells depend on the PI3K but not on ER signaling for survival. Moreover, concurrent silencing of MP1 and ER failed to rescue the observed cell death whereas this phenotype was partly rescued with the expression of a constitutively active AKT1. This suggests that PI3K/AKT activity is not sufficient to counteract the pro-apoptotic signals initiated by MP1 silencing, and that ER is not required for this phenomenon. However, MP1 promotes ER expression and activation in response to estrogen, therefore, the ER signaling pathway may play a contributing role in promoting cell survival. 104   Although our query did not reveal any significant correlations between the levels of MP1 protein expression and disease outcome, this is a biological meaningful molecule that may have a potential clinical relevance for ER-positive breast tumors: • MP1 could be a potential therapeutic target, since ER-positive breast cancer cells are more susceptible to apoptosis upon MP1 depletion. In general, the physiological response of drugs targeting ER-positive tumors involves inhibition of or interference with tumor proliferation and growth. Inhibition of MP1 expression could specifically trigger apoptosis of ER-expressing tumor cells, without affecting non-tumorigenic cells. However, this strategy would be ineffective during treatment with fulvestrant (ICI), since in our cell model, this compound and the alternative cell cycle inhibitor SU9516 were able to prevent apoptosis induced by MP1 silencing. • MP1 expression in ER-positive breast cancer cells could predict the response to specific kinase inhibitors, since these tumors would be more likely to respond to PI3K inhibition than to blockade of MAPK. 105