p130Cas Is Required for Mammary Tumor Growth and Transforming Growth Factor-β-mediated Metastasis through Regulation of Smad2/3 Activity
2009; Elsevier BV; Volume: 284; Issue: 49 Linguagem: Inglês
10.1074/jbc.m109.023614
ISSN1083-351X
AutoresMichael K. Wendt, Jason P. Smith, William P. Schiemann,
Tópico(s)Cell Adhesion Molecules Research
ResumoDuring breast cancer progression, transforming growth factor-β (TGF-β) switches from a tumor suppressor to a pro-metastatic molecule. Several recent studies suggest that this conversion in TGF-β function depends upon fundamental changes in the TGF-β signaling system. We show here that these changes in TGF-β signaling are concomitant with aberrant expression of the focal adhesion protein, p130Cas. Indeed, elevating expression of either the full-length (FL) or just the carboxyl terminus (CT) of p130Cas in mammary epithelial cells (MECs) diminished the ability of TGF-β1 to activate Smad2/3, but increased its coupling to p38 MAPK. This shift in TGF-β signaling evoked (i) resistance to TGF-β-induced growth arrest, and (ii) acinar filling upon three-dimensional organotypic cultures of p130Cas-FL or -CT expressing MECs. Furthermore, rendering metastatic MECs deficient in p130Cas enhanced TGF-β-stimulated Smad2/3 activity, which restored TGF-β-induced growth inhibition both in vitro and in mammary tumors produced in mice. Additionally, whereas elevating TβR-II expression in metastatic MECs had no affect on their phosphorylation of Smad2/3, this event markedly enhanced their activation of p38 MAPK, leading to increased MEC invasion and metastasis. Importantly, depleting p130Cas expression in TβR-II-expressing metastatic MECs significantly increased their activation of Smad2/3, which (i) reestablished the physiologic balance between canonical and noncanonical TGF-β signaling, and (ii) reversed cellular invasion and early mammary tumor cell dissemination stimulated by TGF-β. Collectively, our findings identify p130Cas as a molecular rheostat that regulates the delicate balance between canonical and noncanonical TGF-β signaling, a balance that is critical to maintaining the tumor suppressor function of TGF-β during breast cancer progression. During breast cancer progression, transforming growth factor-β (TGF-β) switches from a tumor suppressor to a pro-metastatic molecule. Several recent studies suggest that this conversion in TGF-β function depends upon fundamental changes in the TGF-β signaling system. We show here that these changes in TGF-β signaling are concomitant with aberrant expression of the focal adhesion protein, p130Cas. Indeed, elevating expression of either the full-length (FL) or just the carboxyl terminus (CT) of p130Cas in mammary epithelial cells (MECs) diminished the ability of TGF-β1 to activate Smad2/3, but increased its coupling to p38 MAPK. This shift in TGF-β signaling evoked (i) resistance to TGF-β-induced growth arrest, and (ii) acinar filling upon three-dimensional organotypic cultures of p130Cas-FL or -CT expressing MECs. Furthermore, rendering metastatic MECs deficient in p130Cas enhanced TGF-β-stimulated Smad2/3 activity, which restored TGF-β-induced growth inhibition both in vitro and in mammary tumors produced in mice. Additionally, whereas elevating TβR-II expression in metastatic MECs had no affect on their phosphorylation of Smad2/3, this event markedly enhanced their activation of p38 MAPK, leading to increased MEC invasion and metastasis. Importantly, depleting p130Cas expression in TβR-II-expressing metastatic MECs significantly increased their activation of Smad2/3, which (i) reestablished the physiologic balance between canonical and noncanonical TGF-β signaling, and (ii) reversed cellular invasion and early mammary tumor cell dissemination stimulated by TGF-β. Collectively, our findings identify p130Cas as a molecular rheostat that regulates the delicate balance between canonical and noncanonical TGF-β signaling, a balance that is critical to maintaining the tumor suppressor function of TGF-β during breast cancer progression. IntroductionInvasion and metastasis are the most lethal characteristics of breast cancer (1.Yoshida B.A. Sokoloff M.M. Welch D.R. Rinker-Schaeffer C.W. J. Natl. Cancer Inst. 2000; 92: 1717-1730Crossref PubMed Scopus (233) Google Scholar, 2.Wakefield L.M. Piek E. Böttinger E.P. J. Mammary Gland Biol. Neoplasia. 2001; 6: 67-82Crossref PubMed Scopus (91) Google Scholar). Transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF-βtransforming growth factor-βFAKfocal adhesion kinaseMECmammary epithelial cellTβR-ITGF-β type I receptorTβR-IITGF-β type II receptorp130CasCrk-associated substratePAI-1plasminogen activator inhibitor-1NmuMGnormal murine mammary epithelialMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinaseCMVcytomegalovirusshRNAshort hairpin RNAβ-galβ-galactosidaseFLfull-lengthCTcarboxyl-terminal. is a powerful suppressor of mammary tumorigenesis, doing so through its ability to repress mammary epithelial cell (MEC) proliferation, as well as through its creation of cell microenvironments that inhibit MEC motility, invasion, and metastasis (2.Wakefield L.M. Piek E. Böttinger E.P. J. Mammary Gland Biol. Neoplasia. 2001; 6: 67-82Crossref PubMed Scopus (91) Google Scholar). During breast cancer progression, the tumor suppressing function of TGF-β is frequently subverted, thus transforming TGF-β from a suppressor of breast cancer formation to a promoter of its growth and metastasis (2.Wakefield L.M. Piek E. Böttinger E.P. J. Mammary Gland Biol. Neoplasia. 2001; 6: 67-82Crossref PubMed Scopus (91) Google Scholar, 3.Buck M.B. Knabbe C. Ann. N. Y. Acad. Sci. 2006; 1089: 119-126Crossref PubMed Scopus (127) Google Scholar, 4.Benson J.R. Lancet Oncol. 2004; 5: 229-239Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Unfortunately, how mammary tumorigenesis overcomes the cytostatic function of TGF-β remains incompletely understood, as does the manner in which developing breast cancers ultimately sense TGF-β as a pro-metastatic factor.Transmembrane signaling by TGF-β commences upon binding to its type II receptor (TβR-II), which recruits and activates its type I receptor (TβR-I), which then phosphorylates and activates Smads 2 and 3. Following their activation, Smads 2 and 3 form heteromeric complexes with Smad4, which collectively translocate to the nucleus to regulate a multitude of transcriptional events and cellular responses (i.e. apoptosis, cytostasis, and homeostasis, (5.Chen R.H. Chang T.Y. Cell Growth Differ. 1997; 8: 821-827PubMed Google Scholar, 6.Blobe G.C. Schiemann W.P. Lodish H.F. N. Engl. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2166) Google Scholar)). In addition to stimulating Smad2/3, TGF-β also activates several noncanonical signaling systems, including members of the MAP kinase family (e.g. ERK1/2, JNK, and p38 MAPK (7.Galliher A.J. Neil J.R. Schiemann W.P. Future Oncol. 2006; 2: 743-763Crossref PubMed Scopus (72) Google Scholar)). Interestingly, several studies suggest that genetic and epigenetic events cooperate with aberrant Smad2/3 activities and functions to facilitate the conversion of TGF-β from tumor suppressor to a tumor promoter (8.Gomis R.R. Alarcón C. Nadal C. Van Poznak C. Massagué J. Cancer Cell. 2006; 10: 203-214Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 9.Adorno M. Cordenonsi M. Montagner M. Dupont S. Wong C. Hann B. Solari A. Bobisse S. Rondina M.B. Guzzardo V. Parenti A.R. Rosato A. Bicciato S. Balmain A. Piccolo S. Cell. 2009; 137: 87-98Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). However, these and other studies also present strong evidence implicating dysregulated activation of several noncanonical TGF-β effectors during this same switch in TGF-β function (10.Tian M. Schiemann W.P. Future Oncol. 2009; 5: 259-271Crossref PubMed Scopus (160) Google Scholar). Thus, deciphering the relative contribution of signaling imbalances that arise between Smad2/3-dependent and -independent TGF-β signaling systems is essential to enhancing our understanding of how TGF-β ultimately promotes the development and progression of mammary tumorigenesis.Recently, we identified a critical αvβ3 integrin:pY284-TβR-II:Grb2 signaling axis that mediates TGF-β stimulation of MAP kinases in normal and malignant MECs, leading to their acquisition of epithelial-mesenchymal transition, invasive, and metastatic phenotypes both in vitro and in vivo (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 12.Galliher A.J. Schiemann W.P. Cancer Res. 2007; 67: 3752-3758Crossref PubMed Scopus (207) Google Scholar, 13.Galliher A.J. Schiemann W.P. Breast Cancer Res. 2006; 8: R42Crossref PubMed Scopus (214) Google Scholar). Moreover, activation of this oncogenic signaling axis by TGF-β requires β3 integrin to form complexes with TβR-II (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 12.Galliher A.J. Schiemann W.P. Cancer Res. 2007; 67: 3752-3758Crossref PubMed Scopus (207) Google Scholar, 13.Galliher A.J. Schiemann W.P. Breast Cancer Res. 2006; 8: R42Crossref PubMed Scopus (214) Google Scholar). Unfortunately, it remains uncertain as to whether this interaction is direct or facilitated through another scaffolding protein. As such, we sought to identify members of focal adhesion complexes as potential integrin effectors capable of contributing to altered TGF-β signaling.p130Cas (Crk-associated substrate) functions as a molecular scaffold within focal adhesion complexes, and is readily phosphorylated by focal adhesion kinase (FAK) and Src (14.Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1026) Google Scholar). Additionally, p130Cas binds stably to a variety of signaling molecules, including the (i) protein-tyrosine kinases FAK, PYK2, Src, Fyn, and Abl; (ii) adaptor molecules Crk, CrkL, Trip6, and AJUBA; (iii) guanine nucleotide exchange factors AND34 and CG3; and (iv) the MAPK family member, JNK (15.Brábek J. Constancio S.S. Shin N.Y. Pozzi A. Weaver A.M. Hanks S.K. Oncogene. 2004; 23: 7406-7415Crossref PubMed Scopus (69) Google Scholar, 16.Geiger B. Cell. 2006; 127: 879-881Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The extensive interactome of p130Cas ideally positions and enables this molecule to interpret and integrate a variety of signaling inputs arising from numerous receptor systems. Indeed, the biological importance of p130Cas is emphasized by studies showing that its genetic ablation in mice elicits embryonic lethality, whereas fibroblasts derived from p130Cas-deficient embryos exhibit drastically altered cytoskeletal architectures (17.Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar). Moreover, fibroblasts transformed by Src become significantly more invasive when engineered to simultaneously overexpress p130Cas (15.Brábek J. Constancio S.S. Shin N.Y. Pozzi A. Weaver A.M. Hanks S.K. Oncogene. 2004; 23: 7406-7415Crossref PubMed Scopus (69) Google Scholar). Patients with primary breast tumors expressing high levels of p130Cas (also known as breast cancer resistance-1) experience a more rapid disease recurrence and have a greater risk of resistance to tamoxifen therapy (18.van der Flier S. Chan C.M. Brinkman A. Smid M. Johnston S.R. Dorssers L.C. Dowsett M. Int. J. Cancer. 2000; 89: 465-468Crossref PubMed Scopus (42) Google Scholar). Recent studies also indicate that specific overexpression of p130Cas/breast cancer resistance-1 expression can confer breast cancer resistance to adriamycin (19.Ta H.Q. Thomas K.S. Schrecengost R.S. Bouton A.H. Cancer Res. 2008; 68: 8796-8804Crossref PubMed Scopus (48) Google Scholar). Moreover, directed overexpression of p130Case in murine MECs significantly increased their proliferative and survival indices in vivo, as well as greatly reduced the latency of mammary tumors arising from murine mammary tumor virus-driven Her2/Neu expression in mice (20.Cabodi S. Tinnirello A. Di Stefano P. Bisarò B. Ambrosino E. Castellano I. Sapino A. Arisio R. Cavallo F. Forni G. Glukhova M. Silengo L. Altruda F. Turco E. Tarone G. Defilippi P. Cancer Res. 2006; 66: 4672-4680Crossref PubMed Scopus (111) Google Scholar). This study also observed the expression of p130Cas to be up-regulated significantly in a subset of human breast cancer samples (20.Cabodi S. Tinnirello A. Di Stefano P. Bisarò B. Ambrosino E. Castellano I. Sapino A. Arisio R. Cavallo F. Forni G. Glukhova M. Silengo L. Altruda F. Turco E. Tarone G. Defilippi P. Cancer Res. 2006; 66: 4672-4680Crossref PubMed Scopus (111) Google Scholar). Collectively, these findings highlight the critical roles played by p130Cas in regulating normal tissue morphogenesis, and in promoting breast cancer progression. With respect to TGF-β, a recent study identified p130Cas as a potential inhibitor of Smad3 function (21.Kim W. Seok Kang Y. Soo Kim J. Shin N.Y. Hanks S.K. Song W.K. Mol. Biol. Cell. 2008; 19: 2135-2146Crossref PubMed Scopus (42) Google Scholar). However, the pathophysiological importance of this event, if any, in mediating the oncogenic activities of TGF-β and/or p130Cas during breast cancer progression remains to be established.The objective of the present study was to determine the role of p130Cas in facilitating the acquisition of oncogenic signaling by TGF-β during breast progression. We show here that p130Cas expression is up-regulated significantly in metastatic breast cancer cells (murine 4T1/human MCF10A-Ca1a) as compared with their nonmetastatic counterparts (murine 67NR/human MCF10A). Moreover, increased p130Cas expression was consistent with a decrease in TGF-β1-induced Smad2/3 signaling. Indeed, overexpression of p130Cas in nonmetastatic MECs led to a decrease in Smad2/3 activity, whereas depletion of p130Cas in metastatic MECs increased Smad2/3 activity. Most importantly, we show for the first time that p130Cas is essential for TGF-β stimulation of breast cancer growth, invasion, and pulmonary dissemination in mice. Taken together, our findings establish p130Cas as a novel molecular rheostat that regulates the balance between canonical and noncanonical TGF-β signaling in developing mammary tumors, whose acquisition of metastatic phenotypes is potentiated by elevated p130Cas expression and its consequential disruption of homeostatic TGF-β signaling.DISCUSSIONTGF-β is a principal player involved in suppressing mammary tumorigenesis, doing so through its ability to maintain the composition of normal MEC microenvironments, and by inhibiting the aberrant proliferation of normal MECs (6.Blobe G.C. Schiemann W.P. Lodish H.F. N. Engl. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2166) Google Scholar, 38.Siegel P.M. Massagué J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1332) Google Scholar). Mammary tumorigenesis has evolved a variety of mechanisms that subvert the tumor suppressing functions of TGF-β, and in doing so, confer oncogenic and metastatic activities upon this multifunctional cytokine (34.Tang B. Vu M. Booker T. Santner S.J. Miller F.R. Anver M.R. Wakefield L.M. J. Clin. Invest. 2003; 112: 1116-1124Crossref PubMed Scopus (338) Google Scholar). Indeed, how TGF-β both suppresses and promotes mammary tumorigenesis remains a fundamental question that directly impacts the ability of science and medicine to effectively target the TGF-β signaling system during the treatment of breast cancer patients. Deciphering this paradox remains the most important question concerning the biological and pathological actions of this multifunctional cytokine (39.Schiemann W.P. Expert Rev. Anticancer Ther. 2007; 7: 609-611Crossref PubMed Scopus (25) Google Scholar).We previously established the importance of aberrant interactions between β3 integrin and TβR-II to promote Src-mediated phosphorylation of TβR-II, which then recruits and binds Grb2. Once bound to phospho-Tyr-284 in TβR-II, Grb2 facilitates TGF-β-mediated activation of noncanonical MAP kinase signaling without affecting the coupling of TGF-β to Smad2/3 (12.Galliher A.J. Schiemann W.P. Cancer Res. 2007; 67: 3752-3758Crossref PubMed Scopus (207) Google Scholar, 13.Galliher A.J. Schiemann W.P. Breast Cancer Res. 2006; 8: R42Crossref PubMed Scopus (214) Google Scholar). Importantly, measures capable of disrupting this signaling axis readily prevent TGF-β from driving breast cancer invasion and metastasis (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 40.Wendt M.K. Schiemann W.P. Breast Cancer Res. 2009; 11: R68Crossref PubMed Scopus (130) Google Scholar). Thus, in addition to establishing the critical importance of p38 MAPK activation in mediating breast cancer metastasis stimulated by TGF-β, these studies also suggested that inappropriate imbalances between canonical and noncanonical TGF-β signaling systems may in fact underlie its prometastatic activities in breast cancer cells. Our findings herein provide the first definitive evidence that (i) canonical and noncanonical signaling imbalances do indeed dictate MEC response to TGF-β, and (ii) p130Cas functions as a novel molecular rheostat that governs the delicate balance between canonical and noncanonical TGF-β effectors. Indeed, overexpression of either full-length or the carboxyl terminus of p130Cas was sufficient to decrease TGF-β-induced Smad2/3 phosphorylation while simultaneously increasing that of p38 MAPK. Moreover, depleting p130Cas significantly increased the activity of Smad2/3 and concomitantly decreased that of p38 MAPK induced by TGF-β, and finally, elevating TβR-II expression amplified the activation of p38 MAPK by TGF-β, which significantly enhanced early metastatic progression of mammary tumors in mice (Fig. 7) (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar). In “hypermetastatic” TβR-II-expressing cells, p130Cas deficiency similarly increased the coupling of TGF-β to Smad2/3, an event that negated the proinvasive and prometastatic activities of p38 MAPK in developing 4T1 tumors. Thus, p130Cas functions in balancing the activation status of canonical and noncanonical effectors targeted by TGF-β, findings that are clinically and medically relevant to the development and progression of mammary tumors regulated by TGF-β.A schematic depicting the function of p130Cas in TGF-β signaling is presented in Fig. 8. Indeed, in normal MECs, TGF-β receptors fail to interact significantly with integrins, which limits TGF-β stimulation of p38 MAPK and the initiation of oncogenic signaling by TGF-β (13.Galliher A.J. Schiemann W.P. Breast Cancer Res. 2006; 8: R42Crossref PubMed Scopus (214) Google Scholar, 40.Wendt M.K. Schiemann W.P. Breast Cancer Res. 2009; 11: R68Crossref PubMed Scopus (130) Google Scholar, 41.Yamashita M. Fatyol K. Jin C. Wang X. Liu Z. Zhang Y.E. Mol. Cell. 2008; 31: 918-924Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 42.Sorrentino A. Thakur N. Grimsby S. Marcusson A. von Bulow V. Schuster N. Zhang S. Heldin C.H. Landström M. Nat. Cell Biol. 2008; 10: 1199-1207Crossref PubMed Scopus (432) Google Scholar). The net effect of these signaling events results in tumor suppression by TGF-β. However, during mammary tumorigenesis, p130Cas expression is up-regulated dramatically, as is the aberrant formation of integrin and TGF-β receptor complexes (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 12.Galliher A.J. Schiemann W.P. Cancer Res. 2007; 67: 3752-3758Crossref PubMed Scopus (207) Google Scholar, 13.Galliher A.J. Schiemann W.P. Breast Cancer Res. 2006; 8: R42Crossref PubMed Scopus (214) Google Scholar, 40.Wendt M.K. Schiemann W.P. Breast Cancer Res. 2009; 11: R68Crossref PubMed Scopus (130) Google Scholar), which collectively decrease the activity of Smad2/3 and increase that of p38 MAPK and other noncanonical effectors that promote breast cancer metastasis stimulated by TGF-β (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 40.Wendt M.K. Schiemann W.P. Breast Cancer Res. 2009; 11: R68Crossref PubMed Scopus (130) Google Scholar). This signaling imbalance can be potentiated by elevated TβR-II expression and its consequential enhancement of p38 MAPK activation and metastasis (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 34.Tang B. Vu M. Booker T. Santner S.J. Miller F.R. Anver M.R. Wakefield L.M. J. Clin. Invest. 2003; 112: 1116-1124Crossref PubMed Scopus (338) Google Scholar, 40.Wendt M.K. Schiemann W.P. Breast Cancer Res. 2009; 11: R68Crossref PubMed Scopus (130) Google Scholar, 43.Muraoka R.S. Dumont N. Ritter C.A. Dugger T.C. Brantley D.M. Chen J. Easterly E. Roebuck L.R. Ryan S. Gotwals P.J. Koteliansky V. Arteaga C.L. J. Clin. Invest. 2002; 109: 1551-1559Crossref PubMed Scopus (449) Google Scholar). In all cases, these various signaling inputs are critically balanced and influenced by the level of p130Cas expression. Indeed, we (see Fig. 1) and others (20.Cabodi S. Tinnirello A. Di Stefano P. Bisarò B. Ambrosino E. Castellano I. Sapino A. Arisio R. Cavallo F. Forni G. Glukhova M. Silengo L. Altruda F. Turco E. Tarone G. Defilippi P. Cancer Res. 2006; 66: 4672-4680Crossref PubMed Scopus (111) Google Scholar) find mammary tumorigenesis to dramatically increase the expression of p130Cas. Based on our findings presented herein, we suggest that this event limits TGF-β stimulation of Smad2/3, which (i) diminishes MEC responsiveness to the cytostatic activities of TGF-β (44.Liu X. Sun Y. Constantinescu S.N. Karam E. Weinberg R.A. Lodish H.F. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 10669-10674Crossref PubMed Scopus (331) Google Scholar); and (ii) promotes amplified coupling of TGF-β to its noncanonical effectors, leading to breast cancer invasion and metastasis. In fact, our findings strongly support the progressive hypothesis that inappropriate imbalances between canonical and noncanonical TGF-β signaling systems underlies the acquisition of metastatic phenotypes in mammary carcinomas, as well as facilitates the oncogenic switch of TGF-β from a tumor suppressor to a prometastatic molecule.Along these lines, a recent report suggests that murine mammary tumor virus-driven p130Cas expression in mice is sufficient to induce mammary gland hyperplasia (20.Cabodi S. Tinnirello A. Di Stefano P. Bisarò B. Ambrosino E. Castellano I. Sapino A. Arisio R. Cavallo F. Forni G. Glukhova M. Silengo L. Altruda F. Turco E. Tarone G. Defilippi P. Cancer Res. 2006; 66: 4672-4680Crossref PubMed Scopus (111) Google Scholar). However, it was necessary to combine transgenic p130Cas expression with that of HER2 to enhance formation of mammary tumors (20.Cabodi S. Tinnirello A. Di Stefano P. Bisarò B. Ambrosino E. Castellano I. Sapino A. Arisio R. Cavallo F. Forni G. Glukhova M. Silengo L. Altruda F. Turco E. Tarone G. Defilippi P. Cancer Res. 2006; 66: 4672-4680Crossref PubMed Scopus (111) Google Scholar). Although specific effects on TGF-β activity and signaling were not examined in this mouse model, these findings do suggest that the tumor promoting properties of p130Cas only manifest in the face of additional oncogenic signaling inputs (i.e. elevated HER2 expression), which mirrors our own results showing that heightened TGF-β signaling (TβR-II expression) requires p130Cas to induce pulmonary dissemination. Moreover, we show that transgenic TβR-II expression led to increased basal and TGF-β-induced production of the prometastatic protein, PAI1, without impacting the phosphorylation of Smad2/3. These findings suggest that (i) p130Cas specifically regulates the activity of Smad2/3 as opposed to that of the TGF-β receptors, and (ii) Smad2/3 expression levels, not those of TGF-β receptors, are rate-limiting during the activation of canonical TGF-β signaling. Thus, p130Cas acts as a molecular rheostat of canonical Smad2/3 and noncanonical p38 MAPK signaling stimulated by TGF-β, and disruption of the balance between these two pathways has dramatic affects on breast cancer growth and progression.In summary, we demonstrated that p130Cas functions to regulate the balance between TGF-β-mediated activation of Smad2/3 and p38 MAPK in normal and metastatic MECs. Moreover, we provide compelling evidence that p130Cas is both necessary and sufficient to drive the oncogenic activities of TGF-β, including its regulation of mammary tumor growth and the initiation of early steps in the metastatic dissemination of breast cancer cells. Collectively, our findings establish p130Cas as an essential mediator that underlies the oncogenic conversion of TGF-β function, thereby enhancing its ability to promote the progression of mammary carcinomas. IntroductionInvasion and metastasis are the most lethal characteristics of breast cancer (1.Yoshida B.A. Sokoloff M.M. Welch D.R. Rinker-Schaeffer C.W. J. Natl. Cancer Inst. 2000; 92: 1717-1730Crossref PubMed Scopus (233) Google Scholar, 2.Wakefield L.M. Piek E. Böttinger E.P. J. Mammary Gland Biol. Neoplasia. 2001; 6: 67-82Crossref PubMed Scopus (91) Google Scholar). Transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF-βtransforming growth factor-βFAKfocal adhesion kinaseMECmammary epithelial cellTβR-ITGF-β type I receptorTβR-IITGF-β type II receptorp130CasCrk-associated substratePAI-1plasminogen activator inhibitor-1NmuMGnormal murine mammary epithelialMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinaseCMVcytomegalovirusshRNAshort hairpin RNAβ-galβ-galactosidaseFLfull-lengthCTcarboxyl-terminal. is a powerful suppressor of mammary tumorigenesis, doing so through its ability to repress mammary epithelial cell (MEC) proliferation, as well as through its creation of cell microenvironments that inhibit MEC motility, invasion, and metastasis (2.Wakefield L.M. Piek E. Böttinger E.P. J. Mammary Gland Biol. Neoplasia. 2001; 6: 67-82Crossref PubMed Scopus (91) Google Scholar). During breast cancer progression, the tumor suppressing function of TGF-β is frequently subverted, thus transforming TGF-β from a suppressor of breast cancer formation to a promoter of its growth and metastasis (2.Wakefield L.M. Piek E. Böttinger E.P. J. Mammary Gland Biol. Neoplasia. 2001; 6: 67-82Crossref PubMed Scopus (91) Google Scholar, 3.Buck M.B. Knabbe C. Ann. N. Y. Acad. Sci. 2006; 1089: 119-126Crossref PubMed Scopus (127) Google Scholar, 4.Benson J.R. Lancet Oncol. 2004; 5: 229-239Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Unfortunately, how mammary tumorigenesis overcomes the cytostatic function of TGF-β remains incompletely understood, as does the manner in which developing breast cancers ultimately sense TGF-β as a pro-metastatic factor.Transmembrane signaling by TGF-β commences upon binding to its type II receptor (TβR-II), which recruits and activates its type I receptor (TβR-I), which then phosphorylates and activates Smads 2 and 3. Following their activation, Smads 2 and 3 form heteromeric complexes with Smad4, which collectively translocate to the nucleus to regulate a multitude of transcriptional events and cellular responses (i.e. apoptosis, cytostasis, and homeostasis, (5.Chen R.H. Chang T.Y. Cell Growth Differ. 1997; 8: 821-827PubMed Google Scholar, 6.Blobe G.C. Schiemann W.P. Lodish H.F. N. Engl. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2166) Google Scholar)). In addition to stimulating Smad2/3, TGF-β also activates several noncanonical signaling systems, including members of the MAP kinase family (e.g. ERK1/2, JNK, and p38 MAPK (7.Galliher A.J. Neil J.R. Schiemann W.P. Future Oncol. 2006; 2: 743-763Crossref PubMed Scopus (72) Google Scholar)). Interestingly, several studies suggest that genetic and epigenetic events cooperate with aberrant Smad2/3 activities and functions to facilitate the conversion of TGF-β from tumor suppressor to a tumor promoter (8.Gomis R.R. Alarcón C. Nadal C. Van Poznak C. Massagué J. Cancer Cell. 2006; 10: 203-214Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 9.Adorno M. Cordenonsi M. Montagner M. Dupont S. Wong C. Hann B. Solari A. Bobisse S. Rondina M.B. Guzzardo V. Parenti A.R. Rosato A. Bicciato S. Balmain A. Piccolo S. Cell. 2009; 137: 87-98Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). However, these and other studies also present strong evidence implicating dysregulated activation of several noncanonical TGF-β effectors during this same switch in TGF-β function (10.Tian M. Schiemann W.P. Future Oncol. 2009; 5: 259-271Crossref PubMed Scopus (160) Google Scholar). Thus, deciphering the relative contribution of signaling imbalances that arise between Smad2/3-dependent and -independent TGF-β signaling systems is essential to enhancing our understanding of how TGF-β ultimately promotes the development and progression of mammary tumorigenesis.Recently, we identified a critical αvβ3 integrin:pY284-TβR-II:Grb2 signaling axis that mediates TGF-β stimulation of MAP kinases in normal and malignant MECs, leading to their acquisition of epithelial-mesenchymal transition, invasive, and metastatic phenotypes both in vitro and in vivo (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 12.Galliher A.J. Schiemann W.P. Cancer Res. 2007; 67: 3752-3758Crossref PubMed Scopus (207) Google Scholar, 13.Galliher A.J. Schiemann W.P. Breast Cancer Res. 2006; 8: R42Crossref PubMed Scopus (214) Google Scholar). Moreover, activation of this oncogenic signaling axis by TGF-β requires β3 integrin to form complexes with TβR-II (11.Galliher-Beckley A.J. Schiemann W.P. Carcinogenesis. 2008; 29: 244-251Crossref PubMed Scopus (72) Google Scholar, 12.Galliher A.J. Schiemann W.P. Cancer Res. 2007; 67: 3752-3758Crossref PubMed Scopus (207) Google Scholar, 13.Galliher A.J. Schiemann W.P. Breast Cancer Res. 2006; 8: R42Crossref PubMed Scopus (214) Google Scholar). Unfortunately, it remains uncertain as to whether this interaction is direct or facilitated through another scaffolding protein. As such, we sought to identify members of focal adhesion complexes as potential integrin effectors capable of contributing to altered TGF-β signaling.p130Cas (Crk-associated substrate) functions as a molecular scaffold within focal adhesion complexes, and is readily phosphorylated by focal adhesion kinase (FAK) and Src (14.Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1026) Google Scholar). Additionally, p130Cas binds stably to a variety of signaling molecules, including the (i) protein-tyrosine kinases FAK, PYK2, Src, Fyn, and Abl; (ii) adaptor molecules Crk, CrkL, Trip6, and AJUBA; (iii) guanine nucleotide exchange factors AND34 and CG3; and (iv) the MAPK family member, JNK (15.Brábek J. Constancio S.S. Shin N.Y. Pozzi A. Weaver A.M. Hanks S.K. Oncogene. 2004; 23: 7406-7415Crossref PubMed Scopus (69) Google Scholar, 16.Geiger B. Cell. 2006; 127: 879-881Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The extensive interactome of p130Cas ideally positions and enables this molecule to interpret and integrate a variety of signaling inputs arising from numerous receptor systems. Indeed, the biological importance of p130Cas is emphasized by studies showing that its genetic ablation in mice elicits embryonic lethality, whereas fibroblasts derived from p130Cas-deficient embryos exhibit drastically altered cytoskeletal architectures (17.Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar). Moreover, fibroblasts transformed by Src become significantly more invasive when engineered to simultaneously overexpress p130Cas (15.Brábek J. Constancio S.S. Shin N.Y. Pozzi A. Weaver A.M. Hanks S.K. Oncogene. 2004; 23: 7406-7415Crossref PubMed Scopus (69) Google Scholar). Patients with primary breast tumors expressing high levels of p130Cas (also known as breast cancer resistance-1) experience a more rapid disease recurrence and have a greater risk of resistance to tamoxifen therapy (18.van der Flier S. Chan C.M. Brinkman A. Smid M. Johnston S.R. Dorssers L.C. Dowsett M. Int. J. Cancer. 2000; 89: 465-468Crossref PubMed Scopus (42) Google Scholar). Recent studies also indicate that specific overexpression of p130Cas/breast cancer resistance-1 expression can confer breast cancer resistance to adriamycin (19.Ta H.Q. Thomas K.S. Schrecengost R.S. Bouton A.H. Cancer Res. 2008; 68: 8796-8804Crossref PubMed Scopus (48) Google Scholar). Moreover, directed overexpression of p130Case in murine MECs significantly increased their proliferative and survival indices in vivo, as well as greatly reduced the latency of mammary tumors arising from murine mammary tumor virus-driven Her2/Neu expression in mice (20.Cabodi S. Tinnirello A. Di Stefano P. Bisarò B. Ambrosino E. Castellano I. Sapino A. Arisio R. Cavallo F. Forni G. Glukhova M. Silengo L. Altruda F. Turco E. Tarone G. Defilippi P. Cancer Res. 2006; 66: 4672-4680Crossref PubMed Scopus (111) Google Scholar). This study also observed the expression of p130Cas to be up-regulated significantly in a subset of human breast cancer samples (20.Cabodi S. Tinnirello A. Di Stefano P. Bisarò B. Ambrosino E. Castellano I. Sapino A. Arisio R. Cavallo F. Forni G. Glukhova M. Silengo L. Altruda F. Turco E. Tarone G. Defilippi P. Cancer Res. 2006; 66: 4672-4680Crossref PubMed Scopus (111) Google Scholar). Collectively, these findings highlight the critical roles played by p130Cas in regulating normal tissue morphogenesis, and in promoting breast cancer progression. With respect to TGF-β, a recent study identified p130Cas as a potential inhibitor of Smad3 function (21.Kim W. Seok Kang Y. Soo Kim J. Shin N.Y. Hanks S.K. Song W.K. Mol. Biol. Cell. 2008; 19: 2135-2146Crossref PubMed Scopus (42) Google Scholar). However, the pathophysiological importance of this event, if any, in mediating the oncogenic activities of TGF-β and/or p130Cas during breast cancer progression remains to be established.The objective of the present study was to determine the role of p130Cas in facilitating the acquisition of oncogenic signaling by TGF-β during breast progression. We show here that p130Cas expression is up-regulated significantly in metastatic breast cancer cells (murine 4T1/human MCF10A-Ca1a) as compared with their nonmetastatic counterparts (murine 67NR/human MCF10A). Moreover, increased p130Cas expression was consistent with a decrease in TGF-β1-induced Smad2/3 signaling. Indeed, overexpression of p130Cas in nonmetastatic MECs led to a decrease in Smad2/3 activity, whereas depletion of p130Cas in metastatic MECs increased Smad2/3 activity. Most importantly, we show for the first time that p130Cas is essential for TGF-β stimulation of breast cancer growth, invasion, and pulmonary dissemination in mice. Taken together, our findings establish p130Cas as a novel molecular rheostat that regulates the balance between canonical and noncanonical TGF-β signaling in developing mammary tumors, whose acquisition of metastatic phenotypes is potentiated by elevated p130Cas expression and its consequential disruption of homeostatic TGF-β signaling.
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