Smad4/DPC4 Silencing and Hyperactive Ras Jointly Disrupt Transforming Growth Factor-β Antiproliferative Responses in Colon Cancer Cells

Abstract

Smad4/DPC4 is a tumor suppressor gene frequently mutated or deleted in pancreatic and metastatic colon cancers. Smad4 acts as a cofactor that binds transforming growth factor-β (TGF-β) receptor-activated Smad2 and Smad3 generating transcriptional complexes. Using SW480.7 colon carcinoma cells, defective in Smad4 function, we have investigated whether this loss plays a role in the resistance of colon cancer cells to the antiproliferative effects of TGF-β. SW480.7 cells contain only one Smad4 allele, which we found encodes a wild type protein that is not expressed. We generated SW480.7 cells conditionally expressing Smad4 via an ecdysone-inducible system. Smad4 expression in these cells failed to rescue TGF-β antiproliferative and gene responses (c-myc down-regulation and induction ofp21/Cip1 and plasminogen activator inhibitor-1). SW480.7 cells contain an activated Ki-ras oncogene. Hyperactivation of Ras can inhibit Smad nuclear accumulation by their phosphorylation at mitogen-activated protein kinase sites. Co-transfection into SW480.7 cells of Smad4 together with a Ras phosphorylation-resistant Smad3 (but not with wild type Smad2, Smad3, adenomatous polyposis coli (APC), or TGF-β type II receptor) restored the TGF-β antiproliferative response. These results suggest that loss of Smad4 function by both deletion and silencing and inhibition of Smad2/3 function by a hyperactive Ras pathway jointly prevent TGF-β antiproliferative responses in SW480.7 colon cancer cells. Smad4/DPC4 is a tumor suppressor gene frequently mutated or deleted in pancreatic and metastatic colon cancers. Smad4 acts as a cofactor that binds transforming growth factor-β (TGF-β) receptor-activated Smad2 and Smad3 generating transcriptional complexes. Using SW480.7 colon carcinoma cells, defective in Smad4 function, we have investigated whether this loss plays a role in the resistance of colon cancer cells to the antiproliferative effects of TGF-β. SW480.7 cells contain only one Smad4 allele, which we found encodes a wild type protein that is not expressed. We generated SW480.7 cells conditionally expressing Smad4 via an ecdysone-inducible system. Smad4 expression in these cells failed to rescue TGF-β antiproliferative and gene responses (c-myc down-regulation and induction ofp21/Cip1 and plasminogen activator inhibitor-1). SW480.7 cells contain an activated Ki-ras oncogene. Hyperactivation of Ras can inhibit Smad nuclear accumulation by their phosphorylation at mitogen-activated protein kinase sites. Co-transfection into SW480.7 cells of Smad4 together with a Ras phosphorylation-resistant Smad3 (but not with wild type Smad2, Smad3, adenomatous polyposis coli (APC), or TGF-β type II receptor) restored the TGF-β antiproliferative response. These results suggest that loss of Smad4 function by both deletion and silencing and inhibition of Smad2/3 function by a hyperactive Ras pathway jointly prevent TGF-β antiproliferative responses in SW480.7 colon cancer cells. adenomatous polyposis coli deleted in pancreatic cancer Forkhead-activated signal transducer-2 ponasterone A polymerase chain reaction transforming growth factor-β The molecular events leading to colon cancer involve the sequential mutation of specific genes that transform a normal colonic epithelium to an adenomatous polyp and, ultimately, to an invasive cancer. Inactivating mutations inAPC 1 (adenomatous polyposis coli) and activating mutations in Ki-ras occur generally early in colon cancer, whereas p53 inactivation occurs generally late, as the adenoma progresses into a carcinoma (1Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4252) Google Scholar). Another relatively late event is loss of responsiveness to TGF-β (2Filmus J. Kerbel R.S. Curr. Opin. Oncol. 1993; 5: 123-129PubMed Google Scholar, 3Fynan T.M. Reiss M. Crit. Rev. Oncog. 1993; 4: 493-540PubMed Google Scholar). Under normal conditions, TGF-β is a potent antimitogen in epithelial, including intestinal, cells (4Massagué J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (2995) Google Scholar, 5Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg. Germany1990: 419-472Google Scholar). Loss of TGF-β antiproliferative responses in colon carcinoma cells may compromise the turnover of the colonic epithelium, thus favoring tumor formation. This loss of responsiveness can result from inactivating mutations in TGF-β receptor genes. The most prominent example of this is the loss of TGF-β type II receptor function in the vast majority of colon cancers with microsatellite instability, which are a small subset (around 10%) of all colon cancers (6Markowitz S. Wang J. Myeroff L. Parsons R. Sun L. Lutterbaugh J. Fan R.S. Zborowska E. Kinzler K.W. Vogelstein B. Brattain M. Wilson J.K.V. Science. 1995; 268: 1336-1338Crossref PubMed Scopus (2134) Google Scholar, 7Grady W.M. Myeroff L.L. Swinler S.E. Rajput A. Thiagalingam S. Lutterbaugh J.D. Neumann A. Brattain M.G. Chang J. Kim S.J. Kinzler K.W. Vogelstein B. Willson J.K. Markowitz S. Cancer Res. 1999; 59: 320-324PubMed Google Scholar, 8MacKay S.L. Auffenberg T. Tannahill C.L. Ksontini R. Josephs M.D. Nowak M. Moldawer L.L. Copeland 3rd, E.M. Ann. Surg. 1998; 227: 781-789Crossref PubMed Scopus (28) Google Scholar). In most other cases, however, loss of TGF-β responsiveness is due to post-receptor defects, the molecular nature of which is only now beginning to emerge.Inactivating mutations in Smad4/DPC4 are the most important of the known TGF-β post-receptor defects in colon cancer. Following the initial identification of Smad4 mutations in half of all pancreatic carcinomas (9Hahn S.A. Schutte M. Hoque A.T. Moskaluk C.A. da Costa L.T. Rozenblum E. Weinstein C.L. Fischer A. Yeo C.J. Hruban R.H. Kern S.E. Science. 1996; 271: 350-353Crossref PubMed Scopus (2157) Google Scholar), Smad4 mutations were reported in colon cancer (10Takagi Y. Kohmura H. Futamura M. Kida H. Tanemura H. Shimokawa K. Saji S. Gastroenterology. 1996; 111: 1369-1372Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 11Thiagalingam S. Lengauer C. Leach F.S. Schutte M. Hahn S.A. Overhauser J. Willson J.K. Markowitz S. Hamilton S.R. Kern S.E. Kinzler K.W. Vogelstein B. Nat. Genet. 1996; 13: 343-346Crossref PubMed Scopus (554) Google Scholar, 12Howe J.R. Roth S. Ringold J.C. Summers R.W. Jarvinen H.J. Sistonen P. Tomlinson I.P. Houlston R.S. Bevan S. Mitros F.A. Stone E.M. Aaltonen L.A. Science. 1998; 280: 1086-1088Crossref PubMed Scopus (755) Google Scholar) and other gastrointestinal cancers (13Powell S.M. Harper J.C. Hamilton S.R. Robinson C.R. Cummings O.W. Cancer Res. 1997; 57: 4221-4224PubMed Google Scholar). In colon cancer, the incidence of Smad4 mutations appears to increase with tumor progression. Although rare as an initiating event (12Howe J.R. Roth S. Ringold J.C. Summers R.W. Jarvinen H.J. Sistonen P. Tomlinson I.P. Houlston R.S. Bevan S. Mitros F.A. Stone E.M. Aaltonen L.A. Science. 1998; 280: 1086-1088Crossref PubMed Scopus (755) Google Scholar) and relatively infrequent (approximately 10% of cases) in colon adenomas and nonmetastatic carcinomas (14Riggins G.J. Kinzler K.W. Vogelstein B. Thiagalingam S. Cancer Res. 1997; 57: 2578-2580PubMed Google Scholar), Smad4 mutations have been reported in more than 30% of invasive metastatic carcinomas and in colon cancer metastases (15Miyaki M. Iijima T. Konishi M. Sakai K. Ishii A. Yasuno M. Hishima T. Koike M. Shitara N. Iwama T. Utsunomiya J. Kuroki T. Mori T. Oncogene. 1999; 18: 3098-3103Crossref PubMed Scopus (351) Google Scholar).Although the importance of Smad4 as a tumor suppressor gene in colon cancer is becoming increasingly apparent, little is know about how the loss of Smad4 function contributes to tumor progression. Smad4 plays a central role in TGF-β signaling by serving as a common partner of other Smad proteins (16Lagna G. Hata A. Hemmati-Brivanlou A. Massagué J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (806) Google Scholar). TGF-β binding brings together two types of transmembrane serine kinases, the type I and type II receptors. In this complex, the type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates Smad2 or the highly related protein Smad3. Other TGF-β family members act in a similar fashion. Activin induces phosphorylation of Smad2 and Smad3, whereas bone morphogenetic proteins induce phosphorylation of the related Smad1, Smad5, and Smad8. Upon phosphorylation, these Smads associate with Smad4 and move into the nucleus where they assemble transcriptional complexes that activate specific sets of genes. Thus, Smad4 is a shared key component of these various signaling pathways.Smad accumulation in the nucleus is a highly regulated process. In addition to the positive effect of the receptor-mediated phosphorylations, which occur at C-terminal sites (17Kretzschmar M. Liu F. Hata A. Doody J. Massagué J. Genes Dev. 1997; 11: 984-995Crossref PubMed Scopus (476) Google Scholar, 18Macias-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar), extracellular signal-regulated kinase and mitogen-activated protein kinase-mediated phosphorylation at other sites, in response to activators of the Ras/mitogen-activated protein kinase pathway, inhibit Smad1, Smad2, and Smad3 accumulation in the nucleus (19Kretzschmar M. Doody J. Massagué J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (766) Google Scholar, 20Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (847) Google Scholar). In mammary epithelial cells transformed by a ras oncogene, this inhibitory effect is sufficiently pronounced to significantly limit the activity of the TGF-β/Smad pathway (20Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (847) Google Scholar).Because Smad4 is a central component of the TGF-β signaling pathway, it has been assumed that the absence of TGF-β antiproliferative responses in Smad4-defective colon carcinoma cells is due to the loss of Smad4 function. However, this hypothesis has not been directly tested, and the possibility that the loss of TGF-β signaling in Smad4-defective cancer cells may be caused by other oncogenic alterations remains open. We have investigated these questions using SW480.7 cells, a colon carcinoma cell line defective in Smad4 function (21Goyette M.C. Cho K. Fasching C.L. Levy D.B. Kinzler K.W. Paraskeva C. Vogelstein B. Stanbridge E.J. Mol. Cell. Biol. 1992; 12: 1387-1395Crossref PubMed Scopus (248) Google Scholar, 22Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (757) Google Scholar, 23Liu F. Pouponnot C. Massagué J. Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (397) Google Scholar). Here we show that restoration of Smad4 function into SW480.7 cells is necessary but not sufficient to rescue the TGF-β antiproliferative response. We provide evidence that, in addition to Smad4, the rescue of this response requires restoration of Smad3 function, which is suppressed by oncogenic Ras. The surprising presence of two distinct Smad inhibitory hits in the same cell raises the possibility that Smad4 has other roles in the maintenance of colonic epithelial homeostasis besides mediating TGF-β antiproliferative responses.DISCUSSIONInhibition of cell cycle progression, facilitation of differentiation, induction of apoptosis, and regulation of cell adhesion and extracellular matrix production are effects of TGF-β through which this factor is thought to contribute to the maintenance of epithelial tissue homeostasis (4Massagué J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (2995) Google Scholar, 5Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg. Germany1990: 419-472Google Scholar). Loss of these TGF-β responses is often observed in carcinoma cells, including colon cancer cells (2Filmus J. Kerbel R.S. Curr. Opin. Oncol. 1993; 5: 123-129PubMed Google Scholar, 3Fynan T.M. Reiss M. Crit. Rev. Oncog. 1993; 4: 493-540PubMed Google Scholar). Different lines of evidence point to Smad4 as a tumor suppressor gene in colon cancer; however, the reasons why its loss contributes to tumorigenesis are not yet understood. Specifically, it has not been clear whether loss of TGF-β antiproliferative responses in Smad4-defective colon cancer cells is due to the loss of Smad4 function or to other oncogenic events. There has not been, to our knowledge, reports that restoration of a wild-type Smad4allele in Smad4-defective tumor-derived cell lines rescues TGF-β antiproliferative responses. We have investigated this question using the SW480.7 colon carcinoma cell line, which is defective in Smad4 function (22Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (757) Google Scholar, 23Liu F. Pouponnot C. Massagué J. Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (397) Google Scholar). One of the oncogenic defects of this cell line is the loss of one copy of chromosome 18 (21Goyette M.C. Cho K. Fasching C.L. Levy D.B. Kinzler K.W. Paraskeva C. Vogelstein B. Stanbridge E.J. Mol. Cell. Biol. 1992; 12: 1387-1395Crossref PubMed Scopus (248) Google Scholar). According to the two-hit model of tumor suppressor gene inactivation (43Knudson Jr., A.G. Hethcote H.W. Brown B.W. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 5116-5220Crossref PubMed Scopus (299) Google Scholar), the loss of Smad4 function in these cells could have arisen from an inactivating mutation in the remaining Smad4 allele. Surprisingly, we found that SW480.7 contain a Smad4 allele that encodes for a wild type protein. However, expression of this allele is strongly suppressed, as its mRNA and protein products are present at very low, nearly undetectable, levels. The mechanism responsible for this defective expression is not known at present, but this finding raises the possibility that Smad4 silencing is another mechanism leading to the loss of Smad4 function in colon cancer, a possibility worthy of future investigation.The lack of detectable Smad4 function allowed us to use SW480.7 cells to study the consequences of restoring expression of this gene. It was recently reported that SW480.7 cells stably transfected with Smad4 are not growth-inhibited by TGF-β (44Schwarte-Waldhoff I. Klein S. Blass-Kampmann S. Hintelmann A. Eilert C. Dreschers S. Kalthoff H. Hahn S.A. Schmiegel W. Oncogene. 1999; 18: 3152-3158Crossref PubMed Scopus (79) Google Scholar). However, if Smad4 is a mediator of antiproliferative responses, its constitutive overexpression in transfected SW480.7 cells could lead to the selection of Smad4-resistant clones due to secondary mutations. To avoid this potential problem, we generated SW480.7 cell lines conditionally expressing a human wild-type Smad4 allele under the control of the ecdysone system. The levels of Smad4 expressed under inducing conditions by the three SW480.7 derivatives used in this study were similar to those of HaCaT keratinocytes, which is a well characterized, TGF-β-responsive cell line (27Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar, 28Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1882) Google Scholar, 38Mulder K.M. Humphrey L.E. Choi H.G. Childress-Fields K.E. Brattain M.G. J. Cell. Physiol. 1990; 145: 501-507Crossref PubMed Scopus (26) Google Scholar, 39Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar). Assays using the highly responsive TGF-β reporter construct 3TP-Lux demonstrated that Smad4 function had been restored in the Smad4-inducible SW480.7 clones. Surprisingly, however, expression of Smad4 in these cells supported only a weak A3-Luc response to TGF-β. The A3-Luc reporter is based on a TGF-β target gene enhancer element (35Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (624) Google Scholar) and is less sensitive to TGF-β than the 3TP-Lux reporter, which is a composite reporter construct empirically assembled to provide high responsiveness to this factor (34Cárcamo J. Zentella A. Massagué J. Mol. Cell. Biol. 1995; 15: 1573-1581Crossref PubMed Google Scholar). The limited signaling ability of Smad4 in these cells was further demonstrated by their failure to show endogenous antiproliferative gene responses, including down-regulation of c-myc or up-regulation of p21/Cip1, which are typical of TGF-β action in colonic and other epithelial cells (37Alexandrow M.G. Moses H.L. Cancer Res. 1995; 55: 1452-1457PubMed Google Scholar,39Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar). The absence of these responses in Smad4-expressing SW480.7 cells was accompanied by a lack of growth inhibition.The absence of robust antiproliferative effects and endogenous gene responses to TGF-β in SW480.7 cells induced to express normal levels of Smad4 raises the possibility that other oncogenic alterations present in these cells are limiting Smad4 signaling function. Our evidence indicates that neither the presence of APCmutations nor limitations in TGF-β type II receptor levels contribute to the lack of TGF-β responsiveness in SW480.7. p53, which is inactivated in SW480.7 cells, is not a candidate because it is also inactivated in HaCaT cells, which are highly responsive to TGF-β (27Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar,28Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1882) Google Scholar, 38Mulder K.M. Humphrey L.E. Choi H.G. Childress-Fields K.E. Brattain M.G. J. Cell. Physiol. 1990; 145: 501-507Crossref PubMed Scopus (26) Google Scholar, 39Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar). The TGF-β receptor-regulated Smads, Smad2, and Smad3 are expressed in SW480.7 cells, and their levels do not appear to be rate-limiting because cotransfection of exogenous Smad2 or Smad3 did not very much improve the antiproliferative response in Smad4-transfected SW480.7 cells.Our results identify the hyperactive Ras pathway present in SW480.7 cells as a rate-limiting event for the signaling function of Smad4. SW480.7 cells harbor a Ki-ras-activating mutation (21Goyette M.C. Cho K. Fasching C.L. Levy D.B. Kinzler K.W. Paraskeva C. Vogelstein B. Stanbridge E.J. Mol. Cell. Biol. 1992; 12: 1387-1395Crossref PubMed Scopus (248) Google Scholar), which is a frequent event in colon cancer (45Bos J.L. Fearon E.R. Hamilton S.R. Verlaan-de Vries M. van Boom J.H. van der Eb A.J. Vogelstein B. Nature. 1987; 327: 293-297Crossref PubMed Scopus (1595) Google Scholar, 46Forrester K. Almoguera C. Han K. Grizzle W.E. Perucho M. Nature. 1987; 327: 298-303Crossref PubMed Scopus (906) Google Scholar). Ras transformation of intestinal and other epithelial cells inhibits TGF-β antiproliferative effects (2Filmus J. Kerbel R.S. Curr. Opin. Oncol. 1993; 5: 123-129PubMed Google Scholar, 47Schwarz L.C. Gingras M.C. Goldberg G. Greenberg A.H. Wright J.A. Cancer Res. 1988; 48: 6999-7003PubMed Google Scholar, 48Valverius E.M. Walker-Jones D. Bates S.E. Stampfer M.R. Clark R. McCormick F. Dickson R.B. Lippman M.E. Cancer Res. 1989; 49: Google Scholar, Oncogene. 1989; 4: Google Scholar, M. B. Howe Oncogene. 1992; Google Scholar). Hyperactivation of the Ras pathway can provide of E. Oncogene. 1998; PubMed Scopus Google Scholar), and this could to the inhibitory effects of TGF-β on these However, we recently that Ras signaling can with Smad signal by Smad accumulation in the nucleus (20Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (847) Google Scholar). accumulation of Smads in response to TGF-β is less in colon cancer cell lines Ki-ras mutations than in colon cancer cell lines wild type Ki-ras (20Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (847) Google Scholar). we show SW480.7 cells also have Smad2/3 nuclear accumulation response. Following this we found that a of Smad3 inhibitory mitogen-activated protein kinase phosphorylation sites can strongly the ability of Smad4 to rescue the antiproliferative response in SW480.7 restoration of Smad4 expression and of Smad3 and also Smad2 inhibition by Ras are for restoration of TGF-β antiproliferative responses in SW480.7 cells. These results provide the evidence that loss of Smad4 function contributes to colon cancer cells of antiproliferative responsiveness to TGF-β. the same our evidence an important of a hyperactive Ras The and Ki-ras mutations is not to SW480.7 the high of Smad4 and Ki-ras mutations in metastatic colon cancer (15Miyaki M. Iijima T. Konishi M. Sakai K. Ishii A. Yasuno M. Hishima T. Koike M. Shitara N. Iwama T. Utsunomiya J. Kuroki T. Mori T. Oncogene. 1999; 18: 3098-3103Crossref PubMed Scopus (351) Google Scholar, J.L. Fearon E.R. Hamilton S.R. Verlaan-de Vries M. van Boom J.H. van der Eb A.J. Vogelstein B. Nature. 1987; 327: 293-297Crossref PubMed Scopus (1595) Google Scholar, 46Forrester K. Almoguera C. Han K. Grizzle W.E. Perucho M. Nature. 1987; 327: 298-303Crossref PubMed Scopus (906) Google Scholar) and pancreatic cancer E. Schutte M. M. Hahn S.A. S. M. Hruban R.H. Yeo C.J. Kern S.E. Cancer Res. 1997; 57: Google Scholar) the of both mutations in These the Smad4 loss and Ras can compromise TGF-β why do both alterations in the same The is at present but may on the in which these mutations occur in a Ki-ras mutations are generally early events in colon cancer (45Bos J.L. Fearon E.R. Hamilton S.R. Verlaan-de Vries M. van Boom J.H. van der Eb A.J. Vogelstein B. Nature. 1987; 327: 293-297Crossref PubMed Scopus (1595) Google Scholar), whereas Smad4 mutations are generally late events (15Miyaki M. Iijima T. Konishi M. Sakai K. Ishii A. Yasuno M. Hishima T. Koike M. Shitara N. Iwama T. Utsunomiya J. Kuroki T. Mori T. Oncogene. 1999; 18: 3098-3103Crossref PubMed Scopus (351) Google Scholar). Ras may TGF-β a loss of Smad4 function on may a more resistance to TGF-β Ras may be sufficient to TGF-β antiproliferative responses but not other responses by Smad4 that tumor and (44Schwarte-Waldhoff I. Klein S. Blass-Kampmann S. Hintelmann A. Eilert C. Dreschers S. Kalthoff H. Hahn S.A. Schmiegel W. Oncogene. 1999; 18: 3152-3158Crossref PubMed Scopus (79) Google Scholar). In this loss of Smad4 function be a specific subset of responses, to the of a more The molecular events leading to colon cancer involve the sequential mutation of specific genes that transform a normal colonic epithelium to an adenomatous polyp and, ultimately, to an invasive cancer. Inactivating mutations inAPC 1 (adenomatous polyposis coli) and activating mutations in Ki-ras occur generally early in colon cancer, whereas p53 inactivation occurs generally late, as the adenoma progresses into a carcinoma (1Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4252) Google Scholar). Another relatively late event is loss of responsiveness to TGF-β (2Filmus J. Kerbel R.S. Curr. Opin. Oncol. 1993; 5: 123-129PubMed Google Scholar, 3Fynan T.M. Reiss M. Crit. Rev. Oncog. 1993; 4: 493-540PubMed Google Scholar). Under normal conditions, TGF-β is a potent antimitogen in epithelial, including intestinal, cells (4Massagué J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (2995) Google Scholar, 5Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg. Germany1990: 419-472Google Scholar). Loss of TGF-β antiproliferative responses in colon carcinoma cells may compromise the turnover of the colonic epithelium, thus favoring tumor formation. This loss of responsiveness can result from inactivating mutations in TGF-β receptor genes. The most prominent example of this is the loss of TGF-β type II receptor function in the vast majority of colon cancers with microsatellite instability, which are a small subset (around 10%) of all colon cancers (6Markowitz S. Wang J. Myeroff L. Parsons R. Sun L. Lutterbaugh J. Fan R.S. Zborowska E. Kinzler K.W. Vogelstein B. Brattain M. Wilson J.K.V. Science. 1995; 268: 1336-1338Crossref PubMed Scopus (2134) Google Scholar, 7Grady W.M. Myeroff L.L. Swinler S.E. Rajput A. Thiagalingam S. Lutterbaugh J.D. Neumann A. Brattain M.G. Chang J. Kim S.J. Kinzler K.W. Vogelstein B. Willson J.K. Markowitz S. Cancer Res. 1999; 59: 320-324PubMed Google Scholar, 8MacKay S.L. Auffenberg T. Tannahill C.L. Ksontini R. Josephs M.D. Nowak M. Moldawer L.L. Copeland 3rd, E.M. Ann. Surg. 1998; 227: 781-789Crossref PubMed Scopus (28) Google Scholar). In most other cases, however, loss of TGF-β responsiveness is due to post-receptor defects, the molecular nature of which is only now beginning to Inactivating mutations in Smad4/DPC4 are the most important of the known TGF-β post-receptor defects in colon cancer. Following the initial identification of Smad4 mutations in half of all pancreatic carcinomas (9Hahn S.A. Schutte M. Hoque A.T. Moskaluk C.A. da Costa L.T. Rozenblum E. Weinstein C.L. Fischer A. Yeo C.J. Hruban R.H. Kern S.E. Science. 1996; 271: 350-353Crossref PubMed Scopus (2157) Google Scholar), Smad4 mutations were reported in colon cancer (10Takagi Y. Kohmura H. Futamura M. Kida H. Tanemura H. Shimokawa K. Saji S. Gastroenterology. 1996; 111: 1369-1372Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 11Thiagalingam S. Lengauer C. Leach F.S. Schutte M. Hahn S.A. Overhauser J. Willson J.K. Markowitz S. Hamilton S.R. Kern S.E. Kinzler K.W. Vogelstein B. Nat. Genet. 1996; 13: 343-346Crossref PubMed Scopus (554) Google Scholar, 12Howe J.R. Roth S. Ringold J.C. Summers R.W. Jarvinen H.J. Sistonen P. Tomlinson I.P. Houlston R.S. Bevan S. Mitros F.A. Stone E.M. Aaltonen L.A. Science. 1998; 280: 1086-1088Crossref PubMed Scopus (755) Google Scholar) and other gastrointestinal cancers (13Powell S.M. Harper J.C. Hamilton S.R. Robinson C.R. Cummings O.W. Cancer Res. 1997; 57: 4221-4224PubMed Google Scholar). In colon cancer, the incidence of Smad4 mutations appears to increase with tumor progression. Although rare as an initiating event (12Howe J.R. Roth S. Ringold J.C. Summers R.W. Jarvinen H.J. Sistonen P. Tomlinson I.P. Houlston R.S. Bevan S. Mitros F.A. Stone E.M. Aaltonen L.A. Science. 1998; 280: 1086-1088Crossref PubMed Scopus (755) Google Scholar) and relatively infrequent (approximately 10% of cases) in colon adenomas and nonmetastatic carcinomas (14Riggins G.J. Kinzler K.W. Vogelstein B. Thiagalingam S. Cancer Res. 1997; 57: 2578-2580PubMed Google Scholar), Smad4 mutations have been reported in more than 30% of invasive metastatic carcinomas and in colon cancer metastases (15Miyaki M. Iijima T. Konishi M. Sakai K. Ishii A. Yasuno M. Hishima T. Koike M. Shitara N. Iwama T. Utsunomiya J. Kuroki T. Mori T. Oncogene. 1999; 18: 3098-3103Crossref PubMed Scopus (351) Google Scholar). Although the importance of Smad4 as a tumor suppressor gene in colon cancer is becoming increasingly apparent, little is know about how the loss of Smad4 function contributes to tumor progression. Smad4 plays a central role in TGF-β signaling by serving as a common partner of other Smad proteins (16Lagna G. Hata A. Hemmati-Brivanlou A. Massagué J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (806) Google Scholar). TGF-β binding brings together two types of transmembrane serine kinases, the type I and type II receptors. In this complex, the type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates Smad2 or the highly related protein Smad3. Other TGF-β family members act in a similar fashion. Activin induces phosphorylation of Smad2 and Smad3, whereas bone morphogenetic proteins induce phosphorylation of the related Smad1, Smad5, and Smad8. Upon phosphorylation, these Smads associate with Smad4 and move into the nucleus where they assemble transcriptional complexes that activate specific sets of genes. Thus, Smad4 is a shared key component of these various signaling Smad accumulation in the nucleus is a highly regulated process. In addition to the positive effect of the receptor-mediated phosphorylations, which occur at C-terminal sites (17Kretzschmar M. Liu F. Hata A. Doody J. Massagué J. Genes Dev. 1997; 11: 984-995Crossref PubMed Scopus (476) Google Scholar,