[CANCER RESEARCH 62, 6045– 6051, November 1, 2002] Restoration of Transforming Growth Factor  Signaling by Functional Expression of Smad4 Induces Anoikis Murali Ramachandra, 1 Isabella Atencio, Amena Rahman, Mei Vaillancourt, Aihua Zou, Jenny Avanzini, Ken Wills, Robert Bookstein, and Paul Shabram Canji, Inc., San Diego, California 92121 ABSTRACT Smad proteins transduce signals carried by the transforming growth factor  (TGF-) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell prolifera- tion, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all pancreatic carcinomas. For functional characterization of Smad4, a recombinant adenovirus encoding Smad4 (Ad-Smad4) was generated. When Smad4 was expressed in Smad4-null breast carcinoma cell line MDA-MB-468 using the recombinant adenovirus, TGF- signaling was restored as determined by TGF--dependent activity of plasminogen activator inhibitor 1 promoter and p21 expression. Infection with Ad- Smad4 in the presence of TGF-1 also resulted in an altered cell mor- phology that coincided with enhanced 1 integrin expression and reduced efficiency of colony formation in soft agar. In agreement with increased p21 expression, Smad4-expressing cells showed modest reduction in S phase. However, Smad4 expression did not lead to induction of apoptosis under normal culture conditions. Interestingly, when Smad4-expressing cells were detached and incubated in suspension, they underwent rapid apoptosis in a TGF--dependent manner. Induction of apoptosis caused by loss of anchorage is known as anoikis. Anoikis is believed to prevent colonization elsewhere of detached cells. Additional characterization re- vealed an increase in the level of focal adhesion kinase 2 (or Pyk2) and activation of caspases 2, 3, 6, and 8 during anoikis because of Smad4 expression and restoration of TGF- signaling. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a functional basis for the strong correlation between defects in Smad4 and development of malignancy. INTRODUCTION The Smad4/DPC4 (deleted in pancreatic carcinoma, locus 4) gene is a candidate tumor suppressor gene (1, 2) that is frequently inacti- vated in pancreatic (1–3), biliary (4, 5), and colorectal tumors (6, 7). Smad4 is homozygously deleted in 30% of pancreatic carcinomas and inactivated by intragenic mutation in another 20% of the tumors. Smad4 belongs to the evolutionarily conserved family of Smad pro- teins (8 –11) that are essential intracellular effectors of signals from the TGF 2 - superfamily of cytokines that regulates a number of cellular processes, including ECM formation, cell proliferation, dif- ferentiation, and apoptosis (12–14). During TGF- and related protein signaling pathways, binding of TGF- to the type II receptor results in recruitment and phosphoryl- ation of the type I receptor (12–14). The activation of the type I receptor then propagates the signal by phosphorylating receptor- activated Smads. Among the receptor-activated Smads, Smad2 and Smad3 can be phosphorylated by binding of TGF- and activin, whereas bone morphogenetic proteins phosphorylate Smad1, Smad5, and Smad8. After phosphorylation, receptor-activated Smads dissoci- ate from the receptor complexes and form a heteromeric complex with a common partner Smad such as Smad4. The Smad complex is then translocated to the nucleus, where it either can directly bind to DNA (15) or associate with other transcription factors (16, 17) to induce TGF--responsive gene activation. Because Smad4 serves as a com- mon partner of other Smad proteins, it is pivotal in signaling pathways originating from TGF- and related proteins. TGF- exhibits potent tumor suppressor properties at early stages of carcinogenesis but promotes tumor progression at late stages (18). Inhibition of epithelial cell growth by TGF- is believed to contribute to a large extent to the tumor suppressor functions of TGF-. How- ever, tumors secrete large amounts of activated TGF-s late during tumor progression, which is thought to facilitate invasion and metas- tasis, induce angiogenesis, and suppress antitumor immune response through effects on cells of both tumor and stromal origin (18). Studies have also suggested that TGF--overexpressing tumors are a partic- ularly aggressive subset, wherein patients whose cancers express TGF- have a prognosis worse than those that do not express TGF- (18). To overcome the growth suppression effects of TGF- during tumorigenesis, tumor cells often acquire mutations in either type II receptors or in the signal transducers Smad4 and Smad2 (18). The extent of TGF- resistance correlates with metastatic progression, and targeted deletion of an essential component of the TGF--signaling cascade, Smad3, promotes metastasis (19). Results from several studies have suggested that defects in Smad4 play a significant role in the malignant progression of tumors. Pan- creatic cancer, which often exhibits defects in Smad4, is highly aggressive, with most of the patients having metastatic disease at the time of diagnosis. In the development of pancreatic adenocarcinoma, inactivation of Smad4 gene occurs late at the stage of in situ or even invasive carcinoma (20). Inactivation of Smad4 in the APC-knockout mouse, a model for human familial adenomatous polyposis, results in more rapid development of malignant tumors than in APC heterozy- gotes (21). On the basis of these results, mutations in Smad4 were suggested to play an important role in malignant progression of colorectal tumors (21). Expression of Smad4 in Smad4-defective tumor cell lines has been shown to restore TGF- signaling and induce cell cycle arrest or apoptosis (22). In addition to the antipro- liferative activity, Smad4 expression in tumor cell lines has been shown to inhibit the expression of vascular endothelial growth factor and enhance the levels of angiogenesis inhibitor thrombospondin-1, causing cells to switch from potently angiogenic to antiangiogenic in vitro and in vivo (23). To additionally characterize the functional consequences of restor- ing TGF- signaling in Smad4-defective cells, we constructed a recombinant Ad encoding human Smad4. Infection of Smad4-defec- tive breast carcinoma cell line MDA-MB-468 with this recombinant Ad vector restored TGF- signaling and growth inhibition, altered cell morphology, and sensitized them to anoikis. Additional characteriza- tion of cells expressing Smad4 revealed increased surface expression of 1 integrin and increased levels of FAK2, but not FAK1, and Received 5/13/02; accepted 8/30/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Canji, Inc., 3525 John Hopkins Court, San Diego, CA 92121-1121. E-mail: murali.ramachandra@canji.com. 2 The abbreviations used are: TGF, transforming growth factor; ECM, extracellular matrix; Ad, adenovirus; FAK, focal adhesion kinase; PAI-1, plasminogen activator inhibitor 1; -gal, -galactosidase; APC, adenomatous polyposis coli; SAPK, stress- activated protein kinase; JNK, c-Jun NH 2 -terminal kinase. 6045 Research. on December 18, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from