Targeted deletion of Smad 4 shows it is required for transforming growth factor b and activin signaling in colorectal cancer cells

Smad4 (DPC4) is a candidate tumor suppressor gene that has been hypothesized to be critical for transmitting signals from transforming growth factor (TGF) b and related ligands. To directly test this hypothesis, the Smad4 gene was deleted through homologous recombination in human colorectal cancer cells. This deletion abrogated signaling from TGF-b, as well as from the TGF-b family member activin. These results provide unequivocal evidence that mutational inactivation of Smad4 causes TGF-b unresponsiveness and provide a basis for understanding the physiologic role of this gene in tumorigenesis. The Smad4 (DPC4) gene was discovered by virtue of its mutational inactivation in a large fraction of pancreatic cancers (1), and has since been found to be mutated in a subset of colorectal cancers (2). Smad4 is homologous to the Drosophila Mad gene, known to be required for signaling by the transforming growth factor (TGF) b family member dpp (3). Based on this homology, it was suggested that the driving force for Smad4 inactivation is the abrogation of TGF-b signaling (1). This hypothesis was attractive because many cancers seem to be unresponsive to TGF-b, the prototype growth-inhibitory polypeptide (4). This simple model became complicated when numerous other homologues of Mad were identified in vertebrate cells (5–19). Some of these homologues appear to stimulate or mimic responses to TGF-b, whereas others inhibit them. The situation is similar in Caenorhabditis elegans, where one of the two Smad4 homologues antagonizes TGF-b-related receptor signaling (20). The Drosophila Mad protein binds to DNA in a sequence-specific manner (21), whereas other Smad proteins contain transcriptional activating domains that function when complexed with DNA-binding partners (22–26). Furthermore, and unlike the situation in Drosophila, there are dozens of TGF-b-like ligands in vertebrates with diverse functions (27). And even the response to TGF-b is variable: whereas many mammalian cells are growth-inhibited by TGF-b, others, including some epithelial cancer cells, are growth stimulated (28–31). Perhaps as a result of these complexities, some experimental observations have supported the idea that Smad4 mediates TGF-b signaling (8, 22, 26, 32–34), whereas others indicated that Smad4 is dispensable for such signaling (J.L.D. and S.E.K., unpublished results). However, experimental approaches to this question have so far been indirect, employing overexpression of exogenously introduced Smad4 constructs. As Smad4 polypeptides have been shown to interact with several other proteins, including other Smads (8, 22, 25, 26), the interpretation of such overexpression experiments is problematical. Moreover, in some studies, overexpression of Smad4 had little effect on TGF-b signaling unless other Smad genes were simultaneously expressed (8, 22, 26, 32). To unambiguously determine whether Smad4 is required for TGF-b signaling in human colorectal cancer cells, we have deleted the Smad4 genes through homologous recombination in cells originally containing two normal Smad4 alleles. MATERIALS AND METHODS Gene Targeting. A promoterless strategy was adapted for targeted disruption of the Smad4 gene (35). A cosmid clone (c417–46) containing human Smad4 was used as the source for homologous arms (36). A 2.2-kb SacIyBglII fragment immediately upstream of exon 3 and a 4.7-kb XbaIyHindIII fragment downstream of exon 4 were assembled in pBluescript surrounding promoterless NEO or HYG cassettes containing simian virus 40 polyadenylation signals (Fig. 1A). Homologous recombination should result in the expression of NEO and HYG genes from the endogenous Smad4 promoter as fusion proteins with the amino-terminal 141 amino acids of Smad4. For first allele targeting, exponentially growing HCT116 human colorectal cancer cells (American Type Culture Collection) were transfected with a NotI-linearized NEO targeting vector. G418 (0.4 mgyml)-resistant clones were screened by a reverse transcriptase–PCR approach, by using the primers ‘‘a’’ (59-CAGCTATAACTACAAATGGAGC) and ‘‘c’’ (59-TTGTTCAATGGCCGATCCCAT) (Fig. 1 A), yielding a 338-bp PCR product. Reverse transcriptase–PCR positive clones were expanded and genomic DNA was prepared from them and used for Southern blot analysis with a hybridization probe located immediately outside the 59 homologous arm (Fig. 1 A). A clone carrying a homologous recombinant and no additional random integrants was then transfected with the HYG targeting vector. The heterozygote clone 5–60 was a subclone derived from the same parent used to derive Smad42y2 clones 5–18 and 5–63, and contained a targeted NEO gene plus a randomly integrated (nontargeted) HYG gene. Hygromycin (0.1 mgyml)-resistant clones were screened by PCR of genomic DNA, by using primers ‘‘a’’ and ‘‘d’’ (59-GCAGGTCTTGCAACGTG) (Fig. 1 A). PCR with primers ‘‘a’’ and ‘‘b’’ (59-CTGCAGTGTTAATCCTGAGAG) (Fig. 1A) and genomic Southern blot analysis were performed to confirm the complete deletion of both Smad4 alleles and the purity of clones (Fig. 1 B and C). Expression Vector Construction. An expression construct for the TGF-b RII gene was constructed by placing a TGF-b RII cDNA (generously provided by H. Lodish and R. WeinThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y952412-5$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations: TGF, transforming growth factor; TbRII, TGF-b type II receptor; aGT, a1,3-galactosyltransferase. §To whom requests for reagents should be addressed. e-mail: kinzlke@welchlink.welch. jhu.edu. ¶To whom reprint requests should be addressed at: The Oncology Center, The Johns Hopkins University School of Medicine, 424 North Bond Street, Baltimore, MD 21231.