Suppression of Tumorigenesis and Induction of p15 by Smad4/DPC4 in Human Pancreatic Cancer Cells

Purpose: The tumor suppressor gene Smad4/DPC4, a key transcription factor in transforming growth factor (TGF) signaling cascades, is inactivated in 50% of pancreatic adenocarcinomas. We seek to determine the role of Smad4/DPC4 in the suppression of tumor cell growth and in the regulation of TGF-mediated expression of cell-cycle regulatory genes p15 and p21. Experimental Design: Smad4/DPC4 is overexpressed by adenoviral infection in CFPac-1 pancreatic cancer cells, in which the Smad4/DPC4 is homozygously deleted, and in Capan-1 pancreatic cancer cells, in which Smad4/DPC4 is not expressed. Expression of the TGFdownstream target gene p21, regulation of the p15 promoter, anchorage-independent growth, and tumorigenesis were examined. Results: We demonstrate that expression of Smad4/DPC4 in Capan-1 cells reduced anchorage-independent growth by more than 50%, and inhibited xenograft tumor growth. However, overexpression of Smad4/DPC4 did not inhibit CFPac-1 cell growth. Interestingly, Smad4/DPC4 induced expression of p15, p21, and TGF-responsive reporter gene in Capan-1 but not in CFPac-1 cells. Furthermore, we found a previously unidentified Smad4 binding element (SBE) located in the region between 356 and 329 bp of the p15 promoter. The p15 promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15 is one of the downstream target genes regulated by Smad/DPC4. Conclusion: These results explain the role of Smad4/ DPC4 in TGF-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/ DPC4-mediated tumor suppression and induction of TGF-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.