Suppression of tumorigenesis and induction of p15^ink4b by Smad4/DPC4 in human pancreatic cancer cells

Purpose: The tumor suppressor gene Smad4/DPC4, a key transcription factor in transforming growth factor β (TGF-β) 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-β-mediated expression of cell-cycle regulatory genes p15^ink4b and p21^waf1. 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 TGF-β downstream target gene p21^waf1, regulation of the p15^ink4b 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^ink4b, p21^waf1, and TGF-β-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^ink4b promoter. The p15^ink4b promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15^ink4b is one of the downstream target genes regulated by Smad/DPC4. Conclusion: These results explain the role of Smad4/DPC4 in TGF-β-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/DPC4-mediated tumor suppression and induction of TGF-β-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.