HINT1 inhibits proteasome-mediated degradation of p21CIP1 and p27KIP1

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 413 Previous studies in our laboratory indicated that HINT1, a member of the evolutionary conserved family of histidine triad proteins, functions as a haplo-insufficient tumor suppressor in mice. Thus, with aging Hint1 deleted mice display an increase in various spontaneous tumors and these mice have a marked increase in susceptibility to induction of gastric tumors by the carcinogen NMBA. Furthermore, both Hint1 +/- and Hint1 -/- mice display a marked increase in the induction of both mammary and ovarian tumors by the carcinogen DMBA (Su,T. et al., Proc. Natl. Acad. Sci. USA 100,7824-7829,2003; Li, H. et al., Oncogene 25,713-721,2006). Several tumor suppressors exect their effects by perturbing cell cycle regulation. Therefore, in the present study we examined possible effects of HINT1 on expression of the cyclin-dependent kinase inhibitors p21CIP1 and p27KIP1. These two proteins were of particular interest since their levels are often reduced in a variety of human cancers. Indeed, we found that overexpression of HINT1 in SW480 human colon cancer cells caused a marked increase (about 3-fold) in cellular levels of both p21CIP1 and p27KIP1 proteins, whereas transfection with a Hint1 RNAi construct decreased the levels of both of these proteins. However, RT-PCR assays indicated that neither HINT1 overexpression nor Hint1 RNAi affected the mRNA levels of p21CIP1 and p27KIP1 in SW480 cells. Furthermore, in transient transfection assays co-transfection with Hint1 did not stimulate p21CIP1 promoter-luciferase or p27KIP1 promoter-luciferase reporter activity. Taken together with the RT-PCR assays, these results indicate that the increased cellular levels of the p21CIP1 and p27KIP1 proteins induced by HINT1 occur at a posttranscriptional level. Assays done in the presence of cycloheximide indicated that overexpression of HINT1 in SW480 cells extended the half lives of both the p21CIP1 and p27KIP1 proteins. Treatment of SW480 cells with the proteasome inhibitor MG132 increased cellular levels of p21CIP1 and p27KIP1 and this effect was augmented by overexpression of HINT1, while Hint1 RNAi impaired MG132 induced upregulation of p21CIP1 and p27KIP1. Co-immunoprecipitation assays indicated that HINT1 does not bind directly to p21CIP1 or p27KIP1. Taken together, our results suggest that HINT1 may play a role in cell cycle regulation by inhibiting proteasome-mediated degradation of p21CIP1 and p27KIP1. Studies are in progress to determine the precise biochemical mechanism. These effects of HINT1 may contribute to its tumor suppressor activity.