HDAC inhibitors induce cell-cycle arrest in the G1 and/or G2 phase, apoptosis and/or differentiation in cancer cells and are showing promise as treatment for a variety of human cancers. Their precise mechanism of action however, has not been fully elucidated. We identified a significant upregulation of PTEN mRNA in a microarray database of Caco-2 colon carcinoma cells treated with the HDAC inhibitor, sodium butyrate. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a dual phosphatase, acting at both serine-threonine and tyrosine sites. PTEN inhibits the activation of Akt/protein kinase B by phosphatidylinositol 3,4,5-triphosphate, thus modulating a major pathway controlling cell proliferation and survival. This study, therefore, aimed to examine the role of PTEN in HDAC inhibitor-induced colon cell maturation. Consistent with the microarray data, we found that butyrate upregulated PTEN mRNA and protein expression in a dose- and time-dependent manner in Caco-2, HT-29, and HCT116 colon cancer cells. PTEN induction was first observed 48-72 hours following butyrate treatment suggesting its upregulation is an indirect effect of butyrate treatment. Upregulated PTEN expression in response to butyrate was also observed in Hela cells, indicating this effect was not specific to colon cancer cells. The structurally related HDAC inhibitor, valproic acid, also induced PTEN expression in colon cancer cells. To determine whether upregulation of PTEN is specific to butyrate induced differentiation or a consistent feature of intestinal cell differentiation, we examined its expression in the Caco-2 in vitro model of colon cell differentiation and along the crypt-villus axis in vivo. PTEN expression was increased during spontaneous differentiation of Caco-2 cells, and along the mouse small intestinal crypt to villus axis in vivo, indicating PTEN is consistently upregulated during intestinal cell differentiation. To directly determine the role of PTEN in butyrate-induced growth inhibition, differentiation and apoptosis in colon cancer cells, PTEN was inhibited using siRNA in colon cancer cells. Western blot analysis confirmed a >80% inhibition of PTEN protein expression following 72-120h exposure to small interfering RNA. Silencing of PTEN partially attenuated butyrate-induced growth inhibition and apoptosis in HCT116 cells, and butyrate-mediated induction of the differentiation marker, alkaline phosphatase activity, in Caco-2 cells. Screening of a panel of 30 colon cancer cell lines identified 2 cell lines, LIM2405 and KM12 that had no detectable PTEN expression, and PTEN was not induced by butyrate in these cell lines. Collectively, these results demonstrate that upregulation of PTEN plays a role in mediating butyrate-induced growth inhibition, apoptosis and differentiation in vitro.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]