Abstract 5008: The H1047R point mutation in p110 alpha of PI3K decrease Bcl2 expression in human colon cancer HCT116 cells

INTRODUCTION: PI3K and anti-apoptotic protein Bcl2 are two important players in regulating cell survival through two distinct cell signaling pathways. Beside the survival function, PI3K is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. At the same time, Bcl2 has also been reported to be involved in actin polymerization and cell motility dissociate from its anti-apoptotic function. Inhibition of PI3K has been shown to result in a 45% decreases in Bcl2 promoter activity. Several reports have shown that loss of Bcl2 expression correlates with tumor recurrence in CRC and high levels of Bcl2 are predictive of relapse-free survival in stage II CRC. Mutations in the catalytic subunit p110α (PIK3CA) of Class 1A PI3K occur in up to 1/3 of human colorectal cancers (CRC) and the kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α and results in a gain of function. In vivo studies have demonstrated that cell lines bearing the p110α mutation are more metastatic than the cells with wild type p110α. The aim of current study was to investigate the effect of H1047R mutation in PI3K on Bcl2 regulation. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous expression level of Bcl2 was detected by immune-blotting analysis in WT and MUT HCT116 cells. To further investigate the effect of WT and MUT p110α on Bcl2 regulation, Bcl2 expression was tested under the condition of ether overexpression or inhibition of p110α. For inhibition experiment, HCT116 WT and MUT cells were treated by the specific inhibitor of p110α A66. While, overexpression of p110α was conducted by stable transfection into HCT116 WT cells with Tet-On inducible plasmids, which contains the whole sequences of coding region of either wild type (PIK3CAWT) or H1047R-p110α (PIK3CAA3140G). RESULTS: 1) Endogenous expression level of Bcl2 in HCT116WT cells was at least 3-times higher than it in HCT116MUT cells. 2) The increase of Bcl2 expression is doxycline dose-dependent in the HCTWT cells which were transfected with pTRE3G-PIK3CAWT. However, overexpression of H1047R-p110α did not affect Bcl2 levesl in HCT116WT cells with pTRE3G-PIK3CAA3166C transfection. 3) Expression level of Bcl2 in HCT116 MUT cells was increased by A66 dose-dependent inhibition of p110α. The down-regulation of Bcl2, however, was observed in HCT WT cells. CONCLUSION: Our results suggested that the wild type and the H1047R p110α have the opposite effect in the regulation of Bcl2 expression: up-regulation by WT- p110α and down- regulation by H1047R p110α. Both PI3K and Bcl2 cannot be ignored in cancer prognosis and therapy. Further studies are needed to reveal the mechanism of PI3K mediated Bcl2-regulation, and the effect of the interaction of Bcl2 and PI3K on cell migration and CRC metastasis. Citation Format: Guanghua Wan, Ashwani Rajput. The H1047R point mutation in p110 alpha of PI3K decrease Bcl2 expression in human colon cancer HCT116 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2015-5008