Abstract 1141: The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells

Abstract

Abstract INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and Inositol polyphosphate-4-phosphatase, type II (INPP4B) are two important enzymes maintaining homeostasis of phosphoinositides in cells. Class IA PI3K produces Phosphatidylinositol-3,4,5-bisphosphate (PIP3) by phosphorylation of Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at D3 position, while INPP4B dephosphorylate Phosphatidylinositol-4,5-bisphosphate (PI(3,4)P2) at the D4 position to generate Phosphatidylinositol 3-phosphate (PI(3)P). All of these phospholipids are important second messengers. PI3K activates its downstream substrate Protein Kinase B (Akt) by PIP3. INPP4B, however, blocks Akt activities in epithelial cells through the turnover of PI(3,4)P2. PI3K/Akt pathway is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. Gain of function mutations in the catalytic subunit p110α of PI3K occur in up to 1/3 of human colorectal cancers (CRC). The kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α. Our previous report showed that this mutation in p110α results in an increase in cellular levels of PIP3 and in turn an enhancement of Akt activity. Since the H1047R mutation and INPP4B have an opposite effect on Akt activities, the interesting question is if there is crosstalk between the two or if H1047R mutation can affect cellular INPP4B expression. We hypothesize that H1047R mutated p110α down-regulates INPP4B and therefore results in increased Akt activity. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous protein expression level of INPP4B was detected by immune-blotting analysis in parental, WT and MUT HCT116 cells. To further investigate the effect on INPP4B of the H1047R mutation, we conducted RNA-sequencing for profiling, quantifying RNA transcripts in WT and MUT HCT116 cells, and compared RNA transcript level of INPP4B between the two isogenic cell lines of HCT116 cells. RESULTS: 1) Endogenous expression level of INPP4B protein in HCT116WT cells was over 10-times higher than in HCT116MUT cells. 2) INPP4B RNA was down regulated by the H1047R mutation in p110α (PIK3CA) of Class 1A PI3K. CONCLUSION: Our results suggested that hyperactivity of Akt in H1047R mutation bearing cells may be also related to low level of cellular INPP4B, and that the H1047R mutated p110α may increase cell migration and cancer metastasis through down regulation of tumor suppressor gene INPP4B. Although further studies are needed to reveal the mechanism of H1047R mutated PI3K mediated INPP4B-regulation, INPP4B expression level may be a potential maker to predict PI3K mutation induced CRC metastasis. Citation Format: Guanghua Wan, Scott A. Ness, Ashwani Rajput. The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1141.