Various cell lines with double mutations of K-Ras/BRAF and PIK3CA were resistant to AKT inhibitors

FACS analysis in a panel of endometrial cancer cell lines revealed a clear dose-dependent effect of NVP-BEZ235 on cell proliferation. NVPBEZ235 induces G1 arrest much more efficiently at a higher concentrationthan at a lower concentration. In contrast, RAD001 does not show evidence of such dose dependency. Previous reports also suggested that NVP-BEZ235 was more effective than rapalogs at higher concentrations. PI3K activity might not be sufficiently suppressed by 100 nM NVP-BEZ235, as indicated by the observation that decreased phosphorylation of Aktis not observed at 50 nM but is observed at 250 nM or higher. In addition, IC50 values were under 100 nM in cells from groups A and B. These data are in agreement with previous reports on other cancers that indicate a discrepancy between the basal activity of the PI3K/Akt pathway and the biochemical activity of NVP-BEZ235. Nevertheless, the dose-dependent antiproliferative activity at concentrations $250 nM suggests that the effect of NVP-BEZ235 was, at least in part, caused by inhibition of the PI3K/Akt pathway. Our data suggest that a dual inhibitor of PI3K/mTOR might be a more promising therapeutic strategy than a single mTOR inhibitor in endometrial cancer. Our in vivo studies in 2 cell lines of xenograft mice support the in vitro findings that inhibition of the PI3K/mTOR axis has an antitumor effect in endometrial cancers. We did not see any superior efficacy of NVP-BEZ235 in the in vivo study. The concentrations we used were 40 mg/kg for NVP-BEZ235 and 5 mg/kg for RAD001, which are equivalent with the previous invivo experiments. In a pharmacodynamic analysis, the levels of p-Akt, p-GSK3beta, p-FOXO1/3a, and p-S6 in tumors returned to the baseline levels within 24 h after administration of NVP-BEZ235, suggesting that inhibition of PI3K signaling by NVP-BEZ235 might not be sufficiently maintained over time. This is compatible with previous data showing that inhibition of p-Aktwas maintained for 16 h, with recovery to baseline levels at 24 h. It remains to be determined which oral dosing schedule is optimal in treatment of endometrial cancer. As well, the mechanisms of in-vivo antitumor effect by these drugs should be more clarified, as inhibition of mTOR might result in anti-angiogenic effect by suppressing HIF1-VEGF pathway. Developing predictive biomarkers in therapeutics targeting the PI3K/mTOR pathway is crucial, as alterations in several molecules are involved in the activation of this pathway. PIK3CA mutation and HER2 amplification have been recommended as useful biomarkers in breast cancer. Mutant oncogenic Ras has been suggested as a dominant determinant of resistance in several solid tumor cells. PTEN deficiency is controversial as a predictive biomarker. The mechanism of resistance in PTEN-deficient tumors might be explained by the predominant activation of p110beta in PTEN mutant tumors, as NVPBEZ235 and most of the other PI3K inhibitors suppress p110beta less preferentially than the other p110 isoforms. However, p110beta is not a predominant isoform in endometrial carcinomas with PTEN mutations. The significance of p110alpha in PTEN mutant endometrial cancer would be helpful to identify patients susceptible to NVP-BEZ235. Thus, the existence of PTEN mutations might be a predictive biomarker for the PI3K/mTOR inhibitors in endometrial carcinomas. Further in vivo analysis, including the anti-tumor effect of NVP-BEA235, RAD001, or a combination of these compounds with a MEK inhibitor on groups C and D tumors would be necessary to evaluate the utility of these factors as biomarkers. She et al reported and suggested that a MEK inhibitor sensitizes these double mutant cells to AKT or PI3K inhibitors.

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