Ipatasertib, an oral AKT inhibitor, effectively inhibits cell proliferation and migration, and induces apoptosis in serous endometrial cancer cell lines in vitro: Endometrial Cancer Molecularly Targeted Therapy Consortium

Abstract

<h3>Objectives:</h3> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. Given that the AKT/mTOR pathway is altered in the vast majority of endometrioid endometrial cancers (ECs), it is logical that IPAT may also have efficacy in EC. Thus, we aimed to evaluate the anti-proliferative effects of IPAT in human endometrioid cell lines as well as in the <i>LKB1fl/flp53fl/fl</i> genetically engineered mouse model of endometrioid EC. <h3>Methods:</h3> The human endometrioid EC cell lines ECC-1 (PTEN mutant) and HEC-1A (PTEN wild type, PIK3CA mutant, KRAS mutant) were exposed to varying concentrations of IPAT (Genentech). Cell proliferation was assessed by MTT and colony assays. Cell cycle progression was measured by Cellometer. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was assessed using tetramethylrhodamine ester (TMRE) and DCFDA assays. Western immunoblotting determined effects of IPAT on BCL-2, MCL-1, Bip, CDK4, CDK6, cyclin D1, pAKT, pS6, PERK, calnexin, and PDI in both cell lines. <i>LKB1fl/flp53fl/fl</i> mice were used to evaluate the effect of IPAT on endometrial tumor growth. The mice were treated with placebo or IPAT (15mg/kg daily intraperitoneally for 4 weeks) starting 8 weeks after tumor induction via AdCre injection. <h3>Results:</h3> IPAT inhibited cellular proliferation in a dose dependent fashion in both cell lines after 72 hours of treatment. Median IC50 was 0.18µM in ECC-1 and 3.5µM in HEC-1A. IPAT induced the activity of cleaved caspase 3 by 15.6-fold in ECC-1 and 1.41-fold in HEC-1A (p<0.01), while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. IPAT inhibited cell cycle progression by arrest in G1 phase in both cell lines after 36 hours of treatment and decreased expression of the cell cycle related proteins, CDK4, CDK6, and cyclin D1. DCFDA assay analysis demonstrated that IPAT induced ROS products by 18% and 19% (p<0.01) in the ECC-1 and HEC-1A cell lines, respectively, and increased Bip and PERK protein expression. In addition, IPAT reduced mitochondrial potential by 12% in the HEC-1A cells and 14% in the ECC-1 cells (p<0.01). Treatment of cells with IPAT at 10µM increased phosphorylated-AKT (pAKT) expression and decreased phosphorylated-S6 (p-S6) expression in a time-dependent manner in both cell lines; however, measurable changes in pAKT and pS6 were observed at concentrations as low as 0.1µM. Notably, an increase in phosphorylated-AKT was expected and is indicative of engagement and binding of IPAT with the phospho-AKT complex. Treatment of <i>LKB1fl/flp53fl/fl</i> mice with IPAT was well-tolerated and reduced tumor weight by 52.1% as compared with control mice (p<0.05). <h3>Conclusions:</h3> IPAT significantly inhibited cell proliferation and induced apoptosis and cellular stress via inhibition of the AKT/mTOR pathway in human endometrioid EC cell lines. Furthermore, IPAT reduced tumor growth in a transgenic mouse model of endometrioid EC. These results suggest that IPAT is worthy of further exploration in clinical trials for EC.