Cytostatic action of the dual PI3K/mTOR inhibitor NVP-BEZ235 in non-functioning pituitary adenomas

NVP-BEZ235 is a novel dual Class I PI3K/mTOR small molecule inhibitor displaying promising antiproliferative activity in various tumor cell lines and several solid cancers. Contrary to the modest response to single PI3K and mTOR inhibitors, NVP-BEZ235 was effective even in those tumors harboring abnormal PI3K signaling due to loss of PTEN function or gain-of-function PI3K mutations. Non-functioning pituitary adenomas (NFPA) display an overactivation of the Akt kinase and were found to poorly respond to the treatment with rapamycin and its analogs. Therefore they could potentially benefit from the concomitant inhibition of the mTOR kinase and PI3 kinase upstream. Indeed NVP-BEZ235 treatment in the rapamycin resistant pituitary tumor cell line AtT-20 potently suppressed cell viability an effect that remained even after overexpression of Akt. Moreover 37 and 35 out of 40 NFPA in primary cell culture responded to 100nM and 10nM NVP-BEZ235 treatment by suppressing cell viability more than 50% (% of cell viability suppression 70±22 and 60±15 respectively). Both concentrations effectively blocked p70 S6K-Thr389 and Akt-Ser473 phosphorylation downstream to mTOR and PI3K respectively. Further investigation in AtT-20 cells revealed that NVP-BEZ235 displays cytostatic action accumulating the cells almost exclusively in the G1 phase. In contrast, no apoptosis was observed by determining cleaved PARP and caspase 3. Cell cycle arrest is associated with decreased Rb phosphorylation and subsequent E2F related transcription and indeed, in AtT-20 cells, NVP-BEZ235 treatment suppressed E2F transcriptional activity by 50%. During cell cycle progression, Rb is phosphorylated by cyclin/cyclin-dependent kinase complexes, such as, cyclin D1–3/Cdk4/6 and cyclin E/Cdk2 complexes. Treatment with high doses (100nM) of NVP-BEZ235 suppressed cyclin D1 and D3 protein levels, while Cdk4 and Cdk6 remained unchanged. In contrast, cyclin E levels decreased even after treatment with lower NVP-BEZ235 concentrations (10nM). In parallel, NVP-BEZ235 treatment increased the protein levels of the cyclin E/Cdk2 inhibitor p27/Kip1. Summarizing, inhibition of the cyclin E/Cdk2 complex by decrease in cyclin E and increase in p27/Kip1 levels may contribute to the suppression in E2F driven transcription and G1 cell cycle arrest observed in NVP-BEZ235 treated pituitary tumor cells. Altogether, these results indicate that the dual PI3K/mTOR inhibitor has a potential as a new antitumor agent for the pharmacological management of NFPA.