Levels of p27 Sensitize to Dual PI3K/mTOR Inhibition

Abstract

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling cascade occurs in a variety of human malignancies, where it sustains tumor cell proliferation and survival. Pharmacologic blockade of this pathway exerts antineoplastic activity by triggering apoptosis and/or cell-cycle arrest. Pituitary adenomas show activation of the PI3K/AKT/mTOR pathway, but only a fraction of them respond in vitro to the antiproliferative action of rapamycin and RAD001 (mTOR inhibitors), possibly because of the described negative feedback loop on AKT which reactivates the signaling cascade. Rats affected by the multiple endocrine neoplasia-like syndrome (MENX) develop pituitary adenomas showing increased activated AKT. In this study, we comparatively investigated the antitumor potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235 and the single mTOR inhibitor RAD001 on rat pituitary adenoma cells in primary culture. NVP-BEZ235 inhibits the PI3K pathway both upstream and downstream of AKT, thereby preventing the negative feedback loop. NVP-BEZ235 was more effective than RAD001 in reducing cell viability of pituitary adenomas. Consistently, NVP-BEZ235 treatment decreased Akt and S6 phosphorylation and triggered apoptosis. Because MENX is caused by a germline loss-of-function mutation in the cell-cycle inhibitor p27Kip1, we investigated the relationship between this defect and response to NVP-BEZ235 treatment. The levels of p27Kip1 positively correlate with the response to NVP-BEZ235 treatment. Combined treatment with NVP-BEZ235 and the proteasome inhibitor bortezomib, which increases p27Kip1 amount, shows synergistic antiproliferative effects on pituitary adenoma cells. Our data suggest that NVP-BEZ235 may represent an effective therapeutic modality for pituitary adenomas and that p27Kip1 levels represent a potential predictor of response to dual PI3K/mTOR inhibition.

The PI3K/AKT/mTOR pathway in the pathophysiology and treatment of pituitary adenomas

“…Caspases are the main protein family effectors of apoptosis and the study by Zatelli et al (2010) demonstrated that the effects of RAD001 on cellular viability of NFPA may be attributed to increased levels of caspase 3/7 activity. Increases in caspase 3/7 activity were also reported by Lee et al (2011) following NVPBEZ235 treatment of MENX rat PAs in primary culture, but did not affect caspase activity in AtT20 cells (Cerovac et al 2010). The use of XL765 and TMZ synergistically increased caspase 3/7 levels and TUNEL-positive aT3-1, MMQ, and GH3 cells compared with either treatment alone (Dai et al 2013).…”

“…Lee et al examined a particular strain of rats with a MENX mutation that results in the development of multiple endocrine tumors, including NFPA. Administration of RAD001 to MENX PA cells in primary culture also resulted in a dose-dependent reduction in cellular viability that reached significance at a dose of 100 nM in 45% of their sample (five out of 11 cultures; Lee et al 2011). Finally, Cerovac et al (2010) exposed 28 human NFPAs in primary culture to rapamycin and reported a small (!20%) reduction in cellular proliferation in 29% (eight of 28 tumors) of their samples.…”

“…This result was significantly more potent that previous studies of RAD001 effects on this cell line. Furthermore, it was noted that incubation with NVPBEZ235 at concentrations that suppress cellular viability also decreased the phosphorylation of AKT and S6, whereas RAD001 decreased phosphorylated S6, but increased phosphorylated AKT (Lee et al 2011). In another study examining NVPBEZ235, NFPA cells in primary cell culture exposed to the dual inhibitor exhibited a 50% decrease in cell viability in 37 of 40 cases (Zatelli et al 2010).…”

“…These findings were attributed to decreased levels of mTOR phosphorylation at the serine-2448 residue, which is a key determinant of mTOR activity. A third study noted a small Greater antiproliferative effect than RAD001; triggers apoptosis Lee et al (2011) but statistically significant antiproliferative effect of 1 nM rapamycin on mouse AtT20 cells and no effect at lower doses (Cerovac et al 2010). Musat et al (2005) have reported that NFPAs have the highest levels of AKT mRNA expression of all PA subtypes.…”

“…One of these agents, NVPBEZ235, is a synthetic small molecule that inhibits both PI3K and mTOR kinase activity by binding to the ATP cleft of these enzymes (Maira et al 2008). In a study by Lee et al (2011), NVPBEZ235 was reported to inhibit the viability of 100% (ten of ten) of MENX rat PAs in primary cell culture. This result was significantly more potent that previous studies of RAD001 effects on this cell line.…”