Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. Further

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. Further investigations indicate that senescence does not contribute to MLN0128-mediated repression of xenograft MCC tumor growth. Finally we also observe robust antitumor effects of MLN0128 when administered as a dual therapy with JQ1 a bromodomain protein BRD4 inhibitor. These results suggest dual blockade of PI3K/mTOR pathway and c-Myc axis is effective in the control of MCC tumor growth. Our results demonstrate that MLN0128 is potent as monotherapy or as a member of combination therapy with JQ1 for advanced MCC. culture experiments with MCC cells followed by Western blot analysis. We first treated MCC cells with or without different concentrations of MLN0128 for 24 hours and then examined the total and phosphorylated protein profile of the targeted pathways by Western blotting. Consistent with published reports on other solid tumors MLN0128 markedly inhibited phosphorylation of both mTOR CI994 (Tacedinaline) and its downstream effectors including 4E-BP1 (Thr37/46) and S6 kinase (Ser235/236) in all three MCV negative MCC cell lines (Figure ?(Figure4A)4A) [21]. As expected MLN0128 also abrogated p-Akt activity (Figure ?(Figure4A)4A) in CI994 (Tacedinaline) these cell lines. These results also correlate well with Western blot data shown in Figure 2B and 2C using xenograft tissues. Figure 4 MLN0128 inhibits mTOR pathway activity and colony formation in MCC cells Blockade of mTOR pathway inhibited the proliferative capacity of tumor cells In Figure ?Figure1 1 we attributed phenotypic reduction of tumor volume after mTOR blockade by MLN0128 to decreased cell proliferation and increased cell death within the tumor. To examine these possibilities we studied effects of mTORC1/2 inhibition by MLN0128 on cell viability and cell proliferation. For this MCC-2 MCC-3 and MCC-5 cells were treated with increasing concentrations of MLN0128 for 12 24 48 and 72 hours respectively and CI994 (Tacedinaline) cell proliferation were analyzed utilizing CCK-8 assay. Results from these experiments with three MCC cell lines showed a decreased cell proliferation over a 72-hr period. The half maximal growth inhibitory concentration (GI50) dose was determined by CCK-8 assay in all three MCC cell lines. The GI50 for MCC-2 MCC-3 and MCC-5 cells is 1200 nM 400 nM and 500 nM respectively (Data not shown). The underlying mechanism for this variation is not clear. In keeping with this non-responder phenotype subsequent experiments were carried out at 800 nM for MCC-2 cell line alone. To complement the results from short-term treatments we performed long-term colony formation assay to determine if the inhibitory effects of MLN0128 were sustained over time. Similarly MLN0128 significantly decreased the number of MCC cell colonies as compared CI994 (Tacedinaline) to that of DMSO controls (Figure ?(Figure4B).4B). Collectively our experiments clearly show that blockade of mTOR by MLN0128 inhibits MCC cell growth which partly accounts for the phenotype reduction of tumor. CI994 (Tacedinaline) Additionally the differential response observed among MCC-2 MCC-3 and MCC-5 cells is further suggestive of the heterogeneous nature of this tumor. While all three cell lines share certain common cellular and molecular characteristics cancer initiation and other changes may be individualistic. Cell cycle arrest and augmentation of cell death with mTOR blockade in MCC cells Because cell proliferation is strictly controlled by cell Tnf cycle checkpoints we analyzed the mTORC1/2 blockade on cell cycle progression using BrdU incorporation labeling method. Cell cycle analysis by flow cytometry showed a significant reduction of cells in the S-phase with a concomitant cell arrest at G0/G1 after MLN0128 treatment in MCC cells (Figure ?(Figure5A).5A). No such cell cycle arrest events were observed in vehicle alone treated control cells. Additionally there was a three-fold increase in sub-G1 population in MCC-3 (2.9% to 9.4%) and MCC-5 (2.1% to 9.5%) which are due to apoptotic cell death (Figure ?(Figure5A).5A). As compared to untreated controls there was only a marginal increased in MCC-2 cells which was less responsive to MLN0128 treatment (7.1% vs 11.2% in treated group) suggesting MCC-2 has a resistant phenotype. When examined by Annexin-V assay for apoptosis the total apoptotic cell death also found increased significantly in all three cell lines and in particular MCC-3 and MCC-5 (Figure ?(Figure5B).5B). To identify.