In accordance with institutional approvals for human study protocols, a total of 22 MCC tumor samples were collected between January 2005 and December 2011 at the Union Hospital, Huazhong University of Science and Technology (Wuhan, China). Written informed consent was obtained from each patient.

MCC MKL-1 cells (Sigma-Aldrich, St. Louis, MO, USA) were grown in RPMI-1640 supplemented (Gibco Life Technologies, Carlsbad, CA, USA) with 10% fetal calf serum, penicillin and streptomycin (Sigma-Aldrich) at 37°C in a humidified 5% CO₂ atmosphere. NVP-BEZ235 was provided by Novartis (Basel, Switzerland) and dissolved in dimethyl sulfoxide (Sigma-Aldrich) to a concentration of 10 mmol/l, stored at −20°C, and further diluted to final concentrations of 50 nM, 100 nM and 200 nM in Dulbecco's modified Eagle's medium at the time of use. Rabbit polyclonal antibodies against phosphorylated mTOR (p-mTOR; Ser2448; cat. no. 2971), phosphorylated Akt (p-Akt; Ser473; cat. no. 4058) and cleaved caspase-3 (cat. no. 9661) used at a dilution of 1:1,000 were purchased from Cell Signaling Technologies (Danvers, MA, USA). Rabbit polyclonal antibodies against p21 (cat. no. sc-397), p27 (cat. no. M-197), p57 (cat. no. C-20) and cyclinD1 (cat. no. sc-753) used at a dilution of 1:250 and mouse monoclonal antibody against tubulin (cat. no. TU-02; dilution, 1:5,000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Additional reagents used in the present study consisted of TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA), puromycin (Sigma-Aldrich), polybrene (Sigma-Aldrich), fibronectin (Sigma-Aldrich), RIPA buffer (Sigma-Aldrich) and enhanced chemiluminescence (ECL) detection reagent (EMD Millipore, Billerica, MD, USA).

The cultured cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in 1X RIPA buffer containing 1 mM DTT and Complete Mini EDTA-free protease inhibitor cocktail. Following incubation on ice for 30 min, the cell lysates were clarified by centrifugation at 16,000 × g for 15 min at 4°C. In total, 20 µg of protein was resolved on 8 or 12% sodium SDS-PAGE gels and transferred electrophoretically onto polyvinylidene difluoride membranes by a semi-dry system (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked in 5% fat-free milk in Tris-buffered saline with 0.1% Tween 20 (Sigma-Aldrich) for 1 h, and incubated with primary antibodies at 4°C overnight, followed by incubation with monoclonal goat anti-mouse (1:15,000; cat. no. SC-2055) or goat anti-rabbit (1:25,000; cat. no. SC-2054) secondary antibodies conjugated with horseradish peroxidase. Subsequent to rinsing three times, the membrane was subjected to western blot analysis with ECL detection reagent.

Cell survival was assessed using cell counting kit-8 (CCK-8; Sigma-Aldrich) according to the manufacturer's instructions. In brief, the cells were plated at a density of 10,000 cells per well in a 96-well plate and exposed to serial dilutions of NVP-BEZ235 for 12, 24, 48 and 72 h, respectively. CCK-8 (10 µl) was added to each well at the end of each time point (12, 24, 48 and 72 h) and incubated at 37°C for 4 h. The absorbance at 450 nm was measured using a microplate spectrophotometer (Bio-Rad Model 680; Bio-Rad Laboratories, Inc.).

Cell death was detected by flow cytometry for Annexin V-fluorescein isothiocyanate, according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). Briefly, 1×10⁶ cells were plated in six-well plates for 12 h followed by treatment with NVP-BEZ235 for 24 h prior to Annexin V and propidium iodide (PI) staining (FACSAria; BD Biosciences). For each dye, appropriate electronic compensation of the FACSAria sorter was performed to avoid overlapping of the two emission spectra. For cell cycle analysis, 1×10⁶ cells were seeded in six-well plates for 12 h, followed by treatment with NVP-BEZ235, and were then labeled with 10 µM bromodeoxyuridine (BrdU; BD Biosciences) for 2 h. BrdU incorporation was detected using Alexa Fluor 488-conjugated mouse anti-BrdU antibody (BD Biosciences), followed by 7-aminoactinomycin D staining for cell cycle analysis, according to the manufacturer's instructions.

Lentivectors directing expression of shRNA specific to p21 (construct no., TRCN0000040123) and p27 (construct no., TRCN0000009856) were purchased from Sigma-Aldrich, and non-targeting pLKO.1 scramble shRNA was obtained from Addgene (plasmid no., 1864). To generate recombinant lentiviruses, 293T/17 cells were co-transfected with gene transfer vectors and virus packaging vectors, ΔH8.2 and VSVG using TransIT-LT1 transfection reagent. Virus supernatants were collected 48 h following transfection. MKL-1 cells were transduced with virus supernatant for 48 h in fibronectin-coated six-well plates in the presence of 8 µg/ml polybrene, subsequent to spinoculation at 800 × g for 30 min, at 32°C. Since the lentiviral shRNA particles also encode a puromycine resistance gene for transduction selection, the cells were then washed and grown in culture media containing 2 µg/ml puromycin dihydrochloride (Sigma-Aldrich) for an additional 72 h. The cells were left to recover and proliferate for at least 2 days prior to undergoing any experimental procedure.

Common tumor suppressor genes and oncogenes, including p53, PTEN, Rb, Ras and B-RAF, are less frequently mutated in MCC in comparison with other skin tumors (14). Growth receptor pathways, such as c-kit, vascular endothelial growth factor and platelet-derived growth factor, are also not highly activated in MCC (14). Previously, mTOR upregulation was found in MCC expressing small T antigens (15). Therefore, the activation of PI3K/Akt/mTOR signaling was investigated in 22 primary human MCC tissue samples prior to examining the effect of the dual PI3K/mTOR inhibitor in MCC cells. Out of the 22 MCC samples tested, 20 out of 22 (90%) were positive for p-mTOR Ser2448, and 19 out of 22 revealed positive staining of p-Akt (Ser473). The findings of the immunohistochemical analysis from a representative sample are revealed in Fig. 1A. In addition, the activation of PI3K/Akt/mTOR signaling was investigated in 5 primary human MCC tissue samples randomly selected by immunoblotting (Fig. 1B), and also confirmed the upregulation of p-mTOR and p-Akt in MCC.

NVP-BEZ235 is an orally bioavailable imidazoquinoline that inhibits PI3K and mTOR kinase activity and has been revealed to inhibit tumor growth in numerous preclinical models. To investigate the in vitro effect of NVP-BEZ235 on the growth of MCC, MKL-1 cells were treated with increasing concentrations of NVP-BEZ235 for 12, 24, 48 and 72 h, respectively, and cell viability was analyzed using the CCK-8 assay. As shown in Fig. 2A, treatment with NVP-BEZ235 reduced the viability of MKL-1 cells in a time- and concentration-dependent manner. The ability of NVP-BEZ235 to modulate the signaling pathway in MKL-1 cells was then assessed by western blotting. As expected, NVP-BEZ235 markedly decreased the levels of Akt and mTOR phosphorylation in a dose-dependent manner in the assessed MKL-1 cells (Fig. 2B), confirming the effect of NVP-BEZ235 on MCC.

To further gain insight into the mechanisms of growth inhibition exerted by NVP-BEZ235, the effect of this agent on the cell cycle and apoptosis in MKL-1 cells was analyzed by flow cytometry. Consistent with the anti-proliferative effects of NVP-BEZ235, a pronounced decrease of cells in the S phase and a concomitant increase in cells in the G0/G1 phase were observed in the treated groups compared with the control group (Fig. 3A), indicating cell cycle arrest in the G0/G1 phase. Notably, NVP-BEZ235 did not induce apoptosis in MKL-1 cells, which was further confirmed by the absence of caspase-3 cleavage and activation (Fig. 3B and C). Collectively, these data indicated that NVP-BEZ235 induced G0/G1 cell cycle arrest, but not apoptosis, in MKL-1 cells.

To explore whether cell cycle regulatory proteins were involved in the cell cycle arrest of MKL-1 cells, the cells were treated with various concentrations of NVP-BEZ235 and cell lysates were subjected to western blot analysis. As revealed in Fig. 3D, downregulation of the cell cycle promoter cyclin D1 and upregulation of the negative cell cycle regulators p21 and p27 were detected subsequent to NVP-BEZ235 treatment in MKL-1 cells. The MKL-1 cells were therefore transfected with shRNA targeting either p21 or p27 (Fig. 4A). As shown in Fig. 4B, knockdown of p21 or p27 expression partially rescued NVP-BEZ235-induced cell cycle arrest to a similar degree, which indicates that NVP-BEZ235-induced suppression of proliferation mainly results from the upregulation of p21 and p27.