oa Inhibition of the Akt Kinase Down-regulates ERK, Bcl-2 and Survivin and Suppresses Proliferation and Survival of Murine VEGF-dependent Angiosarcoma Cells

Introduction: Angiosarcomas are rare and malignant neoplasms that involve abnormal proliferation and migration of cancerous endothelial cells. Angiosarcomas can arise in any region of the body, but tend to be found in the skin, soft tissue, and liver. They are associated with high mortality rate due to their aggressiveness and high rate of metastasis. Therapeutic strategies for treating angiosarcomas involve use of cytotoxic drugs and radiotherapy. However, patient-resistance to these approaches is commonly reported. Hence, there is a need to further understand molecular mechanisms underlying angiosarcomas and explore novel therapeutic targets. Furthermore, because abnormal angiogenesis underlies nearly all types of cancer, a better understanding of cellular pathways regulating cancer endothelial cell function may also lead to development of novel anti-angiogenic therapies. The PI3K/AKT/mTOR signaling pathway plays an important role in regulating cell proliferation and is also regarded as one of the most commonly dis-regulated pathway in cancer. Inhibitors have been developed targeting these signaling proteins and their therapeutic potential has been evaluated in different studies. However, there is little known about the relevance of this signaling pathway to angiosarcomas. Therefore, in this study, we explored the anticancer therapeutic potential of targeting the PI3K/AKT/mTOR signaling pathway using inhibitors of PI3K, AKT or mTOR in a murine VEGF-dependent angiosarcoma cell line. Methods: Cell culture: MS1-VEGF cells (mouse endothelial cells capable of inducing angiosarcomas) were used. Cells were maintained in Dulbecco's Modified Eagle Medium, which was supplemented by 5% fetal bovine serum, 1% penicillin/streptomycin, and 11 mM glucose. Concentration-response experiments: The inhibitors used were: PI3K - LY294002 (10 μM); Akt – AKTi 1/2 inhibitor (AKTi, 10 μM); and mTOR - Temsirolimus (4 μM). LY294002 is a selective inhibitor of PI3K, but is not yet in clinical use. AKTi 1/2 is a non-ATP competitive inhibitor of Akt isoforms 1 and 2. Temsirolimus is a specific inhibitor of mTOR, and has been clinically used for the treatment of advanced renal cell carcinoma. The treatments were conducted over a 48 h period, with DMSO serving as the solvent control. Experiments was repeated at least three times using cells cultured in 12 or 6-well sterile tissue culture plates (Falcon). Cells were incubated in a 5% CO2 incubator at 37 °C. At the end of the treatment period, cell counts were performed and trypan blue dye exclusion was used to assay cell viability. This was done by pipetting a small volume of cell suspension mixed with trypan blue onto a dual chamber counting slide (Bio-Rad TC20 Automated Cell Counter). Proliferation Assay: In order to further assess the effectiveness of the inhibitors, proliferation assays were performed. Cells plated in 96-well plates with the inhibitors for 48 h were treated with the CellTiter 96 Aqueous One Solution reagent (Promega) and incubated in the CO2 incubator at 37 °C for 3 h. A PerkinElmer 2104 plate reader was used (EnVision software) to measure absorbance (at 492 nm). Western blots: Western blots were used to detect the expression of several proteins linked to the PI3K/Akt/mTOR pathway and also to study changes resulting from treatment with inhibitors. Primary antibodies recognizing mTOR, AKT, PI3K, eNOS, LC3B, Beclin-1, ERK, Survivin, and Bcl-2 were used. All proteins probed have known roles in cell proliferation/apoptosis/autophagy. Images were captured using GeneSnap software on a PerkinElmer Geliance 600 imaging system. Quantitiy One (Biorad) was used to analyze Western blot data. Flow cytometry: Cell cycle analysis was performed on the LSR Fortessa analyzer (BD Biosciences) by staining fixed and permeabilized cells with propidium iodide. Data was processed with FACS Diva 8.0 software (BD Biosciences). Data analysis: All the data was analyzed using the statistical software GraphPad Prism 5.0 (GraphPad Software, Inc. CA, USA). Data is presented as mean ±  SEM. Statistical analysis was performed with using ‘t’ test or one-way analysis of variance (ANOVA). Post-hoc comparisons between groups after ANOVA were performed by Tukey's multiple comparison tests. ‘p’ values less than 0.05 were considered to be statistically significant. Results: Incubation of MS1 VEGF cells with LY294002, Akt-i ½ or Temsirolimus caused a reduction in cell number, indicating reduced cell proliferation. The Akt-i ½ was the most effective among the three and also caused a strong reduction ( < 25% viability) in cell viability (unlike LY294002 and Temsirolimus). Proliferation assays performed in 96-well plates indicate a reduction in MS1 VEGF cell proliferation with all three inhibitors, and Akt-i ½ was again the most effective in these assays. Cell cycle analysis revealed a robust increase in ‘sub G0/G1 population’ after treatment with Akt-i ½ (Control - 8% vs Akt-i ½ - 43%), suggesting an increase in cell death. To investigate the mechanisms underlying the actions of the inhibitors, western blot experiments were performed. The data (n = 4) demonstrated down-regulation of the anti-apoptotic Bcl family protein Bcl-2 and the inhibitor of apoptosis protein survivin after treatment with Akt-i ½ (but not LY294002 or Temsirolimus). Treatment with Akt-i ½ also reduced the phosphorylation of Akt and ERK proteins. Furthermore, there was a strong increase in the expression of the autophagy marker LC3B-II after treatment with Akt-i ½. Immuno-staining experiments confirmed aggregation of LC3B-II after Akt-I ½ treatment, suggesting an induction of autophagy. Inhibition of autophagy by 3-methyladenosine (3-MA) reversed Akt-i ½-induced LC3B-II puncta formation and also significantly enhanced Akt-i ½-induced cell toxicity. This suggests that autophagy induction acts as a cell survival mechanism after Akt inhibition. To investigate if Bcl-2 and survivin could be downstream effectors of Akt-i ½, experiments were performed using specific inhibitors: YM155 for survivin and TW37 for Bcl-2. A combination of YM155 and TW37 induced robust changes in cell cycle, increased the ‘sub G0/G1 population’ (Control - 9% vs. TW37 + YM155 - 38%) and reduced the proportion of cells in G0/G1, S as well as G2/M phase. Discussion: The data revealed promising anti-proliferative actions for LY294002, Temsirolimus and Akt-i ½ in MS1 VEGF cells. The drug, Akt-i ½, drug was particularly effective and also substantially reduced cell viability. The data also suggest that Bcl-2 and survivin may be critical components of the anti-proliferative action of Akt-i ½ thus making it a highly effective agent. The data also revealed that the cells induced autophagy as a survival mechanism when Akt was inhibited. In conclusion, multiple signaling pathways and proteins regulate MS1 VEGF cell proliferation and survival, which are targeted by Akt-i ½. Future studies: Future investigations will focus primarily on direct evaluation of apoptosis by flow cytometry and if possible experiments in nude mice to evaluate in vivo drug efficacy.