Expression And Targeted Therapy Of Mtorcl/2Signaling Pathway In Esophageal Squamous Cell Carcinoma

Part I Expression of mTORCl and mTORC2protein in human esophageal squamous cell carcinoma tissue and its clinical significanceObjective:To determine whether mTORC1and mTORC2components are overexpressed and activated in human esophageal squamous cell carcinoma (ESCC) tissue. To analyze the relationship between protein expression and clinical pathologic parameters and provide theoretical evidence for the application of mTOR targeted therapy in the treatment for ESCC.Methods:Immunohistochemistry was used to detect the expression of mTOR, Raptor, Rictor and p-Akt(Ser473) in58tissue samples of ESCC and25tissue sample of normal esophageal mucosa. The positive expression frequencies of these indicators in different pathological conditions of ESCC were analyzed. The function of mTORC1/2signal pathway in carcinogenesis and cancer progression was explored.Results:1. mTOR, mTORC1protein Raptor and mTORC2protein Rictor, were activated in human ESCC tissue. The expression of mTOR, Raptor and Rictor for tumor tissue was significantly higher than normal esophageal tissue (p<0.01), indicating the mTORC1/2signaling pathway may correlate with ESCC carcinogenesis. Meanwhile, the expression of p-Akt(Ser473) was found to be significantly correlated with evaluated Rictor expression (r=0.440, p=0.001), indicating the mTORC2kinase may contribute to the elevated p-Akt(Ser473) expression in ESCC.3. There was a significant correlation between the strong Raptor expression and high tumor stage and poor differentiation (p<0.05). Expression of Rictor and p-Akt(Ser473) was positively correlated with higher tumor stage and lymph node metastasis(p<0.05).Conclusion:1. mTORC1/2signaling pathway components are overexpressed in ESCC. mTORC2kinase activity may correlated with elevated p-Akt(Ser473) expression in ESCC.2. mTORC1activation may associated with high tumor stage and poor differentiation in ESCC. High expression of mTORC2protein may associated with high tumor stage and lymph node metastasis. Therefore, mTORC1/2may represent a therapeutic target for treatment of ESCC. PartⅡ The effect of targeted inhibition of mTGRC1/2signaling by using pharmaceutical inhibitors in esophageal squamous cell carcinoma cellsObjective:To evaluate the effect of targeted inhibition of mTORC1/2signaling on cell proliferation, apoptosis and cell cycle distribution in esophageal squamous cell carcinoma cells, by using two generation of mTOR inhibitors; and to provide experimental evidence for the application of mTORCl inhibitor or mTORC1/2kinase inhibitor in treating esophageal squamous cell carcinoma.Methods:We first observed the activation of mTOR and its downstream proteins p-p70s6k, P-4E-BP1and p-Akt in two ESCC cell lines (Eca-109and TE-1) by Western-blot analysis. And then, the cell proliferation, cell cycle distribution and apoptosis were determined in Eca-109and TE-1by using the first-generation mTOR inhibitor rapamycin and second generation of ATP-competitive dual mTORC1/2kinase inhibitor PP242. The effect of both drugs on cell proliferation was determined by MTT and clone formation assay. The cell cycle distribution was detected by flow cytometry. The effects of both drugs on cell apoptosis were examined by flow cytometry and Western-blot analysis.Results:1. We found that both two cell lines, Eca-109and TE-1, expressed mTOR, p-mTOR and their downstream effectors, including p70S6K, p-4E-BP1and p-Akt. Phosphorylation of mTOR(Ser2448) and p70S6K(Thr389) were suppressed by both rapamycin and PP242. While the phosphorylation of4E-BP1(Thr37/46) and Akt (Ser473) were only decreased by the dual mTORC1/2inhibitor PP242.2. Rapamycin, PP242and Ku-0063794effectively suppressed the cell growth and colony formation ability of both two cell lines. However, the mTORC1/2kinase inhibitor was more effective than rapamycin.3. G0-G1cell cycle arrest was observed in the two cell lines after treatment with rapamycin and PP242.4. PP242, but not rapamycin, induced apoptosis in the two cell lines at relatively higher concentrations.Conclusion:1. mTOR and its downstream effectors are activated in ESCC cells. In contrast to rapamycin, which only partly inhibits mTORC1activity, PP242attenuated the activities of both mTORC1and mTORC2signaling in ESCC cells.2. PP242effectively suppressed ESCC cell proliferation, induced apoptosis, and arrested the cell cycle, which was more effective than rapamycin. Therefore, our study has provided preclinical evidence that targeted inhibition of mTORC1/2signaling is a very effective approach for the treatment of ESCC. PartⅢ The effect of mTORCl/2inhibitor combined with cisplatin in esophageal squamous cell carcinoma cellsObjective:To examine the interaction of the mTORC1/2targeted therapy with cisplatin in ESCC cells, and evaluate the mechanism of mTORC1/2inhibitor which enhancing the effect of cisplatin on ESCC. Methods:Eca-109cells were treated with cisplatin in different concentration alone or combined with rapamycin or PP242. MTT was used to examine the anti-proliferation effect. Flow cytometry and Western-blot analysis were carried out to characterize the apoptosis in Eca109and TE-1cells after treating with different drugs. Akt was examined by Western-blot to investigate the mechnism of the synergistic effect.Results:1. The proliferation of Eca-109cells was inhibited by cisplatin in a dose-dependent manner. The combined treatment with cisplatin and PP242had a synergistic effect on Eca-109cells. In contrast, rapamycin showed only additive interactions with cisplatin in Eca-109cells.2. The percentage of apoptotic cells for the combined treatment was significantly higher than that for PP242or cisplatin alone in both Eca-109and TE-1cells (p<0.05).3. In cisplatin treated Eca-109cells, the phosphorylatio level of Akt(Ser473) was elevated. While combined treatment with cisplatin and PP242reduced the Akt phosphorylation level.Conclusion:1. PP242exerted a synergistic antitumor effect by combining cisplatin in Eca-109cells.2. PP242could promote cisplatin-induced apoptosis in both ESCC cells and modulated cisplatin-induced Akt activity.