Synergistic Inhibition Of BEZ235 And Docetaxel And The Biomarker Effects Of Mortalin Protein In Breast Cancer

Aims:To investigate the anti-tumor effects of BEZ235 in combination with docetaxel in vitro and in vivo, to explore the role of Mortalin in breast cancer, and to find a new biomarker and the possible therapeutic target for breast cancer.Materials and methods:1) Experiments in vitro:MTT and colony formation assay showed that simultaneous administration of BEZ235 and docetaxel could generate the optimal synergistic effect on human breast cancer cell; the motility ability was measured by wound scratch and transwell assay; Hoechst33342 fluorescence staining and western blot assays were used to detect the apoptosis of breast cancer cells after the drug treatment; Western blot assays were used to detect the expression of PI3K/Akt/mTOR signaling pathway and EMT markers of cells treated by BEZ235 and docetaxel. RT-PCR assay used to show the expression level of Mortalin in different breast cancer cells, then detceted the ability of EMT in the breast cancer cells transfected by si-Mortalin.2) Experiments in vivo:MDA-MB231 cells were injected subcutaneously into the female nude mice. And the developing tumors were measured and mice randomly assigned to four different treatment groups (5/group). After 4 weeks of continuous administration, expression of E-cadherin, p-AKT, p-S6, p-4ebp1, cleaved caspase3 protein were analyzed by immunohistochemistry for detecting the synergistic inhibit of drugs. A total of 155 invasive ductal carcinoma of breast tissue were strict follow-up, were selected for immunohistochemical (IHC) staining of Mortalin protein. The correlations between high expression of Mortalin and the clinical features of patients with breast cancer were evaluated and the survival rates were calculated by the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was also analyzed.Results:MTT results exhibited synergistic inhibition of BEZ235 and docetaxel in proliferation of MDA-MB231 and MDA-MB468 cells; colony formation assay, wound scratch assay and transwell assay showed that colony forming ability and the motility were inhibited by combination therapy. Hoechst 33342 stainning and western blot results showed that BEZ235 in combination with docetaxel could activate caspase-3 and induce apoptosis. Western blot and immunofluorescence (IF) staining showed that combination therapy reduced the protein levels related with PI3K/AKT/mTOR signaling pathway. Western blot and IF staining also showed that combination therapy reduced the expression of snail, slug, twist and vimentin and up regulate the expression of E-cadherin. RT-PCR results showed that Mortalin protein was highly expressed in MDA-MB231 cells, and Mortalin protein expression was decreased by transfect siRNA in MDA-MB231 cells. Moreover, knockdown Mortalin expression significantly decreased the migration and invasiveness of breast cancer cells compared with the control group. Furthermore, western blot showed combination therapy was significantly inhibit the expression of Mortalin protein, and the expression of epithelial marker E-cadherin was increased, but the mesenchymal markers were decreased in Mortalin-siRNA-trasfected cell. Combination therapy was significantly inhibite MDA-MB231 cell tumorigenicity in nude mice, and decrease the number of cells positive for p-AKT, p-S6, p-4ebpl and increase in the number of cells with positive cleaved caspase3 and E-cadherin. in treated xenograft tissues.IHC staining and IF staining showed that Mortalin protein mainly cytoplasmic staining pattern in breast cancers. The strongly positive rate of Mortalin protein was 63.9%(99/155) in breast cancers and was significantly higher than in DCIS 34.6%(18/52) and adjacent non-tumor tissues 15.6%(7/45). High expression of Mortalin was closely correlated with histological grade, clinical stage, lymph node metastasis, lower disease free survival (DFS) and overall survival (OS) rates of patients with breast cancer. Moreover, multivariate analysis suggested that Mortalin emerged as a significant independent prognostic factor along with clinical stage and Her2 expression status in patients with breast cancer.Conclusions:1) Combination treatment synergistically inhibit the proliferation of breast cancer cells.2) Combination treatment induces apoptosis and synergistically inhibit the EMT in breast cancer cells.3) Mortalin overexpression in breast cancer cells and induces properties of EMT, and it may be a useful poor prognostic biomarker and a potential therapeutic target for patients with breast cancers.