Study On The Mechanism Of Synergistic Inhibition By Co-treatment With BEZ235and TSA And The Biomarker Effects Of Ezrin Protein For Lung Cancer

Background:Lung cancer is the foremost cause of cancer-related death worldwide, and drug-resistant and metastasis are the leading causes of death in patients with lung cancer. Improving clinical outcomes will require a better understanding of the biological processes that initiate metastasis and finding which target will respond to therapies.PI3K inhibitor BEZ235and histone deacetylase inhibitor TSA are effective therapy drugs for small molecule target, however, which were limited by the side effects and drug resistance after high-dose administration. Recent studies showed that the combination of targeted therapy was significant for reducing the dose, overcoming the drug resistance, and synergistic inhibition in the tumor. Co-treated with BEZ235and TSA was significantly synergy suppressed in pancreatic and prostate cancer, but which role and mechanism in lung cancer is unclear. Our previous studies indicated that Ezrin played an important role for breast and cervical cancer inducing the epithelial-mesenchymal transition.This study is focus on the role of Ezrin in lung cancer treated with BEZ235and TSA.Objectives:To identify the impact on the biological behavior of lung cancer A549cells after treated with TSA and BEZ235in both vitro and vivo, explore the role of Ezrin signaling pathway in lung cancer treated with BEZ235and TSA, and find a new biomarker and therapeutic target for lung cancer.Materials and methods:1) Experiment in vitro:lung cancer cell line A549 proliferation treated by TSA and BEZ235was detected by MTT and colony formation assay; Annexin-V/PI double staining flow cytometry and Hoechst3334fluorescence staining were used to detect the apoptosis of A549cells after the treatment; Western blot assays were used to detect the activity of PI3K/Akt, HDAC, Ezrin signaling pathway, and EMT markers of the cells treated by BEZ235and TSA. Furthermore, we detected the ability of EMT in the cells transfected by si-Ezrin. The motility and invasiveness capacity of A549cell treated by drugs was showed by wound healing assays;2) Experiment in vivo:Nude mices transplanted tumor model of A549cell lines were randomly divided into separately administered group, jointly administered group and the control group. After4weeks of continuous administration, expression of Ki67, Ezrin and p-Ezrin protein was analyzed by immunohistochemistry for detecting the synergistic inhibition abilities of drugs in the tumor were detected. The expression of Ezrin and p-Ezrin mRNA in21cases of lung cancer,15cases of adjacent tissues and8cases of normal lung tissues were measured by RT-PCR; Ezrin and p-Ezrin protein expression in150cases of NSCLC, adjacent tissues and normal tissues were detected by immunohistochemical method, and the correlation between the abnormal expression of protein and clinicopathological characteristics were also analyzed. The survival analysis was used to verify the value of evaluation of Ezrin and p-Ezrin in prognosis of the patient.Results:1) MTT assay and colony formation assays showed that synergistic inhibition of BEZ235and TSA contributed in proliferation of A549cells and colony forming ability; Flow cytometry and Hoechst staining results showed that combination therapy enhanced apoptosis ability in A549cells; Western blot showed that combination of BEZ235and TSA reduced the protein levels related with PI3K/Akt and HDAC signaling pathway; Wound healing assays suggested that the combination therapy inhibited the invasion and motility of A549cells;2) BEZ235and TSA inhibited A549cell tumorigenicity synergistically in nude mice. After the treatment with BEZ235and TSA, the necrosis of tumor was increased, and Ki67, ezrin and p-ezrin levels were down-regulated;3) BEZ235and TSA induced EMT synergisticlly in A549cells, involved E-cad increased and snail down-regulated; Western blot analysis showed that interstitial phenotype of A549cell was enhanced;4) RT-PCR results showed that ezrin mRNA levels in lung cancer were significantly higher than in normal tissues; immunohistochemistry results showed that the positive rates of ezrin, ezrinThr-567and ezrinTyr-353(62.7%,63.3%, and71.3%) significantly higher than the adjacent tissues (31.3%,14.0%, and11.3%) and normal lung tissue (35.7%,14.3%, and7.1%), and were related with clinical stage, pathological differentiation, and lymph node metastasis; Survival analysis showed that abnormal expression ezrinThr-567was closely related with NSCLC survival (p<0.05), whereas in the early NSCLC, ezrin and ezrinThr-567are closely related (p<0.05).Conclusions:1) BEZ235and TSA inhibit the growth of lung cancer A549cells synergistically in both of vivo or vitro;2) BEZ235and TSA inhibit the migration of A549cells via EMT suppressed;3) key molecule of Ezrin signaling pathway plays an important role in EMT of lung cancer cells;4) Ezrin and phosphorylated Ezrin can be used as biomarkers of poor prognosis in NSCLC.