Mutant P53 Regulate Egr-1 Transcription Contributes To Cathepsin L-mediated EMT In Human Lung Cancer

Part I Mutant p53 and Cathepsin L mediated EMTin human lung cancer tissuesObjective: To investigate p53 gene mutations in 78 patients of non-small-cell lung cancers and the relationship between gene mutation and clinicalpathological features. Clarify the relationship between the expression level of Cathepsin L and EMT proteins in tumor tissues and clinical pathological factors.Methods: Tumor and normal lung tissues were obtained from operational samples of 78 patients, p53 gene were automatically sequenced for gene mutation analysis, then compare the results with clinicalpathological features. Western blot and ELISA were used to detect Cathepsin L proteins expression. Analyze the relationship between Cathepsin L with clinical pathological factors. The expression level of E-cadherin and N-cadherin in tumor tissues was analyzed by Western blot and immunohistochemical staining. Analyze respectively the relationship between the expression level of Cathepsin on organization and cell in patients and clinical pathological factors. Analyze respectively the relationship between the mutation p53 and EMT factors.Results: Mutant p53 were detected in 56% of the lung tumor tissues. GCâ†'AT transversions and GCâ†'TA transversions were the major patterns of p53 mutation. The mutant rate was significantly higher in middle-high differentiated tumors than middle-low differentiated tumors. Overexpression of Cathepsin L was associated with tumor differentiation and pathological stage. The Cathepsin L in tumor tissues was significantly higher than that of normal tissues, and the Cathepsin L expression was positively related to tumor differentiation and clinical tumor stage. Mutant p53 may regulate Egr-1 transcription contributes to Cathepsin L-mediated EMT in tumor tissues.Conclusions: The findings indicate that the mutant p53 gain of function plays a pivotal role in EMT of human lung cancer. Overexpression of Cathepsin L was involved in the development of lung cancer progression, and considered to be a prognostic indicator for lung cancer. Part II The mechanism of mutant p53 regulating Cathepsin L mediated EMT in lung cancer cellsObjective: To clarify the mechanism of wild type and mutation p53 regulating Cathepsin L-mediated EMT and explore the role of mutation p53 gain of function in human lung cancer.Methods: Cell lines were stably transfected by different state of p53 gene with lentivirus vector and treated with ionizing radiation. Cell ability to migrate was measured by wound migration assay, and cell invasion was detected by transwell invasion assay. The actin remodeling of tumor cells was assessed with phalloidi. Western blot and immunofluorescence assay were used to detect EMT proteins expression. The DNA binding and transcription activition of Cathepsin L was further determined by chromatin immunoprecipitation assay. Mice in tumor analysis group were sacrificed and tumors were removed for the experiments. The expression level of Cathepsin L and EMT proteins in transplantable tumor was analyzed by Western blot and immunohistochemical staining.Results: Cathepsin L and Egr-1 were up-regulated in mutation type p53 cells after ionizing radiation. Wound migration and transwell invasion assays showed that ionizing radiation induced cell migration, invasion in mutation type p53 cells. In vivo studies, our results showed that ionizing radiation significantly inhibited the xenograft tumor growth. Immunohistochemical staining showed that the expression of E-cadherin was increased and N-cadherin was decreased, and the nucleus Cathepsin L was also increased in p53 mutant tumor tissues.Conclusions: Our findings highlight the critical tumor cell interactions regulated by p53/ Egr-1/ Cathepsin L-dependent EMT that activate intracellular signaling pathways responsible for tumor cell invasion and metastasis. This study will provide Cathepsin L as potential targets for tumor therapy.