Study On Function And Mechanism Of TFEB In The Regulation Of Biological Characteristic Of Pancreatic Cancer

Part Ⅰ Study on relationship between TFEB and biological characteristic of pancreatic cancerBackground: Pancreatic cancer is a highly lethal disease.Many studies have shown that autophagy is involved in the biological characteristic of pancreatic cancer and it is closely related to its invasion and metastasis ability.TFEB is currently considered to be a key factor in the regulation of the autophagic-lysosomal pathway.Therefore,further clarification of the relationship between TFEB and biological characteristic of pancreatic cancer is expected to be the key to improving the current status of treatment of pancreatic cancer.Methods: Immunohistochemistry,real time PCR and Western blot were used to detect the level of TFEB and to explore its relationship with pathological grade,clinical stage and prognosis in pancreatic cancer.The lentiviral technique was used to intervene in the expression of TFEB in pancreatic cancer cells,and its relationship with the biological characteristic of pancreatic cancer was detected by in vitro and in vivo experimental models.Results: We found that the expression level of TFEB in pancreatic cancer tissues was higher than that in normal pancreatic tissues.The expression of TFEB in cancer tissue is closely associated with the differentiation degree.Kaplan-Meier analysis demonstrated the higher level of TFEB predicts a worse overall survival.Using CCK8,clone formation and subcutaneous xenograft experiments,we found that inhibition of TFEB in pancreatic cancer cells suppressed the proliferation of pancreatic cancer cells.Using the wound healing,transwell and in vivo liver metastasis model,we found that inhibition of TFEB suppressed the invasion and migration ability of pancreatic cancer cells.Conclusions: TFEB is highly expressed in pancreatic cancer and is associated with poor pathological differentiation and poor prognosis.Inhibition of TFEB suppresses the proliferation and invasion and migration of pancreatic cancer cells.Part Ⅱ Study on upstream/downstream of TFEB in pancreatic cancerBackground: Pancreatic cancer is one of the most aggressive cancers.Endocytosis and transduction of adhesion signals play important roles in promoting migration.The TFEB-mediated CLEAR network involves extremely broad downstream regulation.However,how TFEB regulates downstream molecules and what factors regulate TFEB deserve further exploration.Methods: Bioinformatics analysis and Western blotting were used to identify the relationship between TGF-β and TFEB.Transmission electron microscopy and confocal microscopy were used to identify that TFEB is the key factors in TGF-β-induced autophagy.The biological effects of TFEB-driven autophagy were investigated in vitro using transwell and wound healing assays and in vivo using liver metastasis mice model.Luciferase assays and motif analysis were used to assess the regulation of RAB5 A gene promoter activity by TGF-β-induced TFEB.Finally,bioinformatics analysis was used to analyze the relationship between RAB5 A and pathological grade,clinical stage and prognosis of pancreatic cancer.Results: We demonstrated that TGF-β induces TFEB expression via the canonical smad pathway in Smad4-positive PC cells and facilitates TFEB-mediated autophagic activation.TFEB-driven autophagy caused by TGF-β regulates RAB5A-dependent endocytosis of Itgα5β1 and promotes the progression of PC cells.We further showed that enhanced TFEB expression and its direct target RAB5 A both predict poor prognosis in PC patients.Conclusions: Our findings reveal TFEB-driven autophagy is required for TGF-β induced migration and metastasis of PC cells by promoting endocytosis of Itgα5β1 and focal adhesion disassembly through the TGF-β-TFEB-RAB5 A axis.Our results highlight the potential utility of suppressing TFEB-driven autophagy to block PC metastasis.Part Ⅲ study on effect of alantolactone,a TFEB inhibitor,on pancreatic cancer cellsBackground: Although surgical resection of pancreatic cancer has made great progress,chemoresistance remains a major obstacle in the treatment of pancreatic cancer.The development of new drugs or a combination of drugs is the key to improving the status of treatment in pancreatic cancer.Methods: We predicted that the natural compound alantolactone is an inhibitor of TFEB by virtual screening.Transmission electron microscopy and confocal microscopy were used to evaluate the effect of alantolactone on autophagy.Cell viability and the proliferation of pancreatic cancer cells treated with alantolactone were assessed by CCK8 assays,cloning formation assays and xenograft experiments.The effect of combination treatment with alantolactone and oxaliplatin was measured by the combination index.Results: We found that alantolactone caused the accumulation of autophagosomes due to inhibition of CTSB/CTSD expression and activity,resulting in lysosomal functional damage.In addition,alantolactone inhibited the proliferation of pancreatic cancer cells in vitro and in vivo.It caused apoptosis and enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin.Conclusions: Our data demonstrated that alantolactone,which caused lysosomal dysfunction leading to inhibition of autophagy degradation,was a pharmacological inhibitor of TFEB in pancreatic cancer cells.It caused apoptosis and enhances the chemosensitivity of pancreatic cancer cells to oxaliplatin.