The Role And Mechanisms Of TFEB-mediated Autophagy In Gastric Cancer Drug Resistance

BackgroundGastric cancer is the second leading cause of cancer deaths worldwide. The incidence of gastric cancer in China accounts for 42.5% of global incidence. There is one Chinese died of gastric cancer every 2 to 3 minutes. Most patients with gastric cancer were at an advanced stage upon diagnosis. Chemotherapy is the main treatment option for the prevention of recurrence and metastasis. However, drug resistance in patients with gastric cancer drugs often causes ineffectiveness of chemotherapy, which may lead to the failure of chemotherapy and poor prognosis. Numerous studies have reported the mechanisms of gastric cancer drug resistance, but its underlying mechanisms have not been yet clarified. These results indicates that the mechanism of drug resistance is very complex and need further investigation from a new prospective. Previous studies suggest that autophagy is an important mechanism that functions as an important mechanism to the clearance of the damaged intracellular proteins and organelles. It is closely related to development of tumors, and have been regarded as a focus of cancer research in recent years. The role of autophagy in cancer is controversial. Some studies show that autophagy can inhibit tumorigenesis, while some studies suggest that tumor cells can harness autophagy as an important survival mechanism in a variety of harsh environments. Then, does autophagy promotes survival of gastric cancer cells upon chemotherapy? How is autophagy regulated? What are the key factor or signaling pathway in autophagy? Previous studies have found that m TOR is the key autophagy regulation pathway. Its downstream effector transcription factor EB, or TFEB is a important molecule that regulates autophagy at the transcriptional level. Its regulation of autophagy have been found to play a key role in a variety of metabolic disorders. Is TFEB involved in gastric cancer malignant phenotype, especially gastric cancer drug resistance? Does TFEB promote gastric cancer drug resistance by regulation of autophagy? How? So far, these have not been clarified.Objective:1、 To clarify the role of autophagy in gastric cancer cell survival during chemotherapy;2、 To investigate the changes of m TORC1/TFEB signaling pathways in chemotherapy-induced autophagy;3、 To clarify the role and mechanisms of TFEB-mediated autophagy in regulating gastric cancer survival.Methods:1、 Western blot, MDC staining, RFP-GFP-LC3 adenoviral transfection, electron microscopy and other in vitro methods were used to detect change of autophagy upon chemotherapy;2、 Immunohistochemistry of LC3 expression was used to evaluate autophagy upon chemotherapy in nude mice tumor transplantation models.3、 MTT, apoptosis experiments were used to evaluate the effect of autophagy in gastric cancer cell survival in vitro.4、 in vivo nude mice tumor transplantation experiments was used to comfirm the role of autophagy in gastric cancer cell survival upon chemotherapy;5、 Western blot was used to observe the changes of m TOR signaling pathway;6、 Western blot, immunofluorescence and other methods were used to observe the changes of m TOR downstream transcription factor TFEB;7、 MTT and apoptosis-related molecular biology techniques were used to evaluate the mechanisms of TFEB in promoting gastric cancer cell survival by transcriptionally activating its downstream target genes Lamp1.Results:1、 Autophagy promotes the gastric cancer cell survival during chemotherapy(1) Chemotherapy induces autophagy in gastric cancer cellsChemotherapy drugs Epirubicin(EPI) and cisplatin(CIS) increased LC3 II expression and p62 degradation. MDC staining showed that EPI can increase the number of acidic vesicles in gastric cancer cells. After RFP-GFP-LC3 adenovirus infection, immunofluorescence showed that EPI increased the number of autophagosomes(RFP + GFP +) significantly. Electron microscopy showed that EPI treatment increased the presence of double membrane structure. Addition of lysosomal inhibitors CQ, vacuolar ATP inhibitors BAF significantly inhibited the autophagic flux induced by chemotherapy. In vivo experiments also showed that intraperitoneal injection of EPI increased expression of tumor LC3 in tumors derived from tumor-bearing nude mice.(2) Inhibition of autophagy increased the therapeutic sensitivity of gastric cancer MTT and apoptosis assay showed that the use of lysosomal inhibitors CMTT and apoptosis assay showed that the use of lysosomal inhibitors CQ can increase the sensitivity of gastric cancer cells to EPI and the CIS by autophagy inhibition. In vivo experiments showed that CQ plus EPI significantly reduced tumor volume and weight in tumor-bearing nude mice.2、m TORC1 / TFEB signaling pathway is the key pathway of chemotherapy induced autophagy(1) Chemotherapeutic agents inhibited m TORC1 pathway.EPI and CIS treatment induced a time-dependent decrease of m TOR phosphorylation at 2448. Similar results was observed in the phosphorylation status of m TOR downstream effector p70S6 K.(2) Chemotherapy drugs induced the dephosphorylation of m TORC1 downstream transcription factors TFEB and subsequent translocation into the nucleus.EPI promoted a time-dependent dephosphorylation of TFEB and nuclear translocation. Immunoprecipitation showed that m TOR, Raptor, YWHA interacted with TFEB under normal condition, and and the interaction diminished during chemotherapy.(3) Immunohistochemistry showed high expression of TFEB was observed in gastric cancer and is closely related with gastric cancer prognosis;3、TFEB promotes the survival of gastric cancer cells by regulating Lamp1 mediated lysosomal function and autophagy.(1) TFEB overexpression increased gastric cancer cell survival by increased lysosomal biogenesis and autophagy.MTT and apoptotic assay confirmed that overexpression of mutant S11 A TFEB reduced chemotherapy-induced apoptosis and promoted cell survival in gastric cancer. TFEB promoted autophagy and transcriptionally activated expression of Lamp1 and other related molecules.(2) Suppression of TFEB increased gastric cancer chemosensitivity by inhibiting chemotherapy-mediated autophagySuppression of TFEB increased gastric cancer chemosensitivity. TFEB suppression inhibited EPI-mediated LC3 II expression and p62 degradation and significantly suppressed an increase in the average number of Lysotracker per cell. Inhibition of TFEB in drug resistant cells reversed its drug resistance;(3) TFEB promotes gastric cancer cells resistant to chemotherapy by regulating Lamp1 mediated lysosomal function.Lamp1 suppression increased chemosensitivity of gastric cancer. Lamp1 inhibition can promote EPI-induced nuclear translocation of TFEB in a feedback mechanism.Conclusion:Protective autophagy is critical for gastric cancer cell survival during chemotherapy. Autophagy inhibition can increase therapeutic sensitivity. m TOR signaling pathway plays an important role in chemotherapy-induced autophagy. TFEB, an important m TOR downstream regulator of autophagy at the transcriptional level is significantly highly expressed in gastric cancer tissues and is closely related with gastric cancer prognosis. Chemotherapy can induce TFEB dephosphorylation and subsequent nuclear transcription. TFEB promotes gastric cancer cell survival by promoting autophagy and Lamp1 mediated lysosomal-mediated function during chemotherapy...