MTORC1/TFEB-mediated Lysosomal Adaptation Induces Cisplatin Resistance In Hepatocellular Carcinoma

BackgroundThe treatment of hepatocellular carcinoma(HCC)has entered the era of comprehensive treatment,however,postoperative recurrence and palliative treatment of advanced HCC are still the bottleneck of clinical HCC treatment.Although in the latest domestic guidelines for the diagnosis and treatment of primary liver cancer,the platinum-based FOLFOX4 chemotherapy regimen is approved for the first-line treatment of unsuitable surgical resection or locally advanced and metastatic liver cancer,but in most international mainstream guidelines,systemic chemotherapy is not recommended as the first-line treatment because HCC itself is not highly sensitive to cytotoxic chemotherapy drugs,which restricts the development of systemic chemotherapy in the treatment of HCC.Studies have found that although various platinum drugs are currently used in tumor treatment,most of the drug resistance mechanisms of tumor cells to the first-generation platinum chemotherapy drug cisplatin(CDDP)are also found in carboplatin,lobaplatin and oxaliplatin and other second-generation and third-generation platinum-based chemotherapy drugs were found.In addition,cisplatin still has important application value and good treatment prospects in local chemotherapy of HCC,including hepatic arterial infusion chemotherapy(HAIC)and transcatheter arterial chemoembolization(TACE).In summary,we selected cisplatin as a tool drug for studying the resistance of HCC to platinum drugs,and explored the potential mechanism of HCC cisplatin resistance,aiming to provide strategies for the improvement of existing platinum-based chemotherapy regimens.Our research group’s previous research found that HCC cells can degrade the cell components damaged by chemotherapy drugs through cytoprotective autophagy,produce nutrients,maintain the survival of HCC,and eventually lead to chemotherapy resistance.In this process,the maintenance of high-level autophagic flux requires the consumption of a large number of lysosomes.Transcription factor EB(TFEB)plays a key role in regulating lysosomal biogenesis and autophagy in tumor cells.After TFEB is translocated into the nucleus,it will initiate transcription of lysosomal biogenesis and autophagy-related genes in a coordinated lysosomal expression and regulatory network,and then maintain autophagic flux,this process of co-regulation of lysosomal biogenesis and autophagy mediated by TFEB is called lysosome adaptation.The classic TFEB activation mode is: when the nutrition is sufficient,the activity of mammalian target of rapamycin C1(mTORC1)on the phosphorylation modification of TFEB is enhanced,preventing TFEB from translocating into the nucleus.In the starvation state,the phosphorylation activity of mTORC1 is reduced,and TFEB is dephosphorylated and translocated into the nucleus.Thus,this lysosomal adaptation often occurs when cellular amino acid levels are low.However,the liver has a dual blood supply system,and under normal circumstances,the vast majority of HCC cells are in a nutrient-adequate state.We thus simulated a state of nutrient adequacy in vitro,and interestingly,cisplatin still induced the initiation of lysosomal adaptation in HCC cells.More and more studies have found that lysosomes not only have the function of digestion and degradation,but also serve as organelles that sense the level of cellular amino acids and reversely regulate the activity of cellular nutrition-related pathways,including the mTORC1/TFEB pathway that we are concerned about.In addition,we initially found that cisplatin can induce HCC cells mitochondrial reactive oxygen species(Mitochondrial reactive oxygen species,mt ROS),lead to LMP,and then cause the efflux of lysosomal content.Therefore,we suspect that cisplatin may affect the accumulation of lysosomal amino acids by inducing LMP,leading to changes in the activity of the mTORC1/TFEB pathway and ultimately initiating lysosomal adaptation.Methods(1)MTT method was used to detect the cell viability of HCC-LM3 and Huh7 cell lines treated with CDDP(0-64 μg/m L)for 24 hours,expressed as half inhibitory concentration(IC50).(2)Immunofluorescence technology was used to observe the translocation of TFEB into the nucleus of HCC-LM3 and Huh7 cell lines after the action of CDDP under the confocal laser microscope;the nuclear protein was extracted,and the expression change of TFEB in the nucleus was detected by Western Blot.(3)Verify whether CDDP can induce lysosome biogenesis and autophagy gene transcription and protein expression in the CLEAR network of HCC-LM3 and Huh7 cell lines by RT-q PCR and Western Blot.(4)Construct TFEB knockdown cell models of HCC-LM3 and Huh7 cell lines,and verify the expression changes of related proteins in CDDP-induced lysosomal adaptation after TFEB knockdown by Western Blot;use flow cytometry to detect TFEB knockdown,apoptotic level of HCC cells under the action of CDDP.In addition,the combined application of autophagy inhibitors 3-methyladenine(3-MA)and chloroquine(CQ),the changes in cell viability of HCC cells after CDDP treatment for 24 hours were detected by MTT.(5)Western Blot was used to detect mTORC1 phosphorylation activity and TFEB phosphorylation level in HCC-LM3 and Huh7 cell lines after CDDP treatment,and explore the changes of mTORC1 phosphorylation activity and its regulation on TFEB during this process.(6)The interaction between Rag GTPases and mTORC1 and TFEB was detected by immunofluorescence experiments and co-immunoprecipitation experiments,and then reflected the changes in the activity of Rag GTPases in HCC cells under the action of CDDP.(7)Using high-performance liquid chromatography-tandem mass spectrometry X(LC-MS/MS)and amino acid content detection kits to detect changes in the relative content of amino acids in lysosomes of HCC cells after the action of CDDP,and then explore the effect of CDDP on the accumulation of amino acids in lysosomes Impact.(8)To verify whether CDDP can induce mitochondrial reactive oxygen species(mt ROS)and lead to changes in lysosomal membrane permeability.First,the fluorescence intensity of Mito SOX was detected by flow cytometry to evaluate the level of mt ROS,and then Western Blot was used to detect the changes in the contents of cathepsin B and cathepsin D in the cytoplasm after removal of lysosomes to confirm whether LMP occurred.(9)Through immunofluorescence and co-immunoprecipitation techniques,the interaction of Rag GTPases with mTORC1 and TFEB was detected after the combined application of mitochondria-specific reducing agent Mito-TEMPO,and the change of Rag GTPases activity was verified after CDDP induced LMP through mt ROS.(10)Through in vivo experiments,wild-type mouse HCC cell line Hep 1-6 and its TFEB stable knockdown cell line were inoculated subcutaneously in mice.After tumor formation,the changes in tumor diameter in mice after CDDP treatment were observed,and tumors in each group were compared.CDDP sensitivity.(11)Immunohistochemical experiments were performed on mouse tumor tissues to detect the expression of lysosome biosynthesis,autophagy and apoptosis-related proteins after CDDP treatment.Results(1)After CDDP was applied to HCC cells,the co-localization of TFEB and the nucleus was significantly increased by confocal laser microscopy,and the expression of TFEB protein in the nucleus was increased by Western Blot.(2)After CDDP was applied to HCC cells,the results of q PCR and Western Blot showed that the transcription levels of lysosome biogenesis and autophagy-related genes such as LAMP1,LAMP2,LAMP3,CLCN7,MCOLN1,CTSB and CTSD in the CLEAR network of HCC cells were significantly up-regulated,The ratio of autophagy-related protein LC3-Ⅱ/LC3-Ⅰ in HCC cells was significantly increased,the level of autophagy substrate p62 was decreased,and the levels of lysosomal membrane proteins LAMP1,cathepsin B and cathepsin D were significantly increased.(3)The results of Western Blot showed that,compared with the non-knockdown group,the ratio of autophagy-related protein LC3-Ⅱ/LC3-I in HCC cells in the TFEB knockdown group was relatively decreased after CDDP treatment,the accumulation of autophagy substrate p62 increased,and the lysate The expressions of enzymosomal membrane proteins LAMP1,cathepsin B,and cathepsin D were significantly reduced;MTT results showed that compared with the non-knockdown group,the TFEB knockdown group and the combined application of autophagy inhibitors such as 3MA and CQ were more effective than CDDP after 24 hours.The IC50 value is lower.The results of Annexin V-FITC/PI double staining flow cytometry showed that the apoptosis level of TFEB knockdown group was significantly increased compared with the non-knockdown group.(4)After CDDP was applied to HCC cells,the co-localization of mTORC1 and TFEB was observed to decrease by confocal laser microscopy,and the protein levels of p70S6 k and 4EBP1 downstream of mTORC1 were detected by Western Blot,while the phosphorylation level of TFEB decreased and the expression of TFEB increased.(5)After CDDP was applied to HCC cells,immunofluorescence experiments showed that the co-localization of TFEB and mTORC1 with Rag B and Rag C decreased,and co-immunoprecipitation experiments showed that the interactions of TFEB and mTORC1 with Rag B and Rag C decreased respectively.(6)After CDDP was applied to HCC cells,the results of LC-MS/MS and amino acid content detection showed that the amino acid content in the lysosome lumen decreased.(7)The results of flow cytometry and Western Blot showed that the fluorescence level of Mito SOX was significantly enhanced after the treatment of CDDP,and the levels of cathepsin B and cathepsin D in the cytoplasm were increased.The fluorescence level is relatively weakened,and the levels of cathepsin B and cathepsin D in the cytoplasm are reduced.(8)The results of co-immunoprecipitation experiments showed that the interaction between TFEB and mTORC1 and Rag B and Rag C respectively decreased after CDDP treatment,and after the combined application of mitochondria-specific reducing agent Mito-TEMPO,TFEB and mTORC1 interacted with Rag B and Rag C respectively.The interaction is relatively increased.(9)In vivo experiments showed that the growth rate of tumors in CDDP-treated mice was significantly slower than that in the non-treated group,and the growth of tumors in TFEB-knockdown mice was more restricted after treatment.The final volume of the tumor was measured after the tumor was taken,and it was found that the tumor volume of the mice in the CDDP treatment group was significantly smaller than that in the non-treatment group,and the tumor volume of the mice in the TFEB knockdown group decreased more significantly after treatment.Conclusions:(1)Lysosome adaptation is involved in the resistance of HCC cells to cisplatin.(2)The lysosomal adaptation initiated by hepatocellular carcinoma to cisplatin stress is mediated by mTORC1/TFEB pathway.(3)Under the action of cisplatin,the change of LMP in hepatoma cells decreased the accumulation of amino acids in lysosome,which led to the inhibition of mTORC1 phosphorylation and the activation of TFEB.