Study Of CAB39 Promotes Cisplatin Resistance In Bladder Cancer Via The LKB1-AMPK-LC3 Pathway

Objective:Bladder cancer is the tenth most common cancer in the world,with about 573,000 new cases and 213,000 deaths per year.Cisplatin-based comprehensive chemotherapy is the first-line regimen of chemotherapy,adjuvant chemotherapy and neoadjuvant chemotherapy for bladder cancer.However,the problem of chemotherapy resistance greatly limits the long-term survival of patients.Although immunocheckpoint inhibitors and FGFR inhibitors have been approved for the treatment of chemotherapy intolerant or resistant advanced bladder cancer,their effectiveness is poor and resistance is likely to develop.It is of great significance to explore the mechanism of cisplatin resistance in bladder cancer and develop chemotherapeutic sensitizing drugs.Therefore,this study aims to explore the molecular mechanism of cisplatin resistance in bladder cancer through proteomics and gene regulation on the basis of the establishment of cisplatin resistant bladder cancer cell lines,providing new ideas for bladder cancer combination therapy.Methods:(1)The cisplatin resistant cell lines T24 r,J82r and UMUC3 r were established by multiple high-dose cisplatin shock to the corresponding bladder cancer cell lines T24,J82 and UMUC3,and the cell viability and proliferation were evaluated by CCK8 assay and clonal formation assay.ROS levels were detected by flow cytometry after H2 DCFDA staining.(2)DIA protein spectroscopy was used to analyze the proteomic differences of T24 and T24 r respectively in the condition of cisplatin shock and no cisplatin shock,and to screen common differential proteins for differential gene functional enrichment analysis.(3)The up-regulated genes in T24 r were knocked down using CRISPR/Cas9 technology to identify cisplatin resistance-related proteins,and verified by Western Blot and gene overexpression.After determining the cisplatin resistance gene,the downstream pathway of cisplatin resistance genes was further explored through gene regulation.Mitochondrial membrane potential was analyzed by JC-1 staining.Mitochondrial morphology and autophagy flux were observed by transmission electron microscopy.Mito-Tracker Red CMXRos were used to label mitochondria and GFP-LC3 to label autophagy.The autophagy of mitochondria was observed by two-photon laser confocal scanning microscope.The levels of CAB39,LKB1,AMPK,ULK1,LC3,PINK1,PARK2 and other related proteins were detected by Western Blot.The subcutaneous bladder cancer models were established in nude mice to evaluate the cytotoxic effect of the autophagy inhibitor chloroquine phosphate combined with cisplatin and gemcitabine(GC)on bladder cancer and the renal toxicity of the drugs.Results:(1)After about 6 months of induction,we successfully established cisplatin resistant bladder cancer cell lines T24 r,J82r,UMUC3r;Compared with their parents,the clonogenesis and cell proliferation of T24 r and UMUC3 r were decreased,while the cell proliferation capacity of J82 r was enhanced compared with that of J82,but the clonogenesis capacity was not significantly changed.After cisplatin shock,intracellular ROS levels of T24 r,J82r and UMUC3 r were significantly lower than those of their parents.(2)DIA protein spectrum analysis showed that 136 proteins were significantly up-regulated and 127 proteins were significantly down-regulated in T24 r cells compared with T24 cells without cisplatin shock.After cisplatin shock,132 proteins and 147 proteins were significantly up-regulated and down-regulated in T24 r cells compared with T24 cells.35 proteins were up-regulated in T24 r with or without cisplatin shock.GO and KEGG functional enrichment analysis of these 35 proteins showed that they were mainly involved in antiviral response,NF-kappa B pathway,mitochondrial activity,membrane transport and AMPK pathway,etc.Through literature research,11 cisplatin resistance candidate genes were further selected from the above35 genes.(3)11 cisplatin resistance candidate genes were knocked down in cisplatin resistant cell lines,and it was found that after CAB39 was knocked down,the sensitivity of T24 r and UMUC3 r to cisplatin was significantly increased,and the intracellular ROS level was significantly increased.After overexpression of CAB39 in T24 and J82,their sensitivity to cisplatin and intracellular ROS levels were significantly reduced.However,CCK8 assay showed that CAB39 negatively regulated the proliferation of bladder cancer cells.Further,the gene LKB1,STK24,STK25,STK26,STK39 and OSR1 downstream of CAB39 was knocked down in T24 r and UMUC3 r.It was found that only after the knockdown of LKB1,the sensitivity of T24 r and UMUC3 r to cisplatin was significantly increased,and the intracellular ROS level was significantly increased.After cisplatin shock,T24 R and UMUC3 R had higher autophagic flux and healthier mitochondrial morphology than their parents.In addition,the autophagy inhibitor chloroquine phosphate significantly enhanced the killing effect of cisplatin on T24 r,J82r and UMUC3 r,and significantly increased the intracellular ROS levels of T24 r,J82r and UMUC3 r.After cisplatin shock,mitochondrial membrane potential of T24 r was significantly higher than that of T24.After cisplatin combined with chloroquine treatment,mitochondrial mode potential of T24 r was similar to that of T24.Autophagy played an important role in maintaining mitochondrial health after cisplatin shock.Western Blot results showed that compared with their parents,the expression level of p-AMPKα and the proportion of LC3II/I in T24 r,J82r and UMUC3 r cells were significantly increased,while the expression levels of LKB1,PINK1,PARK2 and AMPK were not significantly changed.Increased activation of AMPK resulted in increased autophagy flux in drug-resistant cells.After CAB39 was knocked down in T24 r and UMUC3 r,the expression level of p-AMPKα and the proportion of LC3II/I were significantly decreased.After CAB39 was overexpressed in T24 and J82,the expression level of p-AMPKα and the proportion of LC3II/I were significantly increased.The laser confocal imaging showed that autophagy flux decreased significantly after CAB39 was knocked down in T24 r.After cisplatin shock,the mitochondrial membrane potential of CAB39 knockdown cell line was significantly lower than that of control group.After cisplatin shock,CAB39 can significantly enhance autophagy through the LKB1-AMPK-LC3 pathway and maintain the functional health of mitochondria.After knocking down ULK1,a key autophagy gene,in T24 r and UMUC3 r,the sensitivity of them to cisplatin was significantly increased,further demonstrating that autophagy mediated cisplatin resistance in bladder cancer.In animal experiments,compared with simple GC regimen,chloroquine phosphate combined with GC significantly reduced tumor load in nude mice.But there was also an increase in kidney damage.Conclusion: Induction of cisplatin-resistant bladder cancer cell lines by multiple high-dose cisplatin-shock method is a reliable means to study cisplatin-resistant bladder cancer.Enhanced autophagy flow,healthy mitochondrial status,and low intracellular ROS levels are important features of cisplatin-resistant bladder cancer cells.CAB39 enhanced autophagy of bladder cancer cells through LKB1-AMPK-LC3 pathway,maintained healthy mitochondrial structure and function,and promoted cisplatin resistance of bladder cancer.However,CAB39 has a certain inhibitory effect on the proliferation of bladder cancer cells,and its use as a therapeutic target remains to be discussed.Chloroquine phosphate significantly enhances the efficacy of GC regiments for bladder cancer,but may increase the risk of cisplatin intolerance.