Mechanistic Studies Of Inhibitory Role Of Proguanil On The Growth Of Bladder Cancer Via Mediating EGFR Endocytosis And Ubiquitination Degradation

PURPOSE: Muscular invasive bladder cancer(MIBC)is characterized by high invasion,easy metastasis and high mortality.Surgery and traditional chemotherapy are still the current standard treatment for MIBC patients.However,the high mortality rate caused by tumor recurrence and metastasis after treatment is a major clinical problem that urgently needs to be resolved.Therefore,there is an urgent need to find new treatments for MIBC.The antitumor activities of biguanide drugs have attracted considerable attention in recent years.Studies have shown that among the biguanides drugs including phenformin,buformin,metformin and proguanil,proguanil has the strongest inhibitory effect on the growth of seven types of bladder cancer cells.However,the antitumor mechanism of proguanil remains unclear.We previously demonstrated that proguanil strongly binds to EGFR through surface plasmon resonance imaging(SPRi)experiment,and found that proguanil significantly reduces its expression through WB.Therefore,this research aims to explore the specific mechanism of proguanil down-regulating EGFR in bladder cancer cells to provide a theoretical basis for the clinical treatment of bladder cancer patients with proguanil.METHODS: 3D photocrosslinked microarray was used to immobilize proguanil,and then bladder cancer cell lysates were distributed on the surface of the microarray.The captured target proteins on the microarray surface were detected by SPRi assay.The basic expression of EGFR in four types of MIBC cells was detected by WB and RT-PCR assay.The sensitivity of bladder cancer cells to proguanil after stable knockdown or overexpression of EGFR by lentivirus vectors was assessed by MTT clone formation assay,and Transwell assay.Immunofluorescence and Co-IP experiments were used to explore the specific mechanism of proguanil down-regulating the expression of EGFR.Finally,xenograft tumor model was established by injecting T24 and Sh EGFR-T24 cells and was treated or untreated with proguanil.The anti-tumor effect of proguanil in vivo was determined by comparing the difference of tumor size between different groups.The toxicity of proguanil in vivo was evaluated by weight changes and pathological analysis of liver and kidney tissue in mice.RESULTS:(1)EGFR is high expressed in bladder cancer tissues,which is closely related to the poor prognosis of patients.(2)The expression of EGFR in bladder cancer cells was positively correlated with its sensitivity to proguanil.(3)Proguanil mediated EGFR endocytosis in a clathrin-independent manner,and silencing Clathrin(Cla)did not affect the sensitivity of bladder cancer cells to proguanil,while the sensitivity of bladder cancer cells to proguanil was weakened after silencing Caveolin-1(Cav-1).(4)Proguanil mediates endocytosis of EGFR and recruites c-Cbl to promote EGFR ubiquitination and degradation in lysosomal.(5)Vivo studies further confirmed that proguanil inhibits the proliferation of bladder cancer cells by down-regulating the EGFR signaling pathway.CONCLUSION: Proguanil inhibits the proliferation of bladder cancer cells via recruiting c-Cbl to enhance the ubiquitination and degradation of EGFR in the lysosome in Non-Clathrin endocytosis fashion.This study provides a new candidate drug for the individualized treatment of patients with high expression of EGFR.