Pvrvinium Augments The Chemotherapeutic Sensitivity Of Urothelial Carcinoma Of The Bladder

Maintenance of genomic integrity is critical for cell survival and proliferation.Accumulation of DNA damage can lead to cell senescence and apoptosis.As one of the most lethal types of DNA damage,DNA double-strand breaks(DSBs)caused by genotoxic agents have been exploited to kill tumor cells.However,cells suffering DSBs trigger a series of sophisticated DNA repair mechanism to neutralize DNA damage and maintain their genomic stability.This phenomenon is called DNA damage response(DDR).During the DDR process,global gene expression tends to be repressed.However,mRNAs encoding DDR proteins selectively escape from translational repression upon DNA damage.Therefore,decreased mRNA stability of DDR factors may augment chemotherapeutic efficacy.Careful regulation of mRNA stability is one of the fundamental functions of RNA-binding proteins(RBPs).RNA binding proteins interacte with specific subsets of mRNAs and affect their post-transcriptional fate by eliciting turnover and translation regulatory functions.HuR,as one of the best charicterized RBPs,plays a critical role in the stabilization of about 200 short-lived mRNAs.Its cytoplasmic accumulation has been proved to be implicated in the maintenance of genomic stability maintenance and assocatedwith poor prognosis in bladder cancer patients.Since HuR activation may help DDR process,we hypothesize that blockade of these important survival networks of cancer cells should strengthen the toxicity of genotoxic agents.In this study,we show that HuR translocates from the nuclei into cytoplasm in response to chemotherapeutic stimulation.Besides,HuR is required for the efficacy of chemotherapies in urothelial carcinoma of the bladder,as HuR depletion sensitizes bladder cancer cells to chemotherapeutic agents.By employing a luciferase reporter system,we identify pyrvinium pamoate,an FDA-approved anthelminthic drug,as a novel HuR inhibitor that dose-dependently inhibits both HuR-recognized motif containing luciferase readout and UVC-stimulated expression of HuR downstream genes.Immunoblot and immunofluorescence assays further show that cytoplasmic accumulation of HuR could be inhibited by pyrvinium pamoate,with total HuR abundance unaffected.Mechanistically,pyrvinium pamoate decreases cellular ATP production.Immunoblot assay shows that pyrvinium pamoate activates AMP-activated protein kinase(AMPK)in a dose-dependent manner.Besides,the interaction between nuclear transporter importin al and HuR is augmented upon pyrvinium pamotate treatment.Moreover,both an AMPK inhibitor and importin ?1 dead-mutant variant could rescue the impact of pyrvinium pamoate on HuR translocation.Furthermore,chemotherapeutic agents(i.e.,doxorubicin)activates Checkpoint kinase(Chkl)followed by increased Cyclin-dependent kinase(Cdk1)Y15 phosphorylation.However,pyrvinium pamoate treatment reverses this process in both dose-and time-dependent manners.A Cdk1 inhibitor and S202A(phospho-dead mutant)HuR variant abrogate the effect of pyrvinium pamoate on HuR translocation.These results demonstrate that pyrvinium pamoate inhibits HuR cytoplasmic translocation via dual mechanisms where the AMPK/importin ?1 signaling is activated and the Chkl/Cdk1 signaling is inhibited.To verify whether pyrvinium pamoate sensitizes bladder cancer cells to chemotherapies,we combined pyrvinium pamoate with first-line chemotherapeutic agents(cisplatin,doxorubicin,oxaliplatin and vincristine).Combination index(CI)values under different drug concentrations were calculated and significant synergistic effects were observed,as CI values at ED50 and ED75 are less than 1.A 3D colony formation assay further shows that the efficacy of cisplatin is significantly increased in the presence of pyrvinium pamoate,although their doses were both cut by half in the combination group.Besides,our in vivo data also demonstrates a synergistic effect between pyrvinium pamoate and cisplatin,since the combined regimen produces a more significant decrease in both tumor volume and tumor size(P<0.001)compared to either single agent alone.Notably,pyrvinium pamoate augments chemotherapeutic agents-mediated DNA double-strand breaks,as y-H2AX phosphorylation and comet tail moment caused by doxorubicin or cisplatin was increased upon pyrvinium pamoate-additive treatment.These data suggest that the synergism between pyrvinium pamoate and chemotherapeutic agents can be attributed to strengthened DNA damage.We further found that pyrvinium pamoate dramatically downregulated several key DNA repair genes in genotoxically stressed cells,including DNA ligase ? and BRCA2,leading to unbearable genomic instability and cell death.Collectively,our findings are the first to characterize pyrvinium pamoate as a clinical HuR inhibitor and demonstrate that HuR participates DDR process by directly regulating DNA ligase IV and BRCA2 mRNA stability,which provides a novel therapeutically tractable strategy by targeting cytoplasmic translocation of HuR to conquer chemotherapeutic resistance of urothelial carcinoma of the bladder.