Low Doses Of Decitabine Improve The Chemotherapy Efficacy Against Basal-like Bladder Cancer By Targeting Cancer Stem Cells

BackgroundBladder Cancer(BCa)is the most common urinary tumor with a poor prognosis.In addition to surgical treatment,platinum-based chemotherapy remains the current primary treatment for BCa.Cancer stem cells(CSCs)are associated with tumorigenesis,recurrence,metastasis,and drug resistance.Epigenetic regulation such as DNA methylation is crucial for maintaining the stemness of CSCs.DNA methyltransferase(DNMT)is required for DNA methylation,and low-dose treatment with the DNMT inhibitor decitabine(DAC)has been shown to be applicable for the management of certain types of cancer.Therefore,low-dose DAC have promising prospects in the treatment of solid tumors including BCa.However,its antitumor effect and mechanisms are context dependent and its activity has never been systematically studied in BCa treatment.This study explores the efficacy and mechanism of low-dose DAC combined with chemotherapy drugs for BCa,especially BCa stem cells,in vivo and in vitro,and providing novel insights for its clinical use.ObjectiveThis study used BBN-induced BCa mouse model to demonstrate the therapeutic effect and molecular mechanism of low-dose decitabine combined with chemotherapy drugs for BCa.We hope to systematically study the mechanism of cell killing and drug resistance formation of commonly used chemotherapeutic drugs by using the latest methods of life medicine research,and develop effective drug combinations which facilitate accurately guided clinical BCa management.MethodWe started from using the previously established BBN(N-butyl-N-4-hydroxybutyl Nitrosamine)-induced Krt14CreER;RosaEGFP and Sox2CreERT2;R26tdTomato mouse model treated by methyltransferase inhibitor DAC and two chemotherapeutics,gemcitabine and cisplatin.After 26 weeks of BBN induction,mice were injected with DAC combined with/without gemcitabine or cisplatin for treatment,and sacrificed after 30 weeks to evaluate the therapeutic effects of different drug combinations.In addition,we used tamoxifen to induce GFP expression in K14 creGFP mice and Tomato expression in Sox2 creTomato mice for lineage tracing experiments to further analyze the effects of different drug combinations on the self-renewing of CSCs.We also treated BCa cell lines with 100 nM DAC for the following phenotypic assays: apoptosis,sensitivity to cisplatin and gemcitabine(IC50),CSCs staining acquisition,and sphere formation assay,which was to confirm of the effect of low-dose DAC treatment on BCa cells in vitro;we performed methylation DNA immunoprecipitation(MEDIP)sequencing as well as mRNA sequencing to screen target genes and signaling pathways involved in the drug action of DAC in BCa cells.Bisulfite-PCR and real-time quantitative PCR was used to confirm the effect of DAC processing on the target genes.Finally,patient derived xenografts(PDX)were used to evaluate efficacies of different drug combinations for their clinical implications.ResultsIn this study,we demonstrated that low-dose DAC combined with cisplatin or gemcitabine showed better anti-tumor effects than monotherapy in BBN-induced mouse models and cultured BCa cell lines.Importantly,we found that a combination of low-dose DAC and cisplatin or gemcitabine induced a sustained reduction in CSCs,decreased the ratio of ALDH + and CD44V6 + stem cells and their ability of forming sphere,and inhibited the resistance of CSCs to cisplatin and gemcitabine.Methylation DNA immunoprecipitation(MEDIP)sequencing and mRNA-seq has screened the target gene SOCS3,which is the suppressor of STAT3 signaling,and involved in the drug action of DAC in BCa cells.Low-dose DAC can demethylate the promoter of SOCS3 and then activate its expression.It also showed better efficacy in patient derived xenografts(PDX)model,further validating the clinical relevance of the above findings.ConclusionsIn this study,we found for the first time that low dose DAC treatment elevates the efficacy of cisplatin and gemcitabine for managing BCa by inhibiting the stemness of BCa stem cells both in vivo and in vitro.Low dose DAC treatment inhibits the STAT3 pathway to repress the self-renewal capability of CSCs by activating SOCS3 in BCa.Our findings suggest a novel treatment that might be used to promote clinical efficacy of BCa chemotherapy.