FOXA1 Promotes DNA Damage Repairing And The Polarization Of M2 Tumor Associated Macrophages Leading To Cisplatin Resistance In Bladder Cancer

Bladder cancer ranks fourth among male malignancies in the United States.In China,the incidence of bladder cancer ranks the sixth among male malignant tumors.In 2015,about 805,000 cases of bladder cancer were newly diagnosed in China,with about 329,000 deaths.At present,the main treatment methods of bladder cancer are surgical treatment and adjuvant treatment mainly based on chemotherapy.The quality of life of patients after surgery decreases significantly,and the follow-up treatment greatly increases the medical expenditure of patients and the national finance.A study by the university of southern California found that between 1980 and 2010,despite advances in medical techniques and surgical procedures,overall survival for bladder cancer patients did not improve.Since 2010,with the development of neoadjuvant chemotherapy based on cisplatin,the overall survival of patients with bladder cancer has been improved compared with that before.However,the overall response rate of bladder cancer to cisplatin is about 25-40%,and almost all advanced bladder cancers treated with cisplatin will eventually be resistant to cisplatin.To explore the mechanism of cisplatin resistance and to predict the therapeutic effect of cisplatin is a hot research topic of bladder cancer,which is of great significance for reducing the mortality rate of bladder cancer and improving the prognosis of patients.With the popularization of second-generation sequencing technology,after the sequencing of bladder cancer tumor tissues by various institutions,bladder cancer is divided into two subtypes according to different molecular phenotypic patterns.TCGA published articles in Nature and Cell in 2014 and 2017 respectively,indicating that bladder cancer can be divided into two large types according to molecular phenotypes:Luminal type characterized by high FOXA1 expression and Basal type characterized by low FOXA1 expression.Based on the clinical characteristics of the patients,TCGA found that Luminal type bladder cancer was not sensitive to cisplatin chemotherapy,while Basal type bladder cancer was sensitive to cisplatin chemotherapy.FOXA1 may be a key factor affecting cisplatin sensitivity in bladder cancer,but the mechanism is still unclear.FOXA1 is a member of the FOX family of transcription factors.As a"pioneer factor",its main function is to unscrew the compressed chromatin,and promote other transcription factors to bind to DNA and initiate transcription.Using the TCGA and Oncomine databases,we found that patients with bladder cancer with high FOXA1 expression had a 25%higher 5-year survival rate than those with low FOXA1 expression.At the same time,FOXA1 expression was high in early non-invasive bladder cancer and low in late invasive bladder cancer.To further verify the effect of FOXA1 on cisplatin sensitivity of bladder cancer,cisplatin sensitivity tests were performed on different bladder cancer cell lines.The bladder cancer cell lines with high FOXA1 expression(5637,umuc6,um-uc9)were not sensitive to cisplatin,while the cell lines with low FOXA1 expression(um-uc3,um-ucl3)were sensitive to cisplatin.Silencing FOXA1 increased the sensitivity of bladder cancer cell line 5637 to cisplatin.Using mouse bladder cancer cell line MB49 to form tumors subcutaneously in B6 mice followed by intraperitoneal injection of cisplatin for chemotherapy,we found that silencing FOXA1 enhanced the sensitivity of bladder cancer cells to cisplatin chemotherapy.The above preliminary experiments suggested that FOXA1 reduced the sensitivity of bladder cancer to cisplatin chemotherapy.In recent years,DNA damage repair,development and prognosis prediction of bladder cancer have become a major research focus in this field.Cisplatin is a non-specific drug in the cell cycle.The anti-tumor mechanism of cisplatin is that it combines with the DNA bases in the tumor nucleus to form a cross-link,causing DNA damage and destroying DNA replication and transcription.In 2014 the Journal of Clinical Oncology,"and in 2016 the European Urology,there are two new adjuvant chemotherapy for bladder cancer cisplatin Clinical study found that DNA damage repair several key genes such as BRCA1/2,RAD51B,PARP1,ATM,MSH1/2 function defects or missing gene mutations can lead to DNA repair,DNA damage repair gene mutation or missing bladder cancer is more sensitive to cisplatin neoadjuvant chemotherapy.In order to explore the mechanism of FOXA1 affecting the sensitivity of cisplatin to chemotherapy for bladder cancer,we found by immunohistochemistry that after cisplatin treatment,FOXA1 up-regulated the expression of H2AX(a marker protein of DNA double-strand damage)in the bladder cancer tissues of mice.Comet experiments showed that the FOXA1 silencing group produced more DNA fragments than the control group after cisplatin treatment.The above experiments suggested that silencing FOXA1 could enhance the DNA damage of cisplatin on bladder cancer cells.To further verify the effect of FOXA1 on DNA damage repair genes,we silenced FOXA1 bladder cancer cell line 5637 and the control group for second-generation high-throughput rna-seq and conducted GO functional significance enrichment analysis.The results showed that the expression levels of DNA damage repair genes BRCA1,PARP1 and RAD51 decreased in the silenced FOXA1 group compared with the control group.GO analysis showed that homologous recombination repair and mismatch repair pathways were down regulated.It was further suggested that silencing FOXA1 down regulated the expression of BRCA1,PARP1 and RAD51,and reduced the repair function of DNA damage in bladder cancer cells.Tumor associated macrophages(TAM)are the most abundant immune cells in the tumor microenvironment and play an important role in tumor growth,invasion and chemotherapy resistance.It has been reported that M2 type TAM polarization is closely related to cisplatin resistance of bladder cancer.The mechanism is that over-secretion of il-10 by m2type TAM leads to increased expression of anti-apoptotic gene bcl-2,which promotes the development of drug resistance in tumors.Type M2 TAM can also continuously produce angiogenic factors,produce disordered tumor blood vessels,and delay the diffusion of chemotherapy drugs to tumor tissues.After FOXA1 was silenced in mouse bladder cancer cell line MB49,we found that by subcutaneous tumorigenicity experiment in B6 mice,the accumulation of M2 type TAM in tumor tissues was significantly reduced after FOXA1 silencing.Co-culture of wild type bladder cancer cell lines with THP1 cells successfully induced m2-type TAM.After FOXA1 silencing,the ability of m2-type TAM induced by tumor cell medium supernatant was significantly decreased.The above experiments suggested that FOXA1 could influence the polarization of type M2 TAM.Based on the above studies,we conclude that FOXA1 promotes repair of tumor DNA damage and enhances m2-type TAM polarization leading to cisplatin resistance.This study will further reveal the molecular mechanism of cisplatin resistance in bladder cancer.It has certain clinical significance for clinical judgment of prognosis and prediction of curative effect.Part 1FOXA1 affects cisplatin sensitivity in bladder cancer and enhances DNA damage repairObjective:to study the effect of FOXA1 on the biological function and DNA damage repair ability of bladder cancerMethods:the proliferation,invasion,cycle and apoptosis of bladder cancer cells were detected by cck-8,transwell and flow cytometry.The sensitivity of bladder cancer cells to cisplatin was detected by co-culture of bladder cancer cells with cisplatin and intraperitoneal injection of cisplatin after subcutaneous tumor formation in mice.DNA damage in bladder cancer cells was detected by immunohistochemical assay with H2AX and comet assay.Rna-seq was used to explore the signaling pathway that FOXA1 might affect.Results:according to TCGA and ONCOMINE databases,we found that patients with high FOXA1 expression had lower degree of bladder cancer invasion and better prognosis.FOXA1 silencing significantly increased proliferation and invasion of bladder cancer cell line 5647,but significantly increased apoptosis.In the cisplatin sensitivity test,we found that the bladder cancer cell lines with low FOXA1 expression were more sensitive to cisplatin.Silencing FOXA1 increased the efficacy of cisplatin and reduced the volume of subcutaneous tumorigenesis in mice.Immunohistochemistry and comet experiments showed that after FOXA1 silencing,DNA damage fragments generated by cisplatin therapy in bladder cancer cells increased,and the expression of DNA damage marker H2AX increased.Rna-seq results showed that FOXA1 was associated with homologous recombination and mismatch repair signaling pathways.Silencing FOXA1 caused a decrease in the expression of BRCA1,PARP1 and RAD51.Conclusion:FOXA1 decreased cisplatin sensitivity by enhancing DNA repair in bladder cancer.Part ⅡFOXA1 affects immune cell infiltration and M2 tumor associated macrophages polarizationObjective:to investigate the effect of FOXA1 on tumor immune cell invasion and m2-type tumor related macrophage polarization.Methods:TIMER was used to predict the relationship between FOXA1 expression and immune cell infiltration in bladder cancer.Tumor specimens of 83 patients with bladder cancer were collected,and FOXA1,CD8,CD163 and hla-dr immunohistochemistry were performed to determine the relationship between FOXA1 expression and cytotoxic T cells,M2 tumor associated macrophages and dendritic cells.Bladder cancer cell line 5637 was co-cultured with THP1 cells to induce M2 tumor associated macrophages,and then the expression of CD163 and CD206 was detected by RT-pcr to determine the effect of FOXA1 on the polarization of M2 tumor associated macrophages.Results:using the TIMER database,we found that M2 tumor associated macrophages influenced the prognosis of bladder cancer.Furthermore,FOXA1 expression was correlated with the infiltration of m2-type tumor-associated macrophages and CD8+cytotoxic T cells.We collected pathological specimens from 83 patients with bladder cancer.Immunohistochemistry showed that FOXA1 expression was positively correlated with the number of M2 macrophage infiltration,negatively correlated with CD8 cytotoxic T cells,and had no significant relationship with dendritic cells.Co-culture of bladder cancer cell line 5637 with THP1 successfully induced M2 tumor-related macrophages.After FOXA1 silencing,the number of induced significantly decreased,and the expression levels of surface molecules CD163 and CD206 were significantly down regulated in the FOXA1 silencing group.Tumorigenic experiments in mice showed that after FOXA1 silencing,cd163-positive significantly reduced their aggregation in tumor tissues.Conclusion:FOXA1 enhances the polarization of m2-type tumorassociated macrophages in the bladder cancer microenvironment.