Functional And Mechanistic Study Of HMGA2 Role On 5-FU Chemoresistance In Colorectal Cancer

Colorectal cancer(CRC)is one of the most common malignant tumors worldwide,ranking third most commonly diagnosed cancer after lung and breast cancer,and the fourth highest cause of cancer death after lung,liver,and stomach cancer.Patients who have CRC with localized disease is treatable with curative surgical resection.After resection,adjuvant chemotherapy is recommended as standard clinical trial for patients suffered from stage III CRC.Various chemotherapeutic drugs have been used effectively for quite some time;however,5-fluorouracil(5-FU)remains the most widely employed chemotherapeutic agent in CRC treatment.Although 5-FU-based chemotherapy has improved response rates to greater 50%and achieves a median survival rate of up to 2 years,failure of this treatment in>90%of patients is due to drug resistance.Therefore,understanding molecular mechanism of chemoresistance is of great essential and innovative targeted therapies are needed.HMGA2 belongs to the non-histone chromosomal high-mobility group(HMG)family and is located at 12q 13-15.Human HMGA2 consists of five exons and four introns.The first three exons encode three DNA-binding domains responsible for preferential binding of HMGA2 to adenine-thymine(AT)-rich minor grooves of nuclear B-form DNA via its "AT hooks".Therefore,HMGA2 functions as a transcriptional modulator by altering target chromatin structure and through protein-protein interactions.HMGA2 plays an important role in embryo formation,tissue development and tumorigenesis.Recent studies have shown that HMGA2 expression was positive in poorly differentiated tissues and distant liver metastasis of CRC tissues.Also,positive HMGA2 was significantly associated with poor prognosis in CRC.In this study,we aimed to investigate the role of HMGA2 during tumorgenesis,especially in mediating 5-FU chemoresistance in CRC and confirm the mechanism of HMGA2-induced chemoresistance.We previously stably overexpressed HMGA2 in SW620 and HT29 cells(SW620-NC/A2,HT29-NC/A2)and stably knocked down HMGA2 in HCT116 and RKO cells(HCT116-NC/shA2,RKO-NC/shA2).In this study,we employed transwell assay,MTT,Western Blot,plate colony formation,Real-time PCR and flow cytometry assays to assess the role of HMGA2 in mediating invasion,metastasis,EMT,proliferation and 5-FU chemotherapy sensitivity in CRC.The results showed that overexpression of HMGA2 in CRC cells enhanced invasion,metastasis,EMT,proliferation and increased cell viability under 5-FU treatment.Conversely,knockdown of HMGA2 in CRC cells suppressed invasion,metastasis,EMT,proliferation and decreased cell viabilityin response to 5-FU compared with vector control cells.To further explore the role of HMGA2 in xenografts,SW620-NC/A2 cells were subcutaneously injected into the flanks of mice.The results showed that group with HMGA2 overexpression enhanced tumorigenesis with EMT and exhibited marked resistance in the response to 5-FU treatment.All these results indicated that HMGA2 enhanced invasion,metastasis,EMT,proliferation and chemoresistance to 5-FU both in vitro and in vivo.We searched microarray data related to CRC resistance to 5-FU in the Gene Expression Omnibus(GEO)database and selected GSE28702.The results indicated that HMGA2 belonged to a set of genes that were upregulated in the non-responder group compared with the responder group.To determine the signaling pathways that are potentially regulated by HMGA2 in CRC,we performed GSEA using high-throughput RNA-sequencing data from the TCGA database to examine the mode of action of HMGA2.We also performed a microarray analysis in SW620-NC/A2 cells using RNA-Seq to analyze alterations of gene expression due to HMGA2 overexpression.We then focused on genes showing a>2-fold change in expression for functional enrichment analysis using DAVID Bioinformatics Resources 6.7.The results indicated that Wnt pathway was identified as one of signaling pathways showing the strongest association with HMGA2 expression.To further investigate the mechanisms by which HMGA2 executed its function,we adopted a ChIP-on-chip assay for target gene prediction and identified Dvl2 as one of the potential targets of HMGA2 and selected for further analysis.We conducted Western Blot analyses to confirm that Dvl2 was upregulated by HMGA2 overexpression.To test whether Dvl2 may be a direct target of HMGA2,we generated luciferase reporter assays to identify the transcriptional pattern of Dvl2 induced by HMGA2.Our results demonstrated that the promoter region without-264/+217 sequence exhibited a minimal response to HMGA2 stimulation,suggesting that the-264/+217 region of Dvl2 is essentially responsible for the function of HMGA2.Mutation in sequence from-201 to-185 mainly confers the transcriptional activity of Dvl2 promoter in response to HMGA2.ChIP-PCR and ChIP-qPCR analysis further demonstrated that HMGA2 directly bound to the Dvl2 promoter region-264/-100.Hence,HMGA2 may transactivate Dvl2 through directly binding to the promoter site from-201 to-185.We next sought to explore whether Dvl2 affects the chemoresistance of CRC cells induced by HMGA2.Silencing Dvl2 on HMGA2-overexpressing and control cells showed that silencing of Dvl2 significantly reduced MMP7 in SW620-A2 cells compared with that in control SW620-NC cells.All of these data demonstrated that Dvl2 affects the chemoresistance of CRC cells in an HMGA2-dependent manner.To confirm this relationship,we performed qRT-PCR and Western Blot to test whether HMGA2 could activate Wnt signaling.HMGA2 significantly unregulated genes of Wnt/β-catenin pathway compared with the control group,including P-catenin,c-Myc,cyclin D1,MMP7 and CD44.Knockdown of HMGA2 expression in HCT116 cells consistently and significantly decreased the transcriptional activity of Wnt/β-catenin pathway.To further assess the direct regulation of Wnt signaling by HMGA2 in CRC,we used LiCl,a GSK3P inhibitor,to activate the Wnt signaling pathway in HMGA2-knockdown cells.The results confirmed that MMP7 and CD44 were rescued after the activation of Wnt signaling in HCT116-shA2 cells compared with control HCT116-NC cells,which firmly supported the direct regulation of Wnt signaling by HMGA2 in CRC.Finally,we explored the association between HMGA2/Dvl2 axis expression and the chemotherapy response in the 61 patients mentioned above.The results revealed a positive correlation between HMGA2 and Dvl2 expression.In addition,Kaplan-Meier survival analysis indicated that both high HMGA2 and high Dvl2 expression in patients were associated with unfavorable OS.From the above results,the following conclusions could be drawn:1.HMGA2 promoted invasion,metastasis,EMT,proliferation and enhanced chemoresistance to 5-FU in colorectal cancer in vitro and in vivo.2.HMGA2 directly promoted the transcription of Dvl2,and the binding sites between-201 and-185 of Dvl2 promoter contributed to the activity of HMGA2-mediated transcription.3.HMGA2 could enhance chemoresistance to 5-FU through mediating Dvl2/Wnt signfiling pathway.