Research On The Role And Epigenetic Regulatory Mechanisms Of Glutaminase 1(GLS1)in Colorectal Cancer

Background:Colorectal cancer(CRC)is one of the most common malignant tumors.CRC was the fourth most common cancer in the world and the mortality is the third one following lung cancer and breast cancer.Due to the diagnosis at the advanced stages,most of the patients had poor prognosis with extensive tissue invasion and distant metastasis.The lack of effective therapeutic approaches contributes to the high rate of recurrence and morbidity even after surgical resection.In addition to genetic and epigenetic changes,the pathogenesis of tumors contains metabolic abnormalities.As the most important components of amino acid metabolism,glutamine metabolism has played a crucial role in a variety of tumors.However,the role of glutamine metabolism in CRC and its specific regulatory mechanism is still largely unclarified.Thus,elucidating the pathogenesis of CRC will have great scientific value for the treatment and prevention of CRC.Objective:Now many studies have demonstrated the important role of glutamine metabolism in many tumors while we know a little about that in CRC.In this study,we want to illustrate the critical role of glutamine in CRC and explore the specific regulatory mechanism by a series of in vitro and in vivo assays.Specifically,we are inclined to clarify the role of rate-limiting enzyme-glutaminase 1(GLS1)in glutaminolysis in CRC.And we also want to illuminate how GLS1 is regulated.In the end,we wonder if it is useful to target GLS1 for the treatment of CRC.Methods:Firstly,we removed glutamine from the culture medium of CRC cells to detect the effect on the proliferation and metabolism of CRC cells.Then we examined the expression of GLS1 in CRC tissue and cell lines by western blot and Immunohistochemistry(IHC).We detected he effect on the proliferation and metabolism of CRC cells after knockdown of GLS1 by specific siRNA.After that,we tested the effect of Wnt signaling pathway(APC overexpression or ?-catenin knockdown)on the expression of GLS1,And we screened a group of miRNAs which were upregulated after ?-catenin knockdown.Finally we chose miR-137 as the object for our study.Then we used Chromatin Immunoprecipitation(ChIP),Bisulfate genomic sequence(BGS)and Methylated Immunoprecipitation(Med IP)to clarify how HSF1 regulated miR-137.And we used western blot,co-immunoprecipitation(Co-IP)and RNA Immunoprecipitation(RNA-IP)to illustrate the way by which Wnt signaling pathway(?-catenin knockdown)influenced HSF1 expression.At last,we made use of GLS1 inhibitor-compound 968 in the APC min/+ and AOM/DSS mice model to testify the effect of targeting GLS1 on the formation of CRC.Results:We found that glutamine deprivation significantly reduced the cell growth in SW480 and SW620 CRC cells.Meanwhile,the concentration of ATP and a-KG in the cell decreased dramatically,confirming the importance of glutamine on TCA cycle.And protective autophagy occurred after glutamine deprivation.Therefore glutamine deprivation combined with CQ lead to synthetic lethality to cell viability compared with CQ alone.We found that glutaminase 1(GLS1),the rate-limiting enzyme in glutaminolysis,was highly expressed in colorectal carcinoma cells and tissues compared with the normal tissues.Once knockdown of GLS1 by its specific siRNA,the proliferation and metabolism of CRC cells was inhibited.Molecularly,we found inhibiting Wnt signaling pathway(APC overexpression or ?-catenin knockdown)could decrease the protein level of GLS1 but not the mRNA level.In addition,we found GLS1 was the direct target of miR-137 which was epigenetically down-regulated in human colorectal cancer cells and tissues.Moreover,miR-137 down-regulation in colorectal cancer was attributed to promoter methylation and transcription factor HSF1 could interact with DNMT3a and miR-137 promoter,indicating that HSF1 might recruit DNMT3a to miR137 promoter for DNA methylation.Interestingly,?-catenin might regulate HSF1 translation by interacting with HuR which could bind to HSF1 mRNA.To approve this assumption,we examined the expression of GLS1 in the classical CRC mice model-APCmin/+ and AOM/DSS mice.We found a upregulated level of GLS1 protein in these mice.Inhibition of GLS1 activity by compound 968 could inhibit the formation of tumors in APCmin/+ mice.And combination of GLS1 inhibitor compound 968,autophagy inhibitor CQ and asparagine inhibitor L-ASP significantly impaired the development of colitis-associated colorectal cancer.Conclusions:GLS1 has a positive effect on the formation of CRC.The activated Wnt/?-catenin signaling pathway activates HSF1-miR-137-GLS1 axis to promote glutaminolysis.Combination of inhibiting glutaminolysis(968),autophagy inhibition(CQ)and asparagine inhibition(L-ASP)could inhibit the formation of CRC.By doing so,we believe it could facilitate our understanding in the contribution of metabolism to colorectal carcinogenesis and promote the development of novel strategies for the effective prevention and treatment of colorectal cancer and probably other cancers.Innovative points:1.Protective autophagy occurred after glutaminolysis inhibition in CRC cells,And combination of glutaminolysis inhibition,autophagy inhibition(CQ)and asparagine inhibition(L-ASP)confer synthetic lethality to CRC cells.2.Active Wnt signaling stimulates GLS1 expression to drive the pathogenesis of CRC.3.GLS1 is a direct target of miR-137.4.Transcription factor HSF1 could interact with DNMT3a to promote DNA methylation in the miR-137 promoter.5.?-catenin promotes HSF1 translation by interacting with HuR which could bind to HSF1 mRNA.6.GLS1 is high-expressed in the classical CRC mice model-APCmin/+ and AOM/DSS mice.Combination of GLS1 inhibitor compound 968,autophagy inhibitor CQ and asparagine inhibitor L-ASP significantly impaired the development of colorectal cancer.