The Functional Role And Regulatory Mechanism Of ASCT2 In Colorectal Cancer

Background and aims:Deregulated metabolism in cancer cells is widely accepted as a hallmark of cancer,and many tumor cells are fueled by increased glutamine dependence.The high rate of glutamine uptake exhibited by glutamine dependent cells does not appear to result solely from its role as a nitrogen donor in nucleotide and amino acid biosynthesis.Instead,glutamine plays a required role in the uptake of essential amino acids and in maintaining activation of TOR(target of rapamycin)kinase.Moreover,in many cancer cells,glutamine is the primary mitochondrial substrate and is required for maintenance of mitochondrial membrane potential and integrity and for support of the NADPH production needed for redox control and macromolecular synthesis.Therefore,these characteristics make glutamine metabolism an appealing target for new clinical strategies to detect,monitor,and treat cancer.Tumor cells must upregulate one or more of glutamine transporters to satisfy their increased demand for glutamine.If the transporters that specifically serve this purpose in tumor cells are identified,they can be targeted for the development of a brand new class of anticancer drugs.ASCT2 is the most reported glutamine transporter which is upregulated and associated with proliferation of tumor cells,but little is known about its clinical significance and regulatory mechanism in CRC(colorectal cancer).We aim to investigate clinical significance of ASCT2 in CRC and whether silenced miR-137 fosters ASCT2 expression and promotes proliferation of CRC.Methods:Tisssue microarray including CRC tissues and matched non-tumor tissues of 90 patients was for immunohistochemistry analysis.The role of ASCT2 on regulating glutamine metabolism accompanying other metabolites was determined by metabolite profiling.Luciferase reporter analysis was used for identifying the function of miR-137 on ASCT2.Chromatin immunoprecipitation was performing to confirm the regulatory mechanism of miR-137.CRC cells with lentiviral transfection were used for proliferation assay and Xenograft in mice.Data were presented as mean ± SD from at least three independent experiments.Results:To verify the clinic significance of ASCT2 in CRC,we detected ASCT2 expression levels in 90 matched CRC/normal samples.Compared with normal tissues,we found ASCT2 was significantly upregulated in CRC tissues,which was consistent with the results found in lung cancer,prostate cancer and breast cancer.However,the correlation between ASCT2 expression level and TNM stage of CRC is not significant.Neither,the patients with higher ASCT2 level didn't exhibit a worse prognosis than those with lower expression.These results suggest that ASCT2 is much more important in the initiation of tumorigenesis for CRC patients.Based on the results above,we further evaluate ASCT2 function in CRC cell growth and its regulatory mechanism.We depleted ASCT2 expression in multiple CRC cell lines by two specific shRNAs,the growth of all ASCT2-depleted cells were observably reduced,whereas control cells substantially proliferate over time.Examination of cell cycle progression by iodine in HCT116 cells showed that ASCT2 knockdown induces significant increased cell population in G1 phase,with a corresponding decrease of cells in S and G2 phases,which indicated that ASCT2 inhibited cell growth through cell cycle arresting.Mechanistically,we identified ASCT2 is a direct target of miR-137.Compared with negative control,transient expression of miR-137 led to significant decrease in ASCT2 protein and mRNA in CRC cancer cells,whereas the protein and mRNA level of ASCT2 in normal colonic cell transfected with miR-137 inhibitor were increased.Meanwhile,we tested ASCT2 levels in different cancer cells after miR-137 overexpression,and found ASCT2 mRNA and protein in all of them were decreased,which showed that the negative correlatioin between miR-137 and ASCT2 widely existed.To verify a direct binding of miR-137 on ASCT2 3'UTR,a luciferase assay was performed.The luciferase activity with wild type 3'UTR of ASCT2 was markedly inhibited comparing with the control,whereas the luciferase activity with mutant type 3'UTR of ASCT2 was less influenced.The results revealed that miR-137 regulated ASCT2 by targeting its 3'UTR,which was consistent with the mechanism of microRNAs targeting their downstream.To understand how important of ASCT2 in glutamine metabolism,we examined its effect on metabolites by metabolome analysis.ASCT2 depletion caused a substantial decrease in TCA cycle,NEAA production,lipids and nucleotides,but an abundant accumulation of glucose and fructose.All these results above indicated metabolic dysfunction upon ASCT2 depletion.Addditionally,we tested glutamine metabolism of CRC after miR-137 overexpression and found that glutamine consumption,a-KG and ATP productin were all reduced,which was consistent with the results from ASCT2 depletion in CRC cells.So we could deduce that miR-137 exhibited its effect on glutamine metabolism through targeting ASCT2.Colorectal cancer is characterized by global downregulation of miRNAs including miR-137,which arouse our interest in mechanism of miR-137 silencing.ChIP assay revealed that both MeCP2 and DNMT3B selectively bind to the promoter region of miR-137 where was reported to be highly methylated in previous research.We determined miR-137 and ASCT2 levels after treatment of decitabine(DNMTs inhibitor)and MeCP2 shRNAs in HCT116,respectively.MiR-137 mRNA level were found to increase in HCT116 with treatment above,whereas ASCT2 protein level decreased.ChIP assay with decitabine treatment and MeCP2 shRNA further confirmed the binding of DNMT3B and MeCP2 to miR-137 promoter region.Decitabine treatment of HCT116 led to a significant decrease in recruitment of DNMT3B and MeCP2 to the miR-137 promoter when compared with the IgG control and introduction of MeCP2 shRNA into HCT116 likewise decreased the binding of DNMT3B and MeCP2 to miR-137 promoter region.All of these determined that coordination between DNMT3B and MeCP2 promoted miR-137 methylated silencing,which revealed ASCT2 overexpression in tumors was partially through miR-137 downregulation.We finally confirmed the correlation between miR-137 and ASCT2 in 12 normal/CRC paired tissues.Compared with normal colorectal tissues,levels of miR-137 were significantly reduced in CRC tissues from patients,whereas levels of ASCT2 protein increased.Moreover,overexpression of ASCT2 could partially rescue the growth inhibition induced by miR-137,which proved that miR-137 inhibited cell proliferation was partially through downregulating ASCT2.The results above definitely uncovered the negative correlation between miR-137 and ASCT2.Conclusion:ASCT2 expression,which is regulated by miR-137,is markedly elevated in CRC tissue samples compared with normal tissues.