Mechanism And Clinical Significance Of Hypoxia-inducible Factor 1α Promotes Colon Cancer Chemoresistance Through Regulating Glutaminase 2

Colon cancer is one of the most common human gastrointestinal tumor whose incidence and mortality rates remain high for years.Recently,as the chemotherapy regiment’s continuous renewal,therapeutic effect of colon cancer has improved.But patients respond to and benefit from treatment differently,as well as tumor recurrence and metastasis because of chemoresistance are the main obstacle to further improve curative effect.The diversity of metabolic patterns is malignant tumor’s important characteristics,which not only plays a key role in occurrence and progress of tumor,is also a survival guarantee for cancer cells when faced to chemotherapy stress.Therefore,intensive study of tumor metabolism changes and regulation mechanisms in condition to chemoresistance can not only illustrate the molecular mechanisms of colon cancer chemoresistance,but also provide a new therapeutic target for reversing drug-resistance via regulating the specific cell’s metabolism pathway or metabolite.Hypoxia is one of the most important characteristics of solid tumor,of which key mediator,hypoxia-inducible factor 1α（HIF-1α）plays a major regulatory role in tumor metabolism.The transport,synthesis and utilization of Glutamine（Gln）are all increased,of which specific molecular mechanism is still unknown.Gln is deaminized into Glutamate（Glu）by its metabolism key enzyme Glutaminase2’s（GLS2）catalysis.On the one hand,Glu can turn into α-ketoglutarate and enter the tricarboxylic acid cycle（TCA cycle）for anaplerosis in order to supply the decreased median metabolites;on the other hand,under the catalysis of glutathione synthetase,Glu is used to generate glutathione（GSH）which reacts with many chemotherapeutic drugs,enhances drug efflux,decreases cellular drug accumulation,strengthens DNA damage repair,and finally leads to cancer cells chemoresistance.In this study,we find that colon cancer cell is more resistance to chemotherapy under hypoxia,while the expression of HIF-1α and GLS2 are both increased in chemoresistance colon cancer cells.On this basis,we preliminarily discuss HIF-1α influences Gln metabolism through regulating GLS2 and ultimately causes colon cancer chemoresistance,thus can provide a new strategy for reversing drug-resistance.What’s more,immunohistochemical staining is applied for the detection of HIF-1α and GLS2 expression and their clinical significance in colorectal cancer,thus providing a new clue for clinical diagnosis and treatment.METHODS:In part I,（1）Western Blot tests HIF-1α expression in colon cancer cell line Lovo under normoxia（20%O₂）and hypoxia（1%O₂）at different time（12h,24 h,36h,48 h,72h）;（2）Treat Lovo cell with Oxaliplatin（OXA）and 5-fluorouracil（5-Fu）for 48 h,and then Cell counting kit-8（CCK8）tests cell proliferation activity under 20%O₂ and 1%O₂ respectively;（3）Western Blot tests the expression of HIF-1α,hypoxia-inducible factor 2α（HIF-2α）,Glutaminase1（GLS1）and GLS2 in wild colon cancer cell lines HCT-116 and HCT-8,as well as in drug-resistance colon cancer cell lines HCT-116/OXA and HCT-8/5-Fu.In part II,（1）Build HIF-1α knock-down(HIF-1α^KD)Lovo cell line via lentivirus transfection,then Western Blot tests HIF-1α and GLS2 expression under 20%O₂ and 1%O₂ for 12h;（2）Build HIF-1α over expression(HIF-1α^OE)Lovo cell line via lentivirus transfection and then test HIF-1α and GLS2 expression by Western Blot;（3）Analyze the expression correlation between GLS2 and different target genes of HIF-1α by International Cancer Genome Consortium（ICGC）;（4）Analyze the similarity between GLS2 promoter sequence and HIF-1α target gene sequence by TRANSFAC MATRIX TABLE;（5）Luciferase reporter gene tests HIF-1α promotes GLS2 transcription.In part III,（1）Build HIF-1α^OE+GLS2^KD Lovo cell line via lentivirus transfection to knock down GLS2 in HIF-1α^OE Lovo cell line;（2）GSH assay kit respectively detects GSH levels in WT,Ctr,HIF-1α^OE,HIF-1α^OE+GLS2^KD Lovo cell lines;（3）Treat WT,Ctr,HIF-1α^OE,HIF-1α^OE+GLS2^KD,HIF-1α^OE+H2O₂ Lovo cell with OXA and 5-Fu for 48 h respectively,then CCK8 tests cell proliferation activity.In part IV,（1）Immunohistochemical staining is applied for the detection of HIF-1α and GLS2 expression in 108 cases of colorectal cancer and 23 adjacent mucosa tissues through surgical treatment,the correlation between HIF-1α and GLS2 expression and clinicopathologic features of patients with colorectal cancer is analyzed;（2）Spearman rank correlation is used to detect the correlation between HIF-1α expression and GLS2 expression,Kaplan-Meier method is used to evaluate the impact of HIF-1α and GLS2 expression on postoperative survival of the patients with colorectal cancer,Cox univariate and multivariate prognostic analysis is utilized to explore the prognostic factors of the patients with colorectal cancer.RESULTS:1.Hypoxia is connected with colon cancer cell chemoresistance via enhancing HIF-1α’s expressionHIF-1α has the highest expression level under 1%O₂ for 12h;the proliferation activity of Lovo cell under 1%O₂ is stronger than that under 20%O₂ after treated with OXA or 5-Fu;the expression of HIF-1α and GLS2 in drug-resistance colon cancer cells HCT-116/OXA and HCT-8/5-Fu are much higher than their WT cells,while HIF-2α and GLS1’s expression has no significant difference between them.2.GLS2 is a target gene of HIF-1αWhen knock down HIF-1α,the expression of GLS2 is also decreased in WT,Ctr,HIF-1α^KD Lovo cells under 20%O₂ and 1%O₂;when HIF-1α is over expressed under 20%O₂,the expression of GLS2 is also increased;GLS2 has a positive correlation with HIF-1α’ s different target genes analyzed by ICGC;The similarity between GLS2 promoter sequence and HIF-1α target gene sequence is 99.8% analyzed by TRANSFAC MATRIX TABLE;Luciferase reporter gene finds that HIF-1α can promote the transcription of GLS2 and when GLS2 promoter sequence is mutated,the promoting activity of HIF-1α to GLS2 is also decreased.3.HIF-1α promotes colon cancer cell chemoresistance via enhancing cellular GSH levelWhen HIF-1α is over expressed,GSH level is increased,while knock down GLS2,GSH level is decreased as well;the cell proliferation activity is increased when HIF-1α is over expressed,while knock down GLS2 or add H2O₂,the cell proliferation activity is also decreased after treated with OXA or 5-Fu.4.Expression and clinical significance of HIF-1α and GLS2 in colorectal cancerThe expression of HIF-1α and GLS2 are both significantly higher than in adjacent mucosa tissues（P<0.01）.HIF-1α expression is closely correlated with age,TNM stage,lymphatic metastasis and postoperative metastasis（P<0.05）,and GLS2 expression is closely correlated with age,invasion depth and postoperative metastasis（P<0.05）.HIF-1α and GLS2 expression are remarkable positive correlation（r=0.464;P<0.01）,and significantly correlated with prognosis of colorectal cancer,as determined by Spearman rank correlation analysis.Multivariate Cox analysis reveals that TNM stage,postoperative metastasis,HIF-1α and GLS2 expression play a significant role in predicting patient prognosis（P<0.05）.CONCLUSIONS:As an important transcriptional factor,the expression of HIF-1α is significantly increased under hypoxia,and it is closely related to colon cancer chemoresistance.GLS2 is a target gene of HIF-1α,which improves GSH level and leads to colon cancer cell chemoresistance.Moreover,these findings suggest that the patients with high expression of HIF-1α and GLS2 predicts poor prognosis,indicating that they might play an important role in the malignant progression of colorectal cancer.