Glutaminase 1 Regulates The Cancer Stemness Of Hepatocellular Carcinoma And Its Potential Mechanism

Background:Hepatocellular carcinoma(HCC)was ranked as the sixth most common neoplasm and the third leading cause of cancer death.More than half of the world's liver cancers occurred in China.When diagnosed at an early stage,patients generally undergo surgical resection or liver transplantation.However,many cases are diagnosed at an advanced stage and only 20%-30%of liver cancer could be treated surgically.Even after surgical treatment,liver cancer recurs frequently and metastasizes,which are still unsolved problems.Based on the recent literature,the development of cancer recurrence,metastasis,and chemo-and radioresistance in a solid tumor is attributed to the presence of cancer stem cells(CSCs).CSCs are defined as a population of cells that possess characteristics associated with normal stem cells such as self-renewal ability,proliferation and differentiation to produce whole tumors.Cancer stemness contributes to therapy failure and disease progression.Recent advances suggest the existence of CSCs within liver cancer,which are considered to be responsible for tumor formation,metastasis,relapse,and chemoresistance of HCC.Glutamine is an abundant and versatile nutrient that participates in energy formation,macromolecular synthesis,signaling,and provides NADPH(nicotinamide adenine dinucleotide phosphate)and GSH(glutathione)to maintain redox homeostasis.Glutaminolysis begins with its conversion to glutamate catalyzed by the Kidney-type glutaminase(GLS1)in HCC.GLS1 is the rate-limiting enzyme for glutamine catabolism.There is so far no consensus on the metabolic phenotype of CSCs,and little is known about the characteristics of glutamine metabolism of liver CSCs.Here,we aimed to identify the role of GLS1 in stemness,and the feasibility that GLS1 serves as a therapeutic target in HCC for elimination CSCs as well as the possible mechanism.Methods:Publicly-available data from the Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)was mined to unearth the association between GLS1 and stemness phenotype.Using the big data,human tissues and multiple cell lines,we gained a general picture of GLS1 expression in HCC progression.GLS1 mRNA can give rise to two isoforms that differ only in their C-terminal regions,with the longer form named KGA and the shorter form being called GAC.We generated stable cell lines by lentiviral-mediated overexpression KGA or GAC,respectively,and constructed a stable strain of GLS1 knockout by CRISPR/Cas9 system.Using these cell models,we employed qRT-PCR,Western blot,immunofluorescence,flow cytometry and other methods to explore the correlation between the expression of GLS1 and CSC markers.Sphere formation assays and colony formation assays were conducted to analyze the relationship between GLS1 and stemness.A series of bioinformatics analyses and molecular experiments were employed to investigate the role of GLS1 in regulating stemness in silico,in vitro and in vivo and its possible mechanism.Results:In chapter one,we observed that GLS1(both KGA and GAC isoform)was highly expressed in HCC,and that high expression of GAC predicted the poor prognosis of HCC patients.GLS1 is exclusively expressed in the mitochondrial matrix.Upregulation of GLS1 is positively associated with advanced clinicopathological features and stemness phenotype.In chapter two,we found that GLS1 is highly expressed in liver CSCs.We demonstrated that targeting GLS1 reduced the expression of stemness-related genes and suppressed CSC properties such as sphere formation ability,colony formation ability in vitro and tumorigenicity in vivo.In chapter three,we further found that GLS1 regulates stemness properties via ROS/Wnt/?-catenin signaling:targeting GLS1 triggers an increase of ROS,attenuates nuclear translocation of ?-catenin and subsequent inhibits the ?-catenin target genes,leading to the suppression of CSC properties in HCC.Conclusion:Targeting GLS1 attenuates stemness properties in HCC by increasing ROS accumulation and suppressing Wnt/?-catenin pathway,which implied that GLS1 could serve as a therapeutic target for elimination of CSCs.