Oncometabolite L-2-HG-regulated GABPA Transcription Suppresses Malignant Progression Via TGFBR2-SMAD2/3-MYC Axis In Clear Cell Renal Cell Carcinoma

Background:Renal cell carcinoma(RCC)is one of the common malignancies in urological system,accounting for 2%of all adult cancers.The incidence of RCC has been increasing annually worldwide.Clear cell renal cell carcinoma(ccRCC),derived from the epithelial cells in the nephron,is the predominant subtype of RCC(up to 80%of all RCCs).ccRCC is characterized by unique genetic features.Chromosome 3p loss and inactivation of the von Hippel Lindau(VHL)gene are the early events to drive the disease pathogenesis.VHL serves in the E3 ubiquitin ligase complex,which mediates α subunits of hypoxia-inducible factors(HIF1α and 2α)for proteasomal degradation,while the VHL inactivation leads to the stabilization of HIFas even under normoxia,thereby resulting in pseudohypoxic phenotype and metabolic reprogramming.In addition,alterations also occur frequently in other genes encoding metabolic enzymes.All these aberrations together give rise to the abnormal increase of certain metabolites with oncogenic function and drive malignant progression of ccRCC.GA-binding protein A(GABPA)is the ETS family transcription factor,and it forms a complex with its partner either GABPB1 or GABPB2 to regulate target gene transcription.GABPA has long been shown to play oncogenic roles in the pathogenesis of leukemia,prostate cancer,glioblastoma and other malignancies.More recently,GABPA was further identified as a key transcription factor to activate the mutated telomerase reverse transcriptase(TERT)promoter.Thus,GABPA or its partner GABPB1 has been suggested as a therapeutic target for tumors bearing TERT promoter mutations.The mutation of TERT promoter region is one of genetic features and occurs in up to 20%ccRCCs.But it is currently unclear which effects GABPA exerts on ccRCC.The present study is designed to address this issue by combining the ccRCC-specific metabolic reprogramming.Firstly,we observe the differential expression and biological function of GABPA in ccRCC.At the same time,we further explored the molecular mechanisms of GABPA in mediating malignant progression of ccRCC.Lastly,we delve the reason for dysregulation of GABPA expression.Part Ⅰ:The expression and biological function of GABPA in ccRCCMethods:We first compared mRNA expression of GABPA between ccRCC tumors and matched adjacent non-cancerous renal tissues.The result was validated by TCGA cohort of ccRCCs.Protein expression of GABPA was measured by IHC staining in ccRCC tissue microarray cohort.Using KM survival curves and multivariate Cox regression analysis,we compared the relationship between GABPA expression and prognosis.siRNAs and expression vectors were used to manipulate the expression of GABPA.In vitro,cell proliferation ability was examined using CCK-8 assay and flow cytometry.Cell invasion and metastasis capacity was observed by wound healing and Transwell assays.Cell stemness was measured by spheroid formation assay and flow cytometry.In vivo,we further examined the proliferation and metastasis ability using tail vein and subcutaneous tumorigenic xenografts in nude mice.Results:Compared with matched adjacent non-cancerous renal tissues,the expression of GABPA was lower in ccRCC tumor tissues.The patients with low expression of GABPA showed shorter overall survival and disease-free survival.At the same time,GABPA depletion robustly enhanced proliferation,invasion and stemness of ccRCC cells,whereas GABPA overexpression exhibited opposite effects,strongly inhibiting in vivo metastasis and carcinogenesis.Part Ⅱ:GABPA inhibits ccRCC malignant progression via TGFBR2-SMAD2/3-MYC axisMethods:we first looked for potential downstream targets of GABPA using in silico approaches:(1)The RNA sequencing or microarray data analyses of TCGA ccRCC tumors.(2)RNA sequencing analyses of GABPA-depleted 786-O cells.(3)Analyses of available GABPA ChIP-seq data.After GABPA depletion and over-expression,the expression of downstream target was examined by Western blot and Immunofluorescence.Following this,luciferase reporter assays and ChIP-PCR were used to explore the relationship between GABPA and TGFBR2 promoter.The biological function of TGFBR2 in GABPA-mediated phenotypic alterations was observed by a series of rescue experiments.At last,we compared the relationship between TGFBR2 expression and prognosis using KM survival curves and multivariate Cox regression analysis.Results:For sequencing data analysis:(1)TGFBR2 was identified as one of the top genes whose expression was positively correlated with GABPA.Moreover,the GSEA analysis showed that the TGF-β pathway was markedly enriched in tumors expressing higher GABPA.(2)TGFBR2 was among the top downregulated genes,and the TGF-β signal was underrepresented upon GABPA knockdown.(3)The ChIP-seq of GABPA-expressing leukemic K562 and liver cancer HepG2 cells identified a total of 6,810 gene promoters bound by GABPA,and five genes were overlapped based on the integrated analyses above,among which was TGFBR2.GABPA depletion and over-expression down-and up-regulated TGFBR2 expression at protein levels,respectively.GABPA overexpression significantly increased the TGFBR2 promoter activity.When the GABPA binding sites were mutated,the TGFBR2 promoter almost completely lost its response to GABPA.The results of ChIP-PCR showed that GABPA bound to both motifs in TGFBR2 promoter.Ectopic TGFBR2 expression completely erased GABPA depletion-mediated proliferation and invasion acceleration of 786-O cells.Moreover,the patients with low expression of TGFBR2 showed worst prognosis.Part Ⅲ:Oncometabolite L-2-HG suppresses GABPA transcription through epigenetic modificationsMethods:We analyzed the DNA methylation level at GABPA loci between ccRCC tumors and matched adjacent non-cancerous renal tissues in the TCGA cohort of ccRCCs.After treated ccRCC-derived A498 and 786-O cells with the DNA methylation inhibitor 5-azacitidine(5AZA),we measured the alterations of GABPA expression and DNA methylation level using Western blot and Pyrosequencing.A498 and 786-O cells were also incubated with L-2-HG and manipulated the expression of metabolic enzymes,we further observed the changes of GABPA expression、DNA methylation level and malignant phenotype.Results:Compared with matched adjacent non-cancerous renal tissues,the DNA methylation level of GABPA was higher in ccRCC tumor tissues.5-AZA decreased DNA methylation level and increased the expression of GABPA.L-2-HG treatment of 786-O cells significantly facilitated proliferation and invasion,which mimicked the effect of GABPA depletion.In contrast,the restoration of L2HGDH expression and knocking-down of MDH2 or LDHB led to inhibition of cellular proliferation and invasion,while these inhibitory effects were attenuated by addition of L-2-HG.Conclusions:Different from the cancer-promoting function in other cancers,GABPA acts as a tumor suppressor in ccRCC.GABPA activates TGFBR2 transcription,and thereby enhances the TGFβ signaling to inhibit proliferation,stemness and invasion of ccRCC cells.The inhibitory effect of GABPA expression on in vivo metastatic and carcinogenic capacity is even more robust.Oncometabolite L-2-HG epigenetically inhibits GABPA expression,disrupting the GABPATGF-β loop to drive ccRCC aggressiveness.These findings suggest that it should be cautious in targeting GABPA or GABPB1 for cancer intervention.Restoring GABPA expression using DNA methylation inhibitors or other approaches,rather than targeting it,may be a novel strategy for ccRCC therapy.