GABPA-activated TGFBR2 transcription inhibits aggressiveness but is epigenetically erased by oncometabolites in renal cell carcinoma

Renal cell carcinoma (RCC) is diagnosed in up to 300 000 people worldwide each year, with clear cell RCC (ccRCC) as the predominant histological subtype (∼80% of all RCCs) [1‐3]. ccRCC originates from the epithelial cells of the proximal convoluted tubule in the nephron and the inactivation of the von Hippel Lindau (VHL) gene is the early event to drive the disease pathogenesis [4, 5]. 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 HIFαs even under normoxia, thereby resulting in pseudohypoxic phenotype and metabolic reprogramming, including aberrant glycolysis, nucleotide and lipid biosynthesis [5, 6]. In addition, alterations also occur frequently in other genes encoding metabolic enzymes [6]. All these aberrations together give rise to the abnormal increase of certain metabolites with oncogenic function (so-called oncometabolites). For instance, the loss of the L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene, and gain of the malate dehydrogenases (MDHs) or lactate dehydrogenases (LDHs) genes result in the accumulation of L-2-hydroxyglurate (L-2-HG), a bona fide oncometabolite that inhibits a group of enzymes of α-ketoglutarate-dependent dioxygenases including the ten-eleven translocation (TET) family of 5-methylcytosine (5mC) hydroxylases, and histone lysine or RNA demethylases [6‐10]. The L-2-HG-mediated inhibition of TET activity triggers widespread DNA cytosine hypermethylation in ccRCC, thereby promoting aggressive disease [6, 7, 11, 12]. However, it remains poorly defined how such aberrant DNA hypermethylation contributes to ccRCC progression and which genes are targeted by L-2-HG.

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 [13, 14]. GABPA has long been shown to play oncogenic roles in the pathogenesis of leukemia, prostate cancer, glioblastoma and other malignancies [13, 15‐17]. More recently, GABPA was further identified as a key transcription factor to activate the mutated telomerase reverse transcriptase (TERT) promoter [18]. TERT promoter mutations occur in various types of cancer and create de novo ETS binding motifs recognized by the GABPA-containing complex [18]. The interplay between the mutated TERT promoter and GABPA induces the expression of TERT, a rate-limiting component for telomerase activity required for immortalization and malignant transformation [14, 18‐20]. In glioblastoma cells harboring the TERT promoter mutation, GABPB1 depletion led to reduced TERT expression coupled with diminished telomerase activity followed by progressive telomere attrition and eventual loss of oncogenic potential [17]. Given all the above findings, GABPA or its partner GABPB1 has been suggested as a therapeutic target for tumors bearing TERT promoter mutations [17].

TERT promoter mutations occur in up to 20% of RCCs [4, 21], and 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. By doing so, we observe that GABPA acts as a tumor suppressor by stimulating TGFBR2 transcription and TGFβ signaling, while the oncometabolite L-2-HG epigenetically inhibits GABPA expression, disrupting the GABPA-TGFβ loop to drive ccRCC aggressiveness. Clinically, higher GABPA is significantly associated with longer survival of ccRCC patients. These findings may have important implications in understanding ccRCC pathogenesis and precision oncology.

ccRCC as a metabolic disease exhibits extensive metabolic reprogramming including downregulation of the tricarboxylic acid (TCA) cycle and upregulation of fatty acid synthesis, the pentose phosphate pathway and glutamine transporters, thereby giving rise to aggressive disease and poor prognosis [6, 28]. The aberrant accumulation of L-2-HG, a bona fide oncometabolite, has been shown as an important driver for ccRCC progression [6]. Indeed, approximately 1/3 of ccRCC patients already have distant dissemination at presentation, which is in general resistant to conventional chemotherapy and radiotherapy [29]. Although numbers of drugs targeting cellular/molecular pathways have been applied for ccRCC, but patient response to these treatments is limited. Thus, the delineation of ccRCC pathogenesis and identification of new therapeutic targets is demanding tasks. Here we observed a widespread downregulation of GABPA expression in ccRCC and identified TGFBR2 as a direct target of GABPA through which the TGFβ signal was activated to inhibit ccRCC progression. Moreover, GABPA is the downstream effector epigenetically silent by L-2-HG, which disrupted the GABPA-TGFβ loop. Restoring GABPA expression strongly inhibits in vivo metastatic and carcinogenic abilities of ccRCC cells. These results exemplify how oncometabolites rewires a GABPA-mediated signaling and erases its tumor suppressive function for cancer development/progression (Fig. 7G).

TGFBR2 is an established tumor suppressor in several human malignancies [26, 27], however, its role in ccRCC is unclear, although its downregulation has long been observed in ccRCC [30, 31]. The TGFBR2 gene is localized at chromosome 3p24.1 where LOH is widespread in ccRCC [22]. Intriguingly, the TGFBR2 LOH does not affect its expression (Fig. S8), indicating that other regulatory mechanisms are more important. In the present study, we provide strong evidence demonstrating TGFBR2 as the direct target of GABPA: (i) GABPA knockdown and over-expression down- and up-regulates TGFBR2 expression, respectively; (ii) GABPA knockdown and over-expression inhibits and stimulates TGFBR2 promoter activity, respectively; moreover, the GABPA binding motif mutation leads to a dramatic reduction in the basic TFGBR2 promoter activity, indicating a key effect of GABPA on TGFBR2 transcription; (iii) The ChIP assay shows the occupancy of GABPA on the TGFBR2 promoter. Similar results were also obtained from the analysis of ChIP-seq databases. TGFBR2 exhibits a tumor suppressive function in ccRCC and attenuates TGFβ response. Consistently, GABPA depletion significantly inhibits the response of ccRCC cells to TGFβ treatment, which includes diminished SMAD2/3 phosphorylation and increased MYC expression, while reduced CDKN1A expression. These TGFβ downstream effectors consequently result in altered proliferation and invasion of ccRCC cells.

2-HG exists as two isoforms including L-2-HG and D-2-HG, and both and α-KG can be converted with each other by specific enzymes [6]. Isocitrate dehydrogenases (IDH1/2) and MDH1/2 or LDHB catalyze α-KG into D-2-HG and L-2-HG, respectively, whereas D2HGDH and L2HGDH oxidize D-2-HG and L-2-HG to α-KG [6]. Gain-of-function of IDH mutations occurs most frequently in low-grade glioma, cartilaginous tumors, intrahepatic cholangiocarcinoma, and certain hematological malignancies; and thus, the accumulation of D-2-HG occurs in these tumors [6]. In contrast, IDH mutations are rare in ccRCC [22]. Instead, loss of L2GHDH or gain of MDHs and LDHB is widespread, which leads to the generation of excess L-2-HG [10, 12, 22]. In addition, pseudohypoxic phenotype-mediated metabolic reprogramming may also contribute to increased L-2-HG [32, 33]. α-KG is a co-substrate for α-KG/Fe(II)-dependent dioxygenases, while L-2-HG acts as an α-KG antagonist to competitively inhibit α-KG/Fe(II)-dependent dioxygenases among which are the TET family of 5mC hydroxylases [6]. The diminished TET activity subsequently results in aberrant DNA methylation in ccRCC [12]. Our findings demonstrate a causal relationship between GABPA hypermethylation/gene silencing and L-2-HG accumulation; the GABPA hypermethylation is coupled with reduced 5hmC, demonstrating impaired TET activity by L-2-HG in ccRCC cells. Of note, ectopic L2HGDH expression or MDH2 and LDHB depletion robustly increased 5hmC, but only inhibited the methylation of cg08521263 moderately. Likely, the demethylation of other CpGs at the GABPA loci synergizes with the demethylated cg08521263 to promotes GABPA expression. Indeed, there exist other 14 CpGs at the GABPA loci, and 12 of them are largely unmethylated, while the rest two CpGs (cg00106744 and cg21890848) are methylated at low/intermediate levels in tumors. Further studies are required to thoroughly elucidate the role of L-2-HG-mediated methylation in inhibiting GABPA expression. In addition, we recognize the global effect of both 5-AZA and L-2-HG on DNA methylation and 5hmC levels, and specific approaches such as dCas9-TET combined with GABPA promoter targeting have been planned to address these issues in our future studies.

Although we identified that the GABPA-TGFBR2 nexus plays a key suppressive role in ccRCC aggressiveness, it remains possible that GABPA may restrain aggressive ccRCC by regulating other targets. For instance, DICER1, a ribonuclease functioning in the microRNA (miRNA) processing machinery, is transcriptionally activated by GABPA through which the invasive phenotype and metastasis of thyroid cancer are inhibited [34]. In bladder cancer, GABPA activates the Fox1A and GATA3 genes, thereby promoting cancer cell differentiation [19]. Thus, further studies are required to thoroughly delineate various effects of GABPA on ccRCC progression. On the other hand, the strong association between GABPA and TGFBR2 is observed in many types of cancer based on the TCGA data analyses, which indicates a broad implication of the present findings in oncogenesis.

Paradoxically, GABPA has long been documented to act as an oncogenic factor, especially for its critical role in activating the mutated TERT promoter and inducing TERT expression [13, 14, 18]. TERT, as the catalytic component of telomerase, is not only required for infinite proliferation of cancer cells by stabilizing telomere length, but also operative in promoting invasion, metastasis and other cancer hallmarks via a telomere lengthening-independent function [19, 20]. Indeed, glioblastoma cells knocked-out of GABPB1, the GABPA partner required for activation of the mutated TERT promoter, was shown to undergo telomere shortening, senescence or apoptosis and eventual loss of tumorigenesis [17]. Therefore, targeting GABPB1 or GABPA for telomerase-based therapy has been suggested as a novel anti-cancer strategy [17, 35]. However, evidence has recently accumulated that GABPA function may be context-dependent, and it acts as a tumor suppressor in several types of cancer, despite its stimulatory effect on TERT transcription [19, 34, 36‐39]. Similarly, GABPB1 downregulation due to the gene hypermethylation is observed in thyroid carcinoma and its inhibition promotes invasion of thyroid cancer cells, too [40]. These unprecedented findings suggest that it should be cautious in targeting GABPA or GABPB1 for cancer intervention.