The Complex Interplay between Metabolic Reprogramming and Epigenetic Alterations in Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the most common malignancy affecting the kidney. Current therapies are mostly curative for localized disease, but do not completely preclude recurrence and metastization. Thus, it is imperative to develop new therapeutic strategies based on RCC biological properties. Presently, metabolic reprograming and epigenetic alterations are recognized cancer hallmarks and their interactions are still in its infancy concerning RCC. In this review, we explore RCC biology, highlighting genetic and epigenetic alterations that contribute to metabolic deregulation of tumor cells, including high glycolytic phenotype (Warburg effect). Moreover, we critically discuss available data concerning epigenetic enzymes’ regulation by aberrant metabolite accumulation and their consequences in RCC emergence and progression. Finally, we emphasize the clinical relevance of uncovering novel therapeutic targets based on epigenetic reprograming by metabolic features to improve treatment and survival of RCC patients.


Introduction
Renal cancer (RC) is the 16th most incident cancer worldwide according to GLOBOCAN 2018 estimates, accounting for approximately 2% of all cancer-related deaths [1].
Cigarette smoking, obesity and hypertension are the main recognized risk factors for RC [2], although other medical conditions, including renal lithiasis, urinary tract infection and chronic kidney disease, have also been associated with increased risk [3]. Sex also affects RC incidence and mortality, as this malignancy is 50% less frequent in women compared to men [4], which might be due to different smoking habits, occupational risk factors and sex steroid hormones [5]. Moreover, genetics factors might also lead to this two-fold increased risk in men [6]. Approximately 2-3% of all RC are associated with familial history, and several distinct genetic alterations are described [7]. Renal cell carcinoma (RCC) is the most common RC type [7], with clear cell RCC (ccRCC) subtype encompassing approximately 75% of all RCC, followed by papillary RCC (pRCC) and chromophobe RCC (chRCC), which constitute approximately 10% and 5% of all cases, respectively [8]. This review expression associated with hypoxia-related genes accumulation with consequent tumor cell progression and glycolytic metabolism [21,31].

Warburg Effect
Over the last decades, remarkable progress in the knowledge of carcinogenesis and tumor biology has been accomplished, resulting from the recognition of new cancer hallmarks, such as reprogramming of energy metabolism [32]. Indeed, metabolic switch is a well substantiated mechanism essential for tumor initiation and progression, as it supports cell growth and division [32].
Under aerobic conditions, normal quiescent cells of differentiated tissues convert glucose into pyruvate via glycolysis, which is transported from the cytosol to the mitochondria, and subsequently converted to acetyl-coenzyme A (acetyl-CoA). Thereafter, acetyl-CoA enters the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) occurs. In these conditions, cells show basal glycolysis rate and most of adenosine triphosphate (ATP) is provided by OXPHOS [33]. Conversely, in the absence of oxygen, glycolysis is favored and pyruvate is metabolized to lactate [32]. Proliferative cells undergo a metabolic switch, due to bioenergetic and biosynthesis needs. These cells display increased glucose consumption, as well as high glycolytic rates, lactate production, and lipids biosynthesis and of other macromolecules [34].
Tumor cells share the metabolic signature of proliferating cells to fuel cell growth and division [35]. In fact, the highly glycolytic cancer cell metabolism is sustained even in the presence of oxygen, so-called aerobic glycolysis or Warburg effect [35,36]. Thus, in tumor cells, most of the incoming glucose is converted into lactate (approximately 85%), and just a small percentage of pyruvate produced by glycolysis is metabolized in the mitochondria through OXPHOS (about 5%) [34]. Compared to mitochondrial OXPHOS, glycolysis is bioenergetically less efficient, generating less ATP than OXPHOS [37], but allows rapid ATP synthesis in the cytoplasm [38,39]. Furthermore, glycolysis is sustained by preservation of nicotinamide adenine dinucleotide (NAD) + /NADH ratio, since the consumed NAD + is regenerated during pyruvate conversion to lactate [33,40]. In fact, metabolic reprogramming confers several advantages to tumor cells [41]. First, glycolysis supports biosynthesis of lipids, amino acids and also nucleotides by providing intermediates of the glycolytic pathway for anabolic reactions. Cancer energy metabolism promotes cell survival and proliferation under fluctuating oxygen availability. Additionally, Warburg effect protects cells from oxidative stress by reducing reactive oxygen species (ROS) production due to increased levels of antioxidant glutathione. Moreover, increased glycolysis results in augmented lactate production, contributing to microenvironment acidification, which has been associated with invasive phenotype, including increased migration, invasion, metastization, immunosuppression and therapy resistance [38,39,41].
Interestingly, an intra-tumoral symbiosis was described in these tumors. Considering the blood vessels distribution, the tumor cells are exposed to different O2 levels. Hypoxic cancer cells are more distant from the supplying blood vessel and are poorly oxygenated. These cells display high HIF-1α expression that induces glycolytic proteins expression, increasing glucose uptake and lactate production, as well as lactate efflux. On the other hand, well oxygenated tumor cells are able to perform lactate uptake and preferentially use it as the main energy source, by employing it as main substrate for mitochondrial OXPHOS [42,43]. Knowing that both tumor metabolic reprograming and intra-tumoral symbiosis have an important role in tumor growth and therapy resistance, new anti-cancer therapies targeting cancer bioenergetics have been developed.

Metabolic Players in Renal Cell Carcinoma
As described in the previous section, RCC is characterized by mutations in genes related with cell metabolism, namely FH and SDH genes. FH-and SDH-deficient RCC accumulate fumarate or succinate, respectively, which inhibits PHDs, leading to HIF stabilization and accumulation in the cytosol ( Figure 1). In addition to FH and SDH mutations, loss of VHL function is also found in a large proportion of RCCs. Indeed, VHL is responsible for HIF degradation, so that in cells lacking VHL, HIF is stabilized, leading to HIF pathway activation ( Figure 1) [44,45]. Furthermore, increased HIF translation may result from mTOR and MET activation [44]. Consequently, HIF-1α target genes, implicated in glucose metabolism, cell proliferation and angiogenesis, are upregulated, contributing to a glycolytic phenotype and cancer progression [46,47].

Role of Lactate in Tumor Microenvironment
A glycolytic phenotype and high lactate production are common features of RCC [38]. Owing to lactate efflux with co-transport of H + through MCTs, cancer cells maintain the intracellular pH at physiological levels, whereas tumor microenvironment becomes acidic [57], which has been associated with increased tumor aggressiveness [58]. Accordingly, high lactate levels have been RCCs are characterized by a high glycolytic phenotype with increased lactate production, where glucose is the major energy source. Pentose phosphate pathway, fatty acid synthesis and glutaminolysis are also increased to sustain nucleotides, amino acids and lipids biosynthesis. In contrast, TCA cycle activity is decreased in these tumors. In the scheme proteins that were found upregulated in RCC are labeled red, while downregulated proteins are labeled blue. Abbreviations: ACC, Acetyl-coenzyme A carboxylase; Acetyl-CoA, Acetyl-coenzyme A; ALDO, Fructose-bisphosphate aldolase; ENO, Enolase; FAS, Fatty acid synthase; G6PD, Glucose-6-phosphate dehydrogenase; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GLS, Glutaminase; GPI, Glucose phosphatase isomerase; HK, Hexokinase; IDH, Isocitrate dehydrogenase; MCT, Monocarboxylate transporter; OAA, Oxaloacetate; PDH, Pyruvate dehydrogenase; PDK, Pyruvate dehydrogenase kinase; PFK, 6-phosphofructokinase; PGK, Phosphoglycerate kinase; PKM, Pyruvate kinase M; SLC1A5, Solute carrier family 1 member 5; TCA, Tricarboxylic acid cycle; α-KG, Alpha-ketoglutarate.

Role of Lactate in Tumor Microenvironment
A glycolytic phenotype and high lactate production are common features of RCC [38]. Owing to lactate efflux with co-transport of H + through MCTs, cancer cells maintain the intracellular pH at physiological levels, whereas tumor microenvironment becomes acidic [57], which has been associated with increased tumor aggressiveness [58]. Accordingly, high lactate levels have been associated with poor prognosis, high risk for development of metastasis and high tumor recurrence in several solid tumors [59]. Furthermore, lactate plays an important role in tumor microenvironment by supporting tumor growth and proliferation, as well as increasing migration, invasion, sustained angiogenesis, immune escape and therapy resistance [60][61][62], constituting a tumor metabolic fuel [43,63]. Indeed, HIF-1α stabilization by lactate was associated with increased vascular endothelial growth factor (VEGF) and kinase insert domain receptor (VEGFR2) expression levels by tumor and endothelial cells, respectively [64]. Moreover, lactate induces interleukin 8 (IL-8) production by endothelial cells in HIF-1α-independent way [65]. Nonetheless, during long hypoxia periods, N-Myc downstream-regulated (NDRG3) protein expression in cancer cells promotes VEGF, IL-8 and platelet and endothelial cell adhesion molecule 1 (CD31) up-regulation [66].
Remarkably, lactate was shown to impact on immune surveillance, since high microenvironmental levels associated with decreased T cells cytotoxic and natural killer cells activity, along with reduced dendritic cells cytokine release and differentiation [62]. Additionally, lactate-induced HIF-1α stabilization stimulates polarization of tumor-associated macrophages, contributing to immunosuppression [67].
Other lines of evidence regarding lactate role in tumorigenesis include the positive correlation found between tumor lactate content and radioresistance in human solid tumors [68].

Epigenetic Mechanisms
Epigenetics are heritable and reversible changes in gene expression patterns that result from alterations in chromatin and not in DNA sequence [69]. Epigenetic marks are crucial to maintain genomic stability, chromosome imprinting and cell differentiation in normal development [70]. Nevertheless, cellular epigenetic landscape plasticity is affected by genetic, environmental or metabolic stimuli allowing for cellular adaptation but that might lead to neoplastic transformation [71]. Epigenetic deregulation is a cancer hallmark, characterized by global gene expression deregulation with activation of proto-oncogenes and silencing of tumor suppressors genes due to changes in DNA methylation, histone post-translational modifications and chromatin remodeling complexes ( Figure 3) [72,73].
Genes 2019, 10, x FOR PEER REVIEW 6 of 19 associated with poor prognosis, high risk for development of metastasis and high tumor recurrence in several solid tumors [59]. Furthermore, lactate plays an important role in tumor microenvironment by supporting tumor growth and proliferation, as well as increasing migration, invasion, sustained angiogenesis, immune escape and therapy resistance [60][61][62], constituting a tumor metabolic fuel [43,63]. Indeed, HIF-1α stabilization by lactate was associated with increased vascular endothelial growth factor (VEGF) and kinase insert domain receptor (VEGFR2) expression levels by tumor and endothelial cells, respectively [64]. Moreover, lactate induces interleukin 8 (IL-8) production by endothelial cells in HIF-1α-independent way [65]. Nonetheless, during long hypoxia periods, N-Myc downstream-regulated (NDRG3) protein expression in cancer cells promotes VEGF, IL-8 and platelet and endothelial cell adhesion molecule 1 (CD31) up-regulation [66].
Remarkably, lactate was shown to impact on immune surveillance, since high microenvironmental levels associated with decreased T cells cytotoxic and natural killer cells activity, along with reduced dendritic cells cytokine release and differentiation [62]. Additionally, lactateinduced HIF-1 stabilization stimulates polarization of tumor-associated macrophages, contributing to immunosuppression [67].
Other lines of evidence regarding lactate role in tumorigenesis include the positive correlation found between tumor lactate content and radioresistance in human solid tumors [68].

Epigenetic Mechanisms
Epigenetics are heritable and reversible changes in gene expression patterns that result from alterations in chromatin and not in DNA sequence [69]. Epigenetic marks are crucial to maintain genomic stability, chromosome imprinting and cell differentiation in normal development [70]. Nevertheless, cellular epigenetic landscape plasticity is affected by genetic, environmental or metabolic stimuli allowing for cellular adaptation but that might lead to neoplastic transformation [71]. Epigenetic deregulation is a cancer hallmark, characterized by global gene expression deregulation with activation of proto-oncogenes and silencing of tumor suppressors genes due to changes in DNA methylation, histone post-translational modifications and chromatin remodeling complexes ( Figure 3) [72,73].
In normal cells, DNA methylation is associated with repetitive genomic regions, whereas gene promoters are generally unmethylated [79]. Contrarily, cancer cells display global DNA hypomethylation resulting in genome instability with proto-oncogenes activation, loss of imprinting and high mutation rates. Furthermore, aberrant gene promoter hypermethylation in cancer is associated with tumor suppressor genes' transcription silencing [79,80].
Regarding histone methylation, HMTs catalyze the methylation reaction in lysine or arginine residues of histone tails using SAM as a methyl donor. To revert this condition, KDMs remove the methyl group from a methylated lysine by oxidation in a flavin adenine dinucleotide (FAD) or α-ketoglutarate (α-KG) dependent manner [83]. These enzymes have been found deregulated in several tumors. Histone methylation and consequent alteration of transcriptional status is dependent on the methylation degree and the residue modified. Trimethylation of H3K4 is found at transcriptionally active gene promoters, whereas H3K9me and H3K27me3 marks are associated with transcriptional repression [84]. Additionally, H4K20me3, a repressive histone mark, is also frequently found in human cancers [85].
Acetylation of histone lysine residues is established by HATs, using acetyl-CoA as acetyl donor [86]. The addition of acetyl group neutralizes the positive charges of histones decreasing DNA compaction, allowing protein binding and transcription activation [87]. Contrarily, deacetylation leads to DNA condensation by restoring the positive charges of lysine, with consequent transcription abrogation [87]. HDACs are responsible for removing acetyl groups and are categorized into four classes [88]. Classes I, II and IV share a catalytic mechanism in which zinc is used to catalyze hydrolysis of the lysine-amino bond [89]. Conversely, HDAC Class III, or Sirtuins (SIRTs), rely on NAD + for deacetylation activity [90]. HATs and HDACs enzymes' deregulation have been implicated in tumorigenesis and metastasis [91]. Indeed, loss of histone H4 Lys16 acetylation is defined as a common feature in cancer development [85]. Moreover, H3K9 hypoacetylation was associated with a high proliferative profile and higher recurrence rates in ependymal tumors [92].

Epigenetic Alterations in Renal Cell Carcinoma
Epigenetic mechanisms allow cell adaptation to environmental changes, but also fueling cancer cells phenotype by supporting cell proliferation and metabolic reprogramming [93]. In RCC, alterations in DNA methylation profile and deregulation of genes involved in histone modifications and chromatin remodeling are frequently found (Figure 4) [94].
alterations in DNA methylation profile and deregulation of genes involved in histone modifications and chromatin remodeling are frequently found (Figure 4) [94]. RCC commonly display high methylation levels at CpG islands associated with glycolytic phenotype, as well as aggressive behavior [11,21,95]. In RCC, epigenetic silencing through gene promoter hypermethylation often downregulates genes related with Wnt/β-catenin and transforming growth factor (TGF) β pathway, pro-apoptotic genes, cell cycle regulator' genes and genes responsible for cell adhesion [96], implicated in cancer proliferation and metastization [97].
Regarding chromatin remodeling enzymes, ccRCC commonly harbor BAP1 and PBRM1 inactivating mutations (mutated in approximately 15% and 50% of ccRCC cases, respectively) [98]. BAP1-deficient ccRCC tumors are associated with poor prognosis [99], whereas loss of PBRM1 expression increases tumor aggressiveness [100]. Moreover, inactivating mutations in SETD2, KDM5C and KMD6A were also found in RCC (mutated in approximately 5-15% of ccRCC cases) [101]. As a result of HTMs and KDMs deregulation, RCC display a specific histone methylation profile. Indeed, lower global H3K4 methylation levels are associated with poor prognosis and higher recurrence rates in RCC [102,103]. H3K9 and H4K20 global methylation is downregulated in RCC and has been correlated with tumor aggressiveness [103,104]. Furthermore, lower H3K27 methylation levels were found in RCC compared to oncocytomas, associating with higher pathological stage and grade, as well as lymph node and distant metastasis [105].
Globally, HDACs Class I and II seem to act as oncogenes. Namely, HDAC2 overexpression was associated with high proliferation index [106] and high HDAC3 expression levels were found in RCC [107], although an inverse correlation was depicted between pathological stage and HDAC3 levels [106]. Moreover, in vitro assays associated HDAC1 and HDAC6 overexpression with cell invasion, migration and motility modulation, by increasing matrix metalloproteinase expression and decreasing acetylated α-tubulin expression [108]. Furthermore, a The Cancer Genome Atlas (TCGA) dataset analysis associated high HDAC1 mRNA levels with ccRCC worse overall survival [108]. Furthermore, high HDAC6 expression levels were displayed by high grade RCC and were shown to be an independent poor prognostic factor [109]. Paradoxically, although ccRCC samples depict lower HDAC9 levels than normal adjacent tissues, high HDAC9 expression was associated with poor prognosis [110]. Remarkably, global histone acetylation downregulation was found in RCC and associated with patients' outcome [111]. Decreased H3 and H4 global acetylation was associated with high tumor grade, advanced stage, including distant metastasis, and tumor progression [111,112]. RCC commonly display high methylation levels at CpG islands associated with glycolytic phenotype, as well as aggressive behavior [11,21,95]. In RCC, epigenetic silencing through gene promoter hypermethylation often downregulates genes related with Wnt/β-catenin and transforming growth factor (TGF) β pathway, pro-apoptotic genes, cell cycle regulator' genes and genes responsible for cell adhesion [96], implicated in cancer proliferation and metastization [97].
Regarding chromatin remodeling enzymes, ccRCC commonly harbor BAP1 and PBRM1 inactivating mutations (mutated in approximately 15% and 50% of ccRCC cases, respectively) [98]. BAP1-deficient ccRCC tumors are associated with poor prognosis [99], whereas loss of PBRM1 expression increases tumor aggressiveness [100]. Moreover, inactivating mutations in SETD2, KDM5C and KMD6A were also found in RCC (mutated in approximately 5-15% of ccRCC cases) [101]. As a result of HTMs and KDMs deregulation, RCC display a specific histone methylation profile. Indeed, lower global H3K4 methylation levels are associated with poor prognosis and higher recurrence rates in RCC [102,103]. H3K9 and H4K20 global methylation is downregulated in RCC and has been correlated with tumor aggressiveness [103,104]. Furthermore, lower H3K27 methylation levels were found in RCC compared to oncocytomas, associating with higher pathological stage and grade, as well as lymph node and distant metastasis [105].
Globally, HDACs Class I and II seem to act as oncogenes. Namely, HDAC2 overexpression was associated with high proliferation index [106] and high HDAC3 expression levels were found in RCC [107], although an inverse correlation was depicted between pathological stage and HDAC3 levels [106]. Moreover, in vitro assays associated HDAC1 and HDAC6 overexpression with cell invasion, migration and motility modulation, by increasing matrix metalloproteinase expression and decreasing acetylated α-tubulin expression [108]. Furthermore, a The Cancer Genome Atlas (TCGA) dataset analysis associated high HDAC1 mRNA levels with ccRCC worse overall survival [108]. Furthermore, high HDAC6 expression levels were displayed by high grade RCC and were shown to be an independent poor prognostic factor [109]. Paradoxically, although ccRCC samples depict lower HDAC9 levels than normal adjacent tissues, high HDAC9 expression was associated with poor prognosis [110]. Remarkably, global histone acetylation downregulation was found in RCC and associated with patients' outcome [111]. Decreased H3 and H4 global acetylation was associated with high tumor grade, advanced stage, including distant metastasis, and tumor progression [111,112].
Furthermore, in ccRCC cell lines, H3 acetylation restoration inhibited cell proliferation, whereas apoptosis and cell cycle arrest were induced [112].
Regarding HDACs Class III, SIRT1, SIRT3 and SIRT6 were shown downregulated in RCC samples compared with normal kidney tissues, suggesting a putative role as onco-suppressors. Furthermore, in the same study, SIRT3 higher expression levels were correlated with better patients' outcome [113]. SIRTs, in addition to chromatin structure regulation, have been suggested to modulate several cellular processes including metabolism and genomic stability maintenance [114]. Indeed, SIRT1, SIRT3 and SIRT6 downregulation was associated with HIF-1α-mediated glycolytic metabolism activation, tumor growth and glucose homeostasis [115][116][117].

Interplay Between Metabolism and Epigenetic Mechanisms
Cancer cells display several genetic and epigenetic alterations that enable metabolic adaptation and sustain cell proliferation and survival [118]. The cancer metabolic switch leads to metabolites accumulation, which have been suggested to modulate epigenetic factors and consequent gene expression. Indeed, epigenetic enzymes involved in DNA and histone modifications require intermediates of cellular metabolic pathways, such as acetyl-CoA, α-KG, SAM and NAD + as cofactors or substrates [119,120]. Furthermore, metabolic pathways disruption can also lead to accumulation of metabolites such as coenzyme A (CoA-SH), S-adenosylhomocysteine (SAH), β-hydroxybutyrate (β-OHB), 2-hydroxyglutarate (2-HG), fumarate, succinate and lactate, which inhibit specific epigenetic players [121].

Renal Cell Carcinoma Metabolism and Histone Acetylation Modulation
As previously mentioned, RCC display high glycolytic metabolism with increased pyruvate conversion into lactate [45]. Although the effect of these two metabolites in epigenetic landscape has not yet been described for RCC, these two metabolites were shown to act as HDACs inhibitors in other tumors. Accordingly, increased tumor cell apoptosis was associated with HDACs inhibition by pyruvate, suggesting a negative role in tumor formation of the latter [122]. Additionally, most pyruvate is directly converted into lactate to keep the NAD + /NADH ratio, sustaining glycolysis required by cancer cells. Conversely, lactate inhibition of HDACs Classes I and II was reported for colon and cervical cancers. Lactate promotes histone acetylation and gene expression through HDAC-inhibition mechanism contributing to DNA repair and to anticancer therapy resistance [123,124]. Furthermore, increased histone acetylation levels have been found after exposing breast cancer cells to lactate, as well as consequent increased expression on stemness-related genes and an association between lactate induced genes and patient outcome. However, in that study, lactate-associated acetylation was justified not by HDACs inhibition but through HATs activation by acetyl-CoA pool increase [125]. Additionally, other metabolites, such as butyrate and β-OHB, are also well known HDACs inhibitors [126,127]. Interestingly, butyrate plays a dual role in cells' epigenetic regulation. In cancer cells, Warburg effect leads to increased butyrate levels because it is not converted into acetyl-CoA. In these conditions, butyrate accumulation inhibits HDACs Class I, II and IV activity and cell proliferation by transcription of pro-apoptotic genes [128]. Instead, when cells use butyrate as fuel through acetyl-CoA production, proliferation is stimulated and cancer growth is facilitated [128]. Curiously, β-OHB seems to have an effect similar to butyrate. In absence of glycolytic metabolism, β-OHB is used as oxidative energy source instead of epigenetic regulator, inducing tumor growth [129]. In the presence of glycolytic metabolism, however, β-OHB is not converted into acetyl-CoA but it accumulates, leading to HDACs Class I and II inhibition [125]. Additionally, an in vivo study demonstrated specific inhibition of HDACs Class I by β-OHB with consequent increase in global histone acetylation, which was correlated with oxidative stress resistance, inducing transcriptional activity [130]. In parallel, cancer glycolysis decreases NAD + /NADH ratio, essential for SIRTs activity, as these enzymes depend on NAD + as cofactor. Thus, SIRTs inhibition seems to contribute to hyperacetylation and aberrant gene transcription, leading to cancer progression [118]. Nevertheless, additional studies are needed to better understand the interactions between metabolic status and SIRTs activity in cancer.
In RCC, glycolytic metabolism induced by HIF-1α pathway activation leads to TCA cycle suppression by blocking pyruvate conversion into acetyl-CoA [131]. In this situation, tumor cells use other sources to obtain acetyl-CoA essential for lipid synthesis, as well as for HATs function [132]. Cancer cells can reprogram its metabolism to produce mitochondrial citrate, which is transported to cytosol and converted into acetyl-CoA through ATP-citrate lyase [133]. In these conditions, acetyl-CoA plays a role in histone acetylation in response to glucose and growth factor stimulation [134,135]. Cytosolic acetate is another acetyl-CoA source [133]. Under glucose restriction or hypoxic stress, acetate is used as an immediate metabolic precursor for fatty acid synthesis and also induces expression of genes involved in de novo lipid synthesis and cancer cell survival, by promoting histone acetylation at specific promoter regions [136]. Additionally, acetyl-CoA generated from glutamine was shown to be involved in histone acetylation, inducing the expression of genes involved in lipid metabolism [137].
Thus, taking in account the high glycolytic metabolism of RCC, these tumors seem to disclose global histone hyperacetylation and consequent aberrant gene transcription, possibly due to increased HATs activity induced by acetyl-CoA, and also HDACs inhibition by several metabolites that contribute to cancer progression and aggressiveness ( Figure 5A) [118]. Further studies to clarify this hypothesis in this specific model are still needed. In RCC, glycolytic metabolism induced by HIF-1α pathway activation leads to TCA cycle suppression by blocking pyruvate conversion into acetyl-CoA [131]. In this situation, tumor cells use other sources to obtain acetyl-CoA essential for lipid synthesis, as well as for HATs function [132]. Cancer cells can reprogram its metabolism to produce mitochondrial citrate, which is transported to cytosol and converted into acetyl-CoA through ATP-citrate lyase [133]. In these conditions, acetyl-CoA plays a role in histone acetylation in response to glucose and growth factor stimulation [134,135]. Cytosolic acetate is another acetyl-CoA source [133]. Under glucose restriction or hypoxic stress, acetate is used as an immediate metabolic precursor for fatty acid synthesis and also induces expression of genes involved in de novo lipid synthesis and cancer cell survival, by promoting histone acetylation at specific promoter regions [136]. Additionally, acetyl-CoA generated from glutamine was shown to be involved in histone acetylation, inducing the expression of genes involved in lipid metabolism [137].
Thus, taking in account the high glycolytic metabolism of RCC, these tumors seem to disclose global histone hyperacetylation and consequent aberrant gene transcription, possibly due to increased HATs activity induced by acetyl-CoA, and also HDACs inhibition by several metabolites that contribute to cancer progression and aggressiveness ( Figure 5A) [118]. Further studies to clarify this hypothesis in this specific model are still needed.

Renal Cell Carcinoma Metabolism in DNA and Histone Methylation Modulation
Fumarate and succinate accumulation are typically found in FH and SDH-deficient RCC. Both metabolites have been related with α-KG-dependent histone inhibition and DNA demethylases, namely TETs and KDMs [138,139]. In tumors with SDH mutations, succinate accumulation associates with DNA hypermethylation phenotype [140]. Inhibition of histone and DNA demethylases was associated with increased tumor aggressiveness and invasion potential, mediated by epigenetic silencing of genes involved in cell differentiation and epithelial mesenchymal transition (EMT) [140,141]. Moreover, fumarate was also associated with tumor aggressiveness. Sciacovelli et al. demonstrated that intracellular fumarate accumulation drives changes in EMT genes, such as zinc finger E-box binding homeobox (ZEB), snail family transcriptional repressor 1 (SNAI1) and vimentin (VIM), through inhibition of TETs and consequent anti-metastatic microRNA cluster downregulation, in murine renal carcinoma cells [142].
More recently, L enantiomer of 2-hydroxyglutarate (L-2HG) was found to be accumulated in RCC [143]. This metabolite is produced from α-KG by LDHA under hypoxic conditions [144,145] and was demonstrated to be a potent TETs and KDMs inhibitor [146,147]. In RCC, increased L-2HG levels are mediated by L-2HG dehydrogenase (L2HGDH) reduced expression due to copy number loss [143]. Additionally, Shim et al. reported an inverse association between high L-2HG levels and reduced DNA 5hmC levels due to TET activity inhibition [143]. Contrarily, L2HGDH restoration in RCC cells was associated with L-2HG decrease and consequent DNA and histone demethylation by re-activation of TETs and KDMs activity. Moreover, L2HGDH re-expression was able to attenuate the malignant phenotype by inhibiting L-2HG oncogenic role in RCC cell lines [143]. Hence, owing to mutations in FH, SDH and L2HGDH genes, which lead to TET and KMDs inhibition, aberrant accumulation of oncometabolites occurs in RCC, stimulating tumor growth and aggressiveness ( Figure 5B) [148,149].

Conclusions and Perspectives
Although the majority of RCCs are currently diagnosed at initial stages, about 20% of patients already harbor metastases at the time of diagnosis, decreasing the survival rate to less than 20% [150]. Despite advances in targeted therapies, nephrectomy remains the mainstay of RCC treatment [151]. Nevertheless, systemic therapy based on immune system modulators has traditionally been used in metastatic disease, although with modest impact in patient outcome [152]. Over the past decade, significant improvements have been made, and new targeted therapies, including antiangiogenic agents, mTOR and immune checkpoint inhibitors, have emerged as promising alternatives to current therapies [153,154]. Notwithstanding the progress in this field, RCC remains associated with high recurrence rates, and a sizeable proportion of patients with initially localized disease develop metastasis during follow-up [155,156]. The identification of new tumor-associated markers that might allow the design of new therapeutic strategies in management of RCC patients is thus crucial. Cell metabolism deregulation, particularly Warburg effect, plays a major role in RCC development, contributing to accumulation of metabolites that regulate epigenetic factors. Hence, the study and discovery of new metabolic target therapies that might be used in combination with standard therapy might be of clinical value. Furthermore, the usage of drugs that reestablish defects in epigenetic machinery caused by metabolites' accumulation may be a strategy deserving to be explored.

Conflicts of Interest:
The authors declare no conflict of interest.