Metabolic Reprogramming in Renal Cancer: Events of a Metabolic Disease

Abstract

Recent studies have established that tumors can reprogram the pathways involved in nutrient uptake and metabolism to withstand the altered biosynthetic, bioenergetics and redox requirements of cancer cells. This phenomenon is called metabolic reprogramming, which is promoted by the loss of tumor suppressor genes and activation of oncogenes. Because of alterations and perturbations in multiple metabolic pathways, renal cell carcinoma (RCC) is sometimes termed as a “metabolic disease”. The majority of metabolic reprogramming in renal cancer is caused by the inactivation of von Hippel-Lindau (VHL) gene and activation of the Ras-PI3K-AKT-mTOR pathway. Hypoxia-inducible factor (HIF) and Myc are other important players in the metabolic reprogramming of RCC. All types of RCCs are associated with reprogramming of glucose and fatty acid metabolism and the tricarboxylic acid (TCA) cycle. Metabolism of glutamine, tryptophan and arginine is also reprogrammed in renal cancer to favor tumor growth and oncogenesis. Together, understanding these modifications or reprogramming of the metabolic pathways in detail offer ample opportunities for the development of new therapeutic targets and strategies, discovery of biomarkers and identification of effective tumor detection methods.

1. Introduction

Metabolism can be defined as the summation of different biochemical reactions inside the cell to gather energy or ATP and synthesize new biomolecules [1, 2]. Unlike normal cells, cancer cells proliferate rapidly [1, 2]. Therefore, they need a burst of energy and biomolecules to maintain growth and proliferation, which is impossible in the regular metabolic infrastructure of normal cells [1, 2]. Cancer cells remodel or reprogram metabolic pathways to fulfill the high-energy requirement, produce biomolecules on demand, and maintain the redox balance [1, 2].

The development of high-throughput technologies coupled with metabolomics enabled researchers to analyze the real-time status of metabolites in a tissue or body fluid, and relate their concentrations with genetic and physiological changes [3, 4]. These new studies helped us to thoroughly understand the reprogrammed metabolic pathways at different stages of cancer cell growth, and exploit them to develop novel therapeutic approaches [3, 4]. In the context of metabolic reprogramming, there is a newly emerged term, “oncometabolite”, which can be referred as intermediates of metabolism that abnormally accumulate in tumor cells [2, 5]. Oncometabolites are thought to result from genetic mutations associated with cancer development [2, 5].

In this review, we summarize the essential events in tumor-associated metabolic reprogramming and evaluate their contributions to tumor growth and survival, with particular importance to renal cancer.

2. Renal cancer, the metabolic disease

Among all cancers, kidney or renal cancer is an ideal model of metabolic reprogramming [6–9]. The array of genes, which are mutated, inactivated, or hyper-activated in renal tumorigenesis, are reported to be involved in the regulation of various metabolic events, such as glycolysis, TCA cycle, metabolism of glutamine, ATP production and modulation of pathways important for hypoxic condition and redox balance [6–9]. Hence, renal cancer has been duly named as a “Metabolic Disease” [7–10]. Besides the well-established “Warburg effect,” the glutamine-dependent reductive carboxylation occurs in different types of renal cancer cells [9, 11]. Reductive carboxylation induces the reverse flow of TCA cycle flux; and additionally, it promotes the glutathione/oxidized glutathione pathway to buffer oxidative stress [12, 13], and mediate tumor growth [14].

2.1. Reprogramming of metabolic genes in renal cancer

Genomics study and high throughput sequencing have established kidney cancer as a heterogeneous class of diseases rather than a single disease. Renal cancer involves mutation or inactivation of several genes regulating different pathways of cellular metabolism [6, 11]. Consequently, important genetic and molecular variations are observed in different histological subtypes of renal cell carcinoma (RCC), and is considered a continuum of different diseases [6]. According to histology, RCC is classified into three major subtypes, clear cell RCC (ccRCC), papillary RCC (pRCC), and chromophobe RCC (chRCC) [6, 15–17]. Other than that, there are some less prevalent subtypes, e.g., medullary RCC, collecting duct RCC, and hereditary leiomyomatosis and renal cell cancer (HLRCC) [6, 15]. ccRCC is the most prevalent subtype, and it occurs in more than 75% of all the RCC cases [15].

Even before introducing modern metabolomics applications, more than 12 genes involved with vital metabolism were connected with RCC growth and progression [10]. The Cancer Genome Atlas (TCGA) investigated and identified diverse genetic details involved with the three major histological subtypes of renal cancer (clear cell, papillary, and chromophobe) [18–22]. They have analyzed almost 500 samples of ccRCC and found proof of extensive metabolic reprogramming, consisting of down-regulation of the TCA cycle accompanied by up-regulation of the pentose phosphate pathway, fatty acid synthesis, and glutamine transporters [18]. TCGA studies had also shown that metabolic reprogramming contributes to poor prognosis and survival in RCC [18]. Interestingly, subsequent TCGA studies involving the three major histological RCC subtypes, identified somatic mutations and modification of the genes involved with metabolic pathways and associated with RCC progression and survival [18–22]. The essential genes involved in regulating metabolic reprogramming in renal cancer are VHL, PTEN, Akt, mTOR, TSC1/2 and Myc [10, 21, 23].

In most of RCC, with particular importance to ccRCC, the tumor suppressor gene von Hippel-Lindau (VHL) is often mutated or deleted. This leads to dysregulation of the hypoxia-inducible factor (HIF) family of transcription factors and their associated pro-oncogenic mediators, including several growth factors and their receptors [24–26]. The VHL-HIF axis is the most frequently activated pathway in ccRCC, and it acts as a therapeutic target [25, 26]. Inactivation of VHL leads to the stabilization of two VHL E3 ubiquitin ligase complex targets, HIF1α and HIF2α (encoded by HIF1A and EPAS1) [24–26]. Under hypoxic state of cancer cells, HIF1α and HIF2α up-regulate the transcription of several hypoxia-responsive genes involved in tumor growth, angiogenesis, and metastasis, as well as genes associated with glucose transport and metabolism [24, 25]. HIFs are known to drive the expression of several proteins and enzymes involved with glucose uptake and glycolysis like GLUT1(glucose transporter-1), PGK (phosphoglycerate kinase), LDHA (lactate dehydrogenase), PDK1(pyruvate dehydrogenase kinase), and HK (hexokinase) [27–29]. HIFs are also known to mediate suppression of the TCA cycle and oxidative phosphorylation [30].

In RCC cells, frequent mutations of Ras-PI3K–Akt–mTOR pathway genes (including PTEN, mTOR, and PIK3CA) were also observed [31, 32]. The activation of mTOR often influences the metabolic reprogramming in RCC [11]. The studies by TCGA on ccRCC detected several mutations in PTEN, TSC1/2, and PIK3CA, which are all components of the PI3K-AKT-mTOR pathway [32–35]. There are multiple hypotheses and reports over the contribution of mTOR towards metabolic reprogramming in RCC cells [33, 34, 36]. TSC1 and TSC2 code for the proteins hamartin and tuberin to form an inhibitor complex of mTORC1 activation [36]. Besides, inhibition of the tumor suppressor 4E-BP1 [37], mTORC1 augments the expression of HIF-1 and HIF-2 [38].

Like VHL, HIF and mTOR, the oncogene Myc has essential metabolic reprogramming functions in renal cancer [39, 40]. Myc expression is elevated in different types of cancer [39, 40]; and it is a proto-oncogenic transcription factor that up-regulates several genes involved in tumorigenesis [39]. Myc is often overexpressed in RCC cells [40, 41]. Myc plays a vital role in reprogramming glutamine metabolism [40, 42] and fatty acid synthesis in renal cancer [40].

In ccRCC, somatic mutations in chromatin remodeling genes, like PBRM1, SETD2, and BAP1 are common [18, 43]; but there are not many reports available on their involvement with metabolic reprogramming.

3. Reprogrammed metabolic pathways in cancer

The first observation of metabolic reprogramming in cancer cells was made far back in 1927 by Otto Warburg [4]. All of the remodeled metabolic pathways are not equally responsible for the growth and progression of cancer cells, even if they are under the oncogenic influence [1]. The physiological demand controls the onset of anabolism or catabolism for a metabolite [2]. In cancer cells, one or more classical metabolic pathways are reprogrammed according to cellular requirement. For example, in rapidly growing cancer cells, the Warburg effect enables the tumors to develop in a nutrient-deficient condition [4]. In contrast, in the same cells, the pentose phosphate pathway is up-regulated to feed the increased demand for nucleotides [4]. Besides, there could be an up-regulation of glutamine metabolism to protect the cells against oxidative stress and for biosynthesis of lipids [4].

It will be challenging to cover all the aspects of metabolism in cancer cells in a single review as there was a significant boost of research on this area over the last decade. In the following sections, we tried to summarize the reprogramming of major cellular metabolic pathways in cancer cells citing some recent reports. Moreover, we have discussed the importance of these metabolic pathways from the perspective of renal cancer.

3.1. Altered glucose transport and glycolysis

Glucose is the primary source of energy in the biological system [44]. Glucose deprivation in cancer cells results in oxidative stress and cellular cytotoxicity [45]. Glucose homeostasis is regulated by an intricate network of pathways comprised of glucose absorption, glycolysis, glycogenolysis, gluconeogenesis, glucose reabsorption, and glucose excretion [44, 46].

In normal cells, most of the glucose is converted to pyruvate through glycolysis [Figure. 1], which is carried to the mitochondria to enter the TCA cycle. In mitochondria, the pyruvate drives ATP production through the oxidative phosphorylation (OXPHOS)/electron transport (ETC) chain [44, 46]. In contrast to normal cells, cancer cells use lactic acid fermentation with lactate dehydrogenase (LDHA) to convert pyruvate to lactate [Figure. 1]. The catabolism of glucose to lactate yields a lower amount of energy compared to oxidative phosphorylation [46–49]. Therefore, a high glucose consumption rate is necessary to compensate for the energy demand of cancer cells [46–48]. Increased expression of the glucose transporters across the membrane of cancer cells contributes towards high glucose consumption [50]. The expression of passive glucose transporters of the GLUT family is often up-regulated in different cancers [50–52]. In thyroid cancer and colorectal cancer, GLUT1 has been reported to be up-regulated by activated Akt [50, 52]. Oncogenic Ras is also known to enhance GLUT1 expression. GLUT3, another member of the GLUT family, is stimulated by the NF-κB signaling in cancer cells [53].

Figure 1. Reprogramming of glucose transport and glucose metabolism in renal cancer.

Glucose metabolism is comprised of glycolysis and pentose phosphate pathway (PPP) in the cytoplasm and TCA cycle in the mitochondria. Both glycolysis and PPP are upregulated in renal cell carcinoma (RCC). Glucose, transported in to the cells by GLUT and SGLT transporters, undergoes glycolysis to generate pyruvate which then goes through TCA cycle to generate ATP. In cancer cells, the flux of pyruvate entering TCA cycle decreases and the majority of pyruvate undergoes lactic acid fermentation for the rapid production of ATP. The MCT family of lactate transporters are upregulated in cancer cells, leading to increased efflux of lactate; and lactate accumulation results in an immunosuppressive extracellular environment. In cancer cells, increased PPP supplies the ribose sugar for the nucleotide synthesis necessary for rapidly growing cells. The oncogenes like HIF, AKT, STAT3 and EZH2 are known to influence the glucose transport and metabolism at multiple steps in RCC cells. The metabolites are depicted by squares and enzymes or transporters are denoted by ovals. Green arrows: Increase in activity, Red arrows: Decrease in activity. Abbreviations: ALDO, aldolase; ENO, enolase; EZH2, Enhancer of zeste homolog 2; FBP1, fructose-1-bisphophatase; Fructose-1,6-BP, fructose 1,6-bisphosphate; Fructose-6-P, fructose 6-phosphate; Glyceraldehyde-3-P, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; Glyceraldehyde-3-P, glyceraldehyde 3-phosphate; HK, hexokinase; LDH, lactate dehydrogenase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PDK1, pyruvate dehydrogenase kinase; P-Enolpyruvate, phosphoenolpyruvate; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; R5P, ribose-5-phosphate; TKT, transketolase; TPI, triosephosphate isomerase; 1,3-BP-Glycerate, 1,3-bisphospho-D-glycerate; GLUT1, glucose transporter-1; 2-P-Glycerate, 2-phosphoglycerate; 3-P-Glycerate, 3-phosphoglycerate; 6-P-Gluconolactone, 6-phosphoglucono-d-lactone; 6-P-Gluconate, 6-phosphogluconate; 6PGD, 6-phosphogluconate dehydrogenase; G6PD; glucose- 6-phosphate dehydrogenase; HIF, hypoxia inducible factor; MCT1, Monocarboxylate transporter 1; MCT4, Monocarboxylate transporter 4; SGLT2, sodium-glucose cotransporter 2; STAT3, Signal transducer and activator of transcription 3.

Cancer cells have elevated levels of hypoxia-inducible factors or HIFs, irrespective of hypoxia or normoxia [25]. The high levels of HIF induce an increased expression of Lactate Dehydrogenase A (LDHA), which catalyzes the conversion of pyruvate to lactate regardless of oxygen availability [54, 55]. This remodeling of glucose metabolism is known as the “Warburg effect” or aerobic glycolysis [56–58]. Augmentation of aerobic glycolysis expedites the supply of carbon intermediates for the biosynthesis of amino acids, lipids, and nucleic acids [56, 57]. Gene expression of several of the enzymes of the glycolytic pathway like HK (hexokinase), PFK (phosphofructokinase), PGK1(phosphoglycerate kinase), and PKM2 (phosphoglycerate mutase) is up-regulated in different types of cancer [Figure. 1]. On the other hand, the primary byproduct of glycolysis, i.e., lactic acid, is transported out from the cancer cells by the monocarboxylate transporters (MCTs) to facilitate the forward flux of glucose through glycolysis [59, 60]. For that purpose, a high number of MCT transporters are expressed in cancer cells [59, 60]. The glucose dependency of cancer cells is always a subject of interest in developing effective cancer therapies. 2-deoxy-D-glucose (2DG) is a nontoxic structural analog of glucose [61]. 2DG competes with normal glucose for uptake via glucose transporters and enter glycolysis after phosphorylation by hexokinase [61]. Through competing with glucose, 2DG generates an artificial and chemically induced state of glucose deprivation along with suppression of glucose metabolism [61]. 2DG can inhibit glucose metabolism and the growth of breast [62], colon [63], and cervical cancer cells [64].

3.1.1. Glucose transport and Glycolysis in renal cancer

Kidneys play a significant role in glucose homeostasis as they are involved in glucose excretion, gluconeogenesis, and glucose reabsorption [46]. Therefore, kidneys contain a high number of glucose transporters [65]. Courtney et al. (2018) demonstrated through isotope labeling that ccRCC cells show enhanced glycolysis, suppressed pyruvate dehydrogenase flow, and reduced TCA cycle compared to tumors at other anatomic sites [66]. Moreover, they have shown that the rate of glycolysis in ccRCC was significantly higher compared to neighboring kidney cells [66]. ccRCC tumors possess metabolically distinct physiology compared to tumors in the brain and lungs; the glucose oxidation rate was markedly lower in ccRCC cells [66]. Renal cells express multiple members from the GLUT family of passive glucose transporters. e.g., GLUT1 and GLUT2 [65]. Moreover, kidneys also possess a high number of ATP-dependent active glucose transporters from the sodium-dependent glucose co-transporter (SGLT) family [67]. SGLT2, physiologically the most vital member of the SGLT family, is involved in glucose reabsorption in the kidney [67]. The altered expression of glucose transporters is probably one of the major phenotypic characteristics of RCC [Figure. 1]. PBRM1, a tumor suppressor, is the second most frequently mutated gene in ccRCC [68]. Chowdhury et al. have shown that re-expression of PBRM1 in RCC cells led to decreased glucose uptake [68]. Bianchi et al. reported that the inhibition of glycolysis with 2DG (the structural analog of glucose) impaired the viability and proliferation of ccRCC [69].

The VHL mutated ccRCC cells show an increased glucose uptake with enhanced GLUT1 expression [70]. The increase of GLUT1 expression in RCC cells correlates with the reduction of infiltrating CD8⁺ T cells, indicating a role of GLUT1 in the immune-escape machinery of the renal cancer cells [70]. This lower infiltration of CD8⁺ T cells might be caused by increased lactate formation due to enhanced lactic acid fermentation in the RCC cells; and lactate has been described to hinder T-cell activity [71]. Receptor tyrosine kinases (RTKs), like c-Met, EGFR and Axl are often over-expressed in renal cancer cells and play important part(s) in their growth and survival [72–75]. From our laboratory, we have shown that c-Met plays a major role in the growth and survival of renal cancer cells, and it can promote PD-L1-mediated immune escape [73, 76]; and the inhibition of c-Met mediated signaling down-regulates the growth of RCC cells [72, 77]. Renal cancer is often treated with tyrosine kinase inhibitors (TKIs) like sunitinib, sorafenib and others [78]. We have reported that c-Met can down-regulate the growth inhibitory effect of sorafenib in RCC cells [73]. In a study by Nakaigawa et al., it has been demonstrated that an increase in glucose accumulation in RCC is associated with increased resistance to TKIs [78]. Restricting the glucose transport in cancer cells is a potential approach to reduce tumor growth, including renal cancer [79]. Targeting the RCC cells with STF-31, an inhibitor of GLUT1, inhibits tumor growth and promotes cell death [79].

Renal cancer cells are known to display the classical Warburg effect; there is increased cellular expression of all the enzymes involved with glycolysis [80, 81]. Alternatively, the expression of the rate-limiting gluconeogenetic enzyme fructose-1-bisphophatase (FBP1), known to be a tumor suppressor, was almost universally depleted in RCC tumors [82]. Li et al. investigated nearly 600 human RCC tumor samples and found almost 100% depletion of FBP1 in tumor cells compared with normal kidney cells [83]. Interestingly, they reconstructed RCC-like characters in normal kidney cells through genetic manipulation of FBP1; for example, increased glucose uptake and increased production of lactate, GSH, and NADPH [83]. They have demonstrated that FBP1 plays an important antagonistic role towards glycolysis. FBP1 was known to inhibit the glycolytic flux and obstruct the Warburg effect, and ectopic expression of FBP1 inhibited tumor growth in a ccRCC xenograft model. FBP1 also acts as an inhibitor of the transcriptional activity of HIF, and its deficiency promotes a more vigorous HIF activity [83]. A study by Liao et al. further elucidated the epigenetic regulation of FBP1 with the development of renal cancer [84]. Epigenetic chromatin regulator, enhancer of zeste homolog 2 (EZH2), has been shown to suppress FBP1 expression [84], which promotes renal tumor growth [Figure. 1]. Several chemokines can facilitate renal cancer growth [85]. Yakolov et al. reported that CXCL12 and the chemokine receptor CXCR4b, can promote glycolysis during renal tumorigenesis [85]. In RCC, the lactate formation and the expression of LDHA are increased, while the enzyme aldo-keto reductase family 1-member A1 (converts pyruvate to ethanol) is decreased [54]. Genes encoding lactate transporters, e.g., MCT1 and MCT4, are often up-regulated in aggressive renal tumors [54, 86].

The mTOR pathway is also known to influence the glucose metabolism pathway in RCC cells. It acts as a regulator of HIF-mediated gene activation and glycolysis [87]. Düvel et al. have shown that the activation of mTOR in a TSC1/2 knock-out mice model induced a genotypic profile similar to aerobic glycolysis [37]. Akt, which is an upstream activator of mTORC1, is associated with the induction of glucose metabolism components, like GLUT1, HK, and PFK1 [88].

3.2. Pentose phosphate pathway

The pentose phosphate pathway (PPP) detours glycolysis from Glucose-6-phosphate to fructose-6-phosphate [8]. The PPP is a source of reducing equivalents (NADPH) and 5-carbon sugars or pentose phosphates as predecessors for nucleotide synthesis [Figure. 1]. The oxidative branch of PPP regulates the generation of ribonucleotides and NADPH [89]. In contrast, the nonoxidative component governs the generation of pentose phosphates from glycolysis intermediates like fructose-6-phosphate (F6P), glyceraldehyde-3-phosphate (G3P), and vice versa [89]. NADPH plays a vital role in maintaining the redox balance inside the cells by shielding the cells from ROS [90]. Unlike normal cells, cancer cells undergo prolonged metabolic and oxidative stress [90]. Cancer cells reprogram glucose metabolism to generate more NADPH to defend against this chronic oxidative stress [45]. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of pentose phosphate pathway, is often up-regulated in cancer cells [45, 90]. The flux of the PPP in cancer cells is often elevated to encounter the high demand of 5-carbon sugars for the nucleotide biosynthesis and maintain intracellular redox balance for growth and proliferation [89]. Several contemporary studies have shown that in cancer cells PPP is up-regulated by oncogenes, like PI3K, mTORC1, and K-Ras; and inhibited by tumor suppressor genes like p53 and PTEN [91–93].

3.2.1. Pentose phosphate pathway in renal cancer

Renal cancer cells utilize the PPP to counteract the high oxidative stress [94, 95]. This pathway is often modified in kidney ailments like acute kidney injury (AKI) or diabetic kidney disease [94]. Both the oxidative and nonoxidative phases of PPP are augmented in renal cancer. Interestingly, the TCGA correlated up-regulation of the pentose phosphate pathway with aggressive ccRCC and poor patient prognosis. Expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of PPP, is significantly elevated in RCC cells compared with normal kidney cells irrespective of the redox state [94, 95], and correlated with poor patient outcome. Langbein et al. demonstrated an up-regulation of G6PD, transketolase (TKT) and elevated nonoxidative glucose fermentation in metastatic renal cancer [94]. Metabolomic analysis by Lucarelli et al. [96] suggested the involvement of PPP with the metabolic reprogramming of RCC cells as denoted by higher levels of G6PD as well as the presence of PPP-derived metabolites [96]. Zhang et al. demonstrated that G6PD promotes the proliferation of RCC cells through positive feedback regulation of p-Stat3 [95]. In addition, Lucarelli et al. reported that the inhibition of G6PD in RCC cells can trigger a significant decrease in cancer cell survival [96].

3.3. TCA cycle

TCA cycle or citric acid cycle occurs in the mitochondria (28, 55). The pyruvate formed through glycolysis is oxidized to acetyl-CoA by the enzyme pyruvate dehydrogenase (PDH). In cancer cells, PDH gets inhibited by the anti-TCA cycle enzyme pyruvate dehydrogenase kinase (PDK1) [Figure. 1]. The acetyl-CoA enters the TCA cycle through oxaloacetate to form citrate catalyzed by citrate synthase. The citrate undergoes a cyclic mitochondrial route to oxidize the acetyl-CoA to CO₂. The TCA cycle generates succinate and reduced nicotinamide adenine dinucleotide (NADH); and produces ATP through oxidative phosphorylation (OXPHOS) [Figure. 2]. TCA cycle intermediates act as precursors for the biosynthesis of several macromolecules, including heme and fatty acids [51, 97]. The intermediates of TCA cycles are replenished by anaplerotic reactions or anaplerosis [4]. In cancer cells, HIFs are known to suppress the metabolic flux to the TCA cycle through transcriptional activation PDK1, and thereby inhibiting the conversion of pyruvate to acetyl-CoA [28, 55].

Figure 2. Reprogramming of TCA cycle, fatty acid and glutamine metabolism in renal cancer.

In RCC cells, both tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) are down-regulated. The mitochondrial biogenesis is also downregulated in RCC cells. In the context of the fatty acid metabolism, synthesis of fatty acid and lipids are up-regulated whereas β-oxidation of fatty acids is downregulated. Levels of carnitines, the fatty acid carriers, are also increased in RCC. Moreover, metabolites and enzymes in the cholesterol and phospholipid synthesis are also upregulated which leads to accumulation of lipid droplets, which gives the distinct clear histologic phenotype for clear cell renal cancer cells. In terms of glutamine metabolism, increased glutamine and upregulated reductive carboxylation pathway facilitate glutamine-dependent lipogenesis. Also, the glutathione/oxidized glutathione (GSH/GSSG) pathway for glutamine is upregulated to counteract the oxidative stress. The metabolites are depicted by squares and enzymes or transporters are denoted by ovals in the figures. Green arrows: Increase in activity, Red arrows: Decrease in activity. Abbreviations: ABAT, 4-aminobutyrate aminotransferase; ACAT, acetyl-CoA acetyltransferase; COX-2, cyclooxygenase-2; CPT1, carnitine palmitoyltransferase 1; FAS, fatty acid synthase; FH, fumarase; GABA, g-aminobutyric acid; GLS, glutaminase; GGT, γ-glutamyl transpeptidase; GST, glutathione-S-transferase; HADH, hydroxyacyl-CoA dehydrogenase; HETE, hydroxyeicosatetraenoic acid; IDH, isocitrate dehydrogenase; LOX-2, lipoxygenase-2; LPA, lysophosphatidic acid; MCAD, medium-chain specific acyl-CoA dehydrogenase; PA, phosphatidic acid; ROS, reactive oxygen species; SCD1, stearoyl-CoA desaturase-1; SCEH, short-chain enoyl-CoA hydratase; SDH, succinate dehydrogenase. VLCAD, very long-chain specific acyl-CoA dehydrogenase.

The TCA cycle is often perturbed in cancer cells by mutations of the enzymes. For example, mutations in the genes encoding isocitrate dehydrogenase (IDH) [98–100], succinate dehydrogenase (SDH) [101, 102], and fumarate hydratase (FH) [103] can lead to the transformation of normal cells to cancer cells. SDH and FH act as a tumor suppressor in tumors; and loss of function of these two enzymes due to mutation leads to the accumulation of succinate and fumarate that can facilitate tumor-promoting events [101, 103].

As the TCA cycle and subsequent oxidative phosphorylation occur only in mitochondria, the mitochondrial mass and biogenesis play an essential role in tumorigenesis, and it varies according to tumor types [104]. The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) is a crucial regulator of mitochondrial biogenesis [Figure. 2] [104, 105]. Some tumors, like breast cancer, have high PGC-1α expression; and inhibition of PGC-1α can inhibit tumor initiation [106]. Interestingly, PGC-1α can also act as a tumor suppressor in some tumor types, where its overexpression leads to apoptosis of the cancer cells [104]. The pro-oncogenic transcription factor c-Myc, as well as the mTOR signaling, is also known to modulate mitochondrial biogenesis and mitochondrial gene expression in cancer cells [106].

3.3.1. TCA cycle in renal cancer

It has been reported that the TCA cycle is altered in nephrological diseases, like Type 2 diabetes, chronic kidney disease (CKD), and kidney injury [106–108]. In RCC, the enzymes which replenish the metabolic flux to the TCA cycles from other pathways are often down-regulated [Figure. 2]. The pathways involve glycolysis, lipid metabolism, and glutamine metabolism. These enzymes catalyze a reaction to an end product, which can enter the TCA cycle either directly or through biochemical conversion. Pyruvate dehydrogenase (PDH) catalyzes the conversion of pyruvate to acetyl-CoA, which in turn gets inhibited by the anti-TCA cycle enzyme pyruvate dehydrogenase kinase (PDK1) [80, 81]. Pyruvate carboxylase (PC) converts acetyl-CoA to oxaloacetate, the first stable intermediate of the TCA cycle [80, 81]. g-aminobutyric acid transaminase catalyzes the conversion of the neurotransmitter GABA, a byproduct of glutamine metabolism, to succinate, which is another TCA cycle intermediate [80, 81]. Dihydrolipoamide acetyltransferase is a component of the α-ketoglutarate dehydrogenase complex, which regulates the recycling of α-ketoglutarate [80, 81]. Experimental studies found these enzymes are down-regulated in RCC cells compared with normal kidney cells, as supported by proteomics, metabolomics, and transcriptomics findings.

Among the chemical components of the TCA cycle, non-targeted metabolomic analyses demonstrated the amplification of citrate and cis-aconitate, although the levels of malate and fumarate diminished considerably [80, 81]. The decrease in fumarate and malate expression level in ccRCC tissues occurs probably due to reduced succinate dehydrogenase (SDH) in the tumor cells [Figure. 2]. Succinate dehydrogenase catalyzes the conversion of succinate to fumarate, and its deficiency depletes the fumarate and consequently malate [80, 81]. In some ccRCC tumors, the enzyme isocitrate dehydrogenase (IDH) is also decreased compared to the surrounding normal cells. In an atypical case of ccRCC, the gain of functions in the two isoforms of IDH, i.e., IDH1 and IDH2, lead to accumulation of the enantiomer of L-2-hydroxyglutarate as an oncometabolite [109].

Experimental studies involving genetic defects in two critical enzymes of the TCA cycle, fumarase (FH) and succinate dehydrogenase (SDH), were correlated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC) [110, 111]. Consequent studies revealed potential roles of fumarate and succinate in deregulation of the HIF transcription factors through inhibition of prolyl hydroxylation-mediated degradation [112]. As a result, stabilization of HIF1α and HIF2α results in elevated GLUT1 and VEGF levels in HLRCC tumors [112]. In ccRCC cells, an anti-tumorigenic role of fumarase has been suggested as there was retarded cancer cell growth after overexpression of FH [113]. There can also be activation of the transcription factor Nrf2 with the loss of FH activity [114]. The type II papillary renal cell carcinoma (pRCC) has a characteristic inactivating mutation in the FH gene similar to HLRCC [115]. In these cells, there is an activation of the anti-oxidant Nrf2 pathway; we and others have demonstrated that this pathway is associated with the neutralization of excessive reactive oxygen species (ROS) [73, 115]. Nrf2 is one of the essential factors to maintain cellular redox balance [116, 117]. Two potential regulators of Nrf2 activity in FH deficient RCC tumors have been identified [116, 117]. One of them is Abl tyrosine kinase, which modulates Nrf2 nuclear localization, and iASPP, a protein that competes with Nrf2 for binding Keap1, the inhibitor of Nrf2 [116]. A comparatively recent study by Sun et al. found that inhibition of phosphogluconate dehydrogenase (PGD) hinders the proliferation of FH mutant HLRCC cells [118]. They have shown that the inhibition of PGD impedes glycolysis, suppresses reductive carboxylation of glutamine, and increases the NADP+/NADPH ratio to interrupt the cellular redox homeostasis. Their study reveals PGD as a potential target of therapeutics for RCC patients [118].

3.4. Oxidative phosphorylation (OXPHOS)

OXPHOS generates ATP through sequential transport of electrons, known as the electron transport chain (ETC) situated in the mitochondrial membrane [Figure. 2]. In the cancer cells, the quicker ATP production through cytoplasmic glycolysis is favored over mitochondrial OXPHOS [119]. Glycolysis can synthesize ATP up to 100 times faster than OXPHOS, but the total energy yield of glycolysis is very low; it is 18 times lower than OXPHOS [56]. However, there is heterogeneity in the consumption of glucose through glycolysis or OXPHOS depending upon intra-tumoral cell populations, tumor types and the availability of nutrients [120]. PGC-1α, the transcriptional coactivator responsible for mitochondrial biogenesis, also plays an important role in oxidative phosphorylation [105]. PGC-1α increases the number of active mitochondria as well as gene expression of the proteins involved in OXPHOS; and they enhance the oxygen consumption rate (OCR) and ATP production [105, 106]. Glioblastoma multiforme (GBM) one of the most common brain malignancies has impaired mitochondrial activity and incompatible OXPHOS [121]. Due to this bioenergetically unfavorable conditions GBM cells utilizes the mitochondrial substrate-level phosphorylation (mSLP) to generate ATP [121]. They utilize the succinate-CoA ligase (SUCL) reaction of the TCA cycle to generate adequate amount of ATP to withstand the growth and invasion. SUCL catalyzes the conversion of succinyl-CoA and ADP (or GDP) to coenzyme-A, succinate, and ATP (or GTP) [121]. The ATP production through the mSLP in the GBM cells is further supported by increased glutaminolysis which replenishes the succinyl-CoA [121]. Targeting the oxidative phosphorylation in some cancer cells with metformin, the mitochondrial complex-I inhibitor improved clinical efficacy in many cancer types [122].

3.4.1. Oxidative Phosphorylation in renal cancer

Normal kidney cells have a high electron transport chain or oxidative phosphorylation (OXPHOS) activity because the filtration of blood and reabsorption of nutrients are highly ATP dependent [123]. Along with decreased TCA cycle in RCC, the cancer cells also demonstrate diminished OXPHOS activity [124]. Simmonet et al. [124] measured the OXPHOS activity in RCC tissues from patients. They showed mitochondrial impairment was increased from the less aggressive to the most aggressive form of RCCs. And the impairment of OXPHOS was associated with a significantly reduced expression of the OXPHOS complexes. The complex-V of the OXPHOS was downregulated in all the RCC samples of their study [124]. In a more recent study, it has been demonstrated that PGC-1α, the most crucial member of the PPARγ coactivators (PGC) family of transcriptional coactivators, is suppressed in VHL-deficient ccRCC cells by a HIF dependent mechanism [30]. Suppression of PGC-1α leads to decreased expression of TFAM (mitochondrial transcription factor) and retardation of mitochondrial respiration. Whereas the ectopic expression of PGC-1α in VHL null or mutated RCC cells restored mitochondrial function and induced oxidative stress, which resulted impaired tumor growth [30]. They also correlated low PGC-1α expression in ccRCC cells with poor patient outcome, disease advancement, and metastasis [30]. The importance of repression of PGC-1α for the growth of renal cancer is also demonstrated by the study of Felipe-Abrio et al. [125]. MYB-binding protein 1A (MYBBP1A) acts as a repressor of PGC-1α, and inhibits the growth of renal tumors. They have shown that the down-regulation of MYBBP1A in renal cancer cells switches glycolytic metabolism to oxidative phosphorylation by activating PGC-1α.

3.5. Fatty acid metabolism

The ability of tumors to synthesize lipids de novo was discovered as early as in the 1950s, and since then, it has been identified as a significant metabolic condition for some cancers [126–128]. Fatty acid metabolism comprises of both catabolism and anabolism of fatty acids [Figure. 2]. The catabolism of fatty acids involves fatty acid oxidation (FAO or β-oxidation) and the subsequent passage of the produced acetyl-CoA to the TCA cycle. The anabolism or fatty acid synthesis [Figure. 2] consists the generation of fatty acids from acetyl-CoA by fatty acid synthase (FAS) [127, 128]. Fatty acids are also the constituent backbone of several biophysically important biomolecules like hormones, triglycerides, and phospholipids for the rapidly growing tumor cells [128]. For these purposes, the majority of cancer cells overexpress FAS [129]. Cancer cells are capable of synthesizing acetyl-CoA from cytosolic acetate as well as glucose and glutamine [14, 69]. Cancer cells are dependent on acetyl-CoA synthetase 2 (ACSS2), which is often up-regulated during tumorigenesis [130, 131]. The effect of up-regulated fatty acid synthesis on breast cancer advancement and prognosis is accredited to fatty acid synthase back in the 1990s [126]. After that, several anti-cancer drugs targeting FAS underwent clinical trials [132]. The sterol regulatory element-binding protein-1 (SREBP-1) transcription factor often controls the expression of genes involved in fatty acid synthesis in cancer cells [133]. Oncogenic mTORC1 signaling through its mediator S6 kinase (S6k) activates SREBP-1 and related SREBP-2 to enhance the fatty acid and sterol synthesis in cancer cells [37, 134]. Activation of SREBPs was found to initiate the gene expression of Glucose-6-phosphate dehydrogenase (G6PD) and fatty acid synthase (FAS), which are two crucial components to trigger the pentose phosphate pathway and lipid biogenesis [135]. Alternatively, cancer cells have elevated expression of enzymes responsible for lipid storage. One such enzyme, stearoyl-CoA desaturase (SCD1), catalyzes the elongation and desaturation of fatty acids to generate unsaturated fatty acids [136]. SCD1 is also responsible for the synthesis of triglycerides and phospholipids for membrane synthesis [137].

3.5.1. Altered fatty acid metabolism in renal cancer

RCC is often associated with obesity [80, 138]. A higher level of cholesterol ester accumulation has been reported in kidneys of ccRCC patients [139]. Increased levels of fatty acylcarnitines and carnitine were observed in ccRCC tissues [Figure. 2] compared with control cells by metabolomics studies [81]. These variations are directly proportional to the kidney cancer grade. The accumulation of carnitine and acyl-carnitine levels is probably caused by a decrease of β-oxidation enzymes in RCC [Figure. 2]. The exact molecular mechanism for the involvement of carnitine and acyl-carnitines in promoting the RCC phenotype is yet to be explored. Wettersten et al. analyzed several RCC tissues with combined grade-dependent proteomics and metabolomics analysis. They found an impaired β-oxidation pathway in the RCC cells, which leads to increased accumulation of fatty acylcarnitines [81]. Accumulation of “lipid droplets” are considered to be a hallmark of clear-cell renal cell carcinoma (ccRCC) as these lipid droplets in the cytoplasm give rise to the typical clear cell phenotype [81]. The study by Qiu et al. established the accumulation of lipid droplets in the proximity of the endoplasmic reticulum (ER), which helps to maintain the integrity of the ER in the ccRCC cells [140]. The storage of the lipid droplets is caused by the gene perilipin 2 (PLIN2), which is up-regulated in a HIF2 dependent pathway leading to maintenance of ER homeostasis and withstanding cytotoxic stress [140]. Recent studies demonstrated down-regulation of enzymes involved in FAO in ccRCC cells compared to normal kidney cells [81, 141, 142]. SCD1, the enzyme responsible for lipid storage, is highly expressed in ccRCC and plays an essential role in its growth and proliferation [142]. SCD1 inhibition by gene knockdown or using a small-molecule pharmacological inhibitor A939572 reduces RCC growth [142]. Increased expression of fatty acid synthase (FAS) has been shown to be associated with ccRCC tumor aggressiveness and poor patient survival [141]. HIFs are critical players for the growth of RCC cells, and HIF represses carnitine palmitoyltransferase 1A, the enzymatic transporter of fatty acids to the mitochondria from the cytoplasm [143].

The reprogramming of glycerophospholipid metabolism and arachidonic acid metabolism are unique characteristic of renal cancer [8]. Glycerophospholipids (or phosphoglycerides) are the components of cell membranes especially the phospholipid bilayers [Figure. 2]. Glycerophospholipids are also the sources of phosphatidic acid (PA), lysophosphatidic acid (LPA), and triacylglycerol which are building blocks of lipid storages [8]. The expression of autotaxin (ATX), the enzyme that produce LPA is significantly increased in the surrounding tumor endothelial cells of sunitinib-resistant RCC tissues resulting from Protein kinase B (PKB) and extracellular signal-regulated kinase (ERK) activation [144]. The level of phospholipase D2 (PLD2), another enzyme of glycerophospholipid metabolism is also increased in ccRCC cells compared to normal kidney cells and implicated for tumor prognosis and grading [145]. Chemical inhibition of ATX increases the sensitivity of RCC cells to sunitinib suggesting the glycerophospholipid metabolism can be a potential therapeutic target in RCC [144].

Arachidonic acid is another important derivative of membrane phospholipid and the synthesis of different groups of arachidonic acid involves inflammatory enzymes like lipoxygenases (LOXs) and cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) [8]. Expression of the enzymes 5-LOX and 15-LOX2 and 15-hydroxyeicosatetraenoic acid (an immunosuppressive arachidonic acid) is increased in RCC cells compared to normal kidney cells [146, 147]. In RCC cells, LOX also promotes the secretion of immunosuppressive chemokine CXCL2 and cytokine IL10 to regulate immune-evasion of RCC cells [146]. The COX pathway of arachidonic acid metabolism is also involved in tumor-promoting pathways [148]. Prostaglandin E2 (PGE2), a product of COX2, has been reported to promote RCC invasion [148]. Several studies have displayed that levels of COX2 are increased in RCC and associated with tumor size, stage and grade [149, 150]. These results suggest COX2 as a potential target in renal cancer cells.

3.6. Glutamine Metabolism

Glutamine is one of the major nutrients utilized by cancer cells to maintain cellular bioenergetics and biomass. It acts as a building block for protein synthesis and lipid synthesis [Figure. 2]. It is also the precursor of the critical cellular antioxidant glutathione, and in RCC, the glutathione/oxidized glutathione (GSH/GSSG) balance is rigorously regulated [Figure. 2]. Glutamine is imported into the cell through specific transporters like SLC1A5[151]. The enzyme glutaminase (GLS) converts glutamine to glutamate and initiates glutamine catabolism [151, 152]. This glutamate can be exported to the cytoplasm for protein synthesis or converted to α-ketoglutarate by the enzyme glutamate dehydrogenase (GDH) [151, 152]. The enzyme isocitrate dehydrogenase (IDH) catalyzes the “reductive carboxylation” [13, 153, 154] conversion of α-ketoglutarate in the reverse direction to isocitrate to provide acetyl-coenzyme A for lipid synthesis and utilization in the TCA cycle [Figure. 2]. α-ketoglutarate evolved from glutamate contributes to the anaplerosis or replenishment of the TCA cycle [155]. Cancer cells with mitochondrial defects are reported to dependent for growth on the anaplerosis by reductive carboxylation of glutamine [13]. The oncogene c-Myc is known to activate glutaminase expression and consequently positively influence glutamine metabolism in cancer cells [156]. Activating mutations in β-catenin are reported to escalate the expression of glutamine transporters (SLC1A2) and glutamine synthetase in hepatocellular carcinoma [157]. Yang et al. [158] have shown the importance of glutamine catabolism to maintain the TCA cycle flux and cell survival in cancer cells with faulty mitochondria. Glutamine also influences redox control in cancer cells through glutathione (GSH) [159]. Glutathione functions to reduce the levels of reactive oxygen species (ROS) by acting as a substrate for the ROS and getting oxidized to the oxidized glutathione (GSSG). Impairment of glutathione synthesis results in the accumulation of ROS, and consequently hindering the survival and proliferation of cancer cells [160, 161].

3.6.1. Role of glutamine metabolism in renal cancer

Glutamine is known to maintain the urinary pH balance in the renal cortex [162]. Glutamine utilization is increased significantly in ccRCC compared with normal kidney tissues, the GSH/GSSG balance is rigorously regulated [12, 80, 81]. Mullen et al [13] reported that the increased glutamine level is associated with up-regulated free fatty acids in RCC. They further established that reductive carboxylation of glutamine is one of the dominant modes of metabolism in rapidly growing renal carcinoma cells [13]. Dual activation of HIF2 and MYC plays a vital part in glutamine-dependent lipogenesis in RCC cells. Shroff et al. reported an up-regulation of glutaminases (GLS1-2) and glutamine transporters (SLC1A5) along with elevated levels of glutamate and α-ketoglutarate in transgenic mouse models of human RCC after overexpression of MYC [40]. Like MYC, HIF2 is also known to regulate glutamine-dependent lipogenesis [12]. Gameiro et al., in their study, demonstrated that HIF expression is necessary for the induction of reductive carboxylation of α-ketoglutarate in RCC cells [12]. They have shown a reversal of IDH flux to reductive carboxylation of glutamine to citrate, as measured by metabolic flux analysis upon constitutive activation of the HIF2 molecule [12]. Interestingly, there was also an increase of lipogenic acetyl-COA after constitutive activation of HIF2 in RCC cells. HIF2 is also known to promote transcriptional activation of MYC [39]. Overall, these reports suggest important roles of both MYC and HIF2 in glutamine-dependent lipogenesis in renal cancer.

Combined proteomics and metabolomics study demonstrated enrichment of metabolites in the glutamine and GSH/GSSG pathways in ccRCC cells [80, 81]. In ccRCC cells, the expression of glutathione peroxidase 1 (GPX1) was increased. In contrast, the expression of enzymes that inhibit glutamine consumption through the glutathione/oxidized glutathione pathway (glutathione-S-transferase and γ-glutamyl transpeptidase) were decreased, signifying the contribution of glutamine towards maintaining the cellular redox balance by scavenging the ROS [80, 81]. Thus, high-grade, high-stage and metastatic ccRCC are associated with increased glutamine level and glutathione/oxidized glutathione pathways.

Inhibition of glutaminase, the enzyme which catalyzes the conversion of glutamine to glutamate, or the exclusion of glutamine from the cell culture media, resulted in the reduction of cancer cell survival in vitro indicating a dependence of tumor cells on exogenous glutamine, a phenomenon which is also known as glutamine addiction [163, 164]. CB-839, an inhibitor of glutaminase, either alone or combined with the mTOR inhibitor everolimus, showed promising results in ccRCC patients [164–166]. Though another trial involving treatment of advanced or metastatic RCC with a combination of CB-839 and cabozantinib, the c-Met inhibitor failed to achieve significant effect [167]. Glutamine addiction of ccRCC cells promotes their defense against oxidative stress, and it could be exploited for effective imaging of the tumor. Inhibition of glutaminase by pharmacological inhibitors, like CB-839 or BPTES, led to decreased GSH/GSSG ratio; and it increased oxidative stress, DNA damage and tumor cell apoptosis [14]. The glutamine affinity of renal cancer cells was further exploited in an orthotopic mouse model by PET imaging for the detection of the renal tumor, as there was increased uptake of 18F-(2S,4R)4-fluoroglutamine (a glutamine analog radiologic imaging agent). This tumor imaging technique was further registered under an FDA clinical trial for effective detection of tumors [168]. In another interesting study, Miess et al. demonstrated that the inhibition of fatty acid metabolism through impaired β-oxidation made the RCC cells dependent on the glutamine-glutathione pathway to resist lipid peroxidation and ferroptotic cell death [169]. Like ccRCC cells, papillary RCC cells also demonstrate increased glutamine to glutathione conversion rate and down-regulation of gluconeogenesis and oxidative phosphorylation [170]. Proteomics and metabolomics study by Ahmad et al. revealed increased glutathione synthesis, inadequate anabolic glucose synthesis, and compromised oxidative phosphorylation as characteristics of pRCC cells [170].

3.7. Tryptophan Metabolism

Tryptophan (TRP) is an essential amino acid associated with three major downstream metabolic pathways: the serotonin, indoleacetate, and kynurenine (KN) pathways [Figure. 3] [9]. The KN pathway is responsible for the degradation and uptake of the majority of dietary tryptophan. Indoleamine2,3-dioxygenase (IDO) is the rate-limiting enzyme of the KN pathway. Immunosuppression caused by tryptophan depletion and the generation of immunosuppressive metabolites by the KN pathway are the well-studied effects of tryptophan metabolism [Figure. 3]. IDO and tryptophan-2, 3-dioxygenase (TDO2), another enzyme of the kynurenine pathway, are often overexpressed in solid tumors [8]. The accumulated KN is known to promote apoptosis of effector T cells and suppress antitumor immune responses [171]. Also, KN mediates autocrine signaling through the aryl hydrocarbon receptor (AhR) on cancer cells triggering the degradation of extracellular matrix and invasion of cancer cells [172]. Moreover, KN can modulate both the innate and adaptive immune response linked to cancer-associated immunosuppression [173].

Figure 3. Reprogramming of tryptophan metabolism in renal cancer.

Tryptophan is metabolized through three major downstream pathways, the serotonin, indoleacetate, and kynurenine (KN) pathways. The kynurenine pathway of tryptophan metabolism is upregulated. Indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of the KN pathway is upregulated in renal cancer cells; and increased kynurenine in the tumor microenvironment suppresses immune cell activation and facilitates immune evasion of renal cancer cells. The metabolites are depicted by squares and enzymes or transporters are denoted by ovals in the figures. Green arrows: Increase in activity, Red arrows: Decrease in activity. Abbreviations: ALDH2, aldehyde dehydrogenase 2; DDC, DOPA decarboxylase; MAO, monoamine oxidase; QN, quinolinate.

3.7.1. Altered tryptophan metabolism in renal cancer

In RCC, the downstream metabolites of the KN pathway, such as KN and quinolinate, are increased along with a decrease of the TRP level [81]. The expression of IDO is increased in renal tumor endothelial cells compared with normal tissues [174, 175]. In contrast, the enzymes associated with other TRP catabolism routes, like the serotonin and indoleacetate pathways are downregulated. For instance, DOPA decarboxylase (DDC), monoamine oxidase (MAO), and aldehyde dehydrogenase 2 (ALDH2), are decreased in RCC cells indicating an activation of the KN pathway [Figure. 3] [174, 175]. Compared to healthy individuals, quinolinate has been reported as the most amplified urinary metabolite in RCC patients in a urine metabolomics study [81]. Both KN and quinolinate are known to exert immunosuppressive effects in cancer cells [Figure. 3]. Hence, these findings demonstrated increased tryptophan consumption through the KN pathway, which contributed to immunosuppression in RCC cells. This KN mediated immunosuppression boosts renal tumor growth and helps the tumors to evade the natural immune system as well as external immunotherapy. Inhibition of IDO augments the PD-1 inhibitor nivolumab, mediated cytotoxic T cell activation. The combination therapy of the IDO inhibitor epacadostat and the anti-PD-1 antibody pembrolizumab is under clinical trial for different solid tumors [176]; however, the trial of this combination against RCC has recently been halted [177]. Interestingly, the application of immune checkpoint inhibitors against cancer not always yields desirable effects. In fact, the KN/tryptophan ratio is increased in advanced melanoma and RCC patients after treatment with the anti-PD-1 antibody nivolumab [178]. This is probably caused by the adaptive resistance mechanism of the cancer cells with a decreased overall patient survival [178]. However, due to limited reports, the immunosuppressive effect(s) of tryptophan metabolism in RCC leaves many unanswered questions.

3.8. Arginine Metabolism

The semi-essential amino acid arginine plays crucial roles in several metabolic pathways, including protein synthesis and the synthesis of polyamines, nitric oxide, nucleotides, proline, urea, creatine, and glutamate [9]. Citrulline is the precursor of arginine, and arginine is synthesized from citrulline through the urea cycle via two steps [Figure. 4]. Argininosuccinate synthase-1 (ASS1) is the rate-limiting enzyme for arginine synthesis [9]. Arginine auxotrophy is a metabolic state expressed by tumors where the absence or deletion of ASS1 activity renders the cells dependent on extracellular arginine supply [179]. Interestingly, ASS1 and argininosuccinate lyase (ASL), another critical enzyme of the arginine metabolism pathway, are often epigenetically impaired in cancers, like hepatocellular carcinoma, renal cell carcinoma, and melanoma [180–182]. Researchers are exploiting the arginine auxotrophy of tumors to develop effective anticancer therapies [183]. However, the down-regulation of ASL and ASS1 are often associated with chemotherapeutic drug resistance and poor prognosis of cancer [184, 185].

Figure 4. Reprogramming of arginine Metabolism in renal cancer.

Arginine metabolism in cancer cells involves both urea cycle and TCA cycle. Normal cells synthesize arginine from citrulline through the urea cycle. Argininosuccinate synthase-1 (ASS1), the enzyme responsible for arginine synthesis in the urea cycle, is often downregulated in RCC. Thus, renal cancer cells are heavily dependent on external arginine supply for growth and survival. Through the synthesis of Fumarate from Arginosuccinate, an intermediate in the urea cycle, Arginine can enter through TCA cycle; however, this pathway is often downregulated in RCC cells due to lack of arginine synthesis and decreased ASS1 expression. The metabolites are depicted by squares and enzymes or transporters are denoted by ovals in the figures. Red arrows: Decrease in activity. Abbreviations: OCT, ornithine carbamoyl transferase.

3.8.1. Arginine Metabolism in renal cancer

ASS1, the rate-limiting enzyme for arginine synthesis is absent or significantly downregulated in the biopsy samples of ccRCC patients [182]; and hence these cancer cells display arginine auxotrophy or dependency to external arginine supply for growth [182]. In a proteomic study, similar findings were observed in all ccRCC grades [186]. Arginine deprivation can suppress tumor growth in the RENCA mouse model of RCC [182]. As RCC cells are rapid growing, they need an external source of amino acids such as glutamine or arginine to meet the increased demand of essential cellular building blocks like protein and lipids [179]. Arginine deprivation appears to be a promising approach to treat ccRCC and other tumors lacking ASS1 expression. The enzyme arginine deaminase depletes arginine to citrulline through catalytic deamination [182, 187]. A pharmacologically modified (PEGylated) variant of the enzyme arginine deaminase (ADI PEG20) demonstrated significant efficacy against ccRCC tumors [182, 187].

4. Summary and Conclusion

The major reprogrammed metabolic pathways in RCC have been summarized in Figure-5. However, covering all aspects of these pathways that are reprogrammed in renal cancer is beyond the scope of a single review article. The tumor suppressor gene VHL is often inactivated in RCC cells and is one of the key regulators of metabolic reprogramming. The inactivation of VHL triggers oncogenic signaling involving HIF family of transcription factors, which in turn stimulates the reprogramming of multiple metabolic pathways. The Ras-PI3K–Akt–mTOR pathway(s), which is primarily activated through the receptor tyrosine kinases, plays a key role for the altered metabolic events. The RCC cells consume a high amount of glucose, and glucose is metabolized through aerobic glycolysis and lactate fermentation to fulfill the continuous energy demand. The elevated lactate also contributes towards immunosuppression, and the upregulated pentose phosphate pathway accounts for the generation of reducing equivalents (NADPH) to inhibit the oxidative stress and ribose precursors for nucleotide synthesis necessary for the rapidly growing cancer cells. The TCA cycle is downregulated in RCC; and the lipid synthesis outweighs lipid degradation. The β-oxidation pathway of lipids is inhibited. In RCC, the catabolism of amino acids, like arginine and glutamine through the urea cycle is decreased. There is increased uptake of glutamine for the synthesis of fatty acids through reductive carboxylation. The elevated glutamine in RCC also adds to the upregulated glutathione/oxidized glutathione (GSH/GSSG) pathway to neutralize the oxidative stress and reactive oxygen species (ROS). Metabolism of the amino acid tryptophan through the kynurenine (KN) pathway is upregulated to generate elevated levels of immunosuppressants, like kynurenine and quinolinate [Figure. 5].

Figure 5. Overview of altered metabolic events in renal cell carcinoma (RCC).

A summarization of metabolic reprogramming or altered metabolic events in renal cell carcinoma (RCC). Inactivation of the tumor suppressor gene VHL through deletion or mutation is a key factor for the metabolic reprogramming in RCC cells. Loss of VHL leads to the deregulation of the hypoxia-inducible factor (HIF) family of transcription factors and other oncogenic signaling events, including the hyperactivation of Ras-phosphoinositide 3-kinase (PI3K)–AKT–mechanistic target of rapamycin (mTOR) pathway(s); they contribute to the reprogramming of multiple metabolic events in renal cancer cells. In RCC cells, upregulation of glucose transporters (GLUT or SGLT) increase glucose uptake and is catabolized through an elevated aerobic glycolysis (Warburg effect) and lactate fermentation. The upregulated pentose phosphate pathway provides reducing equivalents (NADPH) to inhibit the oxidative stress and ribose precursors for nucleotide synthesis needed for the rapidly growing cells. Increased efflux of lactate contributes to an immunosuppressive tumor microenvironment. The TCA cycle is downregulated in renal cancer cells due to lower conversion rate of pyruvate to acetyl-CoA and suppression of intermediates like fumarate and α-ketoglutarate. In RCC, lipid synthesis predominates over lipid degradation. The β-oxidation pathway of lipids is downregulated and contains the levels of acetyl-CoA to feed the TCA cycle. However, the synthesis of carnitine, fatty acids, phospholipids and cholesterol are upregulated in RCC. Urea cycle is downregulated in renal cancer cells which decreases the catabolism of amino acids like arginine and glutamine. The RCC cells take up increased amounts of glutamine for the synthesis of fatty acids through reductive carboxylation. The elevated glutamine in RCC also adds to the upregulated glutathione/oxidized glutathione (GSH/GSSG) pathway to neutralize the oxidative stress and reactive oxygen species (ROS). Metabolism of the amino acid tryptophan through the kynurenine (KN) pathway is upregulated to generate an elevated level of immunosuppressants like kynurenine and quinolinate. Thus, reprogramming of metabolic pathways produces energy (ATP) and other molecules like lipids, phospholipids and ribose sugars essential for cellular proliferation and enables renal cancer cells to endure hypoxia, nutrient exhaustion and oxidative stress, as well as evade the immune system for survival.

As discussed earlier, renal cancer is often considered to be a metabolic disease for significant reprogramming of the metabolic pathways to maintain cellular bioenergetic balance and undergo evasion from the extracellular stress and immune system. All of these reprogrammed metabolic pathways can be exploited to develop a more effective treatment of RCC through better imaging of tumors and identifying novel therapeutic targets. Development of effective inhibitors or drugs against the reprogrammed metabolic pathways is the basis of numerous anti-cancer targeted therapies; and this always have an advantage as they can be very specific for the tumor cells. Unlike traditional chemotherapy, these targeted therapies are expected to exert lower toxic effects on normal cells. However, targeting the metabolic pathways does have some limitations. The drugs that block the increased metabolism of cancer cells can also affect other rapidly proliferating cells, like bone marrow, intestinal crypts, and hair follicles. The effectiveness of an antimetabolite drug also depends on the genetic mutation pattern of cancer cells as a single metabolic pathway can be affected by multiple oncogenic mutations. Together, metabolomics can provide a better picture to understand the variability of tumor microenvironment for the detection and treatment of specific type of cancer. This can significantly help to develop novel biomarkers and targeted therapies for different cancer types, including renal cancer.