The Metabolic Basis of Kidney Cancer

Familial ccRCC: von Hippel–Lindau

Clear-cell renal cell carcinoma (ccRCC) occurs in both a sporadic (nonfamilial) and a familial form. Patients affected with VHL disease are at risk for the development of tumors in a number of organs, including the kidneys. Patients with VHL have a lifetime risk for ccRCC which can be recurrent, bilateral, and multifocal. Clinical management of VHL-associated RCC involves active surveillance of small renal tumors until the largest tumor reaches 3 cm in size, at which time nephron-sparing surgery is recommended. Complete nephrectomy may be required for larger tumors or tumors invading the renal vasculature (2).

VHL Gene

Genetic linkage analysis was performed in VHL families to localize the VHL gene to the short arm of chromosome 3 (3). Germline alteration of the VHL gene, including point mutation, splice-site mutation, and partial/complete gene deletions, has been identified in nearly 100% of VHL families (4). The VHL gene is a two-hit tumor suppressor gene in which either a germline (in patients with VHL) or sporadic alteration of VHL is associated with loss of chromosome 3p containing the second (wild-type) allele, resulting in complete inactivation of the gene. Multiple studies of the sporadic ccRCC tumors have shown nearly universal loss of chromosome 3p (91%–98%) in combination with either mutation (52%–87%) or promoter hypermethylation (7%–11%) of the remaining VHL allele (5–7).

Oxygen Sensing and Tumorigenesis

The product of the VHL gene, pVHL, is a component of an E3 ubiquitin ligase complex with elongin C (encoded by TCEB1/ELOC), elongin B (encoded by TCEB2/ELOB), Cullin-2 (encoded by CUL2), and RBX1 (encoded by RBX1; Fig. 3; refs. 8–11). The VHL complex targets the hypoxia-inducible factors HIF1α and HIF2α, transcription factors for ubiquitin-mediated degradation in an oxygen-dependent fashion (12–14). In normoxia, the oxygen, iron, and α-ketoglutarate–dependent prolyl hydroxylase (PHD) enzymes transfer hydroxyl groups onto two proline residues in the oxygen-dependent domain of both the HIF1α and HIF2α proteins, enabling recognition by the VHL complex, ubiquitination, and subsequent proteasomal degradation. In oxygen or iron deprivation, loss of PHD enzymic activity results in the stabilization of the HIFα proteins, and the formation of heteromeric dimers with the aryl hydrocarbon receptor nuclear translocator protein (ARNT) that migrate to the nucleus and activate the transcription of genes carrying hypoxia response elements (HRE; refs. 12–15). Stabilized HIF1α and HIF2α regulate the activity of downstream genes and pathways that aid the cell in adapting to low oxygen/iron levels, resulting in alterations such as increased glycolysis via upregulation of the GLUT1 glucose transporter (encoded by SLC2A1), hexokinase 2 (encoded by HK2), and lactate dehydrogenase A (encoded by LDHA); suppression of oxygen usage by reducing pyruvate entry into the TCA cycle via upregulation of pyruvate dehydrogenase kinase 1 (encoded by PDK1); and increased red blood cell production by increased erythropoietin (encoded by EPO) production (Fig. 3). Activation of the hypoxia response is also advantageous in the growth and maintenance of a cancer, including increased angiogenesis via upregulation of vascular endothelial growth factor (encoded by VEGF), and increased cellular growth and transformation via upregulation of platelet-derived growth factor (encoded by PDGF) and transforming growth factor α (encoded by TGFA; Fig. 3; refs. 16, 17).

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Figure 3.

Loss of the VHL/HIF oxygen-sensing pathway in ccRCC. In normoxia, the VHL E3 ubiquitin ligase complex targets the HIF proteins for ubiquitin-mediated degradation once the PHDs have hydroxylated the HIF proteins in an oxygen-dependent manner. In ccRCC renal tumors from patients with VHL disease and the majority of sporadic ccRCCs, the VHL protein is biallelically inactivated, resulting in the loss of protein degradation and stabilization of HIF1α and HIF2α. In sporadic ccRCC tumors that lack VHL loss, the loss of another VHL E3 ubiquitin ligase complex component, TCEB1, has been seen to mimic VHL loss. This mutation-induced stabilization of HIF1α and HIF2α enables binding to their shared dimeric partner HIF1β and localization to the nucleus that allows for transcription activation of the hypoxia response pathway genes irrelevant of the presence of oxygen, a phenomenon referred to as pseudohypoxia. The expression levels of the HIF transcription factors can also be regulated by the PI3K/AKT/mTOR pathway that controls the rate of de novo protein synthesis. The HIFs upregulate the expression of several growth factors, including VEGF, PDGF, and TGFα, that can then bind to their respective receptors and induce angiogenesis and proliferation. The HIF transcription factors also upregulate the expression of several genes that regulate glucose metabolism. Upregulated expression of the GLUT1 glucose transporter and lactate dehydrogenase (LDHA) increase glucose uptake and the conversion of pyruvate to lactate, respectively. In contrast, upregulated PDK1 inhibits the pyruvate dehydrogenase complex and decreases the conversion of pyruvate to acetyl-CoA and entry into the TCA cycle. Current therapeutic approaches to advanced ccRCC target the activated growth factors downstream of the VHL/HIF pathway and the translation of de novo HIF proteins. The neutralizing antibody bevacizumab directly targets VEGF, whereas pazopanib, axitinib, lenvatinib, cabozantinib, sunitinib, and sorafenib target the VEGF receptor or a combination of the VEGF and PDGF receptors. Temsirolimus and everolimus inhibit the PI3K/AKT/mTOR pathway to inhibit de novo HIF protein translation. New small-molecule inhibitors of the interaction between HIF2α and HIF1β, PT2385 and PT2399, are in clinical trial.

Activation of HIFα-dependent pathways is a canonical cellular response to low oxygen/iron that is moderated once normal oxygen/iron levels are achieved. The loss of pVHL function in ccRCCs results in the stability of the HIFα-dependent pathways independent of oxygen/iron levels in a state of “pseudohypoxia.” A subset of sporadic ccRCCs that lack VHL alterations have mutations in other components of the VHL E3 ubiquitin ligase complex, such as elongin C (TCEB1/ELOC), that function like VHL-loss mimics and result in the stabilizationof the HIFα proteins (7, 18). Although some genes are transcriptionally regulated by both HIF1α and HIF2α, each transcription factor has specific targets. In vitro and in vivo studies have indicated that HIF2α may be the more critical factor for tumorigenesis in ccRCC (19, 20). ccRCC tumors frequently lose a segment of chromosome 14q that encodes HIF1α (6, 21). These “HIF2α-only” ccRCCs have increased activity of c-MYC, a known HIF2α-specific transcriptional target (22).

Metabolic Reprogramming

ccRCC tumors lose VHL-dependent oxygen sensing, resulting in stabilized HIFα leading to metabolic reprogramming. Expression and metabolic analyses of ccRCCs have shown upregulated expression of GLUT1, hexokinase, and lactate dehydrogenase A and increased levels of glycolytic metabolites consistent with increased glucose uptake, a dependency on glycolysis, and a shift to aerobic glycolysis (23–25). At the same time, expression of the TCA cycle components is decreased, and entry of pyruvate into the TCA cycle is suppressed, consistent with impaired oxidative phosphorylation. Analysis of TCA cycle metabolites shows decreased levels of fumarate and malate and increased levels of succinate, isocitrate, and citrate (24, 25). These findings are consistent with the reductive carboxylation observed in VHL-deficient ccRCC cell lines and xenografts that converts α-ketoglutarate to citrate by partially reversing the TCA cycle (26–28). Reductive carboxylation can provide the citrate necessary to fuel the increased levels of fatty-acid synthesis present in ccRCC tumors with the required α-ketoglutarate predominantly derived from absorbed glutamine being converted to glutamate by the glutaminase enzymes (26–28). In addition, ccRCC tumors are characterized by increased expression of the oxidative pentose phosphate pathway that produces both the ribose sugars necessary for replication and the NADPH necessary to fuel the reductive carboxylation of isocitrate and maintenance of the reduced glutathione pool (6, 24, 25). Transcriptome-based analysis of ccRCC demonstrates that metabolic reprogramming, including decreased TCA cycle and AMPK complex gene expression and increased pentose phosphate pathway and fatty-acid synthesis gene expression, correlates with patient outcome, i.e., high-grade, high-stage, and low-survival disease (6).

The metabolic shift in ccRCC can be evaluated in vivo using PET imaging to assess the uptake of ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG). ¹⁸F-FDG–PET can assess metastatic disease and can quantitate the effect of therapies targeting glucose metabolism. A recent imaging study based on infusing ¹³C-labeled metabolic substrates, glucose, glutamine, and acetate, into patients with cancer demonstrated the flux of these substrates through the metabolic pathways in tumor cells in vivo (29). The patients with ccRCC infused with [U-¹³C]glucose showed that ¹³C-labeling of glycolytic intermediates in tumors was enhanced, whereas the TCA cycle components had significantly reduced ¹³C-labeling, consistent with aerobic glycolysis and the Warburg effect.

Although upregulation of the VHL/HIF pathway is central to the metabolic reprogramming of ccRCC tumors, other metabolic alterations may also play an important role. ccRCC tumors frequently have mutations in the chromatin-remodeling, chromosome 3p-encoded genes PBRM1, SETD2, and BAP1, as well as in other chromatin remodeling complexes, such as SWI/SNF, that alter gene expression and can affect many aspects of cellular metabolism (6, 7). Mutation of the PI3K/AKT/mTOR pathway, including PTEN, MTOR, PIK3CA, and gain of chromosome 5q are also frequent in ccRCC, with focal amplifications refining the region of interest to 5q35 that includes STSQM1, which encodes p62 that is involved in autophagy and the NRF2 antioxidant response pathway (6, 7, 30).

The metabolic reprogramming in ccRCC is further driven by the intratumoral genetic heterogeneity that characterizes ccRCC tumors (31). Although VHL loss is clonal in ccRCC, additional chromosomal changes and mutations in chromatin remodeling and PI3K pathway genes are often subclonal, potentially resulting in different levels of metabolic reprogramming in different regions of the primary tumor (31, 32). Recent multiregional analysis of primary ccRCC tumors using Dixon-based MRI to evaluate lipid accumulation demonstrated the heterogeneity of fat fraction within some ccRCC tumors (33). Subsequent analysis of these regions after surgical resection by mass spectrometry–based lipidomics and metabolomics confirmed this heterogeneity in metabolic profiles from different regions of the ccRCC tumor (33). A comprehensive evaluation of intratumoral heterogeneity in primary and metastatic tumors from 100 patients with metastatic ccRCC demonstrated that the metastatic sites are characterized by considerably less heterogeneity than primary tumors (34). If metastatic tumors are also found to be characterized by homogeneous metabolic profiles, this could provide unique insight into the most critical metabolic pathways to target in this disease.

Targeting Metabolic Reprogramming in ccRCC

Understanding the metabolic basis of the VHL/HIF pathway provided the foundation for the development of therapeutic approaches targeting this pathway. Since 2005, the FDA has approved nine agents targeting the VHL/HIF pathway in patients with advanced RCC. These therapies include a neutralizing antibody against VEGF, bevacizumab, that directly targets VEGF and six small molecule–based tyrosine kinase inhibitor therapies that target the VEGF receptor: pazopanib, axitinib, lenvatinib, cabozantinib, or a combination of the VEGF and PDGF receptors: sunitinib and sorafenib (Fig. 3; reviewed in ref. 35). Two additional agents, temsirolimus and everolimus, which inhibit the PI3K/AKT/mTOR pathway, lead to reduction of HIFα levels by inhibition of de novo protein translation (Fig. 3). Use of these agents to target metabolic reprogramming has been associated with increased disease-free progression and survival and prolonged disease stability.

Targeting VHL/HIF2

A newly developed therapeutic approach using small-molecule agents, such as PT2977 and PT2399, that directly and specifically inhibit the interaction between HIF2α and its essential binding partner, ARNT, are currently being evaluated in preclinical models as well as in clinical trials in patients with both localized and advanced ccRCC (Fig. 3). In preclinical studies, PT2399 significantly reduced cellular proliferation and survival in ccRCC cell lines as well as in ccRCC patient-derived xenografts (36, 37). Phase II trials are currently under way to evaluate the effectiveness of agents directly targeting the HIF2α pathway in patients (38).

HPRC

HPRC is an autosomal dominant hereditary renal cancer syndrome in which individuals are at risk for developing bilateral, multifocal type 1 papillary RCC (PRCC; ref. 39). The histology of both sporadic and hereditary type 1 PRCC tumors is characterized by papillary or tubular papillary architecture with slender papillae and delicate fibrovascular cores lined with small cells containing basophilic low-grade nuclei and scant amphophilic cytoplasm (40). The median age at diagnosis for patients affected with HPRC is 57 years and, although HPRC-associated type 1 PRCC tumors are malignant and can metastasize, their growth tends to be indolent. Management of patients with HPRC with type 1 PRCC involves active surveillance until the largest tumor reaches the 3 cm threshold, at which time surgical intervention is most often recommended (41).

Genetic linkage analysis in HPRC families localized the disease gene to chromosome 7q31, and germline mutations in the MET proto-oncogene were identified in affected family members (42). Mutations identified in HPRC families have all been missense, located in the tyrosine kinase domain of MET, and predicted to cause constitutive activation of the MET kinase in the absence of MET receptor ligand, hepatocyte growth factor (HGF; ref. 43). Type 1 PRCC tumors are typically characterized by the trisomy of chromosome 7. Duplication of the chromosome 7 bearing the mutant MET allele has been demonstrated in papillary type 1 tumors associated with HPRC, which is predicted to give the cancer cells a growth advantage (43).

Sporadic Type 1 PRCC: MET Alterations

Although MET mutations are found in only 13% to 18% of sporadic papillary type 1 RCC (43, 44), altered MET status (mutation, splice variant, or gene fusion) or increased chromosome 7 copy number was found in 81% of sporadic type 1 PRCC tumors evaluated in The Cancer Genome Atlas (TCGA) study (44), underscoring a central role for MET activation in this kidney tumor subtype.

Type 1 PRCC: MET Pathway

Signaling by growth factors, cytokines, and nutrients through the HGF–MET axis is important for cell proliferation, motility, scattering, differentiation, and morphogenesis during normal embryogenesis and development. HGF binding to MET results in the receptor dimerization and phosphorylation of critical intracellular tyrosines, which recruit adaptor proteins that serve as docking platforms for multiple signal transducers to activate downstream signaling cascades including PI3K–AKT, RAS–RAF–MEK1/2–ERK1/2, JNKs, STAT3, and NF-κB (Figs. 1 and 4; ref. 45). Deregulated MET signaling in HPRC kidney cells due to activating germline MET mutations leads to inappropriate upregulation of cancer-promoting signaling pathways that drive proliferation, motility, and survival of tumor cells.

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Figure 4.

Dysregulation of the PI3K/AKT/mTORC1 pathway by mutation of the PTEN, TSC1, TSC2, and FLCN tumor suppressor genes and the MET oncogene in inherited kidney cancer syndromes. A, Signaling through HGF/MET in normal cells in response to environmental cues activates the PI3K/AKT/mTORC1 pathway, resulting in increased protein synthesis and ribosome biogenesis that are necessary for cell growth and proliferation. HPRC-associated MET mutations lead to constitutive activation of the MET kinase, independent of HGF signaling, and the development of type 1 PRCC. MET-driven PRCC tumors have shown a partial response to MET tyrosine kinase (TK) inhibitors. Tumor suppressors PTEN, TSC1, TSC2, and FLCN function as controls in the mTORC1 pathway to maintain a proper balance of cell growth for cellular homeostasis. FLCN interacts with its binding partners FNIP1 and FNIP2 and, indirectly, in a complex with AMPK. Germline mutations in the PTEN gene in Cowden syndrome, TSC1/2 genes in tuberous sclerosis complex, and the FLCN gene in BHD syndrome result in loss of these controls, leading to upregulated mTORC1 activity and development of renal tumorigenesis. FLCN loss also results in increased PGC1α transcriptional activity, potentially through activated AMPK, which drives mitochondrial biogenesis, causing increased ROS production. Intracellular ROS drives up HIF1α transcriptional activity, leading to the metabolic reprogramming of BHD tumors that favors aerobic glycolysis for energy production. B, Top, FLCN–FNIP localization to the lysosome under conditions of low amino acids requires GDP-loaded RagA/B, which is achieved through GTPase-activating protein (GAP) activity of the GATOR1 complex toward Rag A/B. Bottom, mTORC1 becomes activated on the lysosome in response to amino acids through a lysosome-associated complex that includes v-ATPase, the Ragulator complex, and heterodimers RagA/B and RagC/D. Guanine nucleotide exchange factor (GEF) activity of Ragulator toward RagA/B results in GTP-loaded RagA/B and recruitment of mTORC1 to the lysosome. FLCN/FNIP complex displays GAP activity toward RagC/D, thereby generating GDP-bound RagC/D, which is necessary for mTORC1 activation.

Type I PRCC: Therapeutic Approaches

Agents that target the tyrosine kinase (TK) domain of MET would be predicted to be potentially efficacious in the treatment of HPRC (Fig. 4A). A phase II clinical trial with foretinib, a dual kinase inhibitor that targets the MET and VEGF receptors, was conducted in patients with type 1 papillary renal carcinoma. In patients with germline MET mutation, 50% of patients with HPRC had partial response, and the remaining 50% had stable disease. One of 5 patients with somatic MET mutations had a partial response, and no response was seen in patients with MET amplification (46). In a subsequent phase II clinical trial, treatment with crizotinib, a small-molecule MET TK inhibitor, produced a partial response in 50% (2/4) of patients with MET mutations (47). Eighteen percent of patients with MET-driven PRCC had a partial response in a trial involving another small-molecule MET TK inhibitor, savolitinib (48).

BHD Syndrome

BHD syndrome is an autosomal dominant inherited cancer syndrome in which affected individuals are at risk for the development of benign cutaneous tumors (fibrofolliculomas), pulmonary cysts (often associated with pneumothorax), and kidney tumors. Cutaneous fibrofolliculomas are found in >85% of individuals affected with BHD over the age of 25, and BHD-associated pulmonary cysts occur in 70% to 84% of affected individuals (49, 50). Family members affected with BHD also have a 7-fold increased risk for developing renal tumors (49). BHD-associated renal tumors with variable histologies, including hybrid oncocytic tumors (50%) with features of both chromophobe RCC and oncocytoma, chromophobe RCC (34%), and ccRCC (9%), are found in up to one third of affected individuals (50, 51). Like VHL and HPRC, BHD-associated renal tumors are managed by active surveillance until the largest tumor reaches 3 cm, at which point nephron-sparing surgery is recommended (52). As patients with BHD are at risk for bilateral, multifocal renal tumors, preserving maximum kidney function is a priority.

FLCN Gene

Genetic linkage analysis in BHD families localized the BHD disease gene to chromosome 17p11, and germline mutations in a novel gene, folliculin (FLCN), were found in affected individuals (53). Insertion/deletion, nonsense, splice-site, and missense mutations, as well as partial deletions, have been identified across the FLCN coding region. FLCN is a tumor suppressor gene consistent with the Knudson two-hit model of tumorigenesis (54). Naturally occurring rat and canine models of BHD with germline Flcn mutations develop renal tumors with mutation or loss of the remaining wild-type Flcn allele. The FLCN-null renal tumor cell line UOK257, established from tumor material from a patient with BHD, is tumorigenic in immunocompromised mice and loses its oncogenicity upon the restoration of FLCN expression (50).

FLCN: Regulator of mTOR Activation

Early studies to investigate FLCN function identified two novel binding partners, folliculin interacting protein 1 (FNIP1; ref. 55) and folliculin interacting protein 2 (FNIP2; refs. 56, 57), which interact with the carboxy-terminus of FLCN and with AMPK, a critical energy sensor and negative regulator of mTORC1 (Fig. 4A; ref. 58). Biochemical analysis of the polycystic kidneys and cystic tumors that developed in mice with kidney-targeted Flcn inactivation demonstrated activation of mTORC1 (59–61), and renal tumors that developed in Flcn heterozygous mice subsequent to loss of the wild-type Flcn allele displayed enhanced activity of both mTORC1 and mTORC2, and AKT (62). Taken together, these data establish a role for FLCN as a negative regulator of the AKT–mTOR pathway (Fig. 4A). Evidence supporting the positive regulation of mTOR by FLCN in two other Flcn heterozygous mouse models has suggested that modulation of mTOR activity by FLCN may depend upon cell type or nutritional or energy status (63, 64). The mTOR inhibitor sirolimus (rapamycin) was partially effective in reducing the number and size of cysts and tumors that developed in the kidney-targeted Flcn-deficient mice and allograft tumors (59–61, 65).

FLCN: Amino Acid Sensing for mTOR Activation

mTORC1 is a master regulator of cell growth that responds to environmental cues including amino acids. mTORC1 activation by amino acids requires a lysosome-associated complex that consists of the vacuolar adenosine triphosphatase (v-ATPase), the Ragulator complex, and the Rag GTPases, which exist as obligate heterodimers RagA/B and RagC/D (Fig. 4B). Under conditions of amino acid sufficiency, Ragulator activates RagA/B through its guanine nucleotide exchange factor (GEF) activity resulting in GTP-loaded RagA/B and recruitment of mTORC1 to the lysosome, where it becomes activated by RAS-homolog expressed in brain (RHEB) that is tightly regulated by growth factor signaling and cellular energy status (66). Evidence from multiple laboratories supports a role for the FLCN–FNIP complex in coordinating cellular response to amino acid availability through regulation of the nucleotide states of the Rag heterodimers (Fig. 4B). FLCN in association with FNIP is recruited to the lysosome surface in response to amino acid starvation where the FLCN–FNIP complex acts as a GTPase-activating protein (GAP) toward RagC/D (67, 68), thereby providing the required GDP-loaded RagC for mTOR binding to the Rag heterodimer when amino acids are abundant. FLCN–FNIP localization to the lysosome requires RagA/B to be in the inactive GDP-bound state, which in turn is dependent upon the GAP activity of the GATOR1 (GAP activity toward Rag1) complex toward RagA/B (Fig. 4B; ref. 69). Recently, amino acid sensors for the mTORC1 pathway upstream of GATOR1 have been uncovered (70), but the mechanistic details of how sensing low amino acids facilitates the interaction between GDP-loaded RagA/B and the FLCN–FNIP proteins remain to be determined.

FLCN: PGC1α Activation and Increased Mitochondrial Biogenesis

Early studies have suggested that FLCN inactivation results in metabolic reprogramming in which FLCN-deficient cells undergo a metabolic shift to favor aerobic glycolysis. Elevated HIF transcriptional activity and upregulation of HIF target gene expression were observed in FLCN-null UOK257 renal tumor cells, ACHN renal tumor cells with FLCN knockdown, and BHD-associated chromophobe RCC (71). In subsequent experiments by this group, Flcn-deficient mouse embryonic fibroblasts (Flcn^−/− MEF) displayed a 2-fold increase in HIF transcriptional activity and expression of HIF targets that correlated with increased glucose uptake, lactate production, and extracellular acidification, confirming a “Warburg effect” metabolic transformation in response to Flcn deficiency (72). HIF-dependent elevation of ATP levels in the Flcn^−/− MEFs correlated with enhanced mitochondrial respiration due to increased mitochondrial mass, which led to a significant rise in intracellular reactive oxygen species (ROS). Significantly, this study showed that intracellular ROS was driving the HIF transcriptional activation responsible for the metabolic reprogramming under Flcn deficiency (72). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is a well-known controller of mitochondrial biogenesis through transcriptional upregulation of genes responsible for mitochondrial biosynthesis, and active (phosphorylated) AMPK is known to directly phosphorylate and upregulate the expression of PGC1α (73). Previously, it was demonstrated that the FLCN–FNIP complex interacts directly with AMPK (55, 56), and loss of FLCN was shown to result in the upregulation of PGC1α expression and its transcriptional targets in BHD renal tumors and Flcn-deficient mouse kidney, muscle, and heart (50, 74, 75). In agreement with these previous reports, PGC1α mRNA and protein were increased 3-fold in the Flcn^−/− MEFs with a corresponding increase in PGC1α target genes and coactivators. In addition, AMPK was constitutively activated in Flcn-deficient cells, and upregulation of PGC1α was confirmed to be AMPK-dependent. Importantly, ROS production was shown to be PGC1α-dependent (72). These observations were recapitulated in the human FLCN-deficient UOK257 and FTC-133 cancer cell lines and were reversed by FLCN reexpression. It is notable that advanced VHL-deficient ccRCC and fumarate hydratase–deficient HLRCC-associated type 2 PRCC are both characterized by impaired mitochondrial metabolism (oxidative phosphorylation), although FLCN-deficient BHD-associated RCC is associated with increased oxidative metabolism and activated mitochondrial biogenesis. It is possible that this reflects a difference in the ability of distinct cells of origin to tolerate loss of mitochondrial activity, and/or it could explain the more mitigated aggressiveness of FLCN-deficient tumors in BHD.

Finally, increased mitochondrial mass, nuclear HIF1α staining, and expression of HIF target genes were seen in a BHD-associated chromophobe RCC (72). Taken together, these data support the concept that loss of FLCN constitutively activates AMPK, resulting in PGC1α-driven mitochondrial biogenesis and increased ROS production, leading to HIF transcriptional activity that drives Warburg metabolic reprogramming favoring aerobic glycolysis. Based on this paradigm, therapeutic agents that target the glycolytic pathway may show promise for the treatment of BHD-associated kidney cancer.

TSC

TSC is an autosomal dominant disorder in which affected individuals are at risk for the development of hamartomas in the brain, cutaneous angiofibromas, cardiac rhabdomyomas, pulmonary lymphangioleiomyomatosis (LAM), and kidney neoplasia. TSC is caused by germline loss-of-function mutations in the TSC1 gene on chromosome 9q34, which encodes hamartin, or the TSC2 gene on chromosome 16p13, which encodes tuberin (76, 77). Although one third of TSC cases are thought to be familial, up to two thirds of patients have no family history of TSC, and these mutations are thought to be de novo mutations. Angiomyolipomas, consisting of smooth muscle, fat, and vascular components, are the most frequently observed kidney neoplasm in TSC and demonstrate loss of heterozygosity of the TSC1 or TSC2 allele. However, RCCs that harbor biallelic inactivation of TSC2 are also seen in patients with TSC, and can present with varying histologies, including clear-cell, papillary, and chromophobe RCC (77, 78).

Cowden Syndrome

Cowden syndrome is an autosomal dominant disorder characterized by an increased risk for manifestations in several organs, including tumors of the breast, thyroid, endometrium, and kidney (79). Cowden syndrome is associated with germline mutations of a number of genes, including mutation of the PTEN gene on chromosome 10q23 (79).

PI3K/AKT/mTOR Pathway

The PTEN gene protein product is a phosphatase that catalyzes the conversion of PIP3 to PIP2. In response to the stimulation of growth factor receptors, intracellular levels of PIP3 are increased and activate several downstream pathways, including the PI3K/AKT/mTOR pathway (80). To attenuate and control these pathways, PTEN converts PIP3 back to PIP2. In PTEN-deficient tumors, the increased levels of PIP3 remain constant, leading to continuous activation of AKT that phosphorylates and inhibits the TSC complex, resulting in the upregulation of mTOR (Fig. 4A; ref. 80). The protein products of TSC1 and TSC2, TSC1 and TSC2, are negative regulators of mTORC1 (Fig. 4A). Together, they form a heterodimeric complex with TBC1 domain family member 7 (TBC1D7) that acts on the small GTPase RHEB and stimulates its conversion from GTP-active state to GDP-inactive state, thereby inhibiting the phosphorylation and activation of mTORC1 by RHEB. The TSC1–TSC2 complex responds to nutrients, growth factors, and amino acids, triggering GAP activity toward RHEB. Additionally, in response to cellular energy deficit, TSC2 is phosphorylated and activated by AMPK, leading to suppression of mTORC1 activity. Loss of TSC1 or TSC2 function by mutation, or direct phosphorylation and inactivation of TSC2 by protein kinase B (AKT), leads to RHEB activation and mTOR-dependent phosphorylation of two downstream effectors, p70S6 kinase (which in turn phosphorylates ribosomal protein S6), and 4E-binding protein 1 (4EBP1), resulting in increased protein synthesis, cell growth, and proliferation (Fig. 4A; refs. 76, 80).

A preclinical model using epithelial-specific PTEN-deficient mice demonstrated that use of rapamycin to target the mTOR pathway promoted the rapid regression of advanced mucocutaneous lesions (81). Similarly, mTOR activation in TSC-associated LAM and angiomyolipomas has been successfully targeted therapeutically by treatment with rapalogs (i.e., sirolimus and everolimus; ref. 77). Although the effect on the central nervous system and pulmonary lesions is potentially quite remarkable and possibly paradigm-changing, rapalog treatment of renal lesions is likely cytostatic, because regrowth was observed upon discontinuation of sirolimus treatment (82).

Familial MITF RCC and Melanoma

Carriers of a germline pathogenic variant of MITF (p.E318K) were shown to have a >5-fold increased risk to develop melanoma and RCC, as compared with the general population. The variant MITF protein is affected by impaired sumoylation, differentially regulates DNA binding, and drives enhanced transcriptional activity of genes involved in cell growth, proliferation, and inflammation (Fig. 5; refs. 92, 93). Somatic MITF gene fusions have been reported in RCC, suggesting a role for MITF fusions, similar to TFE3 and TFEB gene fusions, in renal tumorigenesis (91).

Master Regulators of Lysosomal Biogenesis and Autophagy

The transcriptional activity of MiT family members has been shown to affect a number of signaling pathways associated with increased cell proliferation and differentiation. TFE3 and TFEB bind to a 10-bp DNA motif (GTCACGTGAC) termed Coordinated Lysosomal Expression and Regulation (CLEAR) element, thereby increasing the transcription of lysosomal proteins and regulating lysosomal acidification, autophagy, and exo-, endo-, and phagocytosis in a MAPK-dependent mechanism (94). MiT family members are master regulators of lysosomal biogenesis and autophagy, involved in shaping the cell's metabolism and reaction to metabolic stress (95). After tumor initiation, autophagy may be an important mechanism for cancer cell survival in nutrient-deficient environments. A kidney-directed TFEB-overexpressing mouse model develops cystic kidney enlargement, neoplastic lesions, and liver metastasis in an autophagy-independent manner (96). Several genes involved in glucose metabolism, mitochondrial biogenesis (e.g., PPARGC1A encoding PGC1α), and lipid metabolism are under the regulation of a CLEAR element, and constitutive activation of MiT proteins may therefore increase oxidative metabolism (reviewed in ref. 97). A knock-in alveolar soft-part sarcoma mouse model expressing the ASPSCR1–TFE3 fusion gene was shown to express high levels of lactate importers, harbor abundant mitochondria, and metabolize lactate as a metabolic substrate, suggesting a role for lactate as a driver of MiT translocation tumors (98).

MiT Proteins and the mTOR Pathway

Under physiologic and nutrient-rich conditions, TFE3 and TFEB are recruited to lysosomes and phosphorylated by mTORC1 in a mechanism involving FLCN and FNIP (Fig. 5; ref. 67, 99). MiT phosphorylation creates a binding site for 14-3-3, which keeps the transcription factors localized to the cytosol and inactive. Amino acid or serum deprivation causes mTORC1 inactivation, release of TFE3/TFEB into the nucleus, and activation of a transcriptional program that leads to lysosomal biogenesis and autophagy, which initiates a metabolic adaptation of the cell (97). Activation of the PI3K/AKT/mTOR pathway, measured by increased phosphorylation of the S6 ribosomal protein, has been shown in TFE3-fusion cell lines and tumors, and occasional partial responses to mTORC1 inhibitors (e.g., rapamycin and temsirolimus) have been reported in patients with MiT-RCC (100–102). Recent preclinical data suggest promising antitumor effects of dual mTORC1/2 and combined AKT/mTOR inhibitors against translocation RCC cells in vitro and in vivo (2, 102). Such combination treatments may therefore represent a potential therapeutic approach to target a pathway that is constitutively upregulated in MiT-RCC.

HLRCC

HLRCC is an autosomal dominant familial cancer syndrome in which affected individuals are at risk for the development of cutaneous and uterine leiomyomas and an aggressive form of type 2 PRCC (103, 104). HLRCC-associated renal tumors can be early-onset (as early as 10 years of age) and have a propensity to metastasize when the primary tumor is small (as small as 0.5 cm; ref. 104). Early surgical intervention with wide surgical margins is recommended for HLRCC-associated renal tumors due to the aggressive and infiltrative nature of the tumor. Because the renal tumors have such an accelerated growth rate and can spread when the primary tumors are small, annual abdominal imaging is recommended for surveillance of at-risk individuals starting at 8 years of age (105, 106).

Fumarate Hydratase Gene: Aerobic Glycolysis and Impaired Oxidative Phosphorylation

HLRCC is characterized by pathogenic germline variation of the fumarate hydratase (FH) gene (107). The FH gene encodes the gene for the TCA cycle enzyme fumarate hydratase, which catalyzes the interconversion of fumarate and L-malate. HLRCC-associated renal tumors are found to have somatic loss of the wild-type allele of the FH gene. These FH-deficient cells undergo a Warburg metabolic shift characterized by aerobic glycolysis and impaired oxidative phosphorylation (108–110). Stable isotope tracer studies of HLRCC tumor–derived cells, UOK262, demonstrate that very little glucose-derived carbon enters the TCA cycle, whereas glutamine-derived carbon readily enriched TCA cycle intermediates and contributed substantially to the overaccumulation of fumarate (Fig. 6A; ref. 26). The flow of glutamine-derived carbon into the TCA cycle in HLRCC cells presents a metabolic conundrum, as the lack of fumarase activity in the cells prevents the completion of multiple turns of the TCA cycle. HLRCC RCC cells have adapted to this deficiency by reducing their amount of mitochondrial oxidative phosphorylation, instead promoting the formation of citrate directly by reductive carboxylation of α-ketoglutarate (Fig. 6A; refs. 26, 110, 111). In this process, carbon dioxide is conjugated to glutamine-derived α-ketoglutarate by isocitrate dehydrogenase to form citrate, with the concomitant oxidation of NAD(P)H to NAD(P)⁺. Although it is currently unclear whether reductive carboxylation in HLRCC tumor cells occurs preferentially in the mitochondrion, the cytosol, or both, the robust production of cytosolic NADPH catalyzed by enhanced activity of the pentose phosphate pathway (PPP) observed in FH^−/− UOK262 cells suggests that the capacity for cytosolic reductive carboxylation of glutamine-derived α-ketoglutarate is substantial (110).

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Figure 6.

Altered metabolism in FH- and SDH-deficient tumor cells. A, Metabolic remodeling observed using stable isotope-resolved metabolomics in FH- and SDH-deficient human tumor cell lines. Increased aerobic glycolysis and flow of glucose carbons into the oxidative PPP have been observed, as well as very little entry of glucose-derived carbon into the TCA cycle (red circles). Reductive carboxylation of glutamine-derived carbon (blue circles) into citrate has been observed in both FH- and SDH-deficient human tumor cells in vitro. TCA cycle proteins associated with renal tumor formation are highlighted in yellow. B, The DNA CpG island hypermethylation phenotype observed in SDH- and FH-deficient renal tumors is thought to be caused by sustained product-level inhibition of the TET family of methylcytosine dioxygenases by elevated succinate and fumarate. C, Product-level inhibition of PHDs by succinate and fumarate prevents the hydroxylation of proline residues in the oxygen-dependent degradation domains of HIF1/2α, preventing their recognition and targeting for degradation by the VHL ubiquitin ligase complex. D, The family of JmjC domain-containing α-ketoglutarate–dependent histone lysine demethylases (KDM) are inhibited by elevated succinate and fumarate, resulting in histone hypermethylation. Fumarate has been shown to undergo nonenzymatic electrophilic reactions with the free sulfhydryl moiety of reduced glutathione (E) and cysteine thiol groups present in proteins (F).

In addition to the direct metabolic alterations described above, abnormal fumarate accumulation in FH-deficient cells leads to additional alterations of carbon flow through central metabolic pathways. Frezza and colleagues showed that FH^−/− cells rely on the biosynthesis and degradation of heme and the secretion of the heme degradation product bilirubin for their survival, leading to synthetic lethality in cells in which both FH and the heme degradative enzyme heme oxygenase are inactivated (112). Zheng and colleagues found that FH^−/− cells exhibit reversed flux of substrates in the urea cycle enzyme argininosuccinate lyase (ASL) due to elevated fumarate levels, resulting in auxotrophy for arginine and suggesting that arginine depletion may be of therapeutic utility in HLRCC tumors (113).

Decreased AMPK, p53, and DMT1

As noted above, AMPK is an energy sensor that responds to cellular energy status by undergoing phosphorylation and increasing kinase activity in response to rises in the cytosolic AMP/ATP ratio. The result of increased AMPK activation is the suppression of downstream targets that include mediators of cellular proliferation, such as the mTOR pathway, and suppression of anabolic functions, including fatty-acid biosynthesis, via phosphorylation and inactivation of the acetyl-CoA carboxylases ACC1/2. Unexpectedly, AMPK phosphorylation levels were found to be decreased in FH^−/− UOK262 cells despite their strict reliance on glycolysis for production of ATP to fuel anabolic metabolism and cellular proliferation (114). The decreased AMPK activation in UOK262 cells leads to several additional metabolic alterations including decreased levels of p53 as well as the iron importer divalent metal transporter 1 (DMT1). Decreased DMT1 leads to cellular iron deficiency, which promotes the stabilization of HIF1α and the repression of HIF2α translation due to the presence of an iron-responsive element in the 5′ untranslated region of the HIF2A mRNA transcript in FH-deficient HLRCC RCC cells (114). Repression of AMPK phosphorylation along with activation of HIF1α in UOK262 cells promotes invasive and tumorigenic potential (114).

Familial SDH-Deficient RCC

SDH, a tetrameric enzyme complex made up of the products of SDHA, SDHB, SHDC, and SDHD, is found in the inner mitochondrial membrane and is part of both the citric acid cycle and the electron transport chain. SDH catalyzes the oxidation of succinate to fumarate in a coupled reaction with the reduction of ubiquinone to ubiquinol. Patients with loss-of-function pathogenic variations of SDHB, SDHC, and SDHD are at risk for the development of tumors in several organs, including pheochromocytoma, paraganglioma, gastrointestinal stromal tumors, and RCC (115, 116). SDHB-deficient RCCs are characterized by an oncocytic histologic appearance, with eosinophilic cytoplasm consistent with mitochondrial accumulation (117, 118). Patients with germline pathogenic variations in the SDH subunit genes are at risk for the development of early-onset, bilateral, and multifocal RCC that have the propensity to spread when tumors are small. It is recommended that patients harboring germline SDH mutations be screened annually for renal lesions as well as other SDH-related neoplasms (116).

Shift to Aerobic Glycolysis and Impaired Oxidative Phosphorylation

SDH-deficient renal tumor cells are characterized by a Warburg shift to aerobic glycolysis and near-complete impairment of oxidative phosphorylation (118). Isotope-resolved metabolic analysis of tumor-derived SDHB-deficient UOK269 tumor cells revealed robust lactic acid fermentation and very little entry of glucose into TCA cycle metabolites (118). However, glutamine readily labeled TCA cycle intermediates including succinate, which accumulated to extremely high levels in UOK269 cells. Labeling of UOK269 cells with 1-¹³C-glutamine demonstrated that reductive carboxylation of glutamine-derived α-ketoglutarate into citrate occurred in these SDH-deficient tumor cells (118). In a study evaluating SDH-deficient immortalized mouse renal cells, Cardaci and colleagues identified deficiency of additional respiratory chain components, further supporting the observation that oxidative TCA cycle metabolism is impaired in SDH-deficient cells (119).

Mutation Impairs Incorporation of Iron Sulfur Clusters in SDHB

Of the familial SDH mutations that have been found to cause renal tumors, mutations in SDHB are the most prevalent (116). SDHB missense mutations in patients with SDH tumor syndrome are enriched in regions of the SDHB protein that are crucial for ligation of the three Fe–S cluster cofactors that are essential for its function (118). The SDHB protein possesses two tripeptide L(I)YR motifs that are essential for the recruitment of the Fe–S cluster chaperone/cochaperone machinery responsible for insertion of Fe–S clusters into the nascent SDHB polypeptide during complex II assembly (120, 121). Missense mutations in these two L(I)YR motifs in SDHB occur frequently in patients who are at risk for the development of SDH-deficient tumors (118).