The Metabolic Basis of Kidney Cancer...(KDM) enzymes that regulate DNA/histone methylation and acetylation and effect chromatin remodeling. Direct mutation of chromatin remodeling

1006 | CANCER DISCOVERY AUGUST 2019 www.aacrjournals.org

REVIEW

The Metabolic Basis of Kidney Cancer W. Marston Linehan 1 , Laura S. Schmidt 1 , 2 , Daniel R. Crooks 1 , Darmood Wei 1 , Ramaprasad Srinivasan 1 , Martin Lang 1 , and Christopher J. Ricketts 1

1 Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 2 Basic Science Program, Frederick Laboratory for Cancer Research, Frederick, Maryland. Corresponding Author: W. Marston Linehan, National Cancer Institute, 10 Center Drive, MSC 1107, Bethesda, MD 20892. Phone: 240-858-3700; Fax: 301-480-3195; E-mail: [email protected] Cancer Discov 2019;9:1006–21

doi: 10.1158/2159-8290.CD-18-1354 ©2019 American Association for Cancer Research.

ABSTRACT Kidney cancer is not a single disease but represents several distinct types of can-cer that have defi ning histologies and genetic alterations and that follow different

clinical courses and have different responses to therapy. Mutation of genes associated with kidney cancer, such as VHL, FLCN, TFE3, FH, or SDHB , dysregulates the tumor’s responses to changes in oxy-gen, iron, nutrient, or energy levels. The identifi cation of these varying genetic bases of kidney cancer has increased our understanding of the biology of this cancer, allowing the development of targeted therapies and the appreciation that it is a cancer driven by metabolic alterations.

Signifi cance: Kidney cancer is a complex disease composed of different types of cancer that present with different histologies, clinical courses, genetic changes, and responses to therapy. This review describes the known genetic changes within kidney cancer, how they alter tumor metabolism, and how these metabolic changes can be therapeutically targeted.

INTRODUCTION Kidney cancer, or renal cell carcinoma (RCC), affects nearly

300,000 individuals worldwide each year and is responsible for over 100,000 deaths annually. Although patients who present with localized or locally advanced disease have a 5-year survival rate of 20% to 95% depending on the extent of disease, patients with metastatic disease have a 0% to 10% 5-year survival rate ( 1 ). RCC has historically been considered a single disease; patients with renal tumors all underwent the same surgical procedures and patients with advanced disease were treated with similar drugs, none of which were effective. Although there would occasionally be a response, the systemic therapies available did not result in increased survival for patients with advanced disease. An increased understanding of RCC has shown that it consists of a number of different types of cancer that are characterized by different histologies, clinical courses, and responses to therapy and that are caused by different genes ( Fig. 1 ). It is known that at least 17 differ-ent genes can cause RCC and that mutation of these genes can affect the cell’s ability to respond to changes in oxygen, iron, nutrients, or, most notably in the case of mutations in genes for the tricarboxylic acid (TCA) cycle enzymes fumarate hydratase and succinate dehydrogenase (SDH), energy ( Fig. 2 ).

Much of what is known about the genetic and metabolic basis of RCC has come from the study of patients with

inherited RCC susceptibility syndromes, such as von Hippel–Lindau (VHL; VHL gene), hereditary papillary RCC (HPRC; MET gene), Birt–Hogg–Dubé (BHD; FLCN gene), and heredi-tary leiomyomatosis and RCC (HLRCC; FH gene). Studies of familial as well as sporadic (nonfamilial) disease have provided the foundation for the development of therapeutic approaches targeting the metabolic basis of RCC.

CLEAR-CELL RENAL CARCINOMA: VHL/HIF OXYGEN-SENSING PATHWAY 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 recur-rent, 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 identifi ed 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

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The Metabolic Basis of Kidney Cancer REVIEW

Figure 1. The histology and genetics of RCC. RCC is not a single disease; it is separated into different subtypes based on the histology and genetics of the tumors. These histology images show 10 different types of RCC and the genes that are altered in association with these histologies. Mutation of VHL, BAP1, MET, FH, TSC1, TSC2, and PTEN can occur as both germline alterations in inherited disease and somatic alterations in sporadic disease. Mutation of FLCN, SDHB, SDHC, and SDHD occurs as germline alteration in specific inherited disease syndromes. Somatic translocation-induced fusion genes involving TFE3, TFEB, and MITF occur in sporadic disease, whereas mutation of MITF can occur as a germline event. Adapted from Linehan and Ricketts (2) and reprinted with permission from Elsevier.

Clear cellVHL, TCEB1, BAP1

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Papillary epitheloidTFE3, TFEB, MITF

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EosinophilicSDHB, SDHC, SDHD

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ccRCC tumors have shown nearly universal loss of chromo-some 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 TumorigenesisThe 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), Cul-lin-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 ubiqui-tin-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 het-eromeric 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 reduc-ing pyruvate entry into the TCA cycle via upregulation of pyruvate dehydrogenase kinase 1 (encoded by PDK1); and increased red blood cell production by increased erythro-poietin (encoded by EPO) production (Fig. 3). Activation of the hypoxia response is also advantageous in the growth and maintenance of a cancer, including increased angio-genesis via upregulation of vascular endothelial growth fac-tor (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).

Activation of HIFα-dependent pathways is a canonical cellu-lar 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 path-ways independent of oxygen/iron levels in a state of “pseudohy-poxia.” A subset of sporadic ccRCCs that lack VHL alterations have mutations in other components of the VHL E3 ubiq-uitin ligase complex, such as elongin C (TCEB1/ELOC), that function like VHL-loss mimics and result in the stabilization of the HIFα proteins (7, 18). Although some genes are tran-scriptionally regulated by both HIF1α and HIF2α, each tran-scription 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





Linehan et al.REVIEW

Figure 2. Dysregulated pathways in RCC. RCC is a complex disease that can result from germline or somatic mutations that alter the functions of several metabolic pathways. Currently, alteration of 17 genes is specifically associated with RCC. Both germline and somatic alterations of VHL, MET, PTEN, TSC1, TSC2, BAP1, PBRM1, MITF, FH, and CDKN2B are present in RCC. Germline mutations of FLCN, SDHB, SDHC, and SDHD are associated with RCC predisposition syndromes. However, somatic loss of SMARCB1 or alteration of TFE3, TFEB, and MITF occurs in sporadic RCC. Loss of the VHL/HIF oxygen-sensing pathway is critical in several types of RCC. Loss of VHL in tumors results in the inability of the VHL E3 ubiquitin ligase complex to target the HIF transcription factors for degradation, causing the stabilization of HIF1α and HIF2α that activate the hypoxia response. This pseudohypoxia increases the expression of growth factors that induce proliferation, survival, and angiogenesis, including VEGF, PDGF, and TGFα, and increases expres-sion of proteins that regulate glucose metabolism and cell proliferation, including GLUT1, LDHA, PDK1, and CCND1. Activating mutations of MET, inacti-vating mutations of PTEN, TSC1, TSC2, and FLCN, and overactivation of the TFE3, TFEB, and MITF transcription factors in tumors all result in abnormally increased activation of the PI3K/AKT/mTOR pathway. The PI3K/AKT/mTOR pathway normally regulates cell growth, proliferation, and survival and is controlled by many factors, including the availability of metabolic resources, such as amino acids. Dysregulation of the PI3K/AKT/mTOR pathway results in increased protein production that includes the de novo synthesis of the HIF transcription factors, allowing it to influence the VHL/HIF oxygen-sensing pathway. Loss of fumarate hydratase (FH) or components of succinate dehydrogenase (SDHB, SDHC, SDHD) in a tumor directly affects the activity of the TCA cycle, altering metabolism, and results in the accumulation of the oncometabolites fumarate and succinate, respectively. The increased fumarate causes excessive succination of multiple proteins that alters their function, including inactivation of the KEAP1 protein. KEAP1 is a regulator the NRF2/ARE (antioxidant response element) pathway that controls cellular response to oxidative stress. Increased fumarate or succinate both can inhibit α-ketoglutarate–dependent PHD enzymes that regulate the HIF transcription factors, and this results in inhibition of the VHL/HIF oxygen-sensing path-way by an alternative mechanism. Other α-ketoglutarate–dependent enzymes include the Ten-eleven translocation (TET) and Lysine-specific demethylase (KDM) enzymes that regulate DNA/histone methylation and acetylation and effect chromatin remodeling. Direct mutation of chromatin remodeling proteins, such as BAP1 or the SMARCB1 or PBRM1 components of the SWI/SNF complex, also alters gene-expression profiles in RCC. HGF, hepatocyte growth factor; TK, tyrosine kinase domain.

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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 ReprogrammingccRCC tumors lose VHL-dependent oxygen sensing, result-

ing 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,






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 ubiqui-tin-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 con-version 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. Tem-sirolimus 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.

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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 glu-tamine 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, includ-ing decreased TCA cycle and AMPK complex gene expression and increased pentose phosphate pathway and fatty-acid syn-thesis 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-fluorode-oxyglucose (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 infus-ing 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 inter-mediates in tumors was enhanced, whereas the TCA cycle components had significantly reduced 13C-labeling, consist-ent with aerobic glycolysis and the Warburg effect.

Although upregulation of the VHL/HIF pathway is central to the metabolic reprogramming of ccRCC tumors, other meta-bolic alterations may also play an important role. ccRCC tumors frequently have mutations in the chromatin-remodeling, chro-mosome 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 chromo-some 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 chroma-tin remodeling and PI3K pathway genes are often subclonal, potentially resulting in different levels of metabolic repro-gramming 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 lipidom-ics and metabolomics confirmed this heterogeneity in meta-bolic 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 char-acterized by homogeneous metabolic profiles, this could pro-vide unique insight into the most critical metabolic pathways to target in this disease.

Targeting Metabolic Reprogramming in ccRCCUnderstanding the metabolic basis of the VHL/HIF path-

way provided the foundation for the development of thera-peutic approaches targeting this pathway. Since 2005, the FDA has approved nine agents targeting the VHL/HIF path-way 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: paz-opanib, axitinib, lenvatinib, cabozantinib, or a combination of the VEGF and PDGF receptors: sunitinib and sorafenib (Fig. 3; reviewed in ref. 35). Two additional agents, temsiroli-mus 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/HIF2A 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 evalu-ated 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).

TYPE 1 PAPILLARY RCC: MET GENEHPRC

HPRC is an autosomal dominant hereditary renal cancer syn-drome 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 charac-terized by papillary or tubular papillary architecture with slen-der papillae and delicate fibrovascular cores lined with small cells containing basophilic low-grade nuclei and scant ampho-philic 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 surgi-cal intervention is most often recommended (41).

Genetic linkage analysis in HPRC families localized the dis-ease gene to chromosome 7q31, and germline mutations in the MET proto-oncogene were identified in affected family mem-bers (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 chromo-some 7 bearing the mutant MET allele has been demonstrated in papillary type 1 tumors associated with HPRC, which is pre-dicted to give the cancer cells a growth advantage (43).

Sporadic Type 1 PRCC: MET AlterationsAlthough 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 chro-mosome 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 PathwaySignaling by growth factors, cytokines, and nutrients

through the HGF–MET axis is important for cell prolifera-tion, motility, scattering, differentiation, and morphogenesis






during normal embryogenesis and development. HGF bind-ing to MET results in the receptor dimerization and phos-phorylation 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 can-cer-promoting signaling pathways that drive proliferation, motility, and survival of tumor cells.

Type I PRCC: Therapeutic ApproachesAgents 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 crizo-tinib, 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).

BIRT–HOGG–DUBÉ RCC: FLCN NUTRIENT SENSINGBHD Syndrome

BHD syndrome is an autosomal dominant inherited can-cer syndrome in which affected individuals are at risk for

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 com-plex, and the FLCN gene in BHD syndrome result in loss of these controls, leading to upregulated mTORC1 activity and development of renal tumorigen-esis. 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.

HGFLow amino acid levels

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the development of benign cutaneous tumors (fibrofollicu-lomas), pulmonary cysts (often associated with pneumotho-rax), 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, chro-mophobe 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 surveil-lance 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 GeneGenetic 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 ActivationEarly 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 devel-oped 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 (rapa-mycin) 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 ActivationmTORC1 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 triphos-phatase (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 suf-ficiency, 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 sign-aling 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 heter-odimers (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: PGC1` 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 produc-tion, and extracellular acidification, confirming a “Warburg effect” metabolic transformation in response to Flcn defi-ciency (72). HIF-dependent elevation of ATP levels in the Flcn−/− MEFs correlated with enhanced mitochondrial respi-ration 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 coactiva-tor 1-alpha (PGC1α) is a well-known controller of mito-chondrial 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 tran-scriptional 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 recapitu-lated 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 (oxida-tive phosphorylation), although FLCN-deficient BHD-associ-ated 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 ori-gin 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 constitu-tively activates AMPK, resulting in PGC1α-driven mitochon-drial 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 path-way may show promise for the treatment of BHD-associated kidney cancer.

TSC-/- AND PTEN-/- RCC: PI3K/AKT/ mTOR PATHWAY

Two additional RCC susceptibility syndromes, Cowden syn-drome and tuberous sclerosis complex (TSC), are associated with mutations in the PI3K/AKT/mTOR pathway (Fig. 4A).

TSCTSC 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 muta-tions 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 fre-quently 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 histolo-gies, including clear-cell, papillary, and chromophobe RCC (77, 78).

Cowden SyndromeCowden syndrome is an autosomal dominant disorder

characterized by an increased risk for manifestations in sev-eral organs, including tumors of the breast, thyroid, endome-trium, and kidney (79). Cowden syndrome is associated with germline mutations of a number of genes, including muta-tion of the PTEN gene on chromosome 10q23 (79).

PI3K/AKT/mTOR PathwayThe 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 phosphoryla-tion 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 tar-get the mTOR pathway promoted the rapid regression of advanced mucocutaneous lesions (81). Similarly, mTOR acti-vation 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-chang-ing, rapalog treatment of renal lesions is likely cytostatic, because regrowth was observed upon discontinuation of sirolimus treatment (82).

TFE3, TFEB, AND MITF TRANSLOCATION RCC: NUTRIENT SENSING, LYSOSOMAL BIOGENESIS, AND AUTOPHAGY

MiT-RCCs are a subset of sporadic RCC driven by chromo-somal rearrangements creating gene fusions of microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, and MITF (Fig. 5). MiT-RCCs make up approximately 1% to 5% of sporadic RCC tumors and are more common in






children and young adults, representing 42% of all pediatric RCC cases (83). Recent reports suggest that adult-onset trans-location RCCs may be underappreciated partly because they overlap morphologically with the more common papillary and clear-cell types of RCC (84). Among the cohort of adult RCCs described by the TCGA consortium, MiT-RCCs made up approximately 1% of ccRCCs and 12% of papillary type 2 RCCs (44, 85).

TFE3–RCCs are histologically heterogeneous tumors that frequently display a papillary architecture formed by clear and eosinophilic cells with occasional psammomatous calcifi-cations (86, 87). They are relatively aggressive and have a pro-pensity toward early metastasis to regional lymph nodes (84). The less common and generally less aggressive TFEB-fusion RCCs typically present with a biphasic microscopic archi-tecture (84, 88, 89). Diagnosis of MiT-RCC is performed by IHC evaluating nuclear TFE3/TFEB immunoreactivity and “break-apart” FISH to detect chromosomal rearrangements involving TFE3 and TFEB (84). The treatment of localized disease is surgical and includes lymph-node resection, but the possibility of late-onset metastases makes a long clini-cal follow-up necessary (2). Multikinase inhibitors, such as VEGF pathway antagonists (e.g., sunitinib), as well as immu-notherapy with cytokines, such as IL2 and IFNα, have shown limited response in patients with metastatic MiT-RCC. There

is currently no known effective form of therapy for patients with advanced forms of these cancers.

Members of the basic helix–loop–helix and leucine zipper containing MiT transcription factor family share similar pro-tein structures, recognize similar or identical DNA sequences upon homo- and heterodimerization among each other, and drive a transcriptional program involved in the development of melanocytes, osteoclasts, and mast cells. Under physiologic conditions, nuclear localization and transcriptional activity of MiT family proteins is tightly regulated. Chromosomal rearrangements leading to MiT-RCC cause the formation of a chimeric open reading frame with a 3′ part of TFE3/TFEB/MITF and a variable 5′ part of a fusion partner (Fig. 5). MiT fusion isoforms retain the wild-type C-terminus part of the MiT protein with functional DNA binding and dimeriza-tion domains. The fusion partners instead drive increased transcription and/or constitutive nuclear localization of the MiT fusion protein, which results in its dysregulated transcriptional activity that promotes carcinogenesis (90). Translocation of TFE3, located on chromosome Xp11, fre-quently results in fusion with recurrent partners [e.g., PRCC, ASPSCR1, SFPQ (also known as PSF), and NONO], but numer-ous less frequent fusion partners have been described (44, 90, 91). MiT-RCCs characterized by chromosomal transloca-tions of TFEB (chromosome 6p21) mostly involve MALAT1, a

Figure 5. MiT-RCC and regulation of MiT proteins. Subcellular localization of members of the MiT family (TFE3, TFEB, and MITF) is regulated based on nutrient availability. MiT proteins are inactive and sequestered in the cytoplasm by the 14-3-3 chaperone when phosphorylated by active mTORC1 in an FLCN/FNIP-dependent mechanism. Inactivation of mTORC1 allows dephosphorylation and nuclear translocation of the transcription factors, which leads to the upregulation of transcriptional profiles intended to restore nutrient availability of the cell. In MiT-RCC, the physiologic regulation of MiT members is bypassed due to the genetic alterations. The creation of fusion genes due to chromosomal translocations leads to constitutive nuclear localization of the fusion protein (e.g., TFE3), while upregulation of transcription through gene fusions or duplications leads to increased protein product and evasion of cytoplasmic retention (e.g., TFEB). A variant of MITF (p.E318K) leads to its impaired sumoylation and altered transcriptional activity. Constitutive activa-tion of MiT downstream pathways drives tumorigenesis in MiT-RCC.

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nontranslated gene, and result in overexpression of the full-length TFEB protein. Only recently have TFEB translocations with other fusion partners and TFEB amplifications been described (44, 89, 91).

Familial MITF RCC and MelanomaCarriers of a germline pathogenic variant of MITF (p.E318K)

were shown to have a >5-fold increased risk to develop mela-noma 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, prolif-eration, 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 asso-ciated 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 impor-tant 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, mitochon-drial 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 PathwayUnder 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 phos-phorylation 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 tem-sirolimus) have been reported in patients with MiT-RCC (100–102). Recent preclinical data suggest promising antitu-mor 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 rep-resent a potential therapeutic approach to target a pathway that is constitutively upregulated in MiT-RCC.

FUMARATE HYDRATASE–DEFICIENT RCCHLRCC

HLRCC is an autosomal dominant familial cancer syn-drome in which affected individuals are at risk for the develop-ment 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 surveil-lance 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 varia-tion 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 interme-diates 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 meta-bolic 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 reduc-tive carboxylation of α-ketoglutarate (Fig. 6A; refs. 26, 110, 111). In this process, carbon dioxide is conjugated to glu-tamine-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 reduc-tive carboxylation in HLRCC tumor cells occurs preferentially in the mitochondrion, the cytosol, or both, the robust pro-duction of cytosolic NADPH catalyzed by enhanced activ-ity 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).






In addition to the direct metabolic alterations described above, abnormal fumarate accumulation in FH-deficient cells leads to additional alterations of carbon flow through cen-tral 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 biliru-bin 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 sug-gesting that arginine depletion may be of therapeutic utility in HLRCC tumors (113).

Decreased AMPK, p53, and DMT1As 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 inactiva-tion 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 cel-lular proliferation (114). The decreased AMPK activation in UOK262 cells leads to several additional metabolic altera-tions 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 transla-tion due to the presence of an iron-responsive element in the 5′ untranslated region of the HIF2A mRNA transcript in

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 inhi-bition 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).

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FH-deficient HLRCC RCC cells (114). Repression of AMPK phosphorylation along with activation of HIF1α in UOK262 cells promotes invasive and tumorigenic potential (114).

SDH-DEFICIENT RCCFamilial SDH-Deficient RCC

SDH, a tetrameric enzyme complex made up of the prod-ucts 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, paragangli-oma, gastrointestinal stromal tumors, and RCC (115, 116). SDHB-deficient RCCs are characterized by an oncocytic his-tologic 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 harbor-ing 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 interme-diates 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 evaluat-ing 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 preva-lent (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).

ONCOMETABOLITES IN RCCAs outlined above, increased accumulations of fumarate

and succinate occur in tumor cells upon loss of FH and SDH activities, respectively. These metabolite accumulations lead to profound alterations in metabolic cellular processes that extend far beyond intermediary metabolism. Succinate is the metabolic by-product of the PHD enzymes respon-sible for the degradation of HIF1/2α, the Jumonji domain family of histone lysine demethylases (JMJ-KDM), and the ten-eleven translocation (TET) family of hydroxylases that catalyze the hydroxylation of 5-methylcytosine residues in DNA to 5-hydroxymethylcytosine (Fig. 6B–D; ref. 122). The common features of these enzymes are that they are all iron-dependent dioxygenases that catalyze the hydroxylation of their substrates in an enzymatic reaction that consumes molecular oxygen with the concomitant oxidative decarboxy-lation of α-ketoglutarate, producing succinate and CO2. Both succinate and fumarate can exhibit product-level inhibition of the enzymatic reactions of PHDs (123), KDMs (124, 125), and the TET enzymes (125). Among the results of inhibition of these dioxygenases are stabilization of HIF1α, histone, and CpG island hypermethylation, each of which has been shown in SDH- and FH-deficient tumors (Fig. 6B–D; refs. 122, 123, 126). Increased HIF levels are associated with increased VEGF and GLUT1 expression, potentially supplying increased vas-cularity and glucose transport to supply the needs of a rapidly growing cancer.

The oncometabolite 2-hydroxyglutarate (2HG) was recently reported to be elevated in some ccRCC tumors as compared with paired normal renal cortex samples (127). Although accumulation of the D-enantiomer of 2HG is associated with neomorphic gain-of-function mutations in isocitrate dehydrogenases IDH1 and IDH2 in IDH-mutant cancers, the more moderate accumulations of 2HG in the ccRCC tumors were not associated with IDH mutations and involved accumulation of the L-enantiomer of 2HG. The accumulation of L2HG in ccRCC was shown to be associ-ated with decreased expression of the L-2-hydroxyglutarate dehydrogenase (L2HGDH) enzyme and decreased levels of 5-hydroxymethylcytosine in DNA, potentially linking this metabolite to epigenetic changes in ccRCC (127).

In addition to the inhibition of dioxygenases, elevated fumarate plays a role in the biology of FH-deficient tumors due to its propensity for covalent reactions with intracellular substrates, including the sulfhydryl groups present in pro-teins and small molecules (122). Fumarate can react with the sulfur atom of reduced glutathione to produce succinated glutathione (Fig. 6E), which inhibits glutathione function and results in increased oxidative stress in FH−/− cells (128, 129). Fumarate also reacts with cysteine thiols in proteins to produce a covalent modification known as cysteine S-succina-tion (Fig. 6F; ref. 130). Elevated S-succination of proteins in HLRCC tumors can be detected by specific antibodies (131), and is a useful biomarker for the IHC identification of FH-deficient tumors (132). Elevated fumarate has been found to activate the antioxidant response element pathway via the






nuclear factor erythroid 2-related factor 2 (NRF2) transcrip-tion factor, an effect mediated by aberrant S-succination of cysteine residues in the Kelch-like ECH-associated protein 1 (KEAP1) and resulting in its inactivation (e.g., Fig. 6F; refs. 133, 134). Activation of NRF2 transcriptional targets in HLRCC tumor cells likely confers a cellular survival advan-tage in a setting of elevated oxidative stress by activating glutathione metabolism genes as well as other detoxifica-tion enzymes, including HMOX and NQO1 (133, 134). In a recent drug screen, the kinase inhibitor vandetanib emerged as a potent and specific inhibitor of FH-deficient tumor cell growth both in vitro and in vivo (135). The cytotoxic effects of vandetanib on HLRCC cells were associated with inhibition of ABL1 phosphorylation, which resulted in the repression of nuclear translocation of NRF2 and downregulation of anti-oxidant response genes in FH-deficient UOK262 tumor cells. It was also found that ABL1 stimulated mTOR phospho-rylation and could promote aerobic glycolysis in FH−/− cells through increased translation of HIF1α (135). Because of the synergistic effects of the repression of antioxidant defense and aerobic glycolysis, targeting ABL1 with vandetanib pro-vides a potentially promising approach for therapy, and was found to be even more effective both in vitro and in vivo when combined with the AMPK activator metformin (135).

Several major questions about the biology and pathogen-esis in the TCA cycle gene-mutant renal cell cancers remain unanswered. What confers the highly specialized tissue speci-ficity in these disorders, and what are the specific cells of origin of FH- and SDH-deficient neoplasms? What are the initial mechanisms that lead to the aberrant proliferation of these cells upon loss of FH and SDH activity, and what, if any, additional alterations are required for progression to tumor formation, evasion of cellular senescence, and metastasis? Finally, although loss of FH and SDH activity in tumor cells has been shown to impair the flow of metabolites through the TCA cycle (26, 118), the precise mechanisms underlying the profound decreases in cellular respiration and electron transport chain function remain to be determined.

CONCLUSIONSIn summary, kidney cancer is fundamentally a metabolic

disease. Each of the genes known to cause kidney cancer affects the cell’s ability to respond to changes in oxygen, iron, nutrients, or, most notably in the TCA cycle gene mutation renal cancers, energy. The elucidation of these fundamental pathways will hopefully provide the foundation for the devel-opment of effective forms of management and therapy for patients affected with localized, locally advanced, as well as advanced RCC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThis research was supported by the Intramural Research Pro-

gram of the NIH, NCI, Center for Cancer Research. This project has been funded in part with federal funds from the Frederick National Laboratory for Cancer Research, NIH, under contract HHSN261200800001E.

Received November 19, 2018; revised February 19, 2019; accepted March 22, 2019; published first May 14, 2019.

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2019;9:1006-1021. Published OnlineFirst May 14, 2019.Cancer Discov W. Marston Linehan, Laura S. Schmidt, Daniel R. Crooks, et al. The Metabolic Basis of Kidney Cancer

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