REVIEW URRENT C OPINION
Molecular pathways in renal cell carcinoma: recent advances in genetics and molecular biology Daniel Su a, Eric A. Singer b, and Ramaprasad Srinivasan a
Purpose of review Advanced renal cell carcinoma (RCC) remains a largely incurable disease with a grave prognosis despite the availability of a multiplicity of systemic therapies targeted against vascular endothelial growth factor, its receptors, and the mammalian target of rapamycin. Although immune ‘checkpoint inhibitors’ appear to have activity in clear cell RCC based on recent early phase trials, the true magnitude of the benefit conferred by these agents remains to be fully understood. Given the limitations of existing treatment paradigms, ongoing research into new targetable pathways is critical. This review will highlight some of the more promising avenues of investigation into the molecular biology of RCC. Recent findings The hypoxia-inducible factor and mammalian target of rapamycin pathways remain critical targets in clear cell RCC. In addition, genes involved in chromatin remodeling such as polybromo 1 (PBRM1), SET domain containing 2 (SETD2), and BRCA-1-associated protein-1 (BAP1) have been shown to influence tumor biology and predict survival. MET alterations and the Krebs cycle enzyme fumarate hydratase are associated with familial type 1 and type 2 papillary RCC (PRCC), respectively. Alterations in nuclear factor (erythroid-derived 2)-like 2, Kelch-like erythroid-derived cap-n-collar homology-associated protein 1, and cullin 3, components of an oxidative stress response pathway, have been recently recognized in some sporadic papillary tumors as well as in fumarate hydratase-deficient tumor and may serve as additional therapeutic targets. In addition, whole-genome sequencing and integrated genomic analysis strategies are beginning to uncover unique molecular signatures associated with distinct subtypes of RCC, laying the foundation for a molecular classification of RCC and more precise, mechanism-based therapeutic intervention. Summary The complex molecular changes underlying individual RCC variants are yet to be fully elucidated and remain the subject of ongoing investigation. The findings summarized here further exemplify the diversity of RCC and the need to tailor our therapeutic approaches to the unique genetic alterations specific to individual subtypes of RCC. Keywords molecular pathways, renal cell carcinoma, targeted therapy
INTRODUCTION Renal cell carcinoma (RCC) remains among the 10 most common cancers in the United States, with 61 560 new cases and 14 080 deaths estimated in 2015 [1]. The current therapeutic armamentarium against metastatic RCC includes seven agents that target the vascular endothelial growth factor (VEGF) pathway or mammalian target of rapamycin (mTOR) [2]. Despite this seeming plethora of treatment options, patients with unresectable disease are rarely cured. Therefore, continued research into the critical molecular mechanisms driving RCC is clearly needed.
CLEAR CELL RENAL CELL CARCINOMA Clear cell renal cell carcinoma (ccRCC) represents approximately 70–80% of all renal parenchymal
tumors [3]. From a molecular and genetic perspective, ccRCC is also the best-studied subtype of kidney cancer; indeed, most of the agents approved by the
a
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland and b Section of Urologic Oncology, Rutgers Cancer Institute of New Jersey and Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA Correspondence to Ramaprasad Srinivasan, MD, PhD, Head, Molecular Cancer Section, Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, CRC2-5950, Bethesda, MD 20892, USA. Tel: +1 301 496 6353; fax: +1 301 402 0922; e-mail: [email protected] Curr Opin Oncol 2015, 27:217–223 DOI:10.1097/CCO.0000000000000186
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KEY POINTS VEGF, VEGF receptor, and mTOR pathways remain the primary targets in the treatment of metastatic ccRCC. Immune checkpoint inhibitors have shown early promise and are being further evaluated, particularly in ccRCC. Studying the genetics and molecular biology of both ccRCC and non-ccRCC has identified new potential therapeutic targets.
Food and Drug Administration for the treatment of RCC owe their development to our understanding of the biochemical mechanisms underlying this subtype of kidney cancer. Although the emphasis of early genetic and biochemical studies in this disease was on understanding the consequences of von Hippel–Lindau (VHL) inactivation, several other genetic alterations, notably in genes regulating chromatin function, have recently been identified in clear cell renal tumors.
Von Hippel–Lindau and hypoxia-inducible factor Over the past 2 decades, significant advances have been made in elucidating the critical role played by the VHL–hypoxia-inducible factor (HIF) pathway in ccRCC. Germline mutations or deletion of the VHL gene on the short arm of chromosome 3 at 3p21.4 [4] are responsible for VHL syndrome. In addition, inactivating somatic VHL gene mutations or promoter hypermethylation is found in almost 90% of the tumors in patients with nonfamilial clear cell kidney cancer [5]. The majority of the therapeutic agents currently available for treating patients with advanced kidney cancer target downstream alterations resulting from VHL inactivation [6].
Chromatin-remodeling genes Chromatins are inherently dynamic structures composed of DNA complexed with proteins such as histones. Chromatin remodeling is an important epigenetic mechanism that allows selective access of regulatory factors, such as transcription factors, to the chromatin/DNA. This is usually carried out by covalent histone modifications (methylation, demethylation, acetylation, and ubiquitination) or ATP-dependent remodeling complexes [7]. Largescale exome sequencing of ccRCC tumors has identified a high frequency of mutations in several chromatin-remodeling genes [8–10]. Polybromo 1 (PBRM1), located on 3p21, was found to be mutated in up to 41% of ccRCC tumors, 218
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making it the second most commonly mutated gene in ccRCC after VHL [10]. A comprehensive genomic analysis of 446 ccRCC samples conducted by The Cancer Genome Atlas (TCGA) project confirmed the presence of frequent somatic PBRM1 mutations (33%) [11 ]. PBRM1 encodes BAF180, which is a component of a histone nucleosome-remodeling complex. BAF180 belongs to the switching defective/sucrose nonfermenting family of remodelers and is thought to be involved in the targeting of polybromo BRG1-asscoiated factor to a specific pattern of acetylated lysines [12]. The majority of PBRM1 mutations are truncating, resulting in the loss of the protein [10]. Although the exact role played by PBRM1 inactivation in renal oncogenesis is unknown, several studies have implicated it in cell motility and proliferation [13]. The SETD2 gene is found to be somatically mutated in approximately 11.5% of ccRCC [11 ]; like VHL and PBRM1, SETD2 is believed to function as a tumor-suppressor gene, with biallelic inactivation seen in RCC tumors. The protein product of SETD2 is a histone H3 lysine 36 trimethylating enzyme [14], which is linked to DNA mismatch repair and microsatellite instability [15]. BAP1 alterations were found in 8–10% of ccRCC in large studies, including one undertaken by the TCGA [9,11 ]. The BAP1 protein product is a deubiquitinase belonging to the ubiquitin c-terminal hydrolase family and localizes to the nucleus where its exact site of function is cell dependent [16]. Interestingly, the genetic signature of BAP1 mutation is very specific, being characterized by enrichment of the phosphoinositide kinase-3 (PI3K) pathway [13] and mTORC1 activation [17]. Germline mutations of BAP1 were recently identified as being associated with uveal and cutaneous melanoma, mesothelioma, and RCC [18], leading to speculation that loss of BAP1 may be an independent initiating event in a subset of RCC. Mutations in BAP1 and PBRM1 are associated with different histologic features such as Fuhrman grade and sarcomatoid features, biology, and patient outcomes. In one study, survival of 1479 postnephrectomy patients with localized ccRCC was studied in relationship to the BAP1 status of their tumors [19 ]. The authors found that patients with BAP1-negative tumors had an increased risk of ccRCC-related death [hazard ratio (HR) 3.06, 95% confidence interval (CI): 2.28–4.1, P ¼ 6.77 1014]. BAP1 expression was an independent marker of prognosis after adjusting for other, previously described prognostic factors. Another group studied the clinical significance of chromatin-remodeling gene mutations in ccRCC, in which associations between mutation and &&
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clinical and pathologic outcomes were evaluated [20 ]. When comparing BAP1-mutated tumors with PBRM1-mutated tumors, the study found that BAP1 mutation was associated with metastatic disease at presentation (P ¼ 0.059), advanced clinical stage (P ¼ 0.041), and a trend toward shorter recurrencefree survival (P ¼ 0.059). In a third study, Kapur et al. [21] evaluated immunochemistry for BAP1 as a marker to predict poor cancer outcome in 559 patients with nonmetastatic ccRCC who were treated with nephrectomy. They found BAP1 loss was associated with higher Fuhrman grade (P < 0.0001), advanced pT stage (P ¼ 0.0021), sarcomatoid differentiation (P ¼ 0.0001), and necrosis (P < 0.0001). Patients with BAP1-negative tumors had worse disease-free survival (HR 2.9, 95% CI: 1.8–4.7, P < 0.0001) and overall survival (HR 2.0, 95% CI: 1.3–3.1, P ¼ 0.0010) than patients with tumors that stained positive for BAP1. Last, Hakimi et al. [22] studied the effect of PBRM1, SETD2, and BAP1 on cancer-specific survival (CSS). Using two nonoverlapping cohorts of ccRCC patients from Memorial Sloan-Kettering Cancer Center (MKSCC) and TCGA, the authors found that BAP1 mutations were associated with worse CSS in both cohorts (MSKCC, P ¼ 0.002, HR 7.71, 95% CI: 2.08–28.6; TCGA, P ¼ 0.002, HR 2.21, 95% CI: 1.35– 3.63). SETD2 mutations were associated with worse CSS in the TCGA cohort (P ¼ 0.036, HR 1.68, 95% CI: 1.04–2.73), whereas the same association was not detected in the MSKCC cohort, likely because of smaller number of patients. The study found PBRM1 mutations had no impact on CSS [22]. Although mutations in PBRM1 and BAP1 have been described in nonclear cell variants of RCC, they appear to occur much more frequently in ccRCC. Ho et al. [23 ] evaluated a total of 299 RCC tumors (187 ccRCC, 61 papillary, 17 chromophobe, and 34 oncocytoma) for alteration in these chromatin-remodeling genes. Although loss of PBRM1 or BAP1 was seen in 43% and 10% of ccRCC, respectively, PBRM1 mutations occurred only in 3, 6, and 0% of papillary RCC (pRCC), chromophobe RCC, and oncocytoma, respectively (P < 0.0001). BAP1 loss was not observed in any of the non-ccRCC tumors [23 ]. Although the association between alterations in several chromatin-remodeling genes and ccRCC is undeniable, the exact role played by inactivation of these genes in the causation and/or progression of these tumors remains poorly understood and is an area of active investigation. It is hoped that delineating the exact relationship between altered chromatin remodeling and kidney cancer will help identify the relevance of this process as a clinically meaningful therapeutic target in RCC. &
&
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Alterations in the phosphoinositide kinase-3/ Akt/mammalian target of rapamycin pathway and altered metabolic signatures in clear cell renal cell carcinoma In addition to mutations in VHL and chromatinremodeling genes, recurrent alterations leading to activation of the PI3K/Akt/mTOR pathway have been identified in ccRCC tumors. Mutations in PTEN (4%) and MTOR (6%) were identified by whole-exome sequencing by the TCGA, whereas integrated pathway analysis revealed a variety of alterations affecting several components of the PI3K/Akt/mTOR pathway in 28% of ccRCC tumors. The TCGA study also attempted to identify molecular features that were associated with patient survival by analyzing microRNA, DNA methylation, and protein expression. Decreased expression of AMP-activated kinase (AMPK) protein and increased acetyl–CoA carboxylase expression were both correlated with poor overall survival. In addition, a molecular signature reminiscent of the Warburg effect and characterized by reduced AMPK, increased fatty acid synthesis, and upregulation of genes involved in the pentose phosphate pathway and fatty acid synthesis was apparent in tumors associated with poor prognosis [11 ,24]. &&
NONCLEAR CELL RENAL CELL CARCINOMA PRCC accounts for approximately 10–15% of all RCCs and occurs in both sporadic and familial forms. Histologically, pRCC is further classified into type I and type II pRCC. Type I tumors typically have small basophilic cells of low nuclear grade and are often characterized by the presence of macrophages, whereas type II tumors have eosinophilic cells of high nuclear grade [25]. It is important to note that type II pRCC is histologically heterogeneous and likely encompasses many distinct variants. In their familial forms, type I pRCC is associated with hereditary pRCC (HPRC), whereas one form of type II pRCC is associated with hereditary leiomyomatosis and renal cell cancer (HLRCC). Clinically, pRCC can be classified into two groups: an organ-confined group with better survival than ccRCC and a metastatic group that carries a worse prognosis than ccRCC [26]. In the metastatic form, pRCC is resistant to most forms of systemic therapy and its clinical course is almost uniformly fatal [27]. In the modern era of smallmolecule targeted therapy, improved outcomes seen in ccRCC have not translated to nonclear cell variants and survival for patients with advanced pRCC remains inferior compared to those with metastatic ccRCC [28].
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Although the key molecular alterations underlying the majority of pRCC are still not as well understood as ccRCC, recent studies suggest a role for at least two well defined pathways in certain subtypes.
Hepatocyte growth factor/MET pathway The MET gene is located on chromosome 7 and the primary MET product is a transmembrane receptor tyrosine kinase known as Met [29]. Hepatocyte growth factor (HGF) (the gene which is also located on chromosome 7) is primarily secreted by mesenchymal cells [30], is the only known ligand for Met, and acts in a paracrine manner. MET and HGF expression are critical during embryo development [31]. Expression of both proteins continues into adulthood and HGF is upregulated during injury, suggesting activation of this pathway may be important for tissue repair and regeneration [32]. The Met receptor is a heterodimer with an entire extraceullar a-subunit, a large b-subunit with an extracellular region, a transmembrane component, and an intracellular tyrosine kinase domain. Functionally, Met contains five domains, each of which contributes to the complex and multilevel regulation of Met. When activated by HGF, Met is phosphorylated at multiple tyrosine residues. Specifically, Met undergoes autophosphorylation of Y1230, Y1234, and Y1235 located in the activation loop of the tyrosine kinase domain. Y1313 is important in binding of PI3K whereas Y1349 and Y1356 are important in the activation of the multisubstrate-docking site, which interacts with Src homology-2, phosphotyrosine domains, and Metbinding domains [33]. Functionally, Met signaling involves Grb2, Gab1, PI3K, phospholipase C-g, Shc, Src, Shp2, Ship1, and signal transducer and activator of transcription 3 (STAT3) [34]. Binding of Grb2 through the multisubstrate-docking site leads into the RAS/ mitogen-activated protein kinases (MAPK) pathway, which mediates HGF-induced cell scattering and proliferation signals [35]. The PI3K pathway can be activated either downstream via RAS or directly recruited to the multisubstrate-docking site via phosphorylation of Gab1. Activation of the PI3K pathway is associated with cell motility and remodeling of the extracellular matrix. PI3K also triggers activation of the AKT pathway, which is related to cell survival [36]. Last, the STAT3 pathway is implicated in HGF-induced branching morphogenesis [37]. Taken together, the HGF/MET pathway is responsible for various cellular functions that may contribute to cancer survival, invasion, and metastasis when dysregulated. 220
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The HGF/MET pathway can be deranged through various means, including persistent ligand stimulation, aberrant paracrine/autocrine ligand production, MET gene amplification, and MET gene mutations [38,39]. The strongest evidence for activation of the HGF/MET pathway in pRCC comes from patients with HPRC, a familial syndrome wherein affected individuals have a propensity for development of bilateral, multifocal type 1 papillary renal tumors. The hallmark of HPRC is the presence of germline, activating mutations in the tyrosine kinase domain of MET [29]. In addition, renal tumors in patients with HPRC often demonstrate nonrandom duplication of the chromosomal arm on which the mutated MET allele is located. Somatic MET mutations have been described in sporadic forms of pRCC, although the incidence is relatively low (approximately 15%) [40]. Gain of chromosome 7 is a common event in papillary renal tumors [24]. As both MET and HGF are located on chromosome 7, it has been suggested that increased chromosome 7 copy number might lead to activation of the HGF/MET pathway. However, at this time, there is no direct evidence to support the notion that polysomy 7 is associated with MET pathway activation or that MET is an oncogenic driver in tumors bearing this chromosomal alteration. It is hoped that a comprehensive genomic analysis of papillary renal tumors being currently pursued by TCGA will help clarify the role of the MET pathway in these tumors. Although it is unclear to what extent the various alterations involving MET contribute to oncogenesis, the HGF/MET pathway has attracted significant attention as a possible target in patients with advanced pRCC. A recent phase II clinical trial with foretinib, an oral multikinase inhibitor whose targets include Met, demonstrated an overall response rate of 13.5% in patients with pRCC. In this study, germline MET mutation was highly predictive of response to therapy (5/10 with vs. 5/57 without). To explore the possibility that more selective Met inhibition might lead to effective inhibition of the pathway with a better adverse event profile, INC280, a selective Met kinase inhibitor, is being evaluated in a phase II clinical trial in patients with advanced pRCC (NCT02019693).
Fumarate hydratase A second form of hereditary pRCC is associated with alterations in fumarate hydratase (FH), a gene that encodes for the tricarboxylic acid (TCA) cycle enzyme that catalyzes the conversion of fumarate to malate. Germline FH mutation is seen in patients with HLRCC, which is associated with a highly Volume 27 Number 3 May 2015
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aggressive variant of type II pRCC [41]. Loss of FH activity causes a shift in the metabolism of these tumors, characterized by disruption of the TCA cycle and oxidative phosphorylation and a resultant reliance on aerobic glycolysis to meet cellular energy requirements (Warburg effect). In addition, loss of functional FH causes intracellular accumulation of fumarate, with several consequences critical for tumor survival. Fumarate competitively inhibits the 2-oxoglutarate-dependent dioxygenases that catalyze hydroxylation of prolyl residues on HIF, leading to VHL-independent stabilization of HIF-a subunits [42]. Stabilization of HIF-1a leads to increased transcription of both growth and proangiogenic factors [25], as well as genes promoting a glycolytic phenotype [43]. In addition, fumarate also appears to be a key regulator of an oxidative stress response pathway mediated by nuclear factor (erythroid-derived 2)-like 2 (NRF2) in FH/ renal tumors.
Nuclear factor (erythroid-derived 2)-like 2, Kelch-like erythroid-derived cap-n-collar homology-associated protein 1, and cullin 3 Recently, an additional role for intracellular fumarate in activating a cellular oxidative stress response pathway was elucidated. NRF2 is a transcription factor that regulates the expression of a variety of genes whose protein products play an essential role in mitigating cellular damage under conditions of oxidative stress. Intracellular levels and localization of NRF2 are regulated by an E3 ligase complex composed of Kelch-like erythroid-derived cap-n-collar homology-associated protein 1 (KEAP1), cullin 3 (CUL3) and Rbx1; subunits of this E3 ligase complex bind to and target NRF2 for ubiquitin-mediated degradation [44]. In cells with loss of FH function, accumulation of fumarate leads to a posttranslational modification of cysteine residues (succination) in several proteins, including KEAP1 [45]. Modification of KEAP1 leads to impaired NRF2 binding, prevents degradation of NRF2, and leads to upregulation of this molecule [42]. Although NRF2 activation is also seen in sporadic forms of type II pRCC, somatic mutations of FH do not appear to be a common event in these tumors [46]. Instead, a recent study suggests that somatic mutations in NRF2, CUL3, and the sirtuin family of proteins may be responsible for the NRF activation phenotype in a subset of sporadic pRCC [47 ]. Derangements of the NRF2–KEAP1–CUL3 pathway are seen in various malignancies and can occur in multiple ways: somatic mutation in KEAP1 or the KEAP1-binding site for NRF2; epigenetic silencing of KEAP1; accumulation of disruptor proteins such as &
p62; transcriptional induction of NRF2 by oncogenes such as K-Ras, B-Raf, and c-Myc; and succination of KEAP1 in HLRCC tumors as described previously [48–50]. Furthermore, there is evidence that mutations of genes in this pathway confer chemoresistance and radioresistance to certain tumors and are associated with worse outcomes [51–53]. Therefore, this pathway offers a rich variety of targets and mechanisms for possible therapeutic intervention. Several investigators have studied FH-deficient cancer cell lines via synthetic lethality to help identify targets that are indispensable to tumors but not to cells with wild-type FH. Frezza et al. [54] found that heme oxygenase 1 (HMOX1), a member of the heat shock protein family, is synthetically lethal to FH-deficient tumor cells. The authors investigated the escape mechanism through which FH cells are able to survive without an intact TCA cycle and identified a metabolic pathway wherein NADH is generated by glutamine consumption, and excess carbon is disposed of by heme generation via HMOX1 [55]. The dependency of FH-deficient cells on HMOX1 was then confirmed in a mouse model, where HMOX1 was silenced by a short hairpin RNA, leading to reduction in growth of FH-deficient cells and minimal to no effect on control wild-type cells. More recently, investigators at the National Cancer Institute showed that the Abelson murine leukemia viral oncogene homolog 1 (ABL1) plays an important role in both sustaining aerobic glycolysis and mediating oxidative stress response pathways characteristic of FH/ tumors [56 ]. In this study, Sourbier et al. [56 ] used a drug screen to identify the potent activity of vandetanib, a multitargeted tyrosine kinase inhibitor, against FH-deficient tumors both in vitro and in mouse xenograft models. Vandetanib treatment resulted in inhibition of abl activity, and cytotoxicity mediated by this agent was recapitulated by silencing abl, suggesting that the activity of vandetanib in FH-deficient cells is at least partly mediated by inhibition of abl. Furthermore, concurrent pharmacologic activation of AMPK by metformin was synergistic with vandetanib treatment, leading to potent antitumor effects in mouse xenograft models. These data provide novel targets for clinical evaluation in some forms of pRCC. &&
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CONCLUSION Targeted therapies directed against VEGF, VEGF receptor, and mTOR continue to play a crucial role in the management of metastatic ccRCC. With better understanding of the molecular diversity underlying the many distinct subtypes of non-ccRCC, it is hoped
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that more effective, mechanism-based therapeutic strategies can be developed against these entities. When studying the less-common non-ccRCC histologies, multi-institutional trials that employ innovative trial designs are most likely to find success. Acknowledgements None. Financial support and sponsorship This research was funded by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research, Bethesda, Maryland, USA, and by a grant from the National Cancer Institute (P30CA072720). Conflicts of interest There are no conflicts of interest.
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C-Met overexpression in node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu. Int J Cancer 2005; 113:678–682. 40. Schmidt L, Junker K, Nakaigawa N, et al. Novel mutations of the MET protooncogene in papillary renal carcinomas. Oncogene 1999; 18:2343–2350. 41. Linehan WM, Srinivasan R, Schmidt LS. The genetic basis of kidney cancer: a metabolic disease. Nat Rev Urol 2010; 7:277–285. 42. Adam J, Hatipoglu E, O’Flaherty L, et al. Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling. Cancer Cell 2011; 20:524–537. 43. Kim JW, Tchernyshyov I, Semenza GL, et al. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 2006; 3:177–185. 44. Kobayashi A, Kang M-I, Okawa H, et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol 2004; 24:7130–7139. 45. Bardella C, El-Bahrawy M, Frizzell N, et al. Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status. J Pathol 2011; 225:4–11. 46. Kinch L, Grishin NV, Brugarolas J. Succination of Keap1 and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2. Cancer Cell 2011; 20:418–420. 47. Ooi A, Dykema K, Ansari A, et al. CUL3 and NRF2 mutations confer an NRF2 & activation phenotype in a sporadic form of papillary renal cell carcinoma. Cancer Res 2013; 73:2044–2051. CUL2 and NRF2 pathway in sporadic papillary kidney cancer.
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52. Sporn MB, Liby KT. NRF2 and cancer: the good, the bad and the importance of context. Nat Rev Cancer 2012; 12:564–571. 53. Soini Y, Eskelinen M, Juvonen P, et al. Nuclear Nrf2 expression is related to a poor survival in pancreatic adenocarcinoma. Pathol Res Pract 2014; 210:35–39. 54. Frezza C, Zheng L, Folger O, et al. Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature 2011; 477:225–228. 55. Na H-K, Surh Y-J. Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1. Free Radical Biol Med 2014; 67:353–365. 56. Sourbier C, Ricketts Christopher J, Matsumoto S, et al. Targeting ABL1&& mediated oxidative stress adaptation in fumarate hydratase-deficient cancer. Cancer Cell 2014; 26:840–850. ABL1 and its role in HLRCC-associated kidney cancer.
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Molecular pathways in renal cell carcinoma: recent advances in genetics and molecular biology Daniel Su a, Eric A. Singer b, and Ramaprasad Srinivasan a
Purpose of review Advanced renal cell carcinoma (RCC) remains a largely incurable disease with a grave prognosis despite the availability of a multiplicity of systemic therapies targeted against vascular endothelial growth factor, its receptors, and the mammalian target of rapamycin. Although immune ‘checkpoint inhibitors’ appear to have activity in clear cell RCC based on recent early phase trials, the true magnitude of the benefit conferred by these agents remains to be fully understood. Given the limitations of existing treatment paradigms, ongoing research into new targetable pathways is critical. This review will highlight some of the more promising avenues of investigation into the molecular biology of RCC. Recent findings The hypoxia-inducible factor and mammalian target of rapamycin pathways remain critical targets in clear cell RCC. In addition, genes involved in chromatin remodeling such as polybromo 1 (PBRM1), SET domain containing 2 (SETD2), and BRCA-1-associated protein-1 (BAP1) have been shown to influence tumor biology and predict survival. MET alterations and the Krebs cycle enzyme fumarate hydratase are associated with familial type 1 and type 2 papillary RCC (PRCC), respectively. Alterations in nuclear factor (erythroid-derived 2)-like 2, Kelch-like erythroid-derived cap-n-collar homology-associated protein 1, and cullin 3, components of an oxidative stress response pathway, have been recently recognized in some sporadic papillary tumors as well as in fumarate hydratase-deficient tumor and may serve as additional therapeutic targets. In addition, whole-genome sequencing and integrated genomic analysis strategies are beginning to uncover unique molecular signatures associated with distinct subtypes of RCC, laying the foundation for a molecular classification of RCC and more precise, mechanism-based therapeutic intervention. Summary The complex molecular changes underlying individual RCC variants are yet to be fully elucidated and remain the subject of ongoing investigation. The findings summarized here further exemplify the diversity of RCC and the need to tailor our therapeutic approaches to the unique genetic alterations specific to individual subtypes of RCC. Keywords molecular pathways, renal cell carcinoma, targeted therapy
INTRODUCTION Renal cell carcinoma (RCC) remains among the 10 most common cancers in the United States, with 61 560 new cases and 14 080 deaths estimated in 2015 [1]. The current therapeutic armamentarium against metastatic RCC includes seven agents that target the vascular endothelial growth factor (VEGF) pathway or mammalian target of rapamycin (mTOR) [2]. Despite this seeming plethora of treatment options, patients with unresectable disease are rarely cured. Therefore, continued research into the critical molecular mechanisms driving RCC is clearly needed.
CLEAR CELL RENAL CELL CARCINOMA Clear cell renal cell carcinoma (ccRCC) represents approximately 70–80% of all renal parenchymal
tumors [3]. From a molecular and genetic perspective, ccRCC is also the best-studied subtype of kidney cancer; indeed, most of the agents approved by the
a
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland and b Section of Urologic Oncology, Rutgers Cancer Institute of New Jersey and Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA Correspondence to Ramaprasad Srinivasan, MD, PhD, Head, Molecular Cancer Section, Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, CRC2-5950, Bethesda, MD 20892, USA. Tel: +1 301 496 6353; fax: +1 301 402 0922; e-mail: [email protected] Curr Opin Oncol 2015, 27:217–223 DOI:10.1097/CCO.0000000000000186
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KEY POINTS VEGF, VEGF receptor, and mTOR pathways remain the primary targets in the treatment of metastatic ccRCC. Immune checkpoint inhibitors have shown early promise and are being further evaluated, particularly in ccRCC. Studying the genetics and molecular biology of both ccRCC and non-ccRCC has identified new potential therapeutic targets.
Food and Drug Administration for the treatment of RCC owe their development to our understanding of the biochemical mechanisms underlying this subtype of kidney cancer. Although the emphasis of early genetic and biochemical studies in this disease was on understanding the consequences of von Hippel–Lindau (VHL) inactivation, several other genetic alterations, notably in genes regulating chromatin function, have recently been identified in clear cell renal tumors.
Von Hippel–Lindau and hypoxia-inducible factor Over the past 2 decades, significant advances have been made in elucidating the critical role played by the VHL–hypoxia-inducible factor (HIF) pathway in ccRCC. Germline mutations or deletion of the VHL gene on the short arm of chromosome 3 at 3p21.4 [4] are responsible for VHL syndrome. In addition, inactivating somatic VHL gene mutations or promoter hypermethylation is found in almost 90% of the tumors in patients with nonfamilial clear cell kidney cancer [5]. The majority of the therapeutic agents currently available for treating patients with advanced kidney cancer target downstream alterations resulting from VHL inactivation [6].
Chromatin-remodeling genes Chromatins are inherently dynamic structures composed of DNA complexed with proteins such as histones. Chromatin remodeling is an important epigenetic mechanism that allows selective access of regulatory factors, such as transcription factors, to the chromatin/DNA. This is usually carried out by covalent histone modifications (methylation, demethylation, acetylation, and ubiquitination) or ATP-dependent remodeling complexes [7]. Largescale exome sequencing of ccRCC tumors has identified a high frequency of mutations in several chromatin-remodeling genes [8–10]. Polybromo 1 (PBRM1), located on 3p21, was found to be mutated in up to 41% of ccRCC tumors, 218
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making it the second most commonly mutated gene in ccRCC after VHL [10]. A comprehensive genomic analysis of 446 ccRCC samples conducted by The Cancer Genome Atlas (TCGA) project confirmed the presence of frequent somatic PBRM1 mutations (33%) [11 ]. PBRM1 encodes BAF180, which is a component of a histone nucleosome-remodeling complex. BAF180 belongs to the switching defective/sucrose nonfermenting family of remodelers and is thought to be involved in the targeting of polybromo BRG1-asscoiated factor to a specific pattern of acetylated lysines [12]. The majority of PBRM1 mutations are truncating, resulting in the loss of the protein [10]. Although the exact role played by PBRM1 inactivation in renal oncogenesis is unknown, several studies have implicated it in cell motility and proliferation [13]. The SETD2 gene is found to be somatically mutated in approximately 11.5% of ccRCC [11 ]; like VHL and PBRM1, SETD2 is believed to function as a tumor-suppressor gene, with biallelic inactivation seen in RCC tumors. The protein product of SETD2 is a histone H3 lysine 36 trimethylating enzyme [14], which is linked to DNA mismatch repair and microsatellite instability [15]. BAP1 alterations were found in 8–10% of ccRCC in large studies, including one undertaken by the TCGA [9,11 ]. The BAP1 protein product is a deubiquitinase belonging to the ubiquitin c-terminal hydrolase family and localizes to the nucleus where its exact site of function is cell dependent [16]. Interestingly, the genetic signature of BAP1 mutation is very specific, being characterized by enrichment of the phosphoinositide kinase-3 (PI3K) pathway [13] and mTORC1 activation [17]. Germline mutations of BAP1 were recently identified as being associated with uveal and cutaneous melanoma, mesothelioma, and RCC [18], leading to speculation that loss of BAP1 may be an independent initiating event in a subset of RCC. Mutations in BAP1 and PBRM1 are associated with different histologic features such as Fuhrman grade and sarcomatoid features, biology, and patient outcomes. In one study, survival of 1479 postnephrectomy patients with localized ccRCC was studied in relationship to the BAP1 status of their tumors [19 ]. The authors found that patients with BAP1-negative tumors had an increased risk of ccRCC-related death [hazard ratio (HR) 3.06, 95% confidence interval (CI): 2.28–4.1, P ¼ 6.77 1014]. BAP1 expression was an independent marker of prognosis after adjusting for other, previously described prognostic factors. Another group studied the clinical significance of chromatin-remodeling gene mutations in ccRCC, in which associations between mutation and &&
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clinical and pathologic outcomes were evaluated [20 ]. When comparing BAP1-mutated tumors with PBRM1-mutated tumors, the study found that BAP1 mutation was associated with metastatic disease at presentation (P ¼ 0.059), advanced clinical stage (P ¼ 0.041), and a trend toward shorter recurrencefree survival (P ¼ 0.059). In a third study, Kapur et al. [21] evaluated immunochemistry for BAP1 as a marker to predict poor cancer outcome in 559 patients with nonmetastatic ccRCC who were treated with nephrectomy. They found BAP1 loss was associated with higher Fuhrman grade (P < 0.0001), advanced pT stage (P ¼ 0.0021), sarcomatoid differentiation (P ¼ 0.0001), and necrosis (P < 0.0001). Patients with BAP1-negative tumors had worse disease-free survival (HR 2.9, 95% CI: 1.8–4.7, P < 0.0001) and overall survival (HR 2.0, 95% CI: 1.3–3.1, P ¼ 0.0010) than patients with tumors that stained positive for BAP1. Last, Hakimi et al. [22] studied the effect of PBRM1, SETD2, and BAP1 on cancer-specific survival (CSS). Using two nonoverlapping cohorts of ccRCC patients from Memorial Sloan-Kettering Cancer Center (MKSCC) and TCGA, the authors found that BAP1 mutations were associated with worse CSS in both cohorts (MSKCC, P ¼ 0.002, HR 7.71, 95% CI: 2.08–28.6; TCGA, P ¼ 0.002, HR 2.21, 95% CI: 1.35– 3.63). SETD2 mutations were associated with worse CSS in the TCGA cohort (P ¼ 0.036, HR 1.68, 95% CI: 1.04–2.73), whereas the same association was not detected in the MSKCC cohort, likely because of smaller number of patients. The study found PBRM1 mutations had no impact on CSS [22]. Although mutations in PBRM1 and BAP1 have been described in nonclear cell variants of RCC, they appear to occur much more frequently in ccRCC. Ho et al. [23 ] evaluated a total of 299 RCC tumors (187 ccRCC, 61 papillary, 17 chromophobe, and 34 oncocytoma) for alteration in these chromatin-remodeling genes. Although loss of PBRM1 or BAP1 was seen in 43% and 10% of ccRCC, respectively, PBRM1 mutations occurred only in 3, 6, and 0% of papillary RCC (pRCC), chromophobe RCC, and oncocytoma, respectively (P < 0.0001). BAP1 loss was not observed in any of the non-ccRCC tumors [23 ]. Although the association between alterations in several chromatin-remodeling genes and ccRCC is undeniable, the exact role played by inactivation of these genes in the causation and/or progression of these tumors remains poorly understood and is an area of active investigation. It is hoped that delineating the exact relationship between altered chromatin remodeling and kidney cancer will help identify the relevance of this process as a clinically meaningful therapeutic target in RCC. &
&
&
Alterations in the phosphoinositide kinase-3/ Akt/mammalian target of rapamycin pathway and altered metabolic signatures in clear cell renal cell carcinoma In addition to mutations in VHL and chromatinremodeling genes, recurrent alterations leading to activation of the PI3K/Akt/mTOR pathway have been identified in ccRCC tumors. Mutations in PTEN (4%) and MTOR (6%) were identified by whole-exome sequencing by the TCGA, whereas integrated pathway analysis revealed a variety of alterations affecting several components of the PI3K/Akt/mTOR pathway in 28% of ccRCC tumors. The TCGA study also attempted to identify molecular features that were associated with patient survival by analyzing microRNA, DNA methylation, and protein expression. Decreased expression of AMP-activated kinase (AMPK) protein and increased acetyl–CoA carboxylase expression were both correlated with poor overall survival. In addition, a molecular signature reminiscent of the Warburg effect and characterized by reduced AMPK, increased fatty acid synthesis, and upregulation of genes involved in the pentose phosphate pathway and fatty acid synthesis was apparent in tumors associated with poor prognosis [11 ,24]. &&
NONCLEAR CELL RENAL CELL CARCINOMA PRCC accounts for approximately 10–15% of all RCCs and occurs in both sporadic and familial forms. Histologically, pRCC is further classified into type I and type II pRCC. Type I tumors typically have small basophilic cells of low nuclear grade and are often characterized by the presence of macrophages, whereas type II tumors have eosinophilic cells of high nuclear grade [25]. It is important to note that type II pRCC is histologically heterogeneous and likely encompasses many distinct variants. In their familial forms, type I pRCC is associated with hereditary pRCC (HPRC), whereas one form of type II pRCC is associated with hereditary leiomyomatosis and renal cell cancer (HLRCC). Clinically, pRCC can be classified into two groups: an organ-confined group with better survival than ccRCC and a metastatic group that carries a worse prognosis than ccRCC [26]. In the metastatic form, pRCC is resistant to most forms of systemic therapy and its clinical course is almost uniformly fatal [27]. In the modern era of smallmolecule targeted therapy, improved outcomes seen in ccRCC have not translated to nonclear cell variants and survival for patients with advanced pRCC remains inferior compared to those with metastatic ccRCC [28].
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Although the key molecular alterations underlying the majority of pRCC are still not as well understood as ccRCC, recent studies suggest a role for at least two well defined pathways in certain subtypes.
Hepatocyte growth factor/MET pathway The MET gene is located on chromosome 7 and the primary MET product is a transmembrane receptor tyrosine kinase known as Met [29]. Hepatocyte growth factor (HGF) (the gene which is also located on chromosome 7) is primarily secreted by mesenchymal cells [30], is the only known ligand for Met, and acts in a paracrine manner. MET and HGF expression are critical during embryo development [31]. Expression of both proteins continues into adulthood and HGF is upregulated during injury, suggesting activation of this pathway may be important for tissue repair and regeneration [32]. The Met receptor is a heterodimer with an entire extraceullar a-subunit, a large b-subunit with an extracellular region, a transmembrane component, and an intracellular tyrosine kinase domain. Functionally, Met contains five domains, each of which contributes to the complex and multilevel regulation of Met. When activated by HGF, Met is phosphorylated at multiple tyrosine residues. Specifically, Met undergoes autophosphorylation of Y1230, Y1234, and Y1235 located in the activation loop of the tyrosine kinase domain. Y1313 is important in binding of PI3K whereas Y1349 and Y1356 are important in the activation of the multisubstrate-docking site, which interacts with Src homology-2, phosphotyrosine domains, and Metbinding domains [33]. Functionally, Met signaling involves Grb2, Gab1, PI3K, phospholipase C-g, Shc, Src, Shp2, Ship1, and signal transducer and activator of transcription 3 (STAT3) [34]. Binding of Grb2 through the multisubstrate-docking site leads into the RAS/ mitogen-activated protein kinases (MAPK) pathway, which mediates HGF-induced cell scattering and proliferation signals [35]. The PI3K pathway can be activated either downstream via RAS or directly recruited to the multisubstrate-docking site via phosphorylation of Gab1. Activation of the PI3K pathway is associated with cell motility and remodeling of the extracellular matrix. PI3K also triggers activation of the AKT pathway, which is related to cell survival [36]. Last, the STAT3 pathway is implicated in HGF-induced branching morphogenesis [37]. Taken together, the HGF/MET pathway is responsible for various cellular functions that may contribute to cancer survival, invasion, and metastasis when dysregulated. 220
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The HGF/MET pathway can be deranged through various means, including persistent ligand stimulation, aberrant paracrine/autocrine ligand production, MET gene amplification, and MET gene mutations [38,39]. The strongest evidence for activation of the HGF/MET pathway in pRCC comes from patients with HPRC, a familial syndrome wherein affected individuals have a propensity for development of bilateral, multifocal type 1 papillary renal tumors. The hallmark of HPRC is the presence of germline, activating mutations in the tyrosine kinase domain of MET [29]. In addition, renal tumors in patients with HPRC often demonstrate nonrandom duplication of the chromosomal arm on which the mutated MET allele is located. Somatic MET mutations have been described in sporadic forms of pRCC, although the incidence is relatively low (approximately 15%) [40]. Gain of chromosome 7 is a common event in papillary renal tumors [24]. As both MET and HGF are located on chromosome 7, it has been suggested that increased chromosome 7 copy number might lead to activation of the HGF/MET pathway. However, at this time, there is no direct evidence to support the notion that polysomy 7 is associated with MET pathway activation or that MET is an oncogenic driver in tumors bearing this chromosomal alteration. It is hoped that a comprehensive genomic analysis of papillary renal tumors being currently pursued by TCGA will help clarify the role of the MET pathway in these tumors. Although it is unclear to what extent the various alterations involving MET contribute to oncogenesis, the HGF/MET pathway has attracted significant attention as a possible target in patients with advanced pRCC. A recent phase II clinical trial with foretinib, an oral multikinase inhibitor whose targets include Met, demonstrated an overall response rate of 13.5% in patients with pRCC. In this study, germline MET mutation was highly predictive of response to therapy (5/10 with vs. 5/57 without). To explore the possibility that more selective Met inhibition might lead to effective inhibition of the pathway with a better adverse event profile, INC280, a selective Met kinase inhibitor, is being evaluated in a phase II clinical trial in patients with advanced pRCC (NCT02019693).
Fumarate hydratase A second form of hereditary pRCC is associated with alterations in fumarate hydratase (FH), a gene that encodes for the tricarboxylic acid (TCA) cycle enzyme that catalyzes the conversion of fumarate to malate. Germline FH mutation is seen in patients with HLRCC, which is associated with a highly Volume 27 Number 3 May 2015
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aggressive variant of type II pRCC [41]. Loss of FH activity causes a shift in the metabolism of these tumors, characterized by disruption of the TCA cycle and oxidative phosphorylation and a resultant reliance on aerobic glycolysis to meet cellular energy requirements (Warburg effect). In addition, loss of functional FH causes intracellular accumulation of fumarate, with several consequences critical for tumor survival. Fumarate competitively inhibits the 2-oxoglutarate-dependent dioxygenases that catalyze hydroxylation of prolyl residues on HIF, leading to VHL-independent stabilization of HIF-a subunits [42]. Stabilization of HIF-1a leads to increased transcription of both growth and proangiogenic factors [25], as well as genes promoting a glycolytic phenotype [43]. In addition, fumarate also appears to be a key regulator of an oxidative stress response pathway mediated by nuclear factor (erythroid-derived 2)-like 2 (NRF2) in FH/ renal tumors.
Nuclear factor (erythroid-derived 2)-like 2, Kelch-like erythroid-derived cap-n-collar homology-associated protein 1, and cullin 3 Recently, an additional role for intracellular fumarate in activating a cellular oxidative stress response pathway was elucidated. NRF2 is a transcription factor that regulates the expression of a variety of genes whose protein products play an essential role in mitigating cellular damage under conditions of oxidative stress. Intracellular levels and localization of NRF2 are regulated by an E3 ligase complex composed of Kelch-like erythroid-derived cap-n-collar homology-associated protein 1 (KEAP1), cullin 3 (CUL3) and Rbx1; subunits of this E3 ligase complex bind to and target NRF2 for ubiquitin-mediated degradation [44]. In cells with loss of FH function, accumulation of fumarate leads to a posttranslational modification of cysteine residues (succination) in several proteins, including KEAP1 [45]. Modification of KEAP1 leads to impaired NRF2 binding, prevents degradation of NRF2, and leads to upregulation of this molecule [42]. Although NRF2 activation is also seen in sporadic forms of type II pRCC, somatic mutations of FH do not appear to be a common event in these tumors [46]. Instead, a recent study suggests that somatic mutations in NRF2, CUL3, and the sirtuin family of proteins may be responsible for the NRF activation phenotype in a subset of sporadic pRCC [47 ]. Derangements of the NRF2–KEAP1–CUL3 pathway are seen in various malignancies and can occur in multiple ways: somatic mutation in KEAP1 or the KEAP1-binding site for NRF2; epigenetic silencing of KEAP1; accumulation of disruptor proteins such as &
p62; transcriptional induction of NRF2 by oncogenes such as K-Ras, B-Raf, and c-Myc; and succination of KEAP1 in HLRCC tumors as described previously [48–50]. Furthermore, there is evidence that mutations of genes in this pathway confer chemoresistance and radioresistance to certain tumors and are associated with worse outcomes [51–53]. Therefore, this pathway offers a rich variety of targets and mechanisms for possible therapeutic intervention. Several investigators have studied FH-deficient cancer cell lines via synthetic lethality to help identify targets that are indispensable to tumors but not to cells with wild-type FH. Frezza et al. [54] found that heme oxygenase 1 (HMOX1), a member of the heat shock protein family, is synthetically lethal to FH-deficient tumor cells. The authors investigated the escape mechanism through which FH cells are able to survive without an intact TCA cycle and identified a metabolic pathway wherein NADH is generated by glutamine consumption, and excess carbon is disposed of by heme generation via HMOX1 [55]. The dependency of FH-deficient cells on HMOX1 was then confirmed in a mouse model, where HMOX1 was silenced by a short hairpin RNA, leading to reduction in growth of FH-deficient cells and minimal to no effect on control wild-type cells. More recently, investigators at the National Cancer Institute showed that the Abelson murine leukemia viral oncogene homolog 1 (ABL1) plays an important role in both sustaining aerobic glycolysis and mediating oxidative stress response pathways characteristic of FH/ tumors [56 ]. In this study, Sourbier et al. [56 ] used a drug screen to identify the potent activity of vandetanib, a multitargeted tyrosine kinase inhibitor, against FH-deficient tumors both in vitro and in mouse xenograft models. Vandetanib treatment resulted in inhibition of abl activity, and cytotoxicity mediated by this agent was recapitulated by silencing abl, suggesting that the activity of vandetanib in FH-deficient cells is at least partly mediated by inhibition of abl. Furthermore, concurrent pharmacologic activation of AMPK by metformin was synergistic with vandetanib treatment, leading to potent antitumor effects in mouse xenograft models. These data provide novel targets for clinical evaluation in some forms of pRCC. &&
&&
CONCLUSION Targeted therapies directed against VEGF, VEGF receptor, and mTOR continue to play a crucial role in the management of metastatic ccRCC. With better understanding of the molecular diversity underlying the many distinct subtypes of non-ccRCC, it is hoped
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that more effective, mechanism-based therapeutic strategies can be developed against these entities. When studying the less-common non-ccRCC histologies, multi-institutional trials that employ innovative trial designs are most likely to find success. Acknowledgements None. Financial support and sponsorship This research was funded by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research, Bethesda, Maryland, USA, and by a grant from the National Cancer Institute (P30CA072720). Conflicts of interest There are no conflicts of interest.
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Molecular pathways in renal cell carcinoma Su et al. 48. Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1– Nrf2 pathway in stress response and cancer evolution. Genes Cells 2011; 16:123–140. 49. DeNicola GM, Karreth FA, Humpton TJ, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011; 475:106–109. 50. Hanada N, Takahata T, Zhou Q, et al. Methylation of the KEAP1 gene promoter region in human colorectal cancer. BMC Cancer 2012; 12:66. 51. Zhang P, Singh A, Yegnasubramanian S, et al. Loss of Kelch-like ECHassociated protein 1 function in prostate cancer cells causes chemoresistance and radioresistance and promotes tumor growth. Mol Cancer Ther 2010; 9:336–346.
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