Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G0) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.
Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and is often linked to loss of chromosome 3p, which harbors the VHL tumor suppressor gene, loss of chromosome 14q, which includes HIF1A, and gain of chromosome 5q. The relevant target(s) on chromosome 5q is not known. Here we show that 5q amplification leads to overexpression of the SQSTM1 oncogene in ccRCC lines and tumors. Overexpression of SQSTM1 in ccRCC lines promoted resistance to redox stress and increased soft agar growth while downregulation of SQSTM1 decreased resistance to redox stress, impaired cellular fitness, and decreased tumor formation. Therefore the selection pressure to amplify 5q in ccRCC is driven, at least partly, by SQSTM1.
Otto Warburg noted decades ago that cancer cells maintain very high rates of glycolysis, converting glucose to lactate, despite sufficient oxygen to perform oxidative-phosphorylation, which is the more efficient means of generating energy (ATP) from glucose. This conundrum has generated considerable speculation as to whether altered metabolism, including altered glucose metabolism, is a cause or consequence of malignancy and, if the former, what benefits altered metabolism might confer upon cancer cells. Several lines of evidence, including the recent identification of mutations affecting Fumarate Hydratase, Succinate Dehydrogenase, and Isocitrate Dehydrogenase, have strengthened the notion that altered metabolism can cause cancer and a number of non-mutually exclusive models have been put forth to rationalize why cancer cells might benefit from a high rate of glycolysis and decreased oxidative phosphorylation. This chapter will focus on the role of HIF, 2-oxoglutarate, and 2-oxoglutarate-dependent enzymes in cancer and cancer metabolism.
Thalidomide-like drugs such as lenalidomide are clinically important treatments for multiple myeloma and show promise for other B cell malignancies. The biochemical mechanisms underlying their antitumor activity are unknown. Thalidomide was recently shown to bind to, and inhibit, the cereblon ubiquitin ligase. Cereblon loss in zebrafish causes fin defects reminiscent of the limb defects seen in children exposed to thalidomide in utero. Here we show that lenalidomide-bound cereblon acquires the ability to target for proteasomal degradation two specific B cell transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3). Analysis of myeloma cell lines revealed that loss of IKZF1 and IKZF3 is both necessary and sufficient for lenalidomide's therapeutic effect, suggesting that the antitumor and teratogenic activities of thalidomide-like drugs are dissociable.
The evolutionarily conserved protein COP1 has been shown to operate as an E3 ubiquitin ligase complex, and a number of putative substrates have been identified, including the c-JUN oncoprotein and p53 tumor suppressor protein. New work by Migliorini and colleagues described in the current issue of JCI demonstrates that COP1 acts as a tumor suppressor in vivo and does so, at least in part, by promoting the destruction of c-JUN. These findings challenge the view that COP1 regulates p53 stability and call into question the wisdom of developing COP1 inhibitors as potential anticancer agents.
Ischemic cardiomyopathy is the major cause of heart failure and a significant cause of morbidity and mortality. The degree of left ventricular dysfunction in this setting is often out of proportion to the amount of overtly infarcted tissue and how decreased delivery of oxygen and nutrients leads to impaired contractility remains incompletely understood. The PHD prolyl hydroxylases are oxygen-sensitive enzymes that transduce changes in oxygen availability into changes in the stability of the HIF transcription factor, a master regulator of genes that promote survival in a low oxygen-environment.
Methods and Results
We found that cardiac-specific PHD inactivation causes ultrastructural, histological, and functional changes reminiscent of ischemic cardiomyopathy over time. Moreover, chronic expression of a stabilized HIFα variant in cardiomyocytes also led to dilated cardiomyopathy.
Sustained loss of PHD activity and subsequent HIF activation, as would occur in the setting of chronic ischemia, is sufficient to account for many of the changes in the hearts of individuals with chronic coronary artery disease.
cardiomyopathy; hibernation; hypoxia; ischemia; myocardium
IDH1 and IDH2 (Isocitrate Dehydrogenase 1 and 2) mutants are common in several cancers, including leukemias, and overproduce the (R)-enantiomer of 2-hydroxyglutarate [(R)-2-HG]. Elucidating the role of IDH mutations and (R)-2-HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine-independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2-HG, but not (S)-2-HG, despite the fact that (S)-2-HG more potently inhibits enzymes previously linked to the pathogenesis of IDH mutant tumors, such as TET2 (Ten Eleven Translocation 2). We provide evidence that this paradox relates to the ability of (S)-2-HG, but not (R)-2-HG, to inhibit the EglN (Egg-laying Defective Nine) prolyl hydroxylases and, importantly, show that transformation by (R)-2-HG is reversible.
Prior studies have identified recurrent oncogenic mutations in colorectal adenocarcinoma1 and have surveyed exons of protein-coding genes for mutations in 11 affected individuals2,3. Here we report whole-genome sequencing from nine individuals with colorectal cancer, including primary colorectal tumors and matched adjacent non-tumor tissues, at an average of 30.7× and 31.9× coverage, respectively. We identify an average of 75 somatic rearrangements per tumor, including complex networks of translocations between pairs of chromosomes. Eleven rearrangements encode predicted in-frame fusion proteins, including a fusion of VTI1A and TCF7L2 found in 3 out of 97 colorectal cancers. Although TCF7L2 encodes TCF4, which cooperates with β-catenin4 in colorectal carcinogenesis5,6, the fusion lacks the TCF4 β-catenin–binding domain. We found a colorectal carcinoma cell line harboring the fusion gene to be dependent on VTI1A-TCF7L2 for anchorage-independent growth using RNA interference-mediated knockdown. This study shows previously unidentified levels of genomic rearrangements in colorectal carcinoma that can lead to essential gene fusions and other oncogenic events.
Chemical modifications of histones and DNA, such as histone methylation, histone acetylation, and DNA methylation, play critical roles in epigenetic gene regulation. Many of the enzymes that add or remove such chemical modifications are known, or might be suspected, to be sensitive to changes in intracellular metabolism. This knowledge provides a conceptual foundation for understanding how mutations in the metabolic enzymes SDH, FH, and IDH can result in cancer and, more broadly, for how alterations in metabolism and nutrition might contribute to disease. Here, we review literature pertinent to hypothetical connections between metabolic and epigenetic states in eukaryotic cells.
Inactivation of the VHL tumor suppressor protein (pVHL) is a common event in clear cell renal carcinoma, which is the most common form of kidney cancer. pVHL performs many functions, including serving as the substrate recognition module of an ubiquitin ligase complex that targets the alpha subunits of the heterodimeric HIF transcription factor for proteasomal degradation. Deregulation of HIF2α appears to be a driving force in pVHL-defective clear cell renal carcinomas. In contrast, genetic and functional studies suggest that HIF1α serves as a tumor suppressor and is a likely target of the 14q deletions that are characteristic of this tumor type. Drugs that inhibit HIF2α, or its downstream targets such as VEGF, are in various stages of clinical testing. Indeed, clear cell renal carcinomas are exquisitely sensitive to VEGF deprivation and four VEGF inhibitors have now been approved for the treatment of this disease.
Kidney Cancer; von-Hippel-Lindau; hypoxia; angiogenesis; VEGF
The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH), and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EglN prolyl 4-hydroxylases that mark the HIF transcription factor for polyubiquitylation and proteasomal degradation 1. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumors but can suppress tumor growth in some other contexts. IDH1 and IDH2, which catalyze the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumors and leukemias. The resulting mutants display the neomorphic ability to convert 2-OG to the R-enantiomer of 2-hydroxyglutarate (R-2HG) 2, 3. Here we show that R-2HG, but not S-2HG, stimulates EglN activity leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes.
Kidney cancer is one of the 10 most common forms of cancer in both men and women. Ninety percent or more of these cancers are believed to be of epithelial cell origin, and are referred to as renal cell carcinoma (RCC). RCCs can be further subdivided, based on their histologic appearance, into clear-cell renal carcinomas (~75%), papillary renal carcinomas (15%), chromophobe tumors (5%), and oncocytomas (5%).1,2 Studies of hereditary kidney cancer families led to the identification of genes that, when mutated in the germline, confer an increased risk of these various histologic RCC subtypes and hence a glimpse at the molecular circuits that are deregulated in these different forms of RCC.2 In practice, there is some overlap among the histologic subtypes (eg, a tumor with predominantly clear-cell features might contain areas more typical of papillary RCC). Similarly, there are some shared molecular features among these tumor types (see later discussion). This review focuses primarily on the most common form of RCC, clear-cell renal carcinoma, while making note of some recent advances in the other histologic subtypes.
Renal cell carcinoma; Clear-cell renal carcinoma; Hypoxia-inducible factor; HIF-responsive gene products
This report describes clinical characteristics in families with a Type 2A phenotype and functional properties of a novel von Hippel Lindau variant (X214L).
Pedigrees were analyzed. Analysis of VHL coding exons and flanking intronic sequences in DNA from a proband with pheochromocytoma and islet cell tumor was performed. Western blot assays for pVHL, HIFα and Jun B were conducted using VHL null renal clear carcinoma cell lines that were engineered to produce wild type or X214L mutant pVHL.
Pedigree analysis indicated that the variant tracked with disease. The same or similar VHL point mutations were identified in several Type 2A families. The predicted 14 amino acid extended pVHL variant, when reintroduced into VHL null cells, was stable and retained the ability to downregulate HIFα in a hydroxylation-dependent manner. In contrast, the variant was defective with respect to downregulation of JunB.
pVHL X214L, like other pVHL variants associated with a low risk of clear cell renal carcinoma, largely preserves the ability to downregulate HIF. In contrast, this variant, like other pVHL variants linked to Type 2A disease, fails to suppress JunB. This underscores that JunB may play a role in the pathogenesis of Type 2A VHL disease.
germ-line mutation; HIF; Jun B; neuroendocrine; pheochromocytoma; von Hippel Lindau
The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17–50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157–171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.
J. Clin. Invest. 104:1583–1591 (1999).
Kidney cancers often delete chromosome 3p, spanning the VHL tumor suppressor gene, and chromosome 14q, which presumably harbors one or more tumor suppressor genes. pVHL inhibits the HIF transcription factor and HIF2α is a kidney cancer oncoprotein. Here we identify focal, homozygous, deletions of the HIF1α locus on 14q in clear cell renal carcinoma cell lines. Wild-type HIF1α, but not the products of these altered loci, suppress renal carcinoma growth. Conversely, downregulation of HIF1α in HIF1α-proficient lines promote tumor growth. HIF1α activity is diminished in 14q deleted kidney cancers and all of the somatic HIF1α mutations identified in kidney cancers tested to date are loss of function. Therefore HIF1α has the credentials of a kidney cancer suppressor gene.
14q deletion; HIF1α; kidney cancer; tumor suppression; von Hippel-Lindau; hypoxia
mTORC1 is a validated therapeutic target for renal cell carcinoma (RCC). Here, analysis of Tsc1 deficient (mTORC1 hyperactivation) mice uncovered a FoxO-dependent negative feedback circuit constraining mTORC1-mediated renal tumorigenesis. We document robust FoxO activation in Tsc1 deficient benign polycystic kidneys and FoxO extinction upon progression to murine renal tumors; murine renal tumor progression upon genetic deletion of both Tsc1 and FoxOs; and down-regulated FoxO expression in most human renal clear cell and papillary carcinomas, yet continued expression in less aggressive RCCs and benign renal tumor subtypes. Mechanistically, integrated analyses revealed that FoxO-mediated block operates via suppression of Myc through up-regulation of the Myc antagonists, Mxi1-SRα and mir-145, establishing a FoxO-Mxi1-SRα/mir-145 axis as a major progression block in renal tumor development.
Mdm2 is the major negative regulator of the p53 pathway. Here we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by Casein Kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCFβ-TRCP. Inactivation of either β-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging-agents. Moreover, SCFβ-TRCP-dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression.
The multi-subunit Sin3 co-repressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. Here, we have investigated genome-wide binding of mouse Sin3 and RBP2 as well as histone modifications and nucleosome positioning as a function of myogenic differentiation. Remarkably, we find that Sin3 complexes spread immediately downstream of the transcription start site on repressed and transcribed genes during differentiation. We show that RBP2 is part of a Sin3 complex, and on a subset of E2F4 target genes, the coordinated activity of Sin3 and RBP2 leads to deacetylation, demethylation, and repositioning of nucleosomes. Our work provides evidence for coordinated binding of Sin3, chromatin modifications, and chromatin remodeling within discrete regulatory regions, suggesting a model in which spreading of Sin3 binding is ultimately linked to permanent gene silencing on a subset of E2F4 target genes.
Germline mutations in the von Hippel–Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-α subunits (and hence expression of the HIF-α transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (n=82) and inherited RCC (n=64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC.
The challenge in medical oncology has always been to identify compounds that will kill, or at least tame, cancer cells while leaving normal cells unscathed. Most chemotherapeutic agents in use today were selected primarily for their ability to kill rapidly dividing cancer cells grown in cell culture and in mice, with their selectivity determined empirically during subsequent animal and human testing. Unfortunately, most of the drugs developed in this way have relatively low therapeutic indices (low toxic dose relative to the therapeutic dose). Recent advances in genomics are leading to a more complete picture of the range of mutations, both driver and passenger, present in human cancers. Synthetic lethality provides a conceptual framework for using this information to arrive at drugs that will preferentially kill cancer cells relative to normal cells. It also provides a possible way to tackle 'undruggable' targets. Two genes are synthetically lethal if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes leads to death. If one is a cancer-relevant gene, the task is to discover its synthetic lethal interactors, because targeting these would theoretically kill cancer cells mutant in the cancer-relevant gene while sparing cells with a normal copy of that gene. All cancer drugs in use today, including conventional cytotoxic agents and newer 'targeted' agents, target molecules that are present in both normal cells and cancer cells. Their therapeutic indices almost certainly relate to synthetic lethal interactions, even if those interactions are often poorly understood. Recent technical advances enable unbiased screens for synthetic lethal interactors to be undertaken in human cancer cells. These approaches will hopefully facilitate the discovery of safer, more efficacious anticancer drugs that exploit vulnerabilities that are unique to cancer cells by virtue of the mutations they have accrued during tumor progression.
The Third Cambridge Conference on Innovations and Challenges in Renal Cancer, a symposium held in Cambridge, Massachusetts, June 27–28, 2008, and chaired by Michael B. Atkins, was convened to discuss the current state of knowledge in the field, critique new data, stimulate communication among those involved in basic and clinical research, and offer recommendations for further study. Four main topics were discussed: genetics and molecular biology of renal cell cancer, staging and prognosis, systemic therapy, and correlative science and biomarkers in stage IV disease. The conference format combined brief presentations with extended periods of discussion. The conclusions and recommendations are summarized in this paper and presented in more detail in the individual papers that follow.
renal cancer; genetics; molecular biology; staging; prognosis; systemic therapy; correlative science; biomarkers
Recent insights into the role of the VHL tumor suppressor gene in hereditary and sporadic clear cell carcinoma of the kidney (ccRCC) have led to new treatments for patients with metastatic ccRCC, although virtually all patients eventually succumb to the disease. We performed an integrated, genome-wide analysis of copy-number changes and gene expression profiles in 90 tumors, including both sporadic and VHL disease-associated tumors, in hopes of identifying new therapeutic targets in ccRCC. We identified 14 regions of nonrandom copy-number change, including 7 regions of amplification (1q, 2q, 5q, 7q, 8q, 12p, and 20q) and 7 regions of deletion (1p, 3p, 4q, 6q, 8p, 9p, and 14q). An analysis aimed at identifying the relevant genes revealed VHL as one of 3 genes in the 3p deletion peak, CDKN2A and CDKN2B as the only genes in the 9p deletion peak, and MYC as the only gene in the 8q amplification peak. An integrated analysis to identify genes in amplification peaks that are consistently overexpressed among amplified samples confirmed MYC as a potential target of 8q amplification and identified candidate oncogenes in the other regions. A comparison of genomic profiles revealed that VHL disease-associated tumors are similar to a subgroup of sporadic tumors, and thus more homogeneous overall. Sporadic tumors without evidence of biallelic VHL inactivation fell into 2 groups: one group with genomic profiles highly dissimilar to the majority of ccRCC, and a second group with genomic profiles that are much more similar to tumors with biallelic inactivation of VHL.
Renal cancer; VHL; oncogenes; chromosomal changes; SNP arrays