Clear-cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer and its molecular pathogenesis is incompletely understood. Here we report an integrated molecular study of ccRCC in which ≥100 ccRCC cases were fully analyzed by whole-genome and/or whole-exome and RNA sequencing as well as by array-based gene expression, copy number and/or methylation analyses. We identified a full spectrum of genetic lesions and analyzed gene expression and DNA methylation signatures and determined their impact on tumor behavior. Defective VHL-mediated proteolysis was a common feature of ccRCC, which was caused not only by VHL inactivation but also by new hotspot TCEB1 mutations, which abolished Elongin C–VHL binding, leading to HIF accumulation. Other newly identified pathways and components recurrently mutated in ccRCC included PI3K-AKT-mTOR signaling, the KEAP1-NRF2-CUL3 apparatus, DNA methylation, p53-related pathways and mRNA processing. This integrated molecular analysis unmasked new correlations between DNA methylation, gene mutation and/or gene expression and copy number profiles, enabling the stratification of clinical risks for patients with ccRCC.
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Ferlay, J. et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int. J. Cancer 127, 2893–2917 (2010).
Rini, B.I., Campbell, S.C. & Escudier, B. Renal cell carcinoma. Lancet 373, 1119–1132 (2009).
Ljungberg, B. et al. EAU guidelines on renal cell carcinoma: the 2010 update. Eur. Urol. 58, 398–406 (2010).
Gnarra, J.R. et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat. Genet. 7, 85–90 (1994).
Gallou, C. et al. Mutations of the VHL gene in sporadic renal cell carcinoma: definition of a risk factor for VHL patients to develop an RCC. Hum. Mutat. 13, 464–475 (1999).
Schraml, P. et al. VHL mutations and their correlation with tumour cell proliferation, microvessel density, and patient prognosis in clear cell renal cell carcinoma. J. Pathol. 196, 186–193 (2002).
Herman, J.G. et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl. Acad. Sci. USA 91, 9700–9704 (1994).
Varela, I. et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469, 539–542 (2011).
Dalgliesh, G.L. et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 463, 360–363 (2010).
Peña-Llopis, S. et al. BAP1 loss defines a new class of renal cell carcinoma. Nat. Genet. 44, 751–759 (2012).
Guo, G. et al. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma. Nat. Genet. 44, 17–19 (2012).
Greenman, C. et al. Patterns of somatic mutation in human cancer genomes. Nature 446, 153–158 (2007).
Guichard, C. et al. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma. Nat. Genet. 44, 694–698 (2012).
Huang, J. et al. Exome sequencing of hepatitis B virus–associated hepatocellular carcinoma. Nat. Genet. 44, 1117–1121 (2012).
Sung, W.K. et al. Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma. Nat. Genet. 44, 765–769 (2012).
Fujimoto, A. et al. Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators. Nat. Genet. 44, 760–764 (2012).
Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883–892 (2012).
Hakimi, A.A. et al. Adverse outcomes in clear cell renal cell carcinoma with mutations of 3p21 epigenetic regulators BAP1 and SETD2: a report by MSKCC and the KIRC TCGA Research Network. Clin. Cancer Res. 19, 3259–3267 (2013).
Hakimi, A.A. et al. Clinical and pathologic impact of select chromatin-modulating tumor suppressors in clear cell renal cell carcinoma. Eur. Urol. 63, 848–854 (2013).
Kapur, P. et al. Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation. Lancet Oncol. 14, 159–167 (2013).
Aso, T., Lane, W.S., Conaway, J.W. & Conaway, R.C. Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II. Science 269, 1439–1443 (1995).
Kamura, T. et al. Activation of HIF1a ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc. Natl. Acad. Sci. USA 97, 10430–10435 (2000).
Stebbins, C.E., Kaelin, W.G. Jr. & Pavletich, N.P. Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function. Science 284, 455–461 (1999).
Takagi, Y., Pause, A., Conaway, R.C. & Conaway, J.W. Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor suppressor protein. J. Biol. Chem. 272, 27444–27449 (1997).
Delhommeau, F. et al. Mutation in TET2 in myeloid cancers. N. Engl. J. Med. 360, 2289–2301 (2009).
Langemeijer, S.M. et al. Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat. Genet. 41, 838–842 (2009).
Chen, Q., Chen, Y., Bian, C., Fujiki, R. & Yu, X. TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 493, 561–564 (2013).
The Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330–337 (2012).
Zimmerman, E.S., Schulman, B.A. & Zheng, N. Structural assembly of cullin–RING ubiquitin ligase complexes. Curr. Opin. Struct. Biol. 20, 714–721 (2010).
Padmanabhan, B. et al. Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. Mol. Cell 21, 689–700 (2006).
Shibata, T. et al. Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy. Proc. Natl. Acad. Sci. USA 105, 13568–13573 (2008).
Kim, Y.R. et al. Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin. J. Pathol. 220, 446–451 (2010).
Adam, J. 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 20, 524–537 (2011).
Kinch, L., Grishin, N.V. & Brugarolas, J. Succination of Keap1 and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2. Cancer Cell 20, 418–420 (2011).
Ooi, A. et al. CUL3 and NRF2 mutations confer an NRF2 activation phenotype in a sporadic form of papillary renal cell carcinoma. Cancer Res. 73, 2044–2051 (2013).
Kucejova, B. et al. Interplay between pVHL and mTORC1 pathways in clear-cell renal cell carcinoma. Mol. Cancer Res. 9, 1255–1265 (2011).
Yoshida, K. et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature 478, 64–69 (2011).
Kadoch, C. et al. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nat. Genet. 45, 592–601 (2013).
Clark, J. et al. Fusion of splicing factor genes PSF and NonO (p54nrb) to the TFE3 gene in papillary renal cell carcinoma. Oncogene 15, 2233–2239 (1997).
Ross, H. & Argani, P. Xp11 translocation renal cell carcinoma. Pathology 42, 369–373 (2010).
Kuroda, N. et al. Review of renal carcinoma associated with Xp11.2 translocations/TFE3 gene fusions with focus on pathobiological aspect. Histol. Histopathol. 27, 133–140 (2012).
Brannon, A.R. et al. Molecular stratification of clear cell renal cell carcinoma by consensus clustering reveals distinct subtypes and survival patterns. Genes Cancer 1, 152–163 (2010).
Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Kent, W.J. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).
Benson, G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 27, 573–580 (1999).
Shiraishi, Y. et al. An empirical Bayesian framework for somatic mutation detection from cancer genome sequencing data. Nucleic Acids Res. 41, e89 (2013).
Hellmann, I. et al. Why do human diversity levels vary at a megabase scale? Genome Res. 15, 1222–1231 (2005).
Stamatoyannopoulos, J.A. et al. Human mutation rate associated with DNA replication timing. Nat. Genet. 41, 393–395 (2009).
Wendl, M.C. et al. PathScan: a tool for discerning mutational significance in groups of putative cancer genes. Bioinformatics 27, 1595–1602 (2011).
Nannya, Y. et al. A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays. Cancer Res. 65, 6071–6079 (2005).
Yamamoto, G. et al. Highly sensitive method for genomewide detection of allelic composition in nonpaired, primary tumor specimens by use of Affymetrix single-nucleotide-polymorphism genotyping microarrays. Am. J. Hum. Genet. 81, 114–126 (2007).
Tsuji, K., Ishikawa, Y. & Imamura, T. Technique for differentiating alveolar soft part sarcoma from other tumors in paraffin-embedded tissue: comparison of immunohistochemistry for TFE3 and CD147 and of reverse transcription polymerase chain reaction for ASPSCR1-TFE3 fusion transcript. Hum. Pathol. 43, 356–363 (2012).
Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000).
Nabekura, T., Otsu, M., Nagasawa, T., Nakauchi, H. & Onodera, M. Potent vaccine therapy with dendritic cells genetically modified by the gene-silencing-resistant retroviral vector GCDNsap. Mol. Ther. 13, 301–309 (2006).
Kamura, T. et al. VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev. 18, 3055–3065 (2004).
Garrett, K.P. et al. Positive regulation of general transcription factor SIII by a tailed ubiquitin homolog. Proc. Natl. Acad. Sci. USA 92, 7172–7176 (1995).
We thank Y. Mori, M. Nakamura, N. Mizota and S. Ichimura for their technical assistance. We also thank M. Nangaku and N. Takeda for fruitful discussion and comments. We thank T. Kitamura (University of Tokyo) for providing pMXs-puro, M. Onodera (National Center for Child Health and Development, Japan) for providing pGCDNsamIRESEGFP and R.C. Mulligan (Boston Children's Hospital) for providing 293gp cells. This work was supported by KAKENHI (22134006), the Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) (08C46598a) and the Japan Society for the Promotion of Science through the Funding Program for World-Leading Innovative R&D on Science and Technology, initiated by the Council for Science and Technology Policy.
The authors declare no competing financial interests.
Supplementary Figures 1–22, Supplementary Tables 5–9 and 15, and Supplementary Note (PDF 29982 kb)
Characteristics of the patients (XLSX 36 kb)
List of mutations in whole-genome sequencing (XLSX 5326 kb)
List of mutations in exome sequencing (XLSX 525 kb)
Recurrently mutated genes found in whole-exome sequencing (XLSX 64 kb)
Summary of RNA sequencing data (XLSX 15 kb)
Fusion transcripts detected in RNA sequencing (XLSX 15 kb)
Overexpressed annotation terms among differentially methylated genes (XLSX 23 kb)
List of PCR primers for frequently mutated genes (XLSX 13 kb)
List of PCR primers used for deep sequencing (XLSX 63 kb)
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Sato, Y., Yoshizato, T., Shiraishi, Y. et al. Integrated molecular analysis of clear-cell renal cell carcinoma. Nat Genet 45, 860–867 (2013). https://doi.org/10.1038/ng.2699
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