Integrated molecular analysis of clear-cell renal cell carcinoma

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Abstract

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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Figure 1: Mutations in 3p target genes and their impact on survival.
Figure 2: New TCEB1 mutations and HIF accumulation.
Figure 3: Significantly mutated genes and pathways for 106 ccRCC specimens.
Figure 4: Significant copy number alterations in 240 ccRCC specimens.
Figure 5: Significantly mutated pathways for 106 ccRCC specimens.
Figure 6: Correlations between DNA methylation and other genetic lesions.

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Acknowledgements

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.

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Contributions

Y. Sato, S. Maekawa, Y.N., H.S., Y. Suzuki, S.S., K.Y. and A.K. performed DNA sequencing. Y. Shiraishi, Y.O., K.C., H.T., A.F., T.T. and S. Miyano performed bioinformatics analyses of the sequencing data. T.Y., M.S. and T.K. performed the functional analyses of Elongin C mutants. Y. Sato, A.S.-O., A.N. and M.S. performed SNP array and expression array analyses. T.S., G.N. and H.A. performed methylation analysis. H.K. and Y.H. provided specimens and were also involved in planning the project. T.M., D.M. and M.F. confirmed histological diagnosis and performed immunostaining for HIF proteins. Y. Sato, T.Y., Y.O., A.S.-O. and S.O. generated figures and tables and wrote the manuscript. S.O. led the entire project. All authors participated in the discussion and interpretation of data and results.

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Correspondence to Seishi Ogawa.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22, Supplementary Tables 5–9 and 15, and Supplementary Note (PDF 29982 kb)

Supplementary Table 1

Characteristics of the patients (XLSX 36 kb)

Supplementary Table 2

List of mutations in whole-genome sequencing (XLSX 5326 kb)

Supplementary Table 3

List of mutations in exome sequencing (XLSX 525 kb)

Supplementary Table 4

Recurrently mutated genes found in whole-exome sequencing (XLSX 64 kb)

Supplementary Table 10

Summary of RNA sequencing data (XLSX 15 kb)

Supplementary Table 11

Fusion transcripts detected in RNA sequencing (XLSX 15 kb)

Supplementary Table 12

Overexpressed annotation terms among differentially methylated genes (XLSX 23 kb)

Supplementary Table 13

List of PCR primers for frequently mutated genes (XLSX 13 kb)

Supplementary Table 14

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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