Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Epigenetic expansion of VHL-HIF signal output drives multiorgan metastasis in renal cancer

Abstract

Inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC) that leads to the activation of hypoxia-inducible transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response that is specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that loss of Polycomb repressive complex 2 (PRC2)-dependent histone H3 Lys27 trimethylation (H3K27me3) activates HIF-driven chemokine (C-X-C motif) receptor 4 (CXCR4) expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven cytohesin 1 interacting protein (CYTIP) expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Experimental model system and gene expression signature for ccRCC metastasis.
Figure 2: VHL-HIF signal output modulation is associated with ccRCC progression.
Figure 3: VHL-HIF pathway modulation activates functional mediators of ccRCC metastasis.
Figure 4: DNA demethylation activates CYTIP expression in metastatic ccRCC.
Figure 5: Histone modification patterns linked to ccRCC progression.
Figure 6: Loss of PRC2-dependent repression activates CXCR4 expression in metastatic ccRCC.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

References

  1. Kang, Y. et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3, 537–549 (2003).

    CAS  PubMed  Google Scholar 

  2. Nguyen, D.X. et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell 138, 51–62 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Sonoshita, M. et al. Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling. Cancer Cell 19, 125–137 (2011).

    CAS  PubMed  Google Scholar 

  4. Nguyen, D.X., Bos, P.D. & Massague, J. Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer 9, 274–284 (2009).

    CAS  PubMed  Google Scholar 

  5. Kaelin, W.G. Jr. The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat. Rev. Cancer 8, 865–873 (2008).

    CAS  PubMed  Google Scholar 

  6. Staller, P. et al. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature 425, 307–311 (2003).

    CAS  PubMed  Google Scholar 

  7. Kaelin, W.G. Von Hippel-Lindau disease. Annu. Rev. Pathol. 2, 145–173 (2007).

    CAS  PubMed  Google Scholar 

  8. Chauveau, D. et al. Renal involvement in von Hippel-Lindau disease. Kidney Int. 50, 944–951 (1996).

    CAS  PubMed  Google Scholar 

  9. Mandriota, S.J. et al. HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron. Cancer Cell 1, 459–468 (2002).

    CAS  PubMed  Google Scholar 

  10. Gnarra, J.R. et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat. Genet. 7, 85–90 (1994).

    CAS  PubMed  Google Scholar 

  11. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Montani, M. et al. VHL-gene deletion in single renal tubular epithelial cells and renal tubular cysts: further evidence for a cyst-dependent progression pathway of clear cell renal carcinoma in von Hippel-Lindau disease. Am. J. Surg. Pathol. 34, 806–815 (2010).

    PubMed  Google Scholar 

  13. Kapitsinou, P.P. & Haase, V.H. The VHL tumor suppressor and HIF: insights from genetic studies in mice. Cell Death Differ. 15, 650–659 (2008).

    CAS  PubMed  Google Scholar 

  14. Kondo, K., Klco, J., Nakamura, E., Lechpammer, M. & Kaelin, W.G. Jr. Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 1, 237–246 (2002).

    CAS  PubMed  Google Scholar 

  15. Kondo, K., Kim, W.Y., Lechpammer, M. & Kaelin, W.G. Jr. Inhibition of HIF2α is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 1, E83 (2003).

    PubMed  PubMed Central  Google Scholar 

  16. Shen, C. et al. Genetic and functional studies implicate HIF1α as a 14q kidney cancer suppressor gene. Cancer Discov. 1, 222–235 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Müller, A. et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50–56 (2001).

    PubMed  Google Scholar 

  18. Zhang, X.H. et al. Latent bone metastasis in breast cancer tied to Src-dependent survival signals. Cancer Cell 16, 67–78 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Schlesinger-Raab, A., Treiber, U., Zaak, D., Holzel, D. & Engel, J. Metastatic renal cell carcinoma: results of a population-based study with 25 years follow-up. Eur. J. Cancer 44, 2485–2495 (2008).

    PubMed  Google Scholar 

  20. Yao, M. et al. VHL tumor suppressor gene alterations associated with good prognosis in sporadic clear-cell renal carcinoma. J. Natl. Cancer Inst. 94, 1569–1575 (2002).

    CAS  PubMed  Google Scholar 

  21. Young, A.C. et al. Analysis of VHL gene alterations and their relationship to clinical parameters in sporadic conventional renal cell carcinoma. Clin. Cancer Res. 15, 7582–7592 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. D′Alterio, C. et al. Differential role of CD133 and CXCR4 in renal cell carcinoma. Cell Cycle 9, 4492–4500 (2010).

    PubMed  Google Scholar 

  23. Williams, R.D., Elliott, A.Y., Stein, N. & Fraley, E.E. In vitro cultivation of human renal cell cancer. II. Characterization of cell lines. In Vitro 14, 779–786 (1978).

    CAS  PubMed  Google Scholar 

  24. Hess, K.R. et al. Metastatic patterns in adenocarcinoma. Cancer 106, 1624–1633 (2006).

    PubMed  Google Scholar 

  25. Zhao, H. et al. Gene expression profiling predicts survival in conventional renal cell carcinoma. PLoS Med. 3, e13 (2006).

    PubMed  Google Scholar 

  26. Wang, L. et al. Strong expression of chemokine receptor CXCR4 by renal cell carcinoma cells correlates with metastasis. Clin. Exp. Metastasis 26, 1049–1054 (2009).

    CAS  PubMed  Google Scholar 

  27. Zagzag, D. et al. Stromal cell-derived factor-1α and CXCR4 expression in hemangioblastoma and clear cell-renal cell carcinoma: von Hippel-Lindau loss-of-function induces expression of a ligand and its receptor. Cancer Res. 65, 6178–6188 (2005).

    CAS  PubMed  Google Scholar 

  28. Semenza, G.L. Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721–732 (2003).

    CAS  PubMed  Google Scholar 

  29. Mole, D.R. et al. Genome-wide association of hypoxia-inducible factor (HIF)-1α and HIF-2α DNA binding with expression profiling of hypoxia-inducible transcripts. J. Biol. Chem. 284, 16767–16775 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Dalgliesh, G.L. et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 463, 360–363 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen, Q., Coffey, A., Bourgoin, S.G. & Gadina, M. Cytohesin binder and regulator augments T cell receptor–induced nuclear factor of activated T cells.AP-1 activation through regulation of the JNK pathway. J. Biol. Chem. 281, 19985–19994 (2006).

    CAS  PubMed  Google Scholar 

  32. Watford, W.T. et al. Cytohesin binder and regulator (cybr) is not essential for T- and dendritic-cell activation and differentiation. Mol. Cell. Biol. 26, 6623–6632 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Boehm, T. et al. Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration. EMBO J. 22, 1014–1024 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Hofer, S. et al. Dendritic cells regulate T-cell deattachment through the integrin-interacting protein CYTIP. Blood 107, 1003–1009 (2006).

    CAS  PubMed  Google Scholar 

  35. Saharinen, J., Hyytiainen, M., Taipale, J. & Keski-Oja, J. Latent transforming growth factor-β binding proteins (LTBPs)—structural extracellular matrix proteins for targeting TGF-β action. Cytokine Growth Factor Rev. 10, 99–117 (1999).

    CAS  PubMed  Google Scholar 

  36. Bell, O., Tiwari, V.K., Thoma, N.H. & Schubeler, D. Determinants and dynamics of genome accessibility. Nat. Rev. Genet. 12, 554–564 (2011).

    CAS  PubMed  Google Scholar 

  37. Varela, I. et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469, 539–542 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Mikkelsen, T.S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).

    CAS  PubMed  Google Scholar 

  40. Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Margueron, R. et al. Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol. Cell 32, 503–518 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol. Cell 32, 491–502 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Shi, J. et al. The Polycomb complex PRC2 supports aberrant self-renewal in a mouse model of MLL-AF9;Nras(G12D) acute myeloid leukemia. Oncogene 10.1038/onc.2012.110 (2012).

  44. Li, L. et al. Hypoxia-inducible factor linked to differential kidney cancer risk seen with type 2A and type 2B VHL mutations. Mol. Cell. Biol. 27, 5381–5392 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Minn, A.J. et al. Genes that mediate breast cancer metastasis to lung. Nature 436, 518–524 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Ponomarev, V. et al. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur. J. Nucl. Med. Mol. Imaging 31, 740–751 (2004).

    CAS  PubMed  Google Scholar 

  47. Dow, L.E. et al. A pipeline for the generation of shRNA transgenic mice. Nat. Protoc. 7, 374–393 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Banks, R.E. et al. Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer. Cancer Res. 66, 2000–2011 (2006).

    CAS  PubMed  Google Scholar 

  49. McArthur, M., Gerum, S. & Stamatoyannopoulos, G. Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse β-globin LCR. J. Mol. Biol. 313, 27–34 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Song, L. & Crawford, G.E. DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold. Spring. Harb. Protoc. 2010 pdb.prot5384 (2010).

  51. Dai, M. et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 33, e175 (2005).

    PubMed  PubMed Central  Google Scholar 

  52. Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).

    PubMed  Google Scholar 

  53. McShane, L.M. et al. Methods for assessing reproducibility of clustering patterns observed in analyses of microarray data. Bioinformatics 18, 1462–1469 (2002).

    CAS  PubMed  Google Scholar 

  54. Kashyap, V. et al. Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid. J. Biol. Chem. 286, 3250–3260 (2011).

    CAS  PubMed  Google Scholar 

  55. Brenet, F. et al. DNA methylation of the first exon is tightly linked to transcriptional silencing. PLoS ONE 6, e14524 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank members of the Massagué lab for discussion, J. Brooks (Stanford University, California) for clinical annotations, W. Krek (ETH, Swiss Federal Institute of Technology, Zurich, Switzerland) for CXCR4 promoter constructs and C. David and S. Malladi for critical review of the manuscript. Gene expression profiling and high-throughput sequencing were done at the Memorial Sloan-Kettering Cancer Center (MSKCC) Genomics Core Facility, immunohistochemical staining was done at the MSKCC Molecular Cytology Core Facility, and DNA methylation analysis, RNA analysis of clinical samples and Sanger sequencing were done at the MSKCC Geoffrey Beene Translational Oncology Core Facility. The GSE2109 dataset was provided by the International Genomics Consortium and Expression Project for Oncology. S.V. received postdoctoral support from the Maud Kuistila Memorial Foundation, the Emil Aaltonen Foundation, the Paulo Foundation, the Orion-Farmos Research Foundation, the Instrumentarium Science Foundation, The Finnish Medical Foundation and the Academy of Finland. J.M. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

S.V. and J.M. designed experiments. S.V. performed experiments and bioinformatic analysis. W.S. assisted with experiments. F.B. performed STAMP experiments. A.H. supervised Epityper analysis and genomic sequencing. V.E.R. and J.J.-D.H. provided clinical ccRCC specimens. A.A.H. analyzed CXCR4 expression in clinical specimens. A.V. supervised high-throughput sequencing. J.M.S. and S.V. analyzed high-throughput sequencing data. S.V. and J.M. wrote the paper.

Corresponding author

Correspondence to Joan Massagué.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–20 and Supplementary Tables 1–6 (PDF 2030 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vanharanta, S., Shu, W., Brenet, F. et al. Epigenetic expansion of VHL-HIF signal output drives multiorgan metastasis in renal cancer. Nat Med 19, 50–56 (2013). https://doi.org/10.1038/nm.3029

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3029

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer