Skip to main content

Thank you for visiting 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 activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR



Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors.

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

Access options

Buy this article

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

Figure 1: DNA hypomethylation-associated transcriptional activation of a TBC1D16 cryptic isoform in metastatic cancer cells.
Figure 2: TBC1D16-47KD enhances melanoma progression in vitro and in vivo.
Figure 3: TBC1D16-47KD regulates Rab GTPases and EGFR activation in melanoma cells.
Figure 4: Epigenetic reactivation of TBC1D16-47KD predicts response to BRAF inhibition.
Figure 5: Epigenetic reactivation of TBC1D16-47KD predicts response to BRAF and MEK inhibitors by targeting two signaling pathways.
Figure 6: TBC1D16-47KD promoter demethylation is an independent prognostic factor for poor clinical outcome in melanoma patients.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus


  1. Siegel, R., Naishadham, D. & Jemal, A. Cancer statistics. CA Cancer J. Clin. 63, 11–30 (2013).

    Google Scholar 

  2. Jones, P.A. & Baylin, S.B. The epigenomics of cancer. Cell 128, 683–692 (2007).

    Article  CAS  Google Scholar 

  3. Heyn, H. & Esteller, M. DNA methylation profiling in the clinic: applications and challenges. Nat. Rev. Genet. 13, 679–692 (2012).

    Article  CAS  Google Scholar 

  4. Timp, W. & Feinberg, A.P. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. Nat. Rev. Cancer 13, 497–510 (2013).

    Article  CAS  Google Scholar 

  5. Fang, F. et al. Breast cancer methylomes establish an epigenomic foundation for metastasis. Sci. Transl. Med. 3, 75ra25 (2011).

    Article  Google Scholar 

  6. Cunha, S. et al. The RON receptor tyrosine kinase promotes metastasis by triggering MBD4-dependent DNA methylation reprogramming. Cell Reports 6, 141–154 (2014).

    Article  CAS  Google Scholar 

  7. Carmona, F.J. et al. A comprehensive DNA methylation profile of epithelial-to-mesenchymal transition. Cancer Res. 74, 5608–5619 (2014).

    Article  CAS  Google Scholar 

  8. Lujambio, A. et al. A microRNA DNA methylation signature for human cancer metastasis. Proc. Natl. Acad. Sci. USA 105, 13556–13561 (2008).

    Article  CAS  Google Scholar 

  9. Harbst, K. et al. Multiple metastases from cutaneous malignant melanoma patients may display heterogeneous genomic and epigenomic patterns. Melanoma Res. 20, 381–391 (2010).

    CAS  PubMed  Google Scholar 

  10. Marzese, D.M. et al. Epigenome-wide DNA methylation landscape of melanoma progression to brain metastasis reveals aberrations on homeobox D cluster associated with prognosis. Hum. Mol. Genet. 23, 226–238 (2014).

    Article  CAS  Google Scholar 

  11. MacKie, R.M., Hauschild, A. & Eggermont, A.M. Epidemiology of invasive cutaneous melanoma. Ann. Oncol. 20 (suppl. 6), vi1–vi7 (2009).

    PubMed  PubMed Central  Google Scholar 

  12. Villanueva, M.T. Skin cancer: in melanoma ulceration, size matters. Nat. Rev. Clin. Oncol 9, 370 (2012).

    Article  Google Scholar 

  13. Mandalà, M. & Massi, D. Tissue prognostic biomarkers in primary cutaneous melanoma. Virchows Arch. 464, 265–281 (2014).

    Article  Google Scholar 

  14. Tsao, H., Chin, L., Garraway, L.A. & Fisher, D.E. Melanoma: from mutations to medicine. Genes Dev. 26, 1131–1155 (2012).

    Article  CAS  Google Scholar 

  15. Kaufman, H.L. et al. The Society for Immunotherapy of Cancer consensus statement on tumour immunotherapy for the treatment of cutaneous melanoma. Nat. Rev. Clin. Oncol 10, 588–598 (2013).

    Article  CAS  Google Scholar 

  16. Kaufman, H.L. Melanoma as a model for precision medicine in oncology. Lancet Oncol. 15, 251–253 (2014).

    Article  Google Scholar 

  17. Weinreb, A. & Travo, P. Discrimination between human melanoma cell lines by fluorescence anisotropy. Eur. J. Cancer Clin. Oncol. 20, 673–677 (1984).

    Article  CAS  Google Scholar 

  18. Sandoval, J. et al. Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome. Epigenetics 6, 692–702 (2011).

    Article  CAS  Google Scholar 

  19. Akavia, U.D. et al. An integrated approach to uncover drivers of cancer. Cell 143, 1005–1017 (2010).

    Article  CAS  Google Scholar 

  20. Goueli, B.S., Powell, M.B., Finger, E.C. & Pfeffer, S.R. TBC1D16 is a Rab4A GTPase activating protein that regulates receptor recycling and EGF receptor signaling. Proc. Natl. Acad. Sci. USA 109, 15787–15792 (2012).

    Article  CAS  Google Scholar 

  21. El Kasmi, K.C. et al. Cutting edge: a transcriptional repressor and corepressor induced by the STAT3-regulated anti-inflammatory signalling pathway. J. Immunol. 179, 7215–7219 (2007).

    Article  CAS  Google Scholar 

  22. Eisenberg, M.C. et al. Mechanistic modeling of the effects of myoferlin on tumor cell invasion. Proc. Natl. Acad. Sci. USA 108, 20078–20083 (2011).

    Article  CAS  Google Scholar 

  23. Frasa, M.A., Koessmeier, K.T., Ahmadian, M.R. & Braga, V.M. Illuminating the functional and structural repertoire of human TBC/RABGAPs. Nat. Rev. Mol. Cell Biol. 13, 67–73 (2012).

    Article  CAS  Google Scholar 

  24. Onodera, Y. et al. Rab5c promotes AMAP1–PRKD2 complex formation to enhance β1 integrin recycling in EGF-induced cancer invasion. J. Cell Biol. 197, 983–996 (2012).

    Article  CAS  Google Scholar 

  25. Corcoran, R.B. et al. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov. 2, 227–235 (2012).

    Article  CAS  Google Scholar 

  26. Prahallad, A. et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 483, 100–103 (2012).

    Article  CAS  Google Scholar 

  27. Yoon, Y.K. et al. Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells. Mol. Cancer Ther. 8, 2526–2536 (2009).

    Article  CAS  Google Scholar 

  28. Mirzoeva, O.K. et al. Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition. Cancer Res. 69, 565–572 (2009).

    Article  CAS  Google Scholar 

  29. Garnett, M.J. et al. Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature 483, 570–575 (2012).

    Article  CAS  Google Scholar 

  30. Hansen, K.D. et al. Increased methylation variation in epigenetic domains across cancer types. Nat. Genet. 43, 768–775 (2011).

    Article  CAS  Google Scholar 

  31. Hon, G.C. et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res. 22, 246–258 (2012).

    Article  CAS  Google Scholar 

  32. Bert, S.A. et al. Regional activation of the cancer genome by long-range epigenetic remodeling. Cancer Cell 23, 9–22 (2013).

    Article  CAS  Google Scholar 

  33. Kedlaya, R. et al. Interactions between GIPC-APPL and GIPC-TRP1 regulate melanosomal protein trafficking and melanogenesis in human melanocytes. Arch. Biochem. Biophys. 508, 227–233 (2011).

    Article  CAS  Google Scholar 

  34. Huang, Z.M. et al. Targeting protein-trafficking pathways alters melanoma treatment sensitivity. Proc. Natl. Acad. Sci. USA 109, 553–558 (2012).

    Article  CAS  Google Scholar 

  35. Gray-Schopfer, V., Wellbrock, C. & Marais, R. Melanoma biology and new targeted therapy. Nature 445, 851–857 (2007).

    Article  CAS  Google Scholar 

  36. Middleton, M.R. et al. Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma. J. Clin. Oncol. 18, 158–166 (2000).

    Article  CAS  Google Scholar 

  37. Eggermont, A.M., Spatz, A. & Robert, C. Cutaneous melanoma. Lancet 383, 816–827 (2014).

    Article  CAS  Google Scholar 

  38. Catalanotti, F. et al. Phase II trial of MEK inhibitor selumetinib (AZD6244, ARRY-142886) in patients with BRAFV600E/K-mutated melanoma. Clin. Cancer Res. 19, 2257–2264 (2013).

    Article  CAS  Google Scholar 

  39. Girotti, M.R. et al. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. Cancer Discov. 3, 158–167 (2013).

    Article  CAS  Google Scholar 

  40. Altman, D.G. et al. Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK): explanation and elaboration. PLoS Med. 9, e1001216 (2012).

    Article  Google Scholar 

  41. Heyn, H. et al. Distinct DNA methylomes of newborns and centenarians. Proc. Natl. Acad. Sci. USA 109, 10522–10527 (2012).

    Article  CAS  Google Scholar 

  42. Moutinho, C. et al. Epigenetic inactivation of the BRCA1 interactor SRBC and resistance to oxaliplatin in colorectal cancer. J. Natl. Cancer Inst. 106, djt322 (2014).

    Article  Google Scholar 

  43. Lopez-Serra, P. et al. A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect. Nat. Commun. 5, 3608 (2014).

    Article  Google Scholar 

  44. Howard-Jones, N. A CIOMS ethical code for animal experimentation. WHO Chron. 39, 51–56 (1985).

    CAS  PubMed  Google Scholar 

Download references


We thank the patients and their families. The research leading to these results has received funding from the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement no. PIAPP-GA-2009-230614–Target-Melanoma project (F.P., J.O., W.M.G., M.E.), the Worldwide Cancer Research grant reference no. 15-0354 (M.E.), the European Research Council Advanced grant no. 268626–EPINORC project (M.E.), the Ministerio de Ciencia e Innovacion grant numbers SAF2011-22803 (M.E.) and FIS PI13-01339 (A. Villanueva), the CRUK Manchester Institute (C5759/A12328 to R.M.), the Wellcome Trust (100282/Z/12/Z to R.M.), the Cellex Foundation (M.E.) and the Health and Science Departments of the Catalan Government Generalitat de Catalunya 2005-SGR00727 (A. Villanueva) and 2014-SGR 633 (M.E.). M.V. was supported by a Formacion de Profesorado Universitario fellowship from the Spanish Ministry of Education. We thank the staff of the Animal Core Facility of Bellvitge Biomedical Research Institute for mouse care and maintenance. M.E. is an Institucio Catalana de Recerca i Estudis Avançats Research Professor.

Author information

Authors and Affiliations



M.V. and M.E. conceived the study and wrote the manuscript. M.V. performed most experiments with the help of H.J.F., P.L.-S., F.J.C., S.G., C.M., J.L., A.P., H.H. and S.M. A.Vidal and A. Villanueva, together with M.M.-I., performed the mouse studies. A.M.-C., M.R.G., J.L.M., M.T.F.-F., E.E., E.M.-C., R.B.-E., A.B., F.P., J.v.d.O., W.M.G., D.T.F., K.T.F., U.M., P.L. and R.M. analyzed the clinical outcome and drug response data and provided conceptual input. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Manel Esteller.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures and Text

Supplementary Figures 1–12 & Supplementary Tables 1–3 (PDF 3464 kb)

Supplementary Data 1

2,620 CpGs most divergent between primary and metatatic tumor cell lines. (XLSX 322 kb)

Supplementary Data 2

CpGs located outside CpG islands most divergent between primary and metastatic tumor cell lines. (XLSX 12 kb)

Supplementary Data 3

DNA methylation profile of the TBC1D16-45/47KD promoter CpG island according to the DNA methylation microarray values in 36 melanoma cell lines. (XLSX 14 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vizoso, M., Ferreira, H., Lopez-Serra, P. et al. Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR. Nat Med 21, 741–750 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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