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Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR

Nature volume 483, pages 100–103 (2012)Cite this article

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Abstract

Inhibition of the BRAF(V600E) oncoprotein by the small-molecule drug PLX4032 (vemurafenib) is highly effective in the treatment of melanoma1. However, colon cancer patients harbouring the same BRAF(V600E) oncogenic lesion have poor prognosis and show only a very limited response to this drug2,3,4. To investigate the cause of the limited therapeutic effect of PLX4032 in BRAF(V600E) mutant colon tumours, here we performed an RNA-interference-based genetic screen in human cells to search for kinases whose knockdown synergizes with BRAF(V600E) inhibition. We report that blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with BRAF(V600E) inhibition. We find in multiple BRAF(V600E) mutant colon cancers that inhibition of EGFR by the antibody drug cetuximab or the small-molecule drugs gefitinib or erlotinib is strongly synergistic with BRAF(V600E) inhibition, both in vitro and in vivo. Mechanistically, we find that BRAF(V600E) inhibition causes a rapid feedback activation of EGFR, which supports continued proliferation in the presence of BRAF(V600E) inhibition. Melanoma cells express low levels of EGFR and are therefore not subject to this feedback activation. Consistent with this, we find that ectopic expression of EGFR in melanoma cells is sufficient to cause resistance to PLX4032. Our data suggest that BRAF(V600E) mutant colon cancers (approximately 8–10% of all colon cancers2,3,5), for which there are currently no targeted treatment options available, might benefit from combination therapy consisting of BRAF and EGFR inhibitors.

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Figure 1: EGFR inhibition confers sensitivity to BRAF(V600E) inhibition in colon cancer.
Figure 2: Functional and biochemical interaction between BRAF and EGFR inhibition in colon cancer.
Figure 3: Correlation between EGFR levels and response to BRAF inhibition in melanoma and CRC.
Figure 4: EGFR and BRAF(V600E) inhibitors synergize to induce apoptosis of CRC cells and to suppress CRC tumour growth in a xenograft model.

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Acknowledgements

We thank P. Kumar, V. Gambino and W. Grernrum for assistance with experiments. We are grateful to C. Pochet for support. Major financial support for this work was provided by the EU Seventh Framework Programme, grant agreement 259015 (to A.B. and R.B.). Additional funding was provided by a European Research Council grant to R.B.; The Cancer Systems Biology Center grant by the Netherlands Organisation for Scientific Research (NWO); The Dutch Cancer Society; The Netherlands Genomics Initiative (NGI); The Associazione Italiana per la Ricerca sul Cancro (AIRC), 2010 Special Program Molecular Clinical Oncology 5x1000, project 9970; AIRC Investigator Grant (to A.B.); Italian Ministry of University and Research; Italian Ministry of Health, Regione Piemonte (to A.B. and F.D.N.); and Intramural Grant, 5xmille 2008; and Fondazione Piemontese per la Ricerca sul Cancro (to A.B. and F.D.N.).

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Author notes

  1. Anirudh Prahallad, Chong Sun, Sidong Huang and Federica Di Nicolantonio: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Molecular Carcinogenesis, Center for Biomedical Genetics and Cancer Genomics Centre, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands,

    Anirudh Prahallad, Chong Sun, Sidong Huang, Roderick L. Beijersbergen & René Bernards

  2. Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment, University of Torino, Medical School, Str prov 142 Km 3.95, 10060 Candiolo, Torino, Italy,

    Federica Di Nicolantonio, Davide Zecchin & Alberto Bardelli

  3. FIRC-IFOM Institute for Molecular Oncology, Via Adamello 16, 20100 Milan, Italy,

    Federica Di Nicolantonio & Alberto Bardelli

  4. IDIBELL, Institut Català d’Oncologia L’Hospitalet de Llobregat, Gran Via s/n, km 2, 7 08907 L’Hospitalet-Barcelona, Spain,

    Ramon Salazar

Authors
  1. Anirudh Prahallad
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Contributions

R.B. and A.B. conceived the project and supervised all research. R.B., A.P. and S.H. wrote the manuscript. S.H., R.S. and RL.B. designed the experiments. A.P., C.S., S.H., F.D.N. and D.Z. performed the experiments.

Corresponding author

Correspondence to René Bernards.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-6 with legends. (PDF 2490 kb)

Supplementary Table 1

This file shows kinases screened for enhancers of PLX4032 sensitivity. Listed are the gene symbols for the genes tested in the PLX4032 sensitivity screen and the number of shRNAs for each gene present in the library. (XLS 349 kb)

Supplementary Table 2

This file shows the top shRNA candidates from the 'dropout' RNAi screen for enhancers of PLX4032 sensitivity. Listed are the 22 top shRNA candidates that had been sequenced at least 300 times and which were depleted at least five-fold by the drug treatment. (XLS 37 kb)

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Prahallad, A., Sun, C., Huang, S. et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 483, 100–103 (2012). https://doi.org/10.1038/nature10868

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