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A Braf kinase-inactive mutant induces lung adenocarcinoma

Nature volume 548, pages 239–243 (2017)Cite this article

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Abstract

The initiating oncogenic event in almost half of human lung adenocarcinomas is still unknown, a fact that complicates the development of selective targeted therapies. Yet these tumours harbour a number of alterations without obvious oncogenic function including BRAF-inactivating mutations. Inactivating BRAF mutants in lung predominate over the activating V600E mutant that is frequently observed in other tumour types1. Here we demonstrate that the expression of an endogenous Braf(D631A) kinase-inactive isoform in mice (corresponding to the human BRAF(D594A) mutation) triggers lung adenocarcinoma in vivo, indicating that BRAF-inactivating mutations are initiating events in lung oncogenesis. Moreover, inactivating BRAF mutations have also been identified in a subset of KRAS-driven human lung tumours. Co-expression of Kras(G12V) and Braf(D631A) in mouse lung cells markedly enhances tumour initiation, a phenomenon mediated by Craf kinase activity2,3, and effectively accelerates tumour progression when activated in advanced lung adenocarcinomas. We also report a key role for the wild-type Braf kinase in sustaining Kras(G12V)/Braf(D631A)-driven tumours. Ablation of the wild-type Braf allele prevents the development of lung adenocarcinoma by inducing a further increase in MAPK signalling that results in oncogenic toxicity; this effect can be abolished by pharmacological inhibition of Mek to restore tumour growth. However, the loss of wild-type Braf also induces transdifferentiation of club cells, which leads to the rapid development of lethal intrabronchiolar lesions. These observations indicate that the signal intensity of the MAPK pathway is a critical determinant not only in tumour development, but also in dictating the nature of the cancer-initiating cell and ultimately the resulting tumour phenotype.

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Figure 1: Combinations of KrasG12V, Braf D631A and wild-type Braf alleles establish a MAPK activity window that determines cell transformation and oncogene toxicity.
Figure 2: Mice concomitantly expressing Kras(G12V) and Braf(D631A) show reduced survival and increased tumour development mediated by Craf kinase activity.
Figure 3: Hyperactive MAPK signalling triggers bronchiolar carcinoma by transdifferentiation of club cells.
Figure 4: Activation of endogenous Braf D631A results in lung adenocarcinoma development.

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References

  1. Chang, M. T. et al. Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity. Nat. Biotechnol. 34, 155–163 (2016)

    Article  CAS  Google Scholar 

  2. Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010)

    Article  CAS  Google Scholar 

  3. Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010)

    Article  CAS  ADS  Google Scholar 

  4. Sebolt-Leopold, J. S. & Herrera, R. Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat. Rev. Cancer 4, 937–947 (2004)

    Article  CAS  Google Scholar 

  5. Rauen, K. A. The RASopathies. Annu. Rev. Genomics Hum. Genet. 14, 355–369 (2013)

    Article  CAS  Google Scholar 

  6. Hills, S. A. & Diffley, J. F. X. DNA replication and oncogene-induced replicative stress. Curr. Biol. 24, R435–R444 (2014)

    Article  CAS  Google Scholar 

  7. Blasco, R. B. et al. c-Raf, but not Braf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma. Cancer Cell 19, 652–663 (2011)

    Article  CAS  Google Scholar 

  8. Kamata, T. et al. BRAF inactivation drives aneuploidy by deregulating CRAF. Cancer Res. 70, 8475–8486 (2010)

    Article  CAS  Google Scholar 

  9. Guerra, C. et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4, 111–120 (2003)

    Article  CAS  Google Scholar 

  10. Mainardi, S. et al. Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma. Proc. Natl Acad. Sci. USA 111, 255–260 (2014)

    Article  CAS  ADS  Google Scholar 

  11. Karreth, F. A., Frese, K. K., DeNicola, G. M., Baccarini, M. & Tuveson, D. A. C-Raf is required for the initiation of lung cancer by K-Ras(G12D). Cancer Discov. 1, 128–136 (2011)

    Article  CAS  Google Scholar 

  12. Bartkova, J. et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870 (2005)

    Article  CAS  ADS  Google Scholar 

  13. Gorgoulis, V. G. et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434, 907–913 (2005)

    Article  CAS  ADS  Google Scholar 

  14. Dietlein, F. et al. A synergistic interaction between Chk1- and MK2 inhibitors in KRAS-mutant cancer. Cell 162, 146–159 (2015)

    Article  CAS  Google Scholar 

  15. Deschênes-Simard, X. et al. Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation. Genes Dev. 27, 900–915 (2013)

    Article  Google Scholar 

  16. Cheung, W. K. C. & Nguyen, D. X. Lineage factors and differentiation states in lung cancer progression. Oncogene 34, 5771–5780 (2015)

    Article  CAS  Google Scholar 

  17. Andreadi, C. et al. The intermediate-activity (L597V)BRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway. Genes Dev. 26, 1945–1958 (2012)

    Article  CAS  Google Scholar 

  18. Langer, C. J., Besse, B., Gualberto, A., Brambilla, E. & Soria, J.-C. The evolving role of histology in the management of advanced non-small-cell lung cancer. J. Clin. Oncol. 28, 5311–5320 (2010)

    Article  Google Scholar 

  19. Sutherland, K. D. et al. Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung. Cancer Cell 19, 754–764 (2011)

    Article  CAS  Google Scholar 

  20. Xu, X. et al. Evidence for type II cells as cells of origin of K-Ras-induced distal lung adenocarcinoma. Proc. Natl Acad. Sci. USA 109, 4910–4915 (2012)

    Article  CAS  ADS  Google Scholar 

  21. Sutherland, K. D. et al. Multiple cells-of-origin of mutant K-Ras-induced mouse lung adenocarcinoma. Proc. Natl Acad. Sci. USA 111, 4952–4957 (2014)

    Article  CAS  ADS  Google Scholar 

  22. Buchert, M. et al. Genetic dissection of differential signaling threshold requirements for the Wnt/beta-catenin pathway in vivo. PLoS Genet. 6, e1000816 (2010)

    Article  Google Scholar 

  23. Papa, A. et al. Cancer-associated PTEN mutants act in a dominant-negative manner to suppress PTEN protein function. Cell 157, 595–610 (2014)

    Article  CAS  Google Scholar 

  24. Yao, Z. et al. Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS. Naturehttp://dx.doi.org/10.1038/nature23291 (2017)

  25. Doma, E. et al. Skin tumorigenesis stimulated by Raf inhibitors relies upon Raf functions that are dependent and independent of ERK. Cancer Res. 73, 6926–6937 (2013)

    Article  CAS  Google Scholar 

  26. Kammouni, W. et al. Increased K-ras protein and activity in mouse and human lung epithelial cells at confluence. Cell Growth Differ. 13, 441–448 (2002)

    CAS  PubMed  Google Scholar 

  27. Carter, J. et al. Non-p.V600E BRAF mutations are common using a more sensitive and broad detection tool. Am. J. Clin. Pathol. 144, 620–628 (2015)

    Article  CAS  Google Scholar 

  28. Chen, A. P. et al. Forebrain-specific knockout of B-raf kinase leads to deficits in hippocampal long-term potentiation, learning, and memory. J. Neurosci. Res. 83, 28–38 (2006)

    Article  CAS  Google Scholar 

  29. Dankort, D. et al. A new mouse model to explore the initiation, progression, and therapy of BRAFV600E-induced lung tumors. Genes Dev. 21, 379–384 (2007)

    Article  CAS  Google Scholar 

  30. Jonkers, J. et al. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29, 418–425 (2001)

    Article  CAS  Google Scholar 

  31. Jackson, E. L. et al. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res. 65, 10280–10288 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. de Martino for histopathological evaluation of murine lung tumours. This work was supported by grants to M.B. from the European Research Council (ERC-AG/250297-RAS AHEAD), EU-Framework Programme (HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010-260791/EUROCANPLATFORM) and Spanish Ministry of Economy and Competitiveness (SAF2011-30173 and SAF2014-59864-R). M.B. is the recipient of an Endowed Chair from the AXA Research Fund. Funding was also provided by grants to N.R. from the National Institutes of Health (P01 CA129243; R35 CA210085); the Commonwealth Foundation for Cancer Research, the Center for Experimental Therapeutics at Memorial Sloan Kettering Cancer Center and the Stand Up To Cancer – American Cancer Society Lung Cancer Dream Team Translational Research Grant (SU2C-AACR-DT17-15). Support was also received from the NIH MSKCC Cancer Center Support Grant P30 CA008748. Work in the laboratory of R.C. was supported by grants FP7 ERC-2009-StG (242965-Lunely) and Associazione Italiana per la Ricerca sul Cancro (AIRC) grant IG-12023. P.N. was the recipient of an FPU fellowship from the Spanish Ministry of Education. C.A. was the recipient of a postdoctoral fellowship from the Spanish Association Against Cancer (AECC).

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Author notes

  1. Chiara Ambrogio

    Present address: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, 02215, USA

  2. David Santamaría

    Present address: University of Bordeaux, INSERM U1218, ACTION Laboratory, IECB, F-33600, Pessac, France

  3. Mariano Barbacid and David Santamaría: These authors jointly supervised this work.

Authors and Affiliations

  1. Experimental Oncology, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, 28029, Spain

    Patricia Nieto, Chiara Ambrogio, Laura Esteban-Burgos, María Teresa Blasco, Mariano Barbacid & David Santamaría

  2. Bioinformatics Unit, Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, 28029, Spain

    Gonzalo Gómez-López & David G. Pisano

  3. Program in Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, USA

    Zhan Yao & Neal Rosen

  4. Molecular Oncology Group, Cancer Research UK Manchester Institute, Manchester, M20 4BX, UK

    Richard Marais

  5. Department of Pathology, Harvard Medical School, Boston Children’s Hospital, Boston, Massachusetts, USA

    Roberto Chiarle

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Contributions

D.S. and M.B. designed experiments and research aims, analysed data and wrote the manuscript with help from co-authors. P.N. performed experiments and analysed the data with help from C.A., L.E. and M.T.B. R.C. carried out critical interpretation of the tumour phenotype. R.M. provided the Braf+/LSLD631A strain. Z.Y., N.R, R.M. and R.C. contributed critical information and helpful discussions. D.G.P. and G.G.-L. performed the bioinformatic analysis of human datasets.

Corresponding authors

Correspondence to Mariano Barbacid or David Santamaría.

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Competing interests

N.R. is on the scientific advisory board of and receives research funding from Chugai, on the scientific advisory board of and owns stock in Beigene, Wellspring and Kura. N.R. is also on the scientific advisory board of Daiichi-Sankyo, AstraZeneca and Takeda, and is a consultant to Novartis.

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Reviewer Information Nature thanks L. Garraway and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Braf(D631A)-dependent activation of MAPK signalling in vitro.

a, Western blot analysis of the expression levels of the three Raf isoforms in lysates derived from Kras+/G12V;Araf+/+;Braf lox/lox;Craf lox/lox;Trp53–/–;Tg.hUb-cre-ERT2+/T lung adenocarcinoma cell lines. The endogenous Araf alleles were eliminated using CRISPR–Cas9 editing where indicated. Braf lox and Craf lox alleles were eliminated by addition of 4-hydroxytamoxifen (4-OHT) to induce their Cre-mediated recombination where indicated. Gapdh is shown as a loading control. b, Kras+/G12V;A-Raf–/–;Braf lox/lox;Craf lox/lox;Trp53–/–;Tg.hUb-cre-ERT2+/T lung adenocarcinoma cell lines were infected with lentiviral particles expressing combinations of wild-type Braf, kinase-inactive Braf(D631A) and wild-type Craf as indicated before the addition of 4-hydroxytamoxifen to induce elimination of the endogenous conditional Braf and Craf alleles. Cells were serum-starved for 24 h and re-stimulated with serum for the indicated times. The activation of MAPK signalling was assessed by western blot analysis of p-Erk1/2. Expression levels of the exogenous Braf and Craf alleles as well as endogenous Erk1/2 and Gapdh are shown as loading controls. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 Mouse alleles and strains used in the study.

a, Expression of the endogenous KrasG12V oncogene is achieved by Cre-mediated excision of the transcriptional STOP cassette preceding the KrasLSLG12Vgeo allele. Cells expressing the oncogene can be identified by X-gal staining owing to the co-expression of a bicistronic β-galactosidase (β-geo) reporter. Likewise, Cre-mediated excision of the lox-STOP-lox cassette (containing the indicated wild-type exons) induces the expression of the kinase-dead Braf(D631A) and Craf(D468A) mutants instead of the wild-type isoforms. Finally, Cre-mediated excision of the conditional Braf lox allele results in the elimination of the wild-type Braf protein. b, Schematic representation of the proteins expressed in lung cells following intratracheal infection with Ad-Cre virus in the compound strains used in the study.

Extended Data Figure 3 Activation of p-Erk1/2 in lung adenocarcinomas driven by concomitant expression of Kras(G12V) and Braf(D631A) depends on Craf kinase activity.

Representative images of p-Erk1/2 immunostaining of paraffin-embedded lung sections (n = 3 per genotype) from Ad-Cre-infected Kras+/LSLG12Vgeo;Braf+/LSLD631A (KB), Kras+/LSLG12Vgeo;Braf+/LSLD631A;Craf LSLD468A/LSLD468A (KBCKD) and Kras+/LSLG12Vgeo;Braf+/LSLD631A;Craf lox/lox (KBCL). Tumour samples were collected 6 months after Ad-Cre intratracheal infection coincident with the humane end point of the KB strain. Scale bar, 1 mm.

Extended Data Figure 4 MAPK hyperactivation induces transdifferentiation of the bronchiolar epithelium leading to papillary carcinoma.

Immunostaining of paraffin-embedded sections showing transdifferentiation of the bronchiolar epithelium at early stages (1 week) after Ad-Cre infection of Kras+/LSLG12Vgeo;Braf lox/LSLD631A (KBL). Images display staining using antibodies against CC10 (club cell marker, left panels) and SPC (AT2 marker, right panels). Protruding bronchiolar papillary growth is invariably associated with a transdifferentiation process illustrated by the acquisition of SPC+ staining (red arrowheads). Scale bar, 100 μm (top panels), 50 μm (bottom panels).

Extended Data Figure 5 Expression of an endogenous Braf(D631A) inactive mutant allele in mice harbouring KrasG12V-driven lung adenocarcinomas detectable by CT increases tumour growth and reduces survival.

a, Survival of Kras+/FSFG12V;Braf+/LSLD631A;Tg.hUb-cre-ERT2+/T (KFB, solid circles n = 14) and control Kras+/FSFG12V;Tg.hUb-cre-ERT2+/T (KF, empty circles n = 13) mice bearing lung adenocarcinomas detectable by CT. Upon tumour detection mice were maintained on a tamoxifen-containing diet to activate expression of the Braf(D631A) kinase-dead isoform. P < 0.0058, obtained using the log-rank test (Mantel–Cox). b, Waterfall plot representation of the tumour volume increase (measured as fold change) when re-evaluated by CT after 8 weeks of continuous tamoxifen-containing diet in Kras+/FSFG12V;Braf+/LSLD631A;Tg.hUb-cre-ERT2+/T (KFB, n = 29 tumours from 14 mice) and control Kras+/FSFG12V;Tg.hUb-cre-ERT2+/T (KF, n = 38 tumours from 15 mice) cohorts. The same dataset is represented in Fig. 4a.

Extended Data Figure 6 Histology of Braf(D631A)-driven lung adenocarcinoma and evaluation of Braf(D631A)-dependent activation of MAPK signalling in vitro.

a, Haematoxylin and eosin staining of paraffin-embedded lung sections from Ad-Cre-infected Braf+/LSLD631A mice killed at humane end point. Sections display moderately to poorly circumscribed tumours occupying nearly an entire lung lobe, with compressed adjacent lung parenchyma and peripherally infiltrated by mononuclear inflammatory cells, mainly macrophages. Tumour masses are composed by papillary outgrowths or less-differentiated areas with increased cellular pleomorphism. Images correspond to three independent mice. Scale bar, 100 μm. b, Schematic representation of BRAF mutations detected in human tumours with high (top) or low/absent (bottom) kinase activity when compared to the wild-type isoform (see also the accompanying manuscript by Yao et al.24). c, Primary keratinocytes derived from Braf+/LSLD631A (B), Braf lox/LSLD631A (BL) and Braf+/LSLD631A; Craf lox/lox (BC) mice were infected with Ad-Cre (5 MOI) and treated with recombinant EGF (150 ng ml−1 for 1 h) where indicated. The activation of MAPK signalling was assessed by western blot analysis of p-Erk1/2, demonstrating that the activation of MAPK signalling by Braf(D631A) in epithelial cells requires upstream RTK activation and is enhanced by elimination of the wild-type Braf allele. Efficient recombination of the Craf conditional alleles in Ad-Cre-infected BC cells is also shown, demonstrating that MAPK activation by Braf(D631A) is Craf-dependent. Expression levels of Erk1/2 and Gapdh are shown as loading controls. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7 Co-occurring alterations in lung adenocarcinoma patients with BRAF-inactivating mutations.

Whole-genome sequencing data available from the TCGA LUAD study at cBioportal (http://www.cbioportal.org) was used to generate a gene network for the analysis of genetic alterations coincident with BRAF hypoactive mutations. PIK3CA mutations (Q296E, E542K and E545K) were the most frequent co-occurring event. In addition, mutations or gene deletions in known RTK signalling antagonists (DOK2, SPRY2), MAPK scaffolds (KSR1), members of the RAS subfamily (RAP1A, RIT1) or the guanine nucleotide exchange factor TRIO might potentially cooperate with inactive BRAF to sustain MAPK activity.

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Nieto, P., Ambrogio, C., Esteban-Burgos, L. et al. A Braf kinase-inactive mutant induces lung adenocarcinoma. Nature 548, 239–243 (2017). https://doi.org/10.1038/nature23297

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