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Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

Abstract

Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAFV637E knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

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References

  1. Barker N . Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol 2013; 15: 19–33.

    Article  Google Scholar 

  2. Sadanandam A, Lyssiotis CA, Homicsko K, Collisson EA, Gibb WJ, Wullschleger S et al. A colorectal cancer classification system that associates cellular phenotype and responses to therapy. Nat Med 2013; 19: 619–625.

    Article  CAS  Google Scholar 

  3. Fearon ER . Molecular genetics of colorectal cancer. Annu Rev Pathol 2011; 6: 479–507.

    Article  CAS  Google Scholar 

  4. Takahashi M, Nakatsugi S, Sugimura T, Wakabayashi K . Frequent mutations of the beta-catenin gene in mouse colon tumors induced by azoxymethane. Carcinogenesis 2000; 21: 1117–1120.

    CAS  Google Scholar 

  5. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 1992; 256: 668–670.

    Article  CAS  Google Scholar 

  6. Niehrs C . The complex world of WNT receptor signalling. Nat Rev Mol Cell Biol 2012; 13: 767–779.

    Article  CAS  Google Scholar 

  7. van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A et al. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 2002; 111: 241–250.

    Article  CAS  Google Scholar 

  8. van Es JH, Haegebarth A, Kujala P, Itzkovitz S, Koo B-K, Boj SF et al. A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal. Mol Cell Biol 2012; 32: 1918–1927.

    Article  CAS  Google Scholar 

  9. Farrall AL, Riemer P, Leushacke M, Sreekumar A, Grimm C, Herrmann BG et al. Wnt and BMP signals control intestinal adenoma cell fates. Int J Cancer 2012; 131: 2242–2252.

    Article  CAS  Google Scholar 

  10. Snover DC . Update on the serrated pathway to colorectal carcinoma. Hum Pathol 2011; 42: 1–10.

    Article  Google Scholar 

  11. Bettington M, Walker N, Clouston A, Brown I, Leggett B, Whitehall V . The serrated pathway to colorectal carcinoma: current concepts and challenges. Histopathology 2013; 62: 367–386.

    Article  Google Scholar 

  12. Laiho P, Kokko A, Vanharanta S, Salovaara R, Sammalkorpi H, Järvinen H et al. Serrated carcinomas form a subclass of colorectal cancer with distinct molecular basis. Oncogene 2007; 26: 312–320.

    Article  CAS  Google Scholar 

  13. Osaki LH, Gama P . MAPKs and signal transduction in the control of gastrointestinal epithelial cell proliferation and differentiation. Int J Mol Sci 2013; 14: 10143–10161.

    Article  Google Scholar 

  14. Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012; 487: 330–337.

    Article  Google Scholar 

  15. Lao VV, Grady WM . Epigenetics and colorectal cancer. Nat Rev Gastroenterol Hepatol 2011; 8: 686–700.

    Article  CAS  Google Scholar 

  16. Wu JM, Montgomery EA, Iacobuzio-Donahue CA . Frequent beta-catenin nuclear labeling in sessile serrated polyps of the colorectum with neoplastic potential. Am J Clin Pathol 2008; 129: 416–423.

    Article  CAS  Google Scholar 

  17. Yachida S, Mudali S, Martin SA, Montgomery EA, Iacobuzio-Donahue CA . Beta-catenin nuclear labeling is a common feature of sessile serrated adenomas and correlates with early neoplastic progression after BRAF activation. Am J Surg Pathol 2009; 33: 1823–1832.

    Article  Google Scholar 

  18. Rad R, Cadiñanos J, Rad L, Varela I, Strong A, Kriegl L et al. A genetic progression model of Braf(V600E)-induced intestinal tumorigenesis reveals targets for therapeutic intervention. Cancer Cell 2013; 24: 15–29.

    Article  CAS  Google Scholar 

  19. El-Osta H, Falchook G, Tsimberidou A, Hong D, Naing A, Kim K et al. BRAF mutations in advanced cancers: clinical characteristics and outcomes. PLoS ONE 2011; 6: e25806.

    Article  CAS  Google Scholar 

  20. Wan P, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004; 116: 855–867.

    Article  CAS  Google Scholar 

  21. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999; 96: 857–868.

    Article  CAS  Google Scholar 

  22. Vidigal JA, Morkel M, Wittler L, Brouwer-Lehmitz A, Grote P, Macura K et al. An inducible RNA interference system for the functional dissection of mouse embryogenesis. Nucleic Acids Res 2010; 38: e122.

    Article  Google Scholar 

  23. Muñoz J, Stange DE, Schepers AG, van de Wetering M, Koo B-K, Itzkovitz S et al. The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent “+4” cell markers. EMBO J 2012; 31: 3079–3091.

    Article  Google Scholar 

  24. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007; 449: 1003–1007.

    Article  CAS  Google Scholar 

  25. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR . Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 2008; 132: 363–374.

    Article  CAS  Google Scholar 

  26. Carragher LAS, Snell KR, Giblett SM, Aldridge VSS, Patel B, Cook SJ et al. V600EBraf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16INK4a. EMBO Mol Med 2010; 2: 458–471.

    Article  CAS  Google Scholar 

  27. Gonzalo DH, Lai KK, Shadrach B, Goldblum JR, Bennett AE, Downs-Kelly E et al. Gene expression profiling of serrated polyps identifies annexin A10 as a marker of a sessile serrated adenoma/polyp. J Pathol 2013; 230: 420–429.

    Article  CAS  Google Scholar 

  28. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102: 15545–15550.

    Article  CAS  Google Scholar 

  29. Jürchott K, Kuban R-J, Krech T, Blüthgen N, Stein U, Walther W et al. Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway-dependent gene signatures in colorectal cancer cells. PLoS Genet 2010; 6: e1001231.

    Article  Google Scholar 

  30. Azzolin L, Zanconato F, Bresolin S, Forcato M, Basso G, Bicciato S et al. Role of TAZ as mediator of Wnt signaling. Cell 2012; 151: 1443–1456.

    Article  CAS  Google Scholar 

  31. Van der Flier L, Sabates Bellver J, Oving I . The intestinal Wnt/TCF signature. Gastroenterology 2007; 132: 628–632.

    Article  CAS  Google Scholar 

  32. Merlos-Suárez A, Barriga FM, Jung P, Iglesias M, Céspedes MV, Rossell D et al. The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 2011; 8: 511–524.

    Article  Google Scholar 

  33. Sato T, Stange DE, Ferrante M, Vries RGJ, van Es JH, van den Brink S et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 2011; 141: 1762–1772.

    Article  CAS  Google Scholar 

  34. Farin HF, van Es JH, Clevers H . Redundant sources of wnt regulate intestinal stem cells and promote formation of paneth cells. Gastroenterology 2012; 143: e7.

    Article  Google Scholar 

  35. Tsai J, Lee JT, Wang W, Zhang J, Cho H, Mamo S et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci USA 2008; 105: 3041–3046.

    Article  CAS  Google Scholar 

  36. Yeh TC, Marsh V, Bernat BA, Ballard J, Colwell H, Evans RJ et al. Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res 2007; 13: 1576–1583.

    Article  CAS  Google Scholar 

  37. Folkes AJ, Ahmadi K, Alderton WK, Alix S, Baker SJ, Box G et al. The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer. J Med Chem 2008; 51: 5522–5532.

    Article  CAS  Google Scholar 

  38. Murray JT, Campbell DG, Morrice N, Auld GC, Shpiro N, Marquez R et al. Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3. Biochem J 2004; 384: 477–488.

    Article  CAS  Google Scholar 

  39. Ying Q-L, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J et al. The ground state of embryonic stem cell self-renewal. Nature 2008; 453: 519–523.

    Article  CAS  Google Scholar 

  40. Kimura T, Yamamoto E, Yamano H-O, Suzuki H, Kamimae S, Nojima M et al. A novel pit pattern identifies the precursor of colorectal cancer derived from sessile serrated adenoma. Am J Gastroenterol 2012; 107: 460–469.

    Article  CAS  Google Scholar 

  41. Little AS, Balmanno K, Sale MJ, Newman S, Dry JR, Hampson M et al. Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells. Sci Signal 2011; 4: ra17.

    Article  Google Scholar 

  42. Corcoran RB, Dias-Santagata D, Bergethon K, Iafrate AJ, Settleman J, Engelman JA . BRAF gene amplification can promote acquired resistance to MEK inhibitors in cancer cells harboring the BRAF V600E mutation. Sci Signal 2010; 3: ra84.

    Article  CAS  Google Scholar 

  43. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D . RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011; 11: 761–774.

    Article  CAS  Google Scholar 

  44. Fritsche-Guenther R, Witzel F, Sieber A, Herr R, Schmidt N, Braun S et al. Strong negative feedback from Erk to Raf confers robustness to MAPK signalling. Mol Syst Biol 2011; 7: 489.

    Article  Google Scholar 

  45. Bennecke M, Kriegl L, Bajbouj M, Retzlaff K, Robine S, Jung A et al. Ink4a/Arf and oncogene-induced senescence prevent tumor progression during alternative colorectal tumorigenesis. Cancer Cell 2010; 18: 135–146.

    Article  CAS  Google Scholar 

  46. Schwitalla S, Fingerle AA, Cammareri P, Nebelsiek T, Göktuna SI, Ziegler PK et al. Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties. Cell 2012; 152: 25–38.

    Article  Google Scholar 

  47. Vermeulen L, Morrissey E, van der Heijden M, Nicholson AM, Sottoriva A, Buczacki S et al. Defining stem cell dynamics in models of intestinal tumor initiation. Science 2013; 342: 995–998.

    Article  CAS  Google Scholar 

  48. Moon B-S, Jeong W-J, Park J, Kim TI, Min DS, Choi K-Y . Role of oncogenic K-Ras in cancer stem cell activation by aberrant Wnt/β-catenin signaling. J Natl Cancer Inst 2014; 106: djt373.

    Article  Google Scholar 

  49. Phelps RA, Chidester S, Dehghanizadeh S, Phelps J, Sandoval IT, Rai K et al. A two-step model for colon adenoma initiation and progression caused by APC loss. Cell 2009; 137: 623–634.

    Article  CAS  Google Scholar 

  50. Jeong W-J, Yoon J, Park J-C, Lee S-H, Lee S-H, Kaduwal S et al. Ras stabilization through aberrant activation of Wnt/β-catenin signaling promotes intestinal tumorigenesis. Sci Signal 2012; 5: ra30.

    Article  Google Scholar 

  51. Biechele TL, Kulikauskas RM, Toroni RA, Lucero OM, Swift RD, James RG et al. Wnt/β-catenin signaling and AXIN1 regulate apoptosis triggered by inhibition of the mutant kinase BRAFV600E in human melanoma. Sci Signal 2012; 5: ra3.

    PubMed Central  Google Scholar 

  52. Guardavaccaro D, Clevers H . Wnt/β-catenin and MAPK signaling: allies and enemies in different battlefields. Sci Signal 2012; 5: pe15.

    Article  Google Scholar 

  53. Michaloglou C, Vredeveld LCW, Soengas MS, Denoyelle C, Kuilman T, van der Horst CMAM et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 2005; 436: 720–724.

    Article  CAS  Google Scholar 

  54. Dhomen N, Reis-Filho JS, da Rocha Dias S, Hayward R, Savage K, Delmas V et al. Oncogenic Braf induces melanocyte senescence and melanoma in mice. Cancer Cell 2009; 15: 294–303.

    Article  CAS  Google Scholar 

  55. Herr R, Wöhrle FU, Danke C, Berens C, Brummer T . A novel MCF-10A line allowing conditional oncogene expression in 3D culture. Cell Commun Signal 2011; 9: 17.

    CAS  Google Scholar 

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Acknowledgements

This work was funded in part by NGFNplus grant PKM-01GS08105 to MM and BGH and eBio grant 0316184A to MM. We gratefully acknowledge Judith Seidemann (Charité Berlin), Gaby Bläß and Karol Macura (MPI-MG Berlin) for excellent technical assistance, Sonja Banko (animal facility, MPI-MG) for maintainance of the mice, Jan Dörr (Charité Berlin) for SA-β-Galactosidase stainings, Tilman Brummer (University of Freiburg) for the gift of Caco2-tet cells and Julian Heuberger (Max-Delbrück-Center for Molecular Medicine, Berlin) for the gift of the ITF antibody.

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Riemer, P., Sreekumar, A., Reinke, S. et al. Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity. Oncogene 34, 3164–3175 (2015). https://doi.org/10.1038/onc.2014.247

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