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.

  • Original Article
  • Published:

Opposing role of Notch1 and Notch2 in a KrasG12D-driven murine non-small cell lung cancer model

Oncogene volume 34, pages 578–588 (2015)Cite this article

Subjects

Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide. Recently, we have shown that Notch1 inhibition resulted in substantial cell death of non-small cell lung cancer (NSCLC) cells in vitro. New compounds targeting Notch signal transduction have been developed and are now being tested in clinical trials. However, the tumorigenic role of individual Notch receptors in vivo remains largely unclear. Using a KrasG12D-driven endogenous NSCLC mouse model, we analyzed the effect of conditional Notch1 and Notch2 receptor deletion on NSCLC tumorigenesis. Notch1 deficiency led to a reduced early tumor formation and lower activity of MAPK compared with the controls. Unexpectedly, Notch2 deletion resulted in a dramatically increased carcinogenesis and increased MAPK activity. These mice died significantly earlier due to rapidly growing tumor burden. We found that Notch1 regulates Ras/MAPK pathway via HES1-induced repression of the DUSP1 promoter encoding a phosphatase specifically suppressing pERK1/2. Interestingly, Notch1 but not Notch2 ablation leads to decreased HES1 and DUSP1 expression. However, Notch2-depleted tumors showed an appreciable increase in β-catenin expression, a known activator of HES1 and important lung cancer oncogene. Characteristically for β-catenin upregulation, we found that the majority of Notch2-deficient tumors revealed an undifferentiated phenotype as determined by their morphology, E-Cadherin and TTF1 expression levels. In addition, these carcinomas showed aggressive growth patterns with bronchus invasion and obstruction. Together, we show that Notch2 mediates differentiation and has tumor suppressor functions during lung carcinogenesis, whereas Notch1 promotes tumor initiation and progression. These data are further supported by immunohistochemical analysis of human NSCLC samples showing loss or downregulation of Notch2 compared with normal lung tissue. In conclusion, this is the first study characterizing the in vivo functions of Notch1 and Notch2 in KrasG12D-driven NSCLC tumorigenesis. These data highlight the clinical importance of a thorough understanding of Notch signaling especially with regard to Notch-targeted therapies.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Jemal A, Siegel R, Xu J, Ward E . Cancer statistics. CA: a cancer journal for clinicians 2010; 60: 277–300.

    Google Scholar 

  2. Artavanis-Tsakonas S, Rand MD, Lake RJ . Notch signaling: cell fate control and signal integration in development. Science 1999; 284: 770–776.

    Article  CAS  PubMed  Google Scholar 

  3. Bray SJ . Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 2006; 7: 678–689.

    Article  CAS  PubMed  Google Scholar 

  4. Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD et al. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 1991; 66: 649–661.

    Article  CAS  PubMed  Google Scholar 

  5. Baumgart A, Seidl S, Vlachou P, Michel L, Mitova N, Schatz N et al. ADAM17 regulates epidermal growth factor receptor expression through the activation of Notch1 in non-small cell lung cancer. Cancer Res 2010; 70: 5368–5378.

    Article  CAS  PubMed  Google Scholar 

  6. Mazur PK, Gruner BM, Nakhai H, Sipos B, Zimber-Strobl U, Strobl LJ et al. Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo. PLoS One 2010; 5: e13578.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Mazur PK, Einwachter H, Lee M, Sipos B, Nakhai H, Rad R et al. Notch2 is required for progression of pancreatic intraepithelial neoplasia and development of pancreatic ductal adenocarcinoma. Proc Natl Acad Sci USA 2010; 107: 13438–13443.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Weijzen S, Rizzo P, Braid M, Vaishnav R, Jonkheer SM, Zlobin A et al. Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nature Med 2002; 8: 979–986.

    Article  CAS  PubMed  Google Scholar 

  9. Talora C, Sgroi DC, Crum CP, Dotto GP . Specific down-modulation of Notch1 signaling in cervical cancer cells is required for sustained HPV-E6/E7 expression and late steps of malignant transformation. Genes Develop 2002; 16: 2252–2263.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Westhoff B, Colaluca IN, D'Ario G, Donzelli M, Tosoni D, Volorio S et al. Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci USA 2009; 106: 22293–22298.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Konishi J, Kawaguchi KS, Vo H, Haruki N, Gonzalez A, Carbone DP et al. Gamma-secretase inhibitor prevents Notch3 activation and reduces proliferation in human lung cancers. Cancer Res 2007; 67: 8051–8057.

    Article  CAS  PubMed  Google Scholar 

  12. Zheng Y, de la Cruz CC, Sayles LC, Alleyne-Chin C, Vaka D, Knaak TD et al. A rare population of CD24(+)ITGB4(+)Notch(hi) cells drives tumor propagation in NSCLC and requires Notch3 for self-renewal. Cancer Cell 2013; 24: 59–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. ClinicalTrals.gov http://clinicaltrials.gov/.

  14. Maraver A, Serrano M . Notching up a new therapeutic strategy for non-small cell lung carcinoma (NSCLC). Oncotarget 2012; 3: 917–918.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Nicolas M, Wolfer A, Raj K, Kummer JA, Mill P, van Noort M et al. Notch1 functions as a tumor suppressor in mouse skin. Nat Genet 2003; 33: 416–421.

    Article  CAS  PubMed  Google Scholar 

  16. Sriuranpong V, Borges MW, Ravi RK, Arnold DR, Nelkin BD, Baylin SB et al. Notch signaling induces cell cycle arrest in small cell lung cancer cells. Cancer Res 2001; 61: 3200–3205.

    CAS  PubMed  Google Scholar 

  17. Jackson EL, Willis N, Mercer K, Bronson RT, Crowley D, Montoya R et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Develop 2001; 15: 3243–3248.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. DuPage M, Dooley AL, Jacks T . Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat Protocols 2009; 4: 1064–1072.

    Article  CAS  PubMed  Google Scholar 

  19. Kopan R, Ilagan MX . The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 2009; 137: 216–233.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Junttila MR, Karnezis AN, Garcia D, Madriles F, Kortlever RM, Rostker F et al. Selective activation of p53-mediated tumour suppression in high-grade tumours. Nature 2010; 468: 567–571.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Maraver A, Fernandez-Marcos PJ, Herranz D, Canamero M, Munoz-Martin M, Gomez-Lopez G et al. Therapeutic effect of gamma-secretase inhibition in KrasG12V-driven non-small cell lung carcinoma by derepression of DUSP1 and inhibition of ERK. Cancer Cell 2012; 22: 222–234.

    Article  CAS  PubMed  Google Scholar 

  22. Hanlon L, Avila JL, Demarest RM, Troutman S, Allen M, Ratti F et al. Notch1 functions as a tumor suppressor in a model of K-ras-induced pancreatic ductal adenocarcinoma. Cancer Res 2010; 70: 4280–4286.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fan X, Mikolaenko I, Elhassan I, Ni X, Wang Y, Ball D et al. Notch1 and notch2 have opposite effects on embryonal brain tumor growth. Cancer Res 2004; 64: 7787–7793.

    Article  CAS  PubMed  Google Scholar 

  24. Rodenhuis S, Slebos RJ, Boot AJ, Evers SG, Mooi WJ, Wagenaar SS et al. Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res 1988; 48: 5738–5741.

    CAS  PubMed  Google Scholar 

  25. Sato M, Vaughan MB, Girard L, Peyton M, Lee W, Shames DS et al. Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res 2006; 66: 2116–2128.

    Article  CAS  PubMed  Google Scholar 

  26. Radtke F, Raj K . The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nature Rev Cancer 2003; 3: 756–767.

    Article  CAS  Google Scholar 

  27. Besseyrias V, Fiorini E, Strobl LJ, Zimber-Strobl U, Dumortier A, Koch U et al. Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation. J Exp Med 2007; 204: 331–343.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Soriano P . Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999; 21: 70–71.

    Article  CAS  PubMed  Google Scholar 

  29. Feldser DM, Kostova KK, Winslow MM, Taylor SE, Cashman C, Whittaker CA et al. Stage-specific sensitivity to p53 restoration during lung cancer progression. Nature 2010; 468: 572–575.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Haruki N, Kawaguchi KS, Eichenberger S, Massion PP, Olson S, Gonzalez A et al. Dominant-negative Notch3 receptor inhibits mitogen-activated protein kinase pathway and the growth of human lung cancers. Cancer Res 2005; 65: 3555–3561.

    Article  CAS  PubMed  Google Scholar 

  31. Kim MY, Park JH, Choi EJ, Park HS . Presenilin acts as a positive regulator of basal level activity of ERK through the Raf-MEK1 signaling pathway. Biochem Biophys Res Commun 2005; 332: 609–613.

    Article  CAS  PubMed  Google Scholar 

  32. Michie AM, Chan AC, Ciofani M, Carleton M, Lefebvre JM, He Y et al. Constitutive Notch signalling promotes CD4 CD8 thymocyte differentiation in the absence of the pre-TCR complex, by mimicking pre-TCR signals. Int Immunol 2007; 19: 1421–1430.

    Article  CAS  PubMed  Google Scholar 

  33. Kim HA, Koo BK, Cho JH, Kim YY, Seong J, Chang HJ et al. Notch1 counteracts WNT/beta-catenin signaling through chromatin modification in colorectal cancer. J Clin Invest 2012; 122: 3248–3259.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pacheco-Pinedo EC, Durham AC, Stewart KM, Goss AM, Lu MM, Demayo FJ et al. Wnt/beta-catenin signaling accelerates mouse lung tumorigenesis by imposing an embryonic distal progenitor phenotype on lung epithelium. J Clin Invest 2011; 121: 1935–1945.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Peignon G, Durand A, Cacheux W, Ayrault O, Terris B, Laurent-Puig P et al. Complex interplay between beta-catenin signalling and Notch effectors in intestinal tumorigenesis. Gut 2011; 60: 166–176.

    Article  CAS  PubMed  Google Scholar 

  36. Jackson EL, Olive KP, Tuveson DA, Bronson R, Crowley D, Brown M et al. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res 2005; 65: 10280–10288.

    Article  CAS  PubMed  Google Scholar 

  37. Asnaghi L, Vass WC, Quadri R, Day PM, Qian X, Braverman R et al. E-cadherin negatively regulates neoplastic growth in non-small cell lung cancer: role of Rho GTPases. Oncogene 2010; 29: 2760–2771.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Bremnes RM, Veve R, Hirsch FR, Franklin WA . The E-cadherin cell-cell adhesion complex and lung cancer invasion, metastasis, and prognosis. Lung Cancer 2002; 36: 115–124.

    Article  PubMed  Google Scholar 

  39. Wang XQ, Li H, Van Putten V, Winn RA, Heasley LE, Nemenoff RA . Oncogenic K-Ras regulates proliferation and cell junctions in lung epithelial cells through induction of cyclooxygenase-2 and activation of metalloproteinase-9. Mol Biol Cell 2009; 20: 791–800.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ma L, Young J, Prabhala H, Pan E, Mestdagh P, Muth D et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 2010; 12: 247–256.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Saad AG, Yeap BY, Thunnissen FB, Pinkus GS, Pinkus JL, Loda M et al. Immunohistochemical markers associated with brain metastases in patients with nonsmall cell lung carcinoma. Cancer 2008; 113: 2129–2138.

    Article  PubMed  Google Scholar 

  42. von Burstin J, Eser S, Paul MC, Seidler B, Brandl M, Messer M et al. E-cadherin regulates metastasis of pancreatic cancer in vivo and is suppressed by a SNAIL/HDAC1/HDAC2 repressor complex. Gastroenterology 2009; 137: 361–371 371 e361-365.

    Article  CAS  PubMed  Google Scholar 

  43. Myong NH . Reduced expression of E-cadherin in human non-small cell lung carcinoma. Cancer Res Treatment 2004; 36: 56–61.

    Article  Google Scholar 

  44. Sjolund J, Johansson M, Manna S, Norin C, Pietras A, Beckman S et al. Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo. J Clin Invest 2008; 118: 217–228.

    Article  PubMed  Google Scholar 

  45. Huynh C, Poliseno L, Segura MF, Medicherla R, Haimovic A, Menendez S et al. The novel gamma secretase inhibitor RO4929097 reduces the tumor initiating potential of melanoma. PLoS One 2011; 6: e25264.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. van Es JH, van Gijn ME, Riccio O, van den Born M, Vooijs M, Begthel H et al. Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 2005; 435: 959–963.

    Article  CAS  PubMed  Google Scholar 

  47. Wu Y, Cain-Hom C, Choy L, Hagenbeek TJ, de Leon GP, Chen Y et al. Therapeutic antibody targeting of individual Notch receptors. Nature 2010; 464: 1052–1057.

    Article  CAS  PubMed  Google Scholar 

  48. Allen TD, Rodriguez EM, Jones KD, Bishop JM . Activated Notch1 induces lung adenomas in mice and cooperates with Myc in the generation of lung adenocarcinoma. Cancer Res 2011; 71: 6010–6018.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Osanyingbemi-Obidi J, Dobromilskaya I, Illei PB, Hann CL, Rudin CM . Notch signaling contributes to lung cancer clonogenic capacity in vitro but may be circumvented in tumorigenesis in vivo. Mol Cancer Res 2011; 9: 1746–1754.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Eliasz S, Liang S, Chen Y, De Marco MA, Machek O, Skucha S et al. Notch-1 stimulates survival of lung adenocarcinoma cells during hypoxia by activating the IGF-1R pathway. Oncogene 2010; 29: 2488–2498.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Graziani I, Eliasz S, De Marco MA, Chen Y, Pass HI, De May RM et al. Opposite effects of Notch-1 and Notch-2 on mesothelioma cell survival under hypoxia are exerted through the Akt pathway. Cancer Res 2008; 68: 9678–9685.

    Article  CAS  PubMed  Google Scholar 

  52. Cheng HT, Kim M, Valerius MT, Surendran K, Schuster-Gossler K, Gossler A et al. Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron. Development 2007; 134: 801–811.

    Article  CAS  PubMed  Google Scholar 

  53. Chu D, Zheng J, Wang W, Zhao Q, Li Y, Li J et al. Notch2 expression is decreased in colorectal cancer and related to tumor differentiation status. Ann Surg Oncol 2009; 16: 3259–3266.

    Article  PubMed  Google Scholar 

  54. Radtke F, Wilson A, Stark G, Bauer M, van Meerwijk J, MacDonald HR et al. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 1999; 10: 547–558.

    Article  CAS  PubMed  Google Scholar 

  55. Bettstetter M, Berezowska S, Keller G, Walch A, Feuchtinger A, Slotta-Huspenina J et al. Epidermal growth factor receptor, phosphatidylinositol-3-kinase catalytic subunit/PTEN, and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome. Human Pathol 2013; 44: 829–836.

    Article  CAS  Google Scholar 

  56. Slotta-Huspenina J, Koch I, de Leval L, Keller G, Klier M, Bink K et al. The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index. Haematologica 2012; 97: 1422–1430.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank Dr Tibor Schuster for the help with statistical calculations, Nadya Mitova and Birgit Geist for her excellent technical help with the experiments and Joachim Alfer for providing histological pictures. We also thank Freddy Radtke for the Notch1lox/lox mice, Ursula Zimber-Strobl for the Notch2lox/lox mice and David Tuveson and Tyler Jacks for the Kras+/LSL-G12D mice. TD received a grant from the Wilhelm Sander-Stiftung (#2005.158.1). TD and JTS have been supported by grants from the Deutsche Krebshilfe (#109606 to TD and JTS) and the Deutsche Forschungsgemeinschaft (SI 1549/1-1 to JTS). PKM was supported by the Tobacco-Related Disease Research Program, a Dean’s Fellowship from Stanford University and the Child Health Research Institute and Lucile Packard Foundation for Children’s Health at Stanford.

Author Contributions

AB and PKM contributed equally to this work and were responsible for the experimental design, execution, data analysis and manuscript preparation. MA, MR, KS, AF, KB, AW, RB, CP and JD acquired data, provided materials. JTS and TD were equally responsible for supervision of research, data interpretation and manuscript preparation.

Author information

Author notes

  1. A Baumgart and P K Mazur: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine III, Technische Universität München, Munich, Germany

    A Baumgart, K Behnke, C Peschel & T Dechow

  2. Departments of Genetics and Pediatrics, Stanford University, Stanford, CA, USA

    P K Mazur

  3. Department of Experimental Oncology, Technische Universität München, Munich, Germany

    M Anton

  4. Department of Pathology, Technische Universität München, Munich, Germany

    M Rudelius & K Schwamborn

  5. Research Unit Analytical Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

    A Feuchtinger & A Walch

  6. Institute for Diagnostic Imaging, Technische Universität München, Munich, Germany

    R Braren

  7. Department of Internal Medicine I, Universitätsklinikum Freiburg, Freiburg, Germany

    J Duyster

  8. Department of Internal Medicine II, Technische Universität München, Munich, Germany

    J T Siveke

  9. Onkologie Ravensburg, Ravensburg, Germany

    T Dechow

Authors
  1. A Baumgart
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P K Mazur
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M Anton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Rudelius
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K Schwamborn
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A Feuchtinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K Behnke
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A Walch
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. R Braren
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. C Peschel
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. J Duyster
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. J T Siveke
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. T Dechow
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to J T Siveke or T Dechow.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baumgart, A., Mazur, P., Anton, M. et al. Opposing role of Notch1 and Notch2 in a KrasG12D-driven murine non-small cell lung cancer model. Oncogene 34, 578–588 (2015). https://doi.org/10.1038/onc.2013.592

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.592

Keywords

This article is cited by

Search

Advanced search

Quick links