Tumor Biology

, Volume 37, Issue 8, pp 10665–10673 | Cite as

A truncated phosphorylated p130Cas substrate domain is sufficient to drive breast cancer growth and metastasis formation in vivo

  • Joerg Kumbrink
  • Ana de la Cueva
  • Shefali Soni
  • Nadja Sailer
  • Kathrin H. Kirsch
Original Article
  • 163 Downloads

Abstract

Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6–10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6–10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.

Keywords

p130Cas Breast cancer Substrate domain Src*/SD Metastasis Bioluminescence imaging Mouse tumor model 

Notes

Acknowledgments

We gratefully acknowledge Joan Massague and Robert I. Nicholson for cell lines and Amy H. Bouton for the CasB antibody. We thank Matthew D. Layne for critical reading of the manuscript. We greatly appreciate the help of Manish Bais and Tom Balon with bioluminescence imaging and of Kim Bayer in establishing the animal procedures. All bioluminescence imaging was performed at the IVIS Imaging Core of Boston University School of Medicine. This work was supported by the National Cancer Institute grant CA106468, by the National Center for Advancing Translational Sciences through the BU-CTSI grant U54TR001012, both from the National Institute of Health, and the Susan G. Komen for the Cure Breast Cancer Foundation grant KG101208.

Compliance with ethical standards

Animal experiments were performed and the mice euthanized according to guidelines of and approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University.

Supplementary material

13277_2016_4902_Fig5_ESM.gif (213 kb)
Fig. S1

Cell morphology of LM2 cells expressing Src*/SD and control constructs that did not induce significant morphological changes. Cells (A and B) Cells (100,000 cells/well in 6-well plates) stably transduced with the indicated inducible constructs were treated for 24 h with DOX or left untreated. KM, SrcKM; *, Src*. c, control (empty vector). Left panels, expression (anti-HA) and phosphorylation (p-Tyr) of the fusion molecules in WCE (30 μg) was confirmed by IB. n.s., non-specific. Right panels, cell morphology was analyzed by microscopy. Scale, 100 μm. (GIF 212 kb)

13277_2016_4902_MOESM1_ESM.eps (22.2 mb)
High Resolution Image (EPS 22773 kb)
13277_2016_4902_MOESM2_ESM.doc (32 kb)
Table S1 (DOC 32 kb)

References

  1. 1.
    Kumbrink J, Kirsch KH. Targeting Cas family proteins as a novel treatment for breast cancer, breast cancer—current and alternative therapeutic modalities. Esra Gunduz and Mehmet Gunduz (Ed), InTech. 2011;ISBN: 978-953-307-776-5:37-62.Google Scholar
  2. 2.
    Tikhmyanova N, Little JL, Golemis EA. CAS proteins in normal and pathological cell growth control. Cell Mol Life Sci. 2010;67(7):1025–48.CrossRefPubMedGoogle Scholar
  3. 3.
    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74.CrossRefPubMedGoogle Scholar
  4. 4.
    Kumbrink J, Soni S, Laumbacher B, Loesch B, Kirsch KH. Identification of novel Crk-associated substrate (p130Cas) variants with functionally distinct focal adhesion kinase binding activities. J Biol Chem. 2015;290(19):12247–55.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cabodi S, Pilar Camacho-Leal M, Di Stefano P, Defilippi P. Integrin signalling adaptors: not only figurants in the cancer story. Nat Rev Cancer. 2010;10(12):858–70.CrossRefPubMedGoogle Scholar
  6. 6.
    Nikonova AS, Gaponova AV, Kudinov AE, Golemis EA. CAS proteins in health and disease: an update. IUBMB Life. 2014;66(6):387–95.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Dorssers LC, van der Flier S, Brinkman A, van Agthoven T, Veldscholte J, Berns EM, et al. Tamoxifen resistance in breast cancer: elucidating mechanisms. Drugs. 2001;61(12):1721–33.CrossRefPubMedGoogle Scholar
  8. 8.
    Ta HQ, Thomas KS, Schrecengost RS, Bouton AH. A novel association between p130Cas and resistance to the chemotherapeutic drug adriamycin in human breast cancer cells. Cancer Res. 2008;68(21):8796–804.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kirsch KH, Kensinger M, Hanafusa H, August A. A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling. BMC Cell Biol. 2002;3:18.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Soni S, Lin BT, August A, Nicholson RI, Kirsch KH. Expression of a phosphorylated p130(Cas) substrate domain attenuates the phosphatidylinositol 3-kinase/Akt survival pathway in tamoxifen resistant breast cancer cells. J Cell Biochem. 2009;107(2):364–75.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhao Y, Kumbrink J, Lin BT, Bouton AH, Yang S, Toselli PA, et al. Expression of a phosphorylated substrate domain of p130Cas promotes PyMT-induced c-Src-dependent murine breast cancer progression. Carcinogenesis. 2013;34(12):2880–90.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol. 2003;163(5):2113–26.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436(7050):518–24.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kumbrink J, Kirsch KH. Regulation of p130(Cas)/BCAR1 expression in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells by EGR1 and NAB2. Neoplasia. 2012;14(2):108–20.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kumbrink J, Kirsch KH. p130Cas acts as survival factor during PMA-induced apoptosis in HL-60 promyelocytic leukemia cells. Int J Biochem Cell Biol. 2013;45(3):531–5.CrossRefPubMedGoogle Scholar
  16. 16.
    Huang J, Hamasaki H, Nakamoto T, Honda H, Hirai H, Saito M, et al. Differential regulation of cell migration, actin stress fiber organization, and cell transformation by functional domains of Crk-associated substrate. J Biol Chem. 2002;277(30):27265–72.CrossRefPubMedGoogle Scholar
  17. 17.
    Shin NY, Dise RS, Schneider-Mergener J, Ritchie MD, Kilkenny DM, Hanks SK. Subsets of the major tyrosine phosphorylation sites in Crk-associated substrate (CAS) are sufficient to promote cell migration. J Biol Chem. 2004;279(37):38331–7.CrossRefPubMedGoogle Scholar
  18. 18.
    Hornbeck PV, Chabra I, Kornhauser JM, Skrzypek E, Zhang B. PhosphoSite: a bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics. 2004;4(6):1551–61.CrossRefPubMedGoogle Scholar
  19. 19.
    Machiyama H, Hirata H, Loh XK, Kanchi MM, Fujita H, Tan SH, et al. Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration. J Cell Sci. 2014;127(Pt 16):3440–50.CrossRefPubMedGoogle Scholar
  20. 20.
    Cunningham-Edmondson AC, Hanks SK. p130Cas substrate domain signaling promotes migration, invasion, and survival of estrogen receptor-negative breast cancer cells. Breast Cancer (Dove Med Press). 2009;1:39–52.Google Scholar
  21. 21.
    Donato DM, Ryzhova LM, Meenderink LM, Kaverina I, Hanks SK. Dynamics and mechanism of p130Cas localization to focal adhesions. J Biol Chem. 2010;285(27):20769–79.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Camacho Leal MP, Sciortino M, Tornillo G, Colombo S, Defilippi P, Cabodi S. p130Cas/BCAR1 scaffold protein in tissue homeostasis and pathogenesis. Gene. 2015;562(1):1–7.CrossRefGoogle Scholar
  23. 23.
    Janostiak R, Pataki AC, Brabek J, Rosel D. Mechanosensors in integrin signaling: the emerging role of p130Cas. Eur J Cell Biol. 2014;93(10-12):445–54.CrossRefPubMedGoogle Scholar
  24. 24.
    van der Flier S, Brinkman A, Look MP, Kok EM, Meijer-van Gelder ME, Klijn JG, et al. Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment. J Natl Cancer Inst. 2000;92(2):120–7.CrossRefGoogle Scholar
  25. 25.
    Konstantinovsky S, Smith Y, Zilber S, Tuft SH, Becker AM, Nesland JM, et al. Breast carcinoma cells in primary tumors and effusions have different gene array profiles. J Oncol. 2010;2010:969084.CrossRefPubMedGoogle Scholar
  26. 26.
    Tornillo G, Elia AR, Castellano I, Spadaro M, Bernabei P, Bisaro B, et al. p130Cas alters the differentiation potential of mammary luminal progenitors by deregulating c-Kit activity. Stem Cells. 2013;31(7):1422–33.CrossRefPubMedGoogle Scholar
  27. 27.
    Barrett A, Evans IM, Frolov A, Britton G, Pellet-Many C, Yamaji M, et al. A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling. J Cell Sci. 2014;127(Pt 12):2647–58.CrossRefPubMedGoogle Scholar
  28. 28.
    Zheng Y, Asara JM, Tyner AL. Protein-tyrosine kinase 6 promotes peripheral adhesion complex formation and cell migration by phosphorylating p130 CRK-associated substrate. J Biol Chem. 2012;287(1):148–58.CrossRefPubMedGoogle Scholar
  29. 29.
    Cabodi S, Tinnirello A, Bisaro B, Tornillo G, Pilar Camacho-Leal M, Forni G, et al. p130Cas is an essential transducer element in ErbB2 transformation. FASEB J. 2010;24(10):3796–808.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Joerg Kumbrink
    • 1
    • 2
    • 3
  • Ana de la Cueva
    • 1
  • Shefali Soni
    • 1
    • 4
  • Nadja Sailer
    • 1
    • 5
  • Kathrin H. Kirsch
    • 1
  1. 1.Department of BiochemistryBoston University School of MedicineBostonUSA
  2. 2.Department of Medicine III, University Hospital GrosshadernUniversity of MunichMunichGermany
  3. 3.Institute of PathologyUniversity of MunichMunichGermany
  4. 4.The Leona M. and Harry B. Helmsley Charitable TrustNew YorkUSA
  5. 5.Department of BiochemistryUniversity of MunichMunichGermany

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