Advertisement

Inhibition of Epithelial-Mesenchymal Transition and Metastasis by Combined TGFbeta Knockdown and Metformin Treatment in a Canine Mammary Cancer Xenograft Model

  • Camila Leonel
  • Thaiz Ferraz Borin
  • Lívia de Carvalho Ferreira
  • Marina Gobbe Moschetta
  • Marcio Chaim Bajgelman
  • Alicia M. Viloria-Petit
  • Debora Aparecida Pires de Campos ZuccariEmail author
Article

Abstract

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-β) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-β, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-β1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-β1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mice’s lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-β1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-β1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-β1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-β1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-β1 silencing and their effect on EMT.

Keywords

Breast cancer Metastasis Anticarcinogenic agents shRNA TGF-β 

Notes

Acknowledgment

To FAPESP/Fundação de Amparo à Pesquisa do Estado de Sao Paulo for research funding and a studentship. A Canadian Foundation for Innovation (CFI) grant to A.V.P. funded all the equipment used in the intial optimization of these studies.

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no competing interests.

References

  1. 1.
    Simonetti S, Terracciano L, Zlobec I, Kilic E, Stasio L, Quarto M, et al. Immunophenotyping analysis in invasive micropapillary carcinoma of the breast: role of CD24 and CD44 isoforms expression. Breast. 2012;21:165–70.CrossRefPubMedGoogle Scholar
  2. 2.
    Prislei S, Martinelli E, Zannoni GF, Petrillo M, Filippetti F, Mariani M, et al. Role and prognostic significance of the epithelial-mesenchymal transition factor ZEB2 in ovarian cancer. Oncotarget. 2015.Google Scholar
  3. 3.
    Sigurdsson V, Hilmarsdottir B, Sigmundsdottir H, AJR F, Ringnér M, et al. Endothelial induced EMT in breast epithelial cells with stem cell properties. PLoS One. 2011; doi: 10.1371/journal.pone.0023833.Google Scholar
  4. 4.
    Foroni C, Broggini M, Generali D, Damia G. Epithelial–mesenchymal transition and breast cancer: role, molecular mechanisms and clinical impact. Cancer Treat Rev. 2011;38:689–97.CrossRefPubMedGoogle Scholar
  5. 5.
    Yang J, Weinberg RA. Epithelial–mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell. 2008;14:818–29.CrossRefPubMedGoogle Scholar
  6. 6.
    Wen YC, Lee WJ, Tan P, Yang SF, Hsiao M, Lee LM, et al. By inhibiting snail signaling and miR-23a-3p, osthole suppresses the EMT-mediated metastatic ability in prostate cancer. Oncotarget. 2015.Google Scholar
  7. 7.
    Puisieux A, Brabletz T, Caramel J. Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol. 2014;16:488–94.CrossRefPubMedGoogle Scholar
  8. 8.
    Bierie B, Moses HL. TGFb: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer. 2006;6:506–20.CrossRefPubMedGoogle Scholar
  9. 9.
    Ikushima H, Miyazono K. TGFb signalling: a complex web in cancer progression. Nat Rev Cancer. 2010;10:415–24.CrossRefPubMedGoogle Scholar
  10. 10.
    Wu MY, Hill CS. Tgf-beta superfamily signaling in embryonic development and homeostasis. Dev Cell. 2009;16:329–43.CrossRefPubMedGoogle Scholar
  11. 11.
    Siegel PM, Shue W, Cardiff RD, et al. Transforming growth factor h signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis. Proc Natl Acad Sci U S A. 2003;100:8430–5.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bandyopadhyay A, Agyin J, Wang L, et al. Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-h type I receptor kinase inhibitor. Cancer Res. 2006;66:6714–21.CrossRefPubMedGoogle Scholar
  13. 13.
    Padua D, Zhang XH, Wang Q, et al. TGFb primes breast tumors for lung metastasis seeding through angiopoietin-like. Cell. 2008;133:66–77.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Reiman JM, Knutson KL, Radisky DC. Immune promotion of epithelial mesenchymal transition and generation of breast cancer stem cells. Cancer Res. 2010;70(8):3005–8.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wang QZ, Lu YH, Jiang N, Diao Y, Xu RA. The asymmetric division and tumorigenesis of stem cells. Chinese journal of cancer. 2010;29:248–53.CrossRefPubMedGoogle Scholar
  16. 16.
    Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133:704–15.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Short JJ, Curiel DT. Oncolytic adenoviruses targeted to cancer stem cells. Mol Cancer Ther. 2009;8:2096–102.CrossRefPubMedGoogle Scholar
  18. 18.
    Moore LD, Isayeva T, Siegal GP, Ponnazhagan S. Silencing of transforming growth factor-β1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo. Clin Cancer Res. 2008;14:4961.CrossRefPubMedGoogle Scholar
  19. 19.
    Zhao X, Zou Y, Gu Q, Zhao G, Gray H, Pfeffer LM, Yue J. Lentiviral vector mediated Claudin1 silencing inhibits epithelial to mesenchymal transition in breast cancer cells. Viruses. 2015;7:2965–79.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Noto H, Goto A, Tsujimoto T, Noda M. Cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis. PLoS One. 2012;7:33411.CrossRefGoogle Scholar
  21. 21.
    Hatoum D, McGowan EM. Recent advances in the use of metformin: can treating diabetes prevent breast cancer? Biomed Res Int. 2015; doi: 10.1155/2015/548436. Epub 2015 Mar 19.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Camacho L, Dasgupta A, Jiralerspong S. Metformin in breast cancer - an evolving mystery. Breast Cancer Res. 2015;17:88.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Barrière G, Tartary M, Rigaud M. Metformin: a rising star to fight the epithelial mesenchymal transition in oncology. Anti Cancer Agents Med Chem. 2013;13:333–40.CrossRefGoogle Scholar
  24. 24.
    Queiroz EA, Puukila S, Eichler R, Sampaio SC, Forsyth HL, Lees SJ, et al. Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells. PLoS One. 2014;9(5):e98207.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Qu C, Zhang W, Zheng G, Zhang Z, Yin J, He Z. Metformin reverses multidrug resistance and epithelial-mesenchymal transition (EMT) via activating AMP-activated protein kinase (AMPK) in human breast cancer cells. Mol Cell Biochem. 2014;386:63–71.CrossRefPubMedGoogle Scholar
  26. 26.
    Liu Z, Ren L, Liu C, Xia T, Zha X, Wang S. Phenformin induces cell cycle change, apoptosis, and mesenchymal-epithelial transition and regulates the AMPK/mTOR/p70s6k and MAPK/ERK pathways in breast cancer cells. PLoS One. 2015;10(6):e0131207.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells and acts together with chemotherapy to block tumor growth and prolong remission. Cancer Res. 2009;69:7507–11.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Cufí S, Vazquez-Martin A, Oliveras-Ferraros C, Martin-Castillo B, Joven J, Menendez JA. Metformin against TGFb-induced epithelial-to-mesenchymal transition (EMT). Cell Cycle. 2010;9:4461–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Heale BSE, Soifer HS, Bowers C, Rossi JJ. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res. 2005;33:e30.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen ISY, Hahn WC, Sharp PA, Weinberg RA, Novina CD. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003;9:493–501.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22.CrossRefPubMedGoogle Scholar
  32. 32.
    Rattan R, Graham RP, et al. Metformin suppresses ovarian cancer growth and metastasis with enhancement of cisplatin cytotoxicity in vivo. Neoplasia. 2011;13:483–91.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Wang M, Liu X, Guo J, et al. Inhibition of LSD1 by Pargyline inhibited process of EMT and progression of prostate cancer in vivo. Biochem Biophys Res Commun. 2015;467:310–5.CrossRefPubMedGoogle Scholar
  34. 34.
    Hwang YP, Jeong HG. Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase-9 activation through a calcium and protein kinase Calpha-dependent pathway: phorbol-12-myristate-13-acetate-induced/extracellular signal-regulated kinase/activator protein-1. Br J Pharmacol. 2010;160:1195–211.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Cerezo M, Tichet M, Abbe P, et al. Metformin blocks melanoma invasion and metastasis development in AMPK/p53-dependent manner. 2013;12:1605.Google Scholar
  36. 36.
    Zhang J, Shen C, Wang L, Ma Q, Xia P, Qi M, Yang M, Han B. Metformin inhibits epithelial-mesenchymal transition in prostate cancer cells: involvement of the tumor suppressor miR30a and its target gene SOX4. Biochem Biophys Res Commun. 2014;452:746–52.CrossRefPubMedGoogle Scholar
  37. 37.
    Saeki K, Watanabe M, Tsuboi M, et al. Anti-tumour effect of metformin in canine mammary gland tumour cells. Vet J. 2015;205:297–304.CrossRefPubMedGoogle Scholar
  38. 38.
    Barbieri F, Thellung S, Ratto A, et al. In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors. BMC Cancer. 2015;15:228.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kisfalvi K, Aune M, Sinnett-Smith J, et al. Metformin inhibits the growth of human pancreatic cancer xenografts. Pancreas. 2013;42:781–5.CrossRefPubMedGoogle Scholar
  40. 40.
    Burnett JP, Korkaya H, Ouzounova MD, et al. Trastuzumab resistance induces EMT to transform HER2+ PTEN to a triple negative breast cancer that requires unique treatment options. Sci Rep. 2015;5:15821.33.CrossRefGoogle Scholar
  41. 41.
    Pon YL, Zhou HY, Cheung ANY, Ngan HYS, Wong AST. p70 S6 kinase promotes epithelial to mesenchymal transition through snail induction in ovarian cancer cells. Cancer Res. 2008;68:6524–32.CrossRefPubMedGoogle Scholar
  42. 42.
    Rattan R, Fehmi RA, Munkarah A. Metformin: an emerging new therapeutic option for targeting cancer stem cells and metastasis. J Oncol. 2012;2012:928127.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Zarzynska JM. Two faces of TGF-beta1 in breast cancer. Mediat Inflamm. 2014; doi: 10.1155/2014/141747.Google Scholar
  44. 44.
    Siegel PM, Shu W, Cardiff RD, Muller WJ, Massagué J. Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis. Proc Natl Acad Sci U S A. 2003;100:8430–5.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Dunning AM, Ellis PD, McBride S, et al. A transforming growth factorβ1 signal peptide variant increases secretion in vitro and is associated with increased incidence of invasive breast cancer. Cancer Res. 2003;63:2610–5.PubMedGoogle Scholar
  46. 46.
    Bierie B, Moses HL. Transforming growth factor beta (TGF-β) and inflammation in cancer. Cytokine Growth Factor Rev. 2010;21:49–59.CrossRefPubMedGoogle Scholar
  47. 47.
    Sun L, Wu G, Willson JKV, et al. Expression of transforming growth factor β type II receptor leads to reduced malignancy in human breast cancer MCF-7 cells. J Biol Chem. 1994;269:26449–55.PubMedGoogle Scholar
  48. 48.
    Wang J, Sun L, Myeroff L, et al. Demonstration that mutation of the type II transforming growth factor β receptor inactivates its tumor suppressor activity in replication error- positive colon carcinoma cells. J Biol Chem. 1995;270:22044–9.CrossRefPubMedGoogle Scholar
  49. 49.
    Kim SJ, Im YH, Markowitz SD, Bang YJ. Molecular mechanisms of inactivation of TGF-βreceptors during carcinogenesis. Cytokine Growth Factor Rev. 2000;11:159–68.CrossRefPubMedGoogle Scholar
  50. 50.
    Lebrun JJ. The dual role of TGF in human cancer: from tumor suppression to cancer. Metastasis. 2012;2012:28.Google Scholar
  51. 51.
    WANG J, Qiuling Gao Q, Decui Wang D, Wang Z, Hu C. Metformin inhibits growth of lung adenocarcinoma cells by inducing apoptosis via the mitochondria-mediated pathway. Oncol Lett. 2015;10:1343–9.PubMedPubMedCentralGoogle Scholar
  52. 52.
    Tsutsumi Y, Nomiyama T, Kawanami T, et al. Combined treatment with Exendin-4 and metformin attenuates prostate cancer growth. PLoS One. 2015;10:e0139709.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Lipner MB, Marayati R, Deng Y, et al. Metformin treatment does not inhibit growth of pancreatic cancer patient-derived xenografts. PLoS One. 2016;11:e0147113.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Zhang Y, Guan M, Zheng Z, Zhang Q, Gao F, Xue Y. Effects of metformin on CD133+ colorectal cancer cells in diabetic patients. PLoS One. 2013;8:81264.CrossRefGoogle Scholar
  55. 55.
    Cohn A, Lahn MM, Williams KE, Cleverly AL, et al. A phase I dose-escalation study to a predefined dose of a transforming growth factor-β1 monoclonal antibody (TβM1) in patients with metastatic cancer. Int J Oncol. 2014;45:2221–31.PubMedPubMedCentralGoogle Scholar
  56. 56.
    Rice LM, Padilla CM, McLaughlin SR, Mathes A, et al. Fresolimumab treatment decreases biomarkers and improves clinical symptoms in systemic sclerosis patients. J Clin Invest. 2015;125:2795–807.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Wang D, Lu P, Zhang H, et al. Oct-4 and Nanog promote the epithelial-mesenchymal transition of breast cancer stem cells and are associated with poor prognosis in breast cancer patients. Oncotarget. 2014;5:10803–15.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Davis FM, Azimi I, Faville RA, et al. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent. Oncogene. 2014;33:2307–16.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Camila Leonel
    • 1
    • 2
  • Thaiz Ferraz Borin
    • 2
    • 3
  • Lívia de Carvalho Ferreira
    • 1
    • 2
  • Marina Gobbe Moschetta
    • 2
    • 3
  • Marcio Chaim Bajgelman
    • 4
  • Alicia M. Viloria-Petit
    • 5
  • Debora Aparecida Pires de Campos Zuccari
    • 1
    • 2
    Email author
  1. 1.Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP/IBILCE), PostGraduate Program in GeneticsSao Jose do Rio PretoBrazil
  2. 2.Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP), Laboratory of Molecular Investigation of Cancer (LIMC)Sao Jose do Rio PretoBrazil
  3. 3.Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP), PostGraduate Program in Health SciencesSao Jose do Rio PretoBrazil
  4. 4.National Center for Research in Energy and Materials – CNPEM, Brazilian Biosciences National Laboratory – LNBioCampinasBrazil
  5. 5.Department of Biomedical Sciences, Ontario Veterinary CollegeUniversity of GuelphGuelphCanada

Personalised recommendations