Amphiregulin regulates proliferation and migration of HER2-positive breast cancer cells
Tumor initiation and progression rely on cellular proliferation and migration. Many factors are involved in these processes, including growth factors. Amphiregulin (AREG) is involved in normal mammary development and the development of estrogen receptor (ER)-positive breast cancer. The aim of this project was to determine if AREG is involved in the proliferation and progression of HER2-positive breast cancer.
Mouse cell lines MMTV-neu, HC-11 and COMMA-D, as well as human cell lines MCF10A, SKBR3, HCC1954 and BT474 were used. Real-time PCR was used to quantify AREG expression and neutralizing antibodies were used to reduce the autocrine/paracrine effects of AREG. Transfections using siRNA and shRNA were used to knockdown AREG expression in the cancer cell lines. Free-floating sphere formation, colony forming, scratch wound and Transwell assays were used to assess the proliferation, tumor forming and migratory capacities of transfected cancer cells.
We found AREG expression in both normal epithelial cell lines and tumor-derived cell lines. Knockdown of AREG protein expression resulted in reduced sphere sizes and reduced sphere numbers in both mouse and human cancer cells that overexpress erbB2/HER2. AREG was found to be involved in cancer cell migration and invasion. In addition, we found that AREG expression knockdown resulted in different migration capacities in normal and erbB2/HER2 overexpressing cancer cells.
Based on our results we conclude that AREG is involved in regulating the proliferation and migration of erbB2/HER2-positive breast cancer cells.
KeywordsAmphiregulin Breast cancer erbB2 HER2 Migration Transfection
The Institute for Biological Interfaces of Engineering (IBIOE) of Clemson University provided funding for this project. The Calhoun Honors College of Clemson University awarded undergraduate research grants to WMB, JFW, and JMM.
Compliance with ethical standards
Conflict of interest
All authors declare no competing interests with the work described herein.
- 4.D.J. Slamon, W. Godolphon, L.A. Jones, J.A. Holt, S.G. Wong, D.E. Keith, W.J. Levin, S.G. Stuart, J. Udove, A. Ullrich, M.F. Press, Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244, 707–712 (1989). https://doi.org/10.1126/science.2470152 CrossRefPubMedGoogle Scholar
- 5.I.L. Andrulis, S.B. Bull, M.E. Blackstein, D. Sutherland, C. Mak, S. Sidofsky, K.P. Pritzker, R.W. Hartwick, W. Hanna, L. Lickley, R. Wilkinson, A. Qizilbash, U. Ambus, M. Lipa, H. Weizel, A. Katz, M. Baida, S. Mariz, G. Stolk, P. Dacamara, W. Geddie, D. McCready, Neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto breast cancer study group. J. Clin. Oncol. 16, 1340–1349 (1998). https://doi.org/10.1200/JCO.19184.108.40.2060 CrossRefPubMedGoogle Scholar
- 7.P.M. Siegel, E.D. Ryan, R.D. Cardiff, W.J. Muller, Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: Implications for human breast cancer. EMBO J. 18, 2149–2164 (1999). https://doi.org/10.1093/emboj/18.8.2149 CrossRefPubMedCentralPubMedGoogle Scholar
- 10.B.W. Booth, C.A. Boulanger, L.H. Anderson, G.H. Smith, The normal mammary microenvironment suppresses the tumorigenic phenotype of mouse mammary tumor virus-neu-transformed mammary tumor cells. Oncogene 30, 679–689 (2010). https://doi.org/10.1038/onc.2010.439 CrossRefPubMedCentralPubMedGoogle Scholar
- 12.P. Taneja, D.P. Frazier, R.D. Kendig, D. Maglic, T. Sugiyama, F. Kai, N.K. Taneja, K. Inoue, MMTV mouse models and the diagnostic values of MMTV-like sequences in human breast cancer. Expert Rev. Mol. Diagn. 9, 423–440 (2009). https://doi.org/10.1586/erm.09.31 CrossRefPubMedCentralPubMedGoogle Scholar
- 13.M. Shoyab, V.L. McDonald, J.G. Bradley, G.J. Todaro, Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. Proc. Natl. Acad. Sci. USA 85, 6528–6532 (1988). https://doi.org/10.1073/pnas.85.17.6528 CrossRefPubMedCentralPubMedGoogle Scholar
- 16.E. Tzahar, H. Waterman, X. Chen, G. Levkowitz, D. Karunagaran, S. Lavi, B.J. Ratzkin, Y. Yarden, A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol. Cell Biol. 16, 5276–5287 (1996). https://doi.org/10.1128/MCB.16.10.5276 CrossRefPubMedCentralPubMedGoogle Scholar
- 19.M.D. Sternlicht, S.W. Sunnarborg, H. Kouros-Mehr, Y. Yu, D.C. Lee, Z. Werb, Mammary ductal morphogenesis requires paracrine activation of stromal EGFR via ADAM17-dependent shedding of epithelial amphiregulin. Development 132, 3923–3933 (2005). https://doi.org/10.1242/dev.01966 CrossRefPubMedCentralPubMedGoogle Scholar
- 20.N.J. Kenney, R.P. Huang, G.R. Johnson, J.X. Wu, D. Okamura, W. Matheny, E. Kordon, W.J. Gullick, G. Plowman, G.H. Smith, D.S. Salomon, E.D. Andamson, Detection and location of amphiregulin and Cripto-1 expression in the developing postnatal mouse mammary gland. Mol. Reprod. Dev. 41, 277–286 (1995). https://doi.org/10.1002/mrd.1080410302 CrossRefPubMedGoogle Scholar
- 23.B.W. Booth, C.A. Boulanger, L.H. Anderson, L. Jimenez-Rojo, C. Brisken, G.H. Smith, Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics. Exp. Cell Res. 316, 422–432 (2010). https://doi.org/10.1016/j.yexcr.2009.11.006 CrossRefPubMedGoogle Scholar
- 25.E.A. Peterson, E.C. Jenkins, K.A. Lofgren, N. Chandiramani, H. Liu, E. Aranda, M. Barnett, P.A. Kenny, I. Amphiregulin, A critical downstream effector of estrogen signaling in ERalpha-positive breast cancer. Cancer Res. 75, 4830–4838 (2015). https://doi.org/10.1158/0008-5472.CAN-15-0709 CrossRefPubMedCentralPubMedGoogle Scholar
- 33.S. Liu, G. Dontu, I.D. Mantle, S. Patel, N.S. Ahn, K.W. Jackson, P. Suri, M.S. Wicha, Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 66, 6063–6071 (2006). https://doi.org/10.1158/0008-5472.CAN-06-0054 CrossRefPubMedCentralPubMedGoogle Scholar
- 37.J.E. Visvader, G.H. Smith, Murine mammary epithelial stem cells: Discovery, function, and current status. Cold Spring Harb. Perspect. Biol. 3, a004879 (2011)Google Scholar
- 40.K.U. Wagner, B.W. Booth, C.A. Boulanger, G.H. Smith, Multipotent P.I.-M.E.C. are the true targets of MMTV-neu tumorigenesis. Oncogene 32, 1338 (2013). https://doi.org/10.1038/onc.2012.452
- 43.S. Mallepell, A. Krust, P. Chambon, C. Brisken, Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland. Proc. Natl. Acad. Sci. USA 103, 2196–2201 (2006). https://doi.org/10.1073/pnas.0510974103 CrossRefPubMedCentralPubMedGoogle Scholar
- 45.M.J. Ellis, A. Coop, B. Singh, L. Mauriac, A. Llombert-Cussac, F. Janicke, W.R. Miller, D.B. Evans, M. Dugan, C. Brady, E. Quebe-Fehling, M. Borgs, Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: Evidence from a phase III randomized trial. J. Clin. Oncol. 19, 3808–3816 (2001). https://doi.org/10.1200/JCO.2001.19.18.3808 CrossRefPubMedGoogle Scholar
- 46.J.W. Kim, D.K. Kim, A. Min, K.H. Lee, H.J. Nam, J.H. Kim, J.S. Kim, T.Y. Kim, S.A. Im, I.A. Park, Amphiregulin confers trastuzumab resistance via AKT and ERK activation in HER2-positive breast cancer. J. Cancer. Res. Clin. Oncol. 142, 157–165 (2016). https://doi.org/10.1007/s00432-015-2012-4 CrossRefPubMedGoogle Scholar
- 48.L. Ma, F. Lan, Z. Zheng, F. Xie, L. Wang, W. Liu, J. Han, F. Zheng, Y. Xie, Q. Huang, Epidermal growth factor (EGF) and interleukin (IL)-1beta synergistically promote ERK1/2-mediated invasive breast ductal cancer cell migration and invasion. Mol. Cancer 11, 79 (2012). https://doi.org/10.1186/1476-4598-11-79 CrossRefPubMedCentralPubMedGoogle Scholar
- 49.K. Sternke-Hale, A.M. Gonzalez-Angulo, A. Lluch, R.M. Neve, W.L. Kuo, M. Davies, M. Carey, Z. Hu, Y. Guan, A. Sahin, W.F. Symmans, L. Pusztai, L.K. Nolden, H. Horlings, K. Berns, M.C. Hung, M.J. van de Vijver, V. Valero, J.W. Gray, R. Bernards, G.B. Mills, B.T. Hennessy, An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 68, 6084–6091 (2008). https://doi.org/10.1158/0008-5472.CAN-07-6854 CrossRefGoogle Scholar
- 51.K. Subik, J.F. Lee, L. Baxter, T. Strzepek, D. Costello, P. Crowley, L. Xing, M.C. Hung, T. Bonfigio, D.G. Hicks, P. Tang, The expression patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by Immunohistochemical analysis in breast cancer cell lines. Breast Cancer 4, 35–41 (2010)PubMedCentralPubMedGoogle Scholar