Abstract
Overexpression of the oncoprotein erbB2/HER2 is present in 20–30% of breast cancer patients and inversely correlates with patient survival. Reports have demonstrated the deterministic power of the mammary microenvironment where the normal mammary microenvironment redirects cells of non-mammary origin or tumor-derived cells to adopt a mammary phenotype in an in vivo model. This phenomenon is termed tumor cell redirection. Tumor-derived cells that overexpress the erbB2 oncoprotein lose their tumor-forming capacity in this model. In this model, phosphorylation of erbB2 is attenuated thus reducing the tumor cell’s tumor-forming potential. In this report, we describe our results using an in vitro model based on the in vivo model mentioned previously. Tumor-derived cells are mixed in predetermined ratios with normal mammary epithelial cells prior to seeding in vitro. In this in vitro model, the tumor-derived cells are redirected as determined by attenuated phosphorylation of the receptor and reduced sphere and colony formation. These results match those observed in the in vivo model. This in vitro model will allow expanded experimental options in the future to determine additional aspects of tumor cell redirection that can be translated to other types of cancer.
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Andrulis IL, Bull SB, Blackstein ME, Sutherland D, Mak C, Sidlofsky S, Pritzker KP, Hartwick RW, Hanna W, Lickley L, Wilkinson R, Qizibash A, Ambus U, Lipa M, Weizel H, Katz A, Baida M, Mariz S, Stoik G, Dacamara P, Strongitharm D, Geddie W, McCready D (1998) 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
Bocchinfuso WP, Lindzey JK, Hewitt SC, Clark JA, Myers PH, Cooper R, Korach KS (2000) Induction of mammary gland development in estrogen receptor-alpha knockout mice. Endocrinology 141:2982–2994
Booth BW, Boulanger CA, Anderson LH, Jimenez-Rojo L, Brisken C, Smith GH (2010a) Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics. Exp Cell Res 316:422–432
Booth BW, Boulanger CA, Anderson LH, Smith GH (2010b) The normal mammary microenvironment suppresses the tumorigenic phenotype of mouse mammary tumor virus-neu-transformed mammary tumor cells. Oncogene 30:679–689
Booth BW, Boulanger CA, Smith GH (2007) Alveolar progenitor cells develop in mouse mammary glands independent of pregnancy and lactation. J Cell Physiol 212:729–736
Booth BW, Mack DL, Androutsellis-Theotokis A, McKay RD, Boulanger CA, Smith GH (2008) The mammary microenvironment alters the differentiation repertoire of neural stem cells. Proc Natl Acad Sci U S A 105:14891–14896
Boulanger CA, Bruno RD, Mack DL, Gonzales M, Castro NP, Salomon DS, Smith GH (2013) Embryonic stem cells are redirected to non-tumorigenic epithelial cell fate by interaction with the mammary microenvironment. PLoS One 8:e62019
Boulanger CA, Bruno RD, Rosu-Myles M, Smith GH (2012) The mouse mammary microenvironment redirects mesoderm-derived bone marrow cells to a mammary epithelial progenitor cell fate. Stem Cells Dev 21:948–954
Boulanger CA, Mack DL, Booth BW, Smith GH (2007) Interaction with the mammary microenvironment redirects spermatogenic cell fate in vivo. Proc Natl Acad Sci U S A 104:3871–3876
Boulanger CA, Wagner KU, Smith GH (2005) Parity-induced mouse mammary epithelial cells are pluripotent, self-renewing and sensitive to TGF-beta1 expression. Oncogene 24:552–560
Brisken C, Park S, Vass T, Lydon JP, O'Malley BW, Weinberg RA (1998) A paracrine role for the epithelial progesterone receptor in mammary gland development. Proc Natl Acad Sci U S A 95:5076–5081
Bussard KM, Boulanger CA, Booth BW, Bruno RD, Smith GH (2010) Reprogramming human cancer cells in the mouse mammary gland. Cancer Res
Bussard KM, Smith GH (2012) Human breast cancer cells are redirected to mammary epithelial cells upon interaction with the regenerating mammary gland microenvironment in-vivo. PLoS One 7:e49221
Chan SK, Hill ME, Gullick WJ (2006) The role of the epidermal growth factor receptor in breast cancer. J Mammary Gland Biol Neoplasia 11:3–11
Deugnier MA, Faraldo MM, Teuliere J, Thiery JP, Medina D, Glukhova MA (2006) Isolation of mouse mammary epithelial progenitor cells with basal characteristics from the Comma-Dbeta cell line. Dev Biol 293:414–425
Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, Wicha MS (2004) Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res 6:R605–615
Duru N, Fan M, Candas D, Menaa C, Liu HC, Nantajit D, Wen Y, Xiao K, Eldridge A, Chromy BA, Li S, Spitz DR, Lam KS, Wicha MS, Li JJ (2012) HER2-associated radioresistance of breast cancer stem cells isolated from HER2-negative breast cancer cells. Clin Cancer Res 18:6634–6647
Fridriksdottir AJ, Petersen OW, Ronnov-Jessen L (2011) Mammary gland stem cells: current status and future challenges. Int J Dev Biol 55:719–729
Henry MD, Triplett AA, Oh KB, Smith GH, Wagner KU (2004) Parity-induced mammary epithelial cells facilitate tumorigenesis in MMTV-neu transgenic mice. Oncogene 23:6980–6985
Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF 3rd, Hynes NE (2003) The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci U S A 100:8933–8938
Honeth G, Lombardi S, Ginestier C, Hur M, Marlow R, Buchupalli B, Shinomiya I, Gazinska P, Bombelli S, Ramalingam V, Purushotham AD, Pinder SE, Dontu G (2014) Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium. Breast Cancer Res 16:R52
Ithimakin S, Day KC, Malik F, Zen Q, Dawsey SJ, Bersano-Begey TF, Quraishi AA, Ignatoski KW, Daignault S, Davis A, Hall CL, Palanisamy N, Heath AN, Tawakkol N, Luther TK, Clouthier SG, Chadwick WA, Day ML, Kleer CG, Thomas DG, Hayes DF, Korkaya H, Wicha MS (2013) HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab. Cancer Res 73:1635–1646
Jeselsohn R, Brown NE, Arendt L, Klebba I, Hu MG, Kuperwasser C, Hinds PW (2010) Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis. Cancer Cell 17:65–76
Kemper K, de Goeje PL, Peeper DS, van Amerongen R (2014) Phenotype switching: tumor cell plasticity as a resistance mechanism and target for therapy. Cancer Res 74:5937–5941
Kok M, Koornstra RH, Margarido TC, Fles R, Armstrong NJ, Linn SC, Van't Veer LJ, Weigelt B (2009) Mammosphere-derived gene set predicts outcome in patients with ER-positive breast cancer. J Pathol 218:316–326
Korkaya H, Wicha MS (2013) HER2 and breast cancer stem cells: more than meets the eye. Cancer Res 73:3489–3493
Lester J (2007) Breast cancer in 2007: incidence, risk assessment, and risk reduction strategies. Clin J Oncol Nurs 11:619–622
Lo AT, Mori H, Mott J, Bissell MJ (2012) Constructing three-dimensional models to study mammary gland branching morphogenesis and functional differentiation. J Mammary Gland Biol Neoplasia 17:103–110
Mallepell S, Krust A, Chambon P, Brisken C (2006) Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland. Proc Natl Acad Sci U S A 103:2196–2201
Mueller SO, Clark JA, Myers PH, Korach KS (2002) Mammary gland development in adult mice requires epithelial and stromal estrogen receptor alpha. Endocrinology 143:2357–2365
Muller WJ, Sinn E, Pattengale PK, Wallace R, Leder P (1988) Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell 54:105–115
Nguyen-Ngoc KV, Cheung KJ, Brenot A, Shamir ER, Gray RS, Hines WC, Yaswen P, Werb Z, Ewald AJ (2012) ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc Natl Acad Sci U S A 109:E2595–2604
Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406:747–752
Rota LM, Lazzarino DA, Ziegler AN, LeRoith D, Wood TL (2012) Determining mammosphere-forming potential: application of the limiting dilution analysis. J Mammary Gland Biol Neoplasia 17:119–123
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177–182
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, Press MF (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707–712
Stern DF (2008) ERBB3/HER3 and ERBB2/HER2 duet in mammary development and breast cancer. J Mammary Gland Biol Neoplasia 13:215–223
Tome Y, Uehara F, Mii S, Yano S, Zhang L, Sugimoto N, Maehara H, Bouvet M, Tsuchiya H, Kanaya F, Hoffman RM (2014) 3-Dimensional tissue is formed from cancer cells in vitro on Gelfoam(R), but not on Matrigel. J Cell Biochem 115:1362–1367
Wagner KU, Booth BW, Boulanger CA, Smith GH (2013) Multipotent PI-MECs are the true targets of MMTV-neu tumorigenesis. Oncogene 32:1338
Wagner KU, Boulanger CA, Henry MD, Sgagias M, Hennighausen L, Smith GH (2002) An adjunct mammary epithelial cell population in parous females: its role in functional adaptation and tissue renewal. Development 129:1377–1386
Wagner KU, Wall RJ, St-Onge L, Gruss P, Wynshaw-Boris A, Garrett L, Li M, Furth PA, Hennighausen L (1997) Cre-mediated gene deletion in the mammary gland. Nuc Acids Res 25:4323–4330
Zaczek A, Brandt B, Bielawski KP (2005) The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches. Histol Histopathol 20:1005–1015
Acknowledgments
Mammary tumors from MMTV-neu mice from which the MMTV-neu cell lines were established were a gift from Dr. Wen Chen. Funding for this project was provided by the Institute for Biological Interfaces of Engineering of Clemson University and the Calhoun Honors College of Clemson University.
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Supplemental Figure 1
– Co-localization of erbB2 and erbB3. MMTV-neu cells were stained for P-erbB2 (green) and (A) erbB3 (red) or (B) P-erbB3 (red). Arrows indicate co-localization. COMMA-D cells were also stained for P-erbB2 and (C) erbB3 or (D) P-erbB3. No co-localization was found as COMMA-D cells do not express P-erbB2. Nuclei visualized with Hoechst 33342. Scale bar = 100 μm. (TIFF 5274 kb)
Supplemental Figure 2
Immunodetection of erbB4. (A) MMTV-neu cells were stained for P-erbB2 (green) and erbB4 (red). Arrows indicate co-localization. (B) COMMA-D cells were also stained for P-erbB2 and erbB4. No co-localization was found as COMMA-D cells do not express P-erbB2 and very few cells were erbB4+. Nuclei visualized with Hoechst 33342. Scale bar = 100 μm. (TIFF 2905 kb)
Supplemental Figure 3
MMTV-neu and COMMA-Dβgeo cells were grown in 1:50 ratio and immunostained for P-erbB2 (green). COMMA-Dβgeo cells were visualized by Xgal treatment. There is a clear demarcation of P-erbB2 within the MMTV-neu colony that corresponds to a change in cell morphology (represented by the line). Scale bar = 100 μm. (PSD 1819 kb)
Supplemental Figure 4
IPs were conducted on cell lysates from all conditions using anti-erbB3. Western blotting was then performed using anti-erbB2, anti-P-erbB2, anti-P-erbB3, and a second anti-erbB3 antibody. (PSD 1057 kb)
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Park, J.P., Blanding, W.M., Feltracco, J.A. et al. Validation of an in vitro model of erbB2+ cancer cell redirection. In Vitro Cell.Dev.Biol.-Animal 51, 776–786 (2015). https://doi.org/10.1007/s11626-015-9889-8
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DOI: https://doi.org/10.1007/s11626-015-9889-8