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Hormones and Cancer

, Volume 8, Issue 2, pp 69–77 | Cite as

MMTV-PyMT and Derived Met-1 Mouse Mammary Tumor Cells as Models for Studying the Role of the Androgen Receptor in Triple-Negative Breast Cancer Progression

  • Jessica L. Christenson
  • Kiel T. Butterfield
  • Nicole S. Spoelstra
  • John D. Norris
  • Jatinder S. Josan
  • Julie A. Pollock
  • Donald P. McDonnell
  • Benita S. Katzenellenbogen
  • John A. Katzenellenbogen
  • Jennifer K. Richer
Original Article

Abstract

Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

Keywords

Breast Cancer Androgen Receptor Enzalutamide Androgen Receptor Expression TNBC Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

The authors thank Susan Kane from the City of Hope for providing MMTV-PyMT tissue; Alexander Borowsky from the University of California – Davis for permission to use the Met-1 cell line, the University of Colorado Denver Tissue Biobanking and Processing Core and the University of Colorado Cancer Center Tissue Culture Core, both supported by the National Institute of Health/National Cancer Institute Cancer Core Support Grant P30 CA046934, for experimental assistance; and Britta Jacobsen for critical reading of the manuscript. Enzalutamide was provided by Astellas, Inc. and Medivation, Inc. (Medivation, Inc. was acquired by Pfizer, Inc. in September 2016).

Compliance with Ethical Standards

Funding

This study was funded by a National Institute of Health R01 CA187733-01A1 to JKR; NIH NRSA T32 CA190216-01A1 postdoctoral fellowship to JLC; US Army Synergistic Idea Development Awards W81XWH-10-1-0179 and W81XWH-15-1 to JAK, JSJ, JAP, JN, and DPMcD; Breast Cancer Research Foundation grant to BSK; and enzalutamide was provided by Astellas, Inc. and Medivation, Inc. (Medivation, Inc. was acquired by Pfizer, Inc. in September 2016).

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

12672_2017_285_MOESM1_ESM.pdf (146 kb)
Supplemental Fig. 1 AR expression in micro- and macrometastases from MMTV-PyMT mice. Metastatic lungs were formalin-fixed and paraffin embedded. 5 μm sections were stained for PyMT, AR and H&E. Shown are representative images of a micrometastasis (top, arrow) and a macrometastasis (bottom); 10× and 40× objectives, scale bar = 100 μm, inset zoom ×2.5 (PDF 146 kb)
12672_2017_285_MOESM2_ESM.pdf (45 kb)
Supplemental Fig. 2 PyMT expression in response to AR agonist treatment. Met-1 cells were grown in media with hormone-stripped serum (10% DCC) for 48 hours then treated for 3 days with 0.1% EtOH or 10 nM dihydrotestosterone (DHT). Cells were formalin-fixed and paraffin embedded. 5 μm sections were stained for PyMT. Shown are representative images; 40× objective. The percentage of PyMT positive cells per field in five nonoverlapping fields per sample; mean (PDF 44 kb)
12672_2017_285_MOESM3_ESM.pdf (163 kb)
Supplemental Table S1 (PDF 162 kb)

References

  1. 1.
    Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (2016) SEER Cancer Statistics Review, 1975–2013. November 2015 SEER data submission. National Cancer Institute, BethesdaGoogle Scholar
  2. 2.
    Foulkes WD, Smith IE, Reis-Filho JS (2010) Triple-negative breast cancer. N Engl J Med 363:1938–1948CrossRefPubMedGoogle Scholar
  3. 3.
    Howlader N, Altekruse SF, Li CI, Chen VW, Clarke CA, Ries LA, Cronin KA (2014) US incidence of breast cancer subtypes defined by joint hormone receptor and HER2 status. J Natl Cancer Inst 106Google Scholar
  4. 4.
    McNamara KM, Yoda T, Takagi K, Miki Y, Suzuki T, Sasano H (2013) Androgen receptor in triple negative breast cancer. J Steroid Biochem Mol Biol 133:66–76CrossRefPubMedGoogle Scholar
  5. 5.
    Barton VN, D’Amato NC, Gordon MA, Lind HT, Spoelstra NS, Babbs BL, Heinz RE, Elias A, Jedlicka P, Jacobsen BM, Richer JK (2015) Multiple molecular subtypes of triple-negative breast cancer critically rely on androgen receptor and respond to enzalutamide in vivo. Mol Cancer Ther 14:769–778CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Traina TA, Miller K, Yardley DA, O’Shaughnessy J, Cortes J, Awada A, Kelly CM, Trudeau ME, Schmid P, Gianni L, Garcia-Estevez L, Nanda R, Ademuyiwa FO, Chan S, Steinberg JL, Blaney ME, Tudor IC, Uppal H, Peterson AC, Hudis CA (2015) Results from a phase 2 study of enzalutamide, and androgen receptor inhibitor, in advanced AR+ triple-negative breast cancer. J Clin Oncol 33(suppl; abstr 1003)Google Scholar
  7. 7.
    Lawson DA, Bhakta NR, Kessenbrock K, Prummel KD, Yu Y, Takai K, Zhou A, Eyob H, Balakrishnan S, Wang CY, Yaswen P, Goga A, Werb Z (2015) Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 526:131–135CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Hattori Y, Yoshida A, Yoshida M, Takahashi M, Tsuta K (2015) Evaluation of androgen receptor and GATA binding protein 3 as immunohistochemical markers in the diagnosis of metastatic breast carcinoma to the lung. Pathol Int 65:286–292CrossRefPubMedGoogle Scholar
  9. 9.
    McNamara KM, Yoda T, Miki Y, Nakamura Y, Suzuki T, Nemoto N, Miyashita M, Nishimura R, Arima N, Tamaki K, Ishida T, Ohuchi N, Sasano H (2015) Androgen receptor and enzymes in lymph node metastasis and cancer reoccurrence in triple-negative breast cancer. Int J Biol Markers 30:184–189CrossRefGoogle Scholar
  10. 10.
    Cimino-Mathews A, Hicks JL, Illei PB, Halushka MK, Fetting JH, De Marzo AM, Park BH, Argani P (2012) Androgen receptor expression is usually maintained in initial surgically resected breast cancer metastases but is often lost in end-stage metastases found at autopsy. Hum Pathol 43:1003–1011CrossRefPubMedGoogle Scholar
  11. 11.
    Quail DF, Joyce JA (2013) Microenvironmental regulation of tumor progression and metastasis. Nat Med 19:1423–1437CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fluck MM, Schaffhausen BS (2009) Lessons in signaling and tumorigenesis from polyomavirus middle T antigen. Microbiol Mol Biol Rev 73:542–563, Table of ContentsCrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Guy CT, Cardiff RD, Muller WJ (1992) Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol 12:954–961CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, Pollard JW (2003) Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 163:2113–2126CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Shoushtari AN, Michalowska AM, Green JE (2007) Comparing genetically engineered mouse mammary cancer models with human breast cancer by expression profiling. Breast Dis 28:39–51CrossRefPubMedGoogle Scholar
  16. 16.
    Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121:2750–2767CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Doane AS, Danso M, Lal P, Donaton M, Zhang L, Hudis C, Gerald WL (2006) An estrogen receptor-negative breast cancer subset characterized by a hormonally regulated transcriptional program and response to androgen. Oncogene 25:3994–4008CrossRefPubMedGoogle Scholar
  18. 18.
    Farmer P, Bonnefoi H, Becette V, Tubiana-Hulin M, Fumoleau P, Larsimont D, Macgrogan G, Bergh J, Cameron D, Goldstein D, Duss S, Nicoulaz AL, Brisken C, Fiche M, Delorenzi M, Iggo R (2005) Identification of molecular apocrine breast tumours by microarray analysis. Oncogene 24:4660–4671CrossRefPubMedGoogle Scholar
  19. 19.
    Borowsky AD, Namba R, Young LJ, Hunter KW, Hodgson JG, Tepper CG, McGoldrick ET, Muller WJ, Cardiff RD, Gregg JP (2005) Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior. Clin Exp Metastasis 22:47–59CrossRefPubMedGoogle Scholar
  20. 20.
    Christenson JL, Kane SE (2014) Darpp-32 and t-Darpp are differentially expressed in normal and malignant mouse mammary tissue. Mol Cancer 13:192CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Borowicz S, Van Scoyk M, Avasarala S, Karuppusamy Rathinam MK, Tauler J, Bikkavilli RK, Winn RA (2014) The soft agar colony formation assay. J Vis Exp e51998Google Scholar
  22. 22.
    Cochrane DR, Bernales S, Jacobsen BM, Cittelly DM, Howe EN, D’Amato NC, Spoelstra NS, Edgerton SM, Jean A, Guerrero J, Gomez F, Medicherla S, Alfaro IE, McCullagh E, Jedlicka P, Torkko KC, Thor AD, Elias AD, Protter AA, Richer JK (2014) Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide. Breast Cancer Res 16:R7CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Mori S, Chang JT, Andrechek ER, Matsumura N, Baba T, Yao G, Kim JW, Gatza M, Murphy S, Nevins JR (2009) Anchorage-independent cell growth signature identifies tumors with metastatic potential. Oncogene 28:2796–2805CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Liao X, Thrasher JB, Pelling J, Holzbeierlein J, Sang QX, Li B (2003) Androgen stimulates matrix metalloproteinase-2 expression in human prostate cancer. Endocrinology 144:1656–1663CrossRefPubMedGoogle Scholar
  25. 25.
    Zhang BG, Du T, Zang MD, Chang Q, Fan ZY, Li JF, Yu BQ, Su LP, Li C, Yan C, Gu QL, Zhu ZG, Yan M, Liu B (2014) Androgen receptor promotes gastric cancer cell migration and invasion via AKT-phosphorylation dependent upregulation of matrix metalloproteinase 9. Oncotarget 5:10584–10595CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Zhang Y, Shen Y, Cao B, Yan A, Ji H (2015) Elevated expression levels of androgen receptors and matrix metalloproteinase-2 and -9 in 30 cases of hepatocellular carcinoma compared with adjacent tissues as predictors of cancer invasion and staging. Exp Ther Med 9:905–908PubMedGoogle Scholar
  27. 27.
    Gonzalez LO, Corte MD, Vazquez J, Junquera S, Sanchez R, Alvarez AC, Rodriguez JC, Lamelas ML, Vizoso FJ (2008) Androgen receptor expression in breast cancer: relationship with clinicopathological characteristics of the tumors, prognosis, and expression of metalloproteases and their inhibitors. BMC Cancer 8:149CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Nelson CC, Hendy SC, Shukin RJ, Cheng H, Bruchovsky N, Koop BF, Rennie PS (1999) Determinants of DNA sequence specificity of the androgen, progesterone, and glucocorticoid receptors: evidence for differential steroid receptor response elements. Mol Endocrinol 13:2090–2107CrossRefPubMedGoogle Scholar
  29. 29.
    Archer TK, Fryer CJ, Lee HL, Zaniewski E, Liang T, Mymryk JS (1995) Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo. J Steroid Biochem Mol Biol 53:421–429CrossRefPubMedGoogle Scholar
  30. 30.
    Otten AD, Sanders MM, McKnight GS (1988) The MMTV LTR promoter is induced by progesterone and dihydrotestosterone but not by estrogen. Mol Endocrinol 2:143–147CrossRefPubMedGoogle Scholar
  31. 31.
    Lai JJ, Lai KP, Zeng W, Chuang KH, Altuwaijri S, Chang C (2012) Androgen receptor influences on body defense system via modulation of innate and adaptive immune systems: lessons from conditional AR knockout mice. Am J Pathol 181:1504–1512CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Mikkonen L, Pihlajamaa P, Sahu B, Zhang FP, Janne OA (2010) Androgen receptor and androgen-dependent gene expression in lung. Mol Cell Endocrinol 317:14–24CrossRefPubMedGoogle Scholar
  33. 33.
    Ardiani A, Farsaci B, Rogers CJ, Protter A, Guo Z, King TH, Apelian D, Hodge JW (2013) Combination therapy with a second-generation androgen receptor antagonist and a metastasis vaccine improves survival in a spontaneous prostate cancer model. Clin Cancer Res 19:6205–6218CrossRefPubMedGoogle Scholar
  34. 34.
    Ardiani A, Gameiro SR, Kwilas AR, Donahue RN, Hodge JW (2014) Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway. Oncotarget 5:9335–9348CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Pu Y, Xu M, Liang Y, Yang K, Guo Y, Yang X, Fu YX (2016) Androgen receptor antagonists compromise T cell response against prostate cancer leading to early tumor relapse. Sci Transl Med 8:333ra347CrossRefGoogle Scholar
  36. 36.
    Kwilas AR, Ardiani A, Gameiro SR, Richards J, Hall AB, Hodge JW (2016) Androgen deprivation therapy sensitizes triple negative breast cancer cells to immune-mediated lysis through androgen receptor independent modulation of osteoprotegerin. Oncotarget 7:23498–23511PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Jessica L. Christenson
    • 1
  • Kiel T. Butterfield
    • 1
  • Nicole S. Spoelstra
    • 1
  • John D. Norris
    • 2
  • Jatinder S. Josan
    • 3
  • Julie A. Pollock
    • 4
  • Donald P. McDonnell
    • 2
  • Benita S. Katzenellenbogen
    • 5
  • John A. Katzenellenbogen
    • 6
  • Jennifer K. Richer
    • 1
  1. 1.Department of PathologyUniversity of ColoradoAuroraUSA
  2. 2.Department of Pharmacology and Cancer BiologyDuke UniversityDurhamUSA
  3. 3.Department of ChemistryVirginia Tech UniversityBlacksburgUSA
  4. 4.Department of ChemistryUniversity of RichmondRichmondUSA
  5. 5.Department of Molecular and Integrative PhysiologyUniversity of IllinoisUrbanaUSA
  6. 6.Department of ChemistryUniversity of IllinoisUrbanaUSA

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