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Breast Cancer Research and Treatment

, Volume 143, Issue 3, pp 423–433 | Cite as

Progesterone stimulates progenitor cells in normal human breast and breast cancer cells

  • Heidi N. HiltonEmail author
  • N. Santucci
  • A. Silvestri
  • S. Kantimm
  • L. I. Huschtscha
  • J. D. Graham
  • C. L. Clarke
Preclinical study

Abstract

The epithelium of the human breast is made up of a branching ductal–lobular system, which is lined by a single layer of luminal cells surrounded by a contractile basal cell layer. The co-ordinated development of stem/progenitor cells into these luminal and basal cells is fundamentally important for breast morphogenesis. The ovarian steroid hormones, progesterone (P) and 17β-estradiol, are critical in driving this normal breast development, yet ovarian activity has also been shown to be a major driver of breast cancer risk. We previously demonstrated that P treatment increases proliferation and augments the number of progenitor-like cells, and that the progesterone receptor (PR) is also expressed in the bipotent progenitor-enriched subfraction. Here we demonstrate that PR is expressed in a subset of CD10+ basal cells and that P stimulates this CD10+ cell compartment, which is enriched for bipotent progenitor activity. In addition, we have shown that P stimulates progenitor cells in human breast cancer cell lines and expands the cancer stem cell population via increasing the stem-like CD44+ population. As changes in cell type composition are one of the hallmark features of breast cancer progression, the demonstration that progenitor cells are stimulated by P in both normal breast and in breast cancer cells has critical implications in discerning the mechanisms of how P increases breast cancer risk.

Keywords

Progesterone Progenitor Breast cancer Cell lineage 

Abbreviations

ALDH

Aldehyde dehydrogenase

CFC

Colony forming cell

CK14

Cytokeratin-14

CK18

Cytokeratin-18

CSC

Cancer stem cell

E

17β-estradiol

ER

Estrogen receptor

FACS

Fluorescence-activated cell sorter

FITC

Fluorescein isothiocyanate

HRT

Hormone replacement therapy

IF

Immunofluorescence

IPX

Immunoperoxidase

MEC

Mammary epithelial cell

miRNA

MicroRNA

qPCR

Quantitative PCR

P

Progesterone

PE

Phycoerythrin

PR

Progesterone receptor

SD

Standard deviation

SE

Standard error

Notes

Acknowledgments

We wish to thank Arno Therapeutics, Inc. for generously donating the onapristone. We wish to thank Dr Xin Maggie Wang for assistance with flow cytometry, performed in the Flow Cytometry Centre at Westmead Millennium Institute which is supported by the National Health and Medical Research Council of Australia (NHMRC) and Cancer Institute New South Wales. This study was supported by an NHMRC project grant (1011496), Cure Cancer Australia Foundation and the National Breast Cancer Foundation. We gratefully acknowledge the advice and assistance of Dr. Karen Byth, Westmead Millennium Institute, with statistical analysis.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

10549_2013_2817_MOESM1_ESM.tif (1.3 mb)
Supp Fig. 1 a Live and single cell gating strategy for detection of MUC1+ and CD10+ cells. b Relative mRNA expression levels of (i) CK14 and (ii) CK18 in each subpopulation, normalised to TBP (chart represents mean + SD). (TIFF 1,336 kb)
10549_2013_2817_MOESM2_ESM.tif (874 kb)
Supp Fig. 2 a Day 10 of CFC assay performed using sorted CD10 + and MUC1 + cells b Percentage of CFUs yielded from 2,000 cells seeded c Proportions of different colony types yielded from CFC assays using CD10 + and MUC1 + sorted cells. c Fold change in proportion of MUC1 + cells with indicated hormone treatments (n = 3). d Fold change in proportion of (i) luminal, or (ii) basal acini with indicated hormone treatments. Data are representative of the mean + SE for 3 independent experiments using tissue from 2 individuals. (TIFF 873 kb)
10549_2013_2817_MOESM3_ESM.tif (458 kb)
Supp Fig. 3a Average proportion of CSCs in T47Ds following treatment with ORG (1nM) or MPA (1nM), ± onapristone (ONP; 100nM). Chart represents mean + SE (n = 3). b Relative mRNA levels of CD44 were normalised to TBP in T47D cells following 48 h treatment with P (100nM) ± onapristone (1uM) or progestins (1nM) ± onapristone (100nM). Graph represents mean + SE from 3 biological replicates. (TIFF 457 kb)
10549_2013_2817_MOESM4_ESM.tif (1 mb)
Supp Fig. 4 a (i) Representative image of IPX staining for PR in HCC1428 cells. Scale bar represents 50 μm (ii) Gating strategy for detection and collection of CD44hi and CD44- cell fractions. b Relative transcript numbers of PR and CD44 in each subfraction. Chart represents mean + SD (TIFF 1,028 kb)

References

  1. 1.
    Anderson TJ, Ferguson DJ, Raab GM (1982) Cell turnover in the “resting” human breast: influence of parity, contraceptive pill, age and laterality. Br J Cancer 46(3):376–382PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Ferguson DJ, Anderson TJ (1981) Morphological evaluation of cell turnover in relation to the menstrual cycle in the “resting” human breast. Br J Cancer 44(2):177–181PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Longacre TA, Bartow SA (1986) A correlative morphologic study of human breast and endometrium in the menstrual cycle. Am J Surg Pathol 10(6):382–393PubMedCrossRefGoogle Scholar
  4. 4.
    Hunter DJ, Colditz GA, Hankinson SE, Malspeis S, Spiegelman D, Chen W, Stampfer MJ, Willett WC (2010) Oral contraceptive use and breast cancer: a prospective study of young women. Cancer Epidemiol Biomarkers Prev 19(10):2496–2502PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Beral V, Reeves G, Bull D, Green J (2011) Breast cancer risk in relation to the interval between menopause and starting hormone therapy. J Natl Cancer Inst 103(4):296–305PubMedCrossRefGoogle Scholar
  6. 6.
    Chlebowski RT, Manson JE, Anderson GL, Cauley JA, Aragaki AK, Stefanick ML, Lane DS, Johnson KC, Wactawski-Wende J, Chen C, Qi L, Yasmeen S, Newcomb PA, Prentice RL (2013) Estrogen plus progestin and breast cancer incidence and mortality in the women’s health initiative observational study. J Natl Cancer Inst 105(8):526–535PubMedCrossRefGoogle Scholar
  7. 7.
    Howard BA, Gusterson BA (2000) Human breast development. J Mammary Gland Biol Neoplasia 5(2):119–137PubMedCrossRefGoogle Scholar
  8. 8.
    Asselin-Labat ML, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER, Yasuda H, Smyth GK, Martin TJ, Lindeman GJ, Visvader JE (2010) Control of mammary stem cell function by steroid hormone signalling. Nature 465(7299):798–802PubMedCrossRefGoogle Scholar
  9. 9.
    Joshi PA, Jackson HW, Beristain AG, Di Grappa MA, Mote P, Clarke C, Stingl J, Waterhouse PD, Khokha R (2010) Progesterone induces adult mammary stem cell expansion. Nature 465(7299):803–807PubMedCrossRefGoogle Scholar
  10. 10.
    Visvader JE (2011) Cells of origin in cancer. Nature 469(7330):314–322PubMedCrossRefGoogle Scholar
  11. 11.
    Abd El-Rehim DM, Pinder SE, Paish CE, Bell J, Blamey RW, Robertson JF, Nicholson RI, Ellis IO (2004) Expression of luminal and basal cytokeratins in human breast carcinoma. J Pathol 203(2):661–671PubMedCrossRefGoogle Scholar
  12. 12.
    Taylor-Papadimitriou J, Stampfer M, Bartek J, Lewis A, Boshell M, Lane EB, Leigh IM (1989) Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J Cell Sci 94(Pt 3):403–413PubMedGoogle Scholar
  13. 13.
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100(7):3983–3988PubMedCrossRefGoogle Scholar
  14. 14.
    Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5):555–567PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414(6859):105–111PubMedCrossRefGoogle Scholar
  16. 16.
    Villadsen R, Fridriksdottir AJ, Rønnov-Jessen L, Gudjonsson T, Rank F, LaBarge MA, Bissell MJ, Petersen OW (2007) Evidence for a stem cell hierarchy in the adult human breast. J Cell Biol 177(1):87–101PubMedCrossRefGoogle Scholar
  17. 17.
    Raouf A, Zhao Y, To K, Stingl J, Delaney A, Barbara M, Iscove N, Jones S, McKinney S, Emerman J, Aparicio S, Marra M, Eaves C (2008) Transcriptome analysis of the normal human mammary cell commitment and differentiation process. Cell Stem Cell 3(1):109–118PubMedCrossRefGoogle Scholar
  18. 18.
    Nakshatri H, Srour EF, Badve S (2009) Breast cancer stem cells and intrinsic subtypes: controversies rage on. Curr Stem Cell Res Ther 4(1):50–60PubMedCrossRefGoogle Scholar
  19. 19.
    Bachelard-Cascales E, Chapellier M, Delay E, Pochon G, Voeltzel T, Puisieux A, Caron de Fromentel C, Maguer-Satta V (2010) The CD10 enzyme is a key player to identify and regulate human mammary stem cells. Stem Cells 28(6):1081–1088PubMedCrossRefGoogle Scholar
  20. 20.
    Garbe JC, Pepin F, Pelissier FA, Sputova K, Fridriksdottir AJ, Guo DE, Villadsen R, Park M, Petersen OW, Borowsky AD, Stampfer MR, LaBarge MA (2012) Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia. Cancer Res 72(14):3687–3701PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Keller PJ, Arendt LM, Skibinski A, Logvinenko T, Klebba I, Dong S, Smith AE, Prat A, Perou CM, Gilmore H, Schnitt S, Naber SP, Garlick JA, Kuperwasser C (2012) Defining the cellular precursors to human breast cancer. Proc Natl Acad Sci USA 109(8):2772–2777PubMedCrossRefGoogle Scholar
  22. 22.
    Stingl J, Eaves CJ, Kuusk U, Emerman JT (1998) Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast. Differentiation 63(4):201–213PubMedCrossRefGoogle Scholar
  23. 23.
    Gusterson BA, Monaghan P, Mahendran R, Ellis J, O’Hare MJ (1986) Identification of myoepithelial cells in human and rat-breasts by anti-common acute lymphoblastic leukemia antigen antibody A12. J Natl Cancer Inst 77(2):343–349PubMedGoogle Scholar
  24. 24.
    Clarke RB, Howell A, Potten CS, Anderson E (1997) Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Res 57(22):4987–4991PubMedGoogle Scholar
  25. 25.
    Hilton HN, Graham JD, Kantimm S, Santucci N, Cloosterman D, Huschtscha LI, Mote PA, Clarke CL (2012) Progesterone and estrogen receptors segregate into different cell subpopulations in the normal human breast. Mol Cell Endocrinol 361(1–2):191–201PubMedCrossRefGoogle Scholar
  26. 26.
    Graham JD, Mote PA, Salagame U, van Dijk JH, Balleine RL, Huschtscha LI, Reddel RR, Clarke CL (2009) DNA replication licensing and progenitor numbers are increased by progesterone in normal human breast. Endocrinology 150(7):3318–3326PubMedCrossRefGoogle Scholar
  27. 27.
    Axlund S, Yoo B, Rosen R, Schaack J, Kabos P, LaBarbera D, Sartorius C (2013) Progesterone-inducible cytokeratin 5-positive cells in luminal breast cancer exhibit progenitor properties. Horm Cancer 4(1):36–49. doi: 10.1007/s12672-012-0127-5 PubMedCrossRefGoogle Scholar
  28. 28.
    Horwitz KB, Sartorius CA (2008) Progestins in hormone replacement therapies reactivate cancer stem cells in women with preexisting breast cancers: a hypothesis. J Clin Endocrinol Metab 93(9):3295–3298PubMedCrossRefGoogle Scholar
  29. 29.
    Stingl J, Raouf A, Emerman JT, Eaves CJ (2005) Epithelial progenitors in the normal human mammary gland. J Mammary Gland Biol Neoplasia 10(1):49–59PubMedCrossRefGoogle Scholar
  30. 30.
    Borgna S, Armellin M, di Gennaro A, Maestro R, Santarosa M (2012) Mesenchymal traits are selected along with stem features in breast cancer cells grown as mammospheres. Cell Cycle 11(22):4242–4251PubMedCrossRefGoogle Scholar
  31. 31.
    Fillmore CM, Gupta PB, Rudnick JA, Caballero S, Keller PJ, Lander ESKC (2010) Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling. Proc Natl Acad Sci USA 107(50):21737–21742PubMedCrossRefGoogle Scholar
  32. 32.
    Wang X-Y, Penalva L, Yuan H, Linnoila RI, Lu J, Okano H, Glazer R (2010) Musashi1 regulates breast tumor cell proliferation and is a prognostic indicator of poor survival. Mol Cancer 9(1):221PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Sutherland RL, Hall RE, Pang GY, Musgrove EA, Clarke CL (1988) Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells. Cancer Res 48(18):5084–5091PubMedGoogle Scholar
  34. 34.
    Brisken C (2013) Progesterone signalling in breast cancer: a neglected hormone coming into the limelight. Nat Rev Cancer 13(6):385–396PubMedCrossRefGoogle Scholar
  35. 35.
    Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, Hanada R, Joshi PA, Aliprantis A, Glimcher L, Pasparakis M, Khokha R, Ormandy CJ, Widschwendter M, Schett G, Penninger JM (2010) Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 468(7320):98–102PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Asselin-Labat ML, Shackleton M, Stingl J, Vaillant F, Forrest NC, Eaves CJ, Visvader JE, Lindeman GJ (2006) Steroid hormone receptor status of mouse mammary stem cells. J Natl Cancer Inst 98(14):1011–1014PubMedCrossRefGoogle Scholar
  37. 37.
    Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, Asselin-Labat ML, Gyorki DE, Ward T, Partanen A, Feleppa F, Huschtscha LI, Thorne HJ, kConFab, Fox SB, Yan M, French JD, Brown MA, Smyth GK, Visvader JE, Lindeman GJ (2009) Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat Med 15(8):907–913PubMedCrossRefGoogle Scholar
  38. 38.
    Hilton HN, Kantimm S, Graham JD, Clarke CL (2013) Changed lineage composition is an early event in breast carcinogenesis. Histol Histopathol 28(9):1197–1204PubMedGoogle Scholar
  39. 39.
    Maguer-Satta V, Besançon R, Bachelard-Cascales E (2011) Concise review: neutral endopeptidase (CD10): A multifaceted environment actor in stem cells, physiological mechanisms, and cancer. Stem Cells 29(3):389–396PubMedCrossRefGoogle Scholar
  40. 40.
    Huggins C, Moon RC, Sotokichi M (1962) Extinction of experimental mammary cancer, I. Estradiol-17β and progesterone. Proc Natl Acad Sci USA 48(3):379–386PubMedCrossRefGoogle Scholar
  41. 41.
    Maillot G, Lacroix-Triki M, Pierredon S, Gratadou L, Schmidt S, Bénès V, Roché H, Dalenc F, Auboeuf D, Millevoi S, Vagner S (2009) Widespread estrogen-dependent repression of microRNAs involved in breast tumor cell growth. Cancer Res 69(21):8332–8340PubMedCrossRefGoogle Scholar
  42. 42.
    Cui W, Li Q, Feng L, Ding W (2011) MiR-126-3p regulates progesterone receptors and involves development and lactation of mouse mammary gland. Mol Cell Biochem 355(1–2):17–25PubMedCrossRefGoogle Scholar
  43. 43.
    Cochrane DR, Jacobsen BM, Connaghan KD, Howe EN, Bain DL, Richer JK (2012) Progestin regulated miRNAs that mediate progesterone receptor action in breast cancer. Mol Cell Endocrinol 355(1):15–24PubMedCrossRefGoogle Scholar
  44. 44.
    Cittelly DM, Finlay-Schultz J, Howe EN, Spoelstra NS, Axlund SD, Hendricks P, Jacobsen BM, Sartorius CA, Richer JK (2013) Progestin suppression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4. Oncogene 32(20):2555–2564PubMedCrossRefGoogle Scholar
  45. 45.
    Li M, Zhao D, Ma G, Zhang B, Fu X, Zhu Z, Fu L, Sun X, Dong J-T (2013) Upregulation of ATBF1 by progesterone-PR signaling and its functional implication in mammary epithelial cells. Biochem Biophys Res Commun 430(1):358–363PubMedCrossRefGoogle Scholar
  46. 46.
    Vares G, Cui X, Wang B, Nakajima T, Nenoi M (2013) Generation of breast cancer stem cells by steroid hormones in irradiated human mammary cell lines. PLoS One 8(10):e77124PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Palafox M, Ferrer I, Pellegrini P, Vila S, Hernandez-Ortega S, Urruticoechea A, Climent F, Soler MT, Muñoz P, Viñals F, Tometsko M, Branstetter D, Dougall WC, González-Suárez E (2012) RANK induces epithelial–mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis. Cancer Res 72(11):2879–2888PubMedCrossRefGoogle Scholar
  48. 48.
    Shipitsin M, Campbell LL, Argani P, Weremowicz S, Bloushtain-Qimron N, Yao J, Nikolskaya T, Serebryiskaya T, Beroukhim R, Hu M, Halushka MK, Sukumar S, Parker LM, Anderson KS, Harris LN, Garber JE, Richardson AL, Schnitt SJ, Nikolsky Y, Gelman RS, Polyak K (2007) Molecular definition of breast tumor heterogeneity. Cancer Cell 11(3):259–273PubMedCrossRefGoogle Scholar
  49. 49.
    Tsang JS, Huang Y-H, Luo M-H, Ni Y-B, Chan S-K, Lui PW, Yu AC, Tan P, Tse G (2012) Cancer stem cell markers are associated with adverse biomarker profiles and molecular subtypes of breast cancer. Breast Cancer Res Treat 136(2):407–417PubMedCrossRefGoogle Scholar
  50. 50.
    Ghebeh H, Sleiman G, Manogaran P, Al-Mazrou A, Barhoush E, Al-Mohanna F, Tulbah A, Al-Faqeeh K, Adra C (2013) Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers. BMC Cancer 13(1):289PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Graham JD, Mote PA, Salagame U, Balleine RL, Huschtscha LI, Clarke CL (2009) Hormone-responsive model of primary human breast epithelium. J Mammary Gland Biol Neoplasia 14(4):367–379PubMedCrossRefGoogle Scholar
  52. 52.
    Mote PA, Leary JA, Clarke CL (1998) Immunohistochemical detection of progesterone receptors in archival breast cancer. Biotech Histochem 73(3):117–127PubMedCrossRefGoogle Scholar
  53. 53.
    Mote PA, Balleine RL, McGowan EM, Clarke CL (1999) Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle. J Clin Endocrinol Metab 84(8):2963–2971PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Heidi N. Hilton
    • 1
    Email author
  • N. Santucci
    • 1
  • A. Silvestri
    • 1
  • S. Kantimm
    • 1
  • L. I. Huschtscha
    • 2
  • J. D. Graham
    • 1
  • C. L. Clarke
    • 1
  1. 1.Westmead Institute for Cancer Research, Sydney Medical SchoolUniversity of Sydney at Westmead Millennium InstituteWestmeadAustralia
  2. 2.Children’s Medical Research InstituteWestmeadAustralia

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