Hormones and Cancer

, Volume 6, Issue 4, pp 142–152 | Cite as

Granulosa Cell-Specific Brca1 Loss Alone or Combined with Trp53 Haploinsufficiency and Transgenic FSH Expression Fails to Induce Ovarian Tumors

  • Dannielle H. Upton
  • Emily S. Fuller
  • Emily K. Colvin
  • Kirsty A. Walters
  • Mark Jimenez
  • Reena Desai
  • David J. Handelsman
  • Viive M. Howell
  • Charles M. AllanEmail author
Original Paper


BRCA1 mutations are associated with ovarian cancer. Previous studies reported that murine granulosa cell (GC) Brca1 loss caused ovarian-uterine tumors resembling serous cystadenomas, but the pathogenesis of these tumors may have been confounded by ectopic Brca1 expression and altered estrous cycling. We have used Tg.AMH.Cre conferring proven ovarian and GC-specific Cre activity to selectively target Brca1 disruption, denoted Brca1 GC−/−. Furthermore, ovary-specific Brca1 GC−/− was combined with global Trp53 haploinsufficiency (Trp53 +/−) and transgenic follicle-stimulating hormone (Tg.FSH) overexpression as a multi-hit strategy to investigate additional genetic and hormonal ovarian tumorigenesis mechanisms. However, 12-month-old Brca1 GC−/− mice had no detectable ovarian or uterine tumors. Brca1 GC−/− mice had significantly increased ovary weights, follicles exhibiting more pyknotic granulosa cells, and fewer corpora lutea with regular estrous cycling compared to controls. Isolated Brca1 GC−/− mutation lengthened the estrous cycle and proestrus stage; however, ovarian cystadenomas were not observed, even when Brca1 GC−/− was combined with Trp53 +/− and overexpressed Tg.FSH. Our Brca1 GC−/− models reveal that specific intra-follicular Brca1 loss alone, or combined with cancer-promoting genetic (Trp53 loss) and endocrine (high serum FSH) changes, was not sufficient to cause ovarian tumors. Our findings show that the ovary is remarkably resistant to oncogenesis, and support the emerging view of an extragonadal, multi-hit origin for ovarian tumorigenesis.


Ovarian Cancer Granulosa Cell BRCA1 Mutation Corpus Luteum Estrous Cycle 
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.



We thank Mamdouh Khalil and the staff of the ANZAC and Kolling Institute animal facilities.

Conflict of Interest

The authors declare that they have no competing interests.


This work was supported by the National Health and Medical Research Council (Australia) Project Grant (APP1008160) (CMA, DJH, KAW, VMH), Cancer council NSW project grant (VMH), and Cancer Institute NSW Fellowship (VMH).


  1. 1.
    Berchuck A, Heron KA, Carney ME et al (1998) Frequency of germline and somatic BRCA1 mutations in ovarian cancer. Clin Cancer Res 4:2433–2437PubMedGoogle Scholar
  2. 2.
    Pal T, Permuth-Wey J, Betts JA et al (2005) BRCA1 and BRCA2 mutations account for a large proportion of ovarian carcinoma cases. Cancer 104:2807–2816PubMedCrossRefGoogle Scholar
  3. 3.
    Brose MS, Rebbeck TR, Calzone KA, Stopfer JE, Nathanson KL, Weber BL (2002) Cancer risk estimates for BRCA1 mutation carriers identified in a risk evaluation program. J Natl Cancer Inst 94:1365–1372PubMedCrossRefGoogle Scholar
  4. 4.
    Leeper K, Garcia R, Swisher E, Goff B, Greer B, Paley P (2002) Pathologic findings in prophylactic oophorectomy specimens in high-risk women. Gynecol Oncol 87:52–56PubMedCrossRefGoogle Scholar
  5. 5.
    Piek JMJ, van Diest PJ, Zweemer RP et al (2001) Dysplastic changes in prophylactically removed fallopian tubes of women predisposed to developing ovarian cancer. J Pathol 195:451–456PubMedCrossRefGoogle Scholar
  6. 6.
    Geisler JP, Hatterman-Zogg MA, Rathe JA, Buller RE (2002) Frequency of BRCA1 dysfunction in ovarian cancer. J Natl Cancer Inst 94:61–67PubMedCrossRefGoogle Scholar
  7. 7.
    Russell PA, Pharoah PDP, De Foy K et al (2000) Frequent loss of BRCA1 mRNA and protein expression in sporadic ovarian cancers. Int J Cancer 87:317–321PubMedCrossRefGoogle Scholar
  8. 8.
    Chan KYK, Ozçelik H, Cheung ANY, Ngan HYS, Khoo US (2002) Epigenetic factors controlling the BRCA1 and BRCA2 genes in sporadic ovarian cancer. Cancer Res 62:4151–4156PubMedGoogle Scholar
  9. 9.
    Schuijer M, Berns EM (2003) TP53 and ovarian cancer. Hum Mutat 21:285–291PubMedCrossRefGoogle Scholar
  10. 10.
    The Cancer Genome Atlas Research N (2011) Integrated genomic analyses of ovarian carcinoma. Nature 474:609–615CrossRefGoogle Scholar
  11. 11.
    Kupryjańczyk J, Thor AD, Beauchamp R et al (1993) p53 gene mutations and protein accumulation in human ovarian cancer. Proc Natl Acad Sci U S A 90:4961–4965PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Bosari S, Viale G, Radaelli U, Bossi P, Bonoldi E, Coggi G (1993) p53 accumulation in ovarian carcinomas and its prognostic implications. Hum Pathol 24:1175–1179PubMedCrossRefGoogle Scholar
  13. 13.
    Zweemer RP, Shaw PA, Verheijen RM et al (1999) Accumulation of p53 protein is frequent in ovarian cancers associated with BRCA1 and BRCA2 germline mutations. J Clin Pathol 52:372–375PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Quinn BA, Brake T, Hua X et al (2009) Induction of ovarian leiomyosarcomas in mice by conditional inactivation of Brca1 and p53. PLoS One 4:e8404PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Clark-Knowles KV, Senterman MK, Collins O, Vanderhyden BC (2009) Conditional inactivation of Brca1, p53 and Rb in mouse ovaries results in the development of leiomyosarcomas. PLoS One 4:e8534Google Scholar
  16. 16.
    Clark-Knowles KV, Garson K, Jonkers J, Vanderhyden BC (2007) Conditional inactivation of Brca1 in the mouse ovarian surface epithelium results in an increase in preneoplastic changes. Exp Cell Res 313:133–145PubMedCrossRefGoogle Scholar
  17. 17.
    Chodankar R, Kwang S, Sangiorgi F et al (2005) Cell-nonautonomous induction of ovarian and uterine serous cystadenomas in mice lacking a functional Brca1 in ovarian granulosa cells. Curr Biol 15:561–565PubMedCrossRefGoogle Scholar
  18. 18.
    Hong H, Yen H-Y, Brockmeyer A et al (2010) Changes in the mouse estrus cycle in response to Brca1 inactivation suggest a potential link between risk factors for familial and sporadic ovarian cancer. Cancer Res 70:221–228PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Parrott JA, Doraiswamy V, Kim G, Mosher R, Skinner MK (2001) Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer. Mol Cell Endocrinol 172:213–222PubMedCrossRefGoogle Scholar
  20. 20.
    Radu A, Pichon C, Camparo P et al (2010) Expression of follicle-stimulating hormone receptor in tumor blood vessels. N Engl J Med 363:1621–1630PubMedCrossRefGoogle Scholar
  21. 21.
    Walters KA, Middleton LJ, Joseph SR et al (2012) Targeted loss of androgen receptor signaling in murine granulosa cells of preantral and antral follicles causes female subfertility. Biol Reprod 87(151):1–11Google Scholar
  22. 22.
    Lécureuil C, Fontaine I, Crepieux P, Guillou F (2002) Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice. Genesis 33:114–118PubMedCrossRefGoogle Scholar
  23. 23.
    Allan CM, Kalak R, Dunstan CR et al (2010) Follicle-stimulating hormone increases bone mass in female mice. Proc Natl Acad Sci U S A 107:22629–22634PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Fathalla MF (1971) Incessant ovulation? A factor in ovarian neoplasia? Lancet 298:163CrossRefGoogle Scholar
  25. 25.
    Cramer D, Welch WR (1983) Determinants of ovarian cancer rsik II. Inferences regarding pathogenesis. J Natl Cancer Inst 71:717–721PubMedGoogle Scholar
  26. 26.
    Lim P, Robson M, Spaliviero J et al (2009) Sertoli cell androgen receptor DNA binding domain is essential for the completion of spermatogenesis. Endocrinology 150:4755–4765PubMedCrossRefGoogle Scholar
  27. 27.
    Liu X, Holstege H, van der Gulden H et al (2007) Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proc Natl Acad Sci U S A 104:12111–12116PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Jacks T, Remington L, Williams BO et al (1994) Tumor spectrum analysis in p53-mutant mice. Curr Biol 4:1–7PubMedCrossRefGoogle Scholar
  29. 29.
    Allan CM, Haywood M, Swaraj S et al (2001) A novel transgenic model to characterize the specific effects of follicle-stimulating hormone on gonadal physiology in the absence of luteinizing hormone actions. Endocrinology 142:2213–2220PubMedGoogle Scholar
  30. 30.
    Singh J, O’Neill C, Handelsman DJ (1995) Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. Endocrinology 136:5311–5321PubMedGoogle Scholar
  31. 31.
    Schwenk F, Baron U, Rajewsky K (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res 23:5080–5081PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    McTavish KJ, Jimenez M, Walters KA et al (2007) Rising follicle-stimulating hormone levels with age accelerate female reproductive failure. Endocrinology 148:4432–4439PubMedCrossRefGoogle Scholar
  33. 33.
    Mettus RV, Rane SG (2003) Characterization of the abnormal pancreatic development, reduced growth and infertility in Cdk4 mutant mice. Oncogene 22:8413–8421PubMedCrossRefGoogle Scholar
  34. 34.
    Jimenez M, Spaliviero JA, Grootenhuis AJ, Verhagen J, Allan CM, Handelsman DJ (2005) Validation of an ultrasensitive and specific immunofluorometric assay for mouse follicle-stimulating hormone. Biol Reprod 72:78–85PubMedCrossRefGoogle Scholar
  35. 35.
    Desmeules P, Devine PJ (2006) Characterizing the ovotoxicity of cyclophosphamide metabolites on cultured mouse ovaries. Toxicol Sci 90:500–509PubMedCrossRefGoogle Scholar
  36. 36.
    Braw RH, Tsafriri A (1980) Effect of PMSG on follicular atresia in the immature rat ovary. J Reprod Fertil 59:267–272PubMedCrossRefGoogle Scholar
  37. 37.
    Labiche A, Heutte N, Herlin P, Chasle J, Gauduchon P, Elie N (2010) Stromal compartment as a survival prognostic factor in advanced ovarian carcinoma. Int J Gynecol Cancer 20:28–33PubMedCrossRefGoogle Scholar
  38. 38.
    Elie N, Labiche A, Michels J-J, Herlin P (2011) Control of low-resolution scanning of ovarian tumor stromal compartment. Image Anal Stereol 24:85–93CrossRefGoogle Scholar
  39. 39.
    McNamara KM, Harwood DT, Simanainen U, Walters KA, Jimenez M, Handelsman DJ (2010) Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography-tandem mass spectrometry. J Steroid Biochem Mol Biol 121:611–618PubMedCrossRefGoogle Scholar
  40. 40.
    Harwood DT, Handelsman DJ (2009) Development and validation of a sensitive liquid chromatography–tandem mass spectrometry assay to simultaneously measure androgens and estrogens in serum without derivatization. Clin Chim Acta 409:78–84PubMedCrossRefGoogle Scholar
  41. 41.
    Cheng G, Weihua Z, Mäkinen S et al (2002) A role for the androgen receptor in follicular atresia of estrogen receptor beta knockout mouse ovary. Biol Reprod 66:77–84PubMedCrossRefGoogle Scholar
  42. 42.
    Yu V (2000) Caretaker Brca1: keeping the genome in the straight and narrow. Breast Cancer Res 2:82–85PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Yoshida K, Miki Y (2004) Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage. Cancer Sci 95:866–871PubMedCrossRefGoogle Scholar
  44. 44.
    Larson JS, Tonkinson JL, Lai MT (1997) A BRCA1 mutant alters G2-M cell cycle control in human mammary epithelial cells. Cancer Res 57:3351–3355PubMedGoogle Scholar
  45. 45.
    Yan Y, Spieker RS, Kim M, Stoeger SM, Cowan KH (2005) BRCA1-mediated G2/M cell cycle arrest requires ERK1/2 kinase activation. Oncogene 24:3285–3296PubMedCrossRefGoogle Scholar
  46. 46.
    Shao N, Chai YL, Shyam E, Reddy P, Rao VN (1996) Induction of apoptosis by the tumor suppressor protein BRCA1. Oncogene 13:1–7PubMedGoogle Scholar
  47. 47.
    Deng C-X (2006) BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and cancer evolution. Nucleic Acids Res 34:1416–1426PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Xu X, Wagner KU, Larson D et al (1999) Conditional mutation of Brca 1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat Genet 22:37–43PubMedCrossRefGoogle Scholar
  49. 49.
    Dierich A, Sairam MR, Monaco L et al (1998) Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad Sci U S A 95:13612–13617PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Colgan TJ, Murphy J, Cole DEC, Narod S, Rosen B (2001) Occult carcinoma in prophylactic oophorectomy specimens: prevalence and association with BRCA germline mutation status. Am J Surg Pathol 25:1283–1289PubMedCrossRefGoogle Scholar
  51. 51.
    Kurman RJ, Shih I-M (2010) The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory. Am J Surg Pathol 34:433–443Google Scholar
  52. 52.
    Kurman RJ, Shih I-M (2011) Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer—shifting the paradigm. Hum Pathol 42:918–931PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Dubeau L (2008) The cell of origin of ovarian epithelial tumours. Lancet Oncol 9:1191–1197PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Ouchi M, Fujiuchi N, Sasai K et al (2004) BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition. J Biol Chem 279:19643–19648PubMedCrossRefGoogle Scholar
  55. 55.
    George J, Alsop K, Etemadmoghadam D et al (2013) Nonequivalent gene expression and copy number alterations in high-grade serous ovarian cancers with BRCA1 and BRCA2 mutations. Clin Cancer Res 19:3474–3484PubMedCrossRefGoogle Scholar
  56. 56.
    Esteller M, Silva JM, Dominguez G et al (2000) Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 92:564–569PubMedCrossRefGoogle Scholar
  57. 57.
    Cornelis R, Neuhausen S, Arason A et al (1995) Allele loss rate at 17q12-q21 in breast and ovarian tumors from 52 germ line BRCA1-mutation carriers. Genes Chromosom Cancer 13:203–210PubMedCrossRefGoogle Scholar
  58. 58.
    Cressman VL, Backlund DC, Hicks EM, Gowen LC, Godfrey V, Koller BH (1999) Mammary tumor formation in p53- and BRCA1-deficient mice. Cell Growth Differ Mol Biol J Am Assoc Cancer Res 10:1–10Google Scholar
  59. 59.
    Jonkers J, Meuwissen R, van der Gulden H, Peterse H, van der Valk M, Berns A (2001) Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat Genet 29:418–425PubMedCrossRefGoogle Scholar
  60. 60.
    Banerji S, Cibulskis K, Rangel-Escareno C et al (2012) Sequence analysis of mutations and translocations across breast cancer subtypes. Nature 486:405–409PubMedCentralPubMedCrossRefGoogle Scholar
  61. 61.
    Flesken-Nikitin A, Choi K-C, Eng JP, Shmidt EN, Nikitin AY (2003) Induction of carcinogenesis by concurrent inactivation of p53 and Rb1 in the mouse ovarian surface epithelium. Cancer Res 63:3459–3463PubMedGoogle Scholar
  62. 62.
    Miki Y, Swensen J, Shattuck-Eidens D et al (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:66–71PubMedCrossRefGoogle Scholar
  63. 63.
    Szabova L, Yin C, Bupp S et al (2012) Perturbation of Rb, p53, and Brca1 or Brca2 cooperate in inducing metastatic serous epithelial ovarian cancer. Cancer Res 72:4141–4153PubMedCentralPubMedCrossRefGoogle Scholar
  64. 64.
    Perets R, Wyant GA, Muto KW et al (2013) Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian cancer in brca;Tp53;pten models. Cancer Cell 24:751–765PubMedCentralPubMedCrossRefGoogle Scholar
  65. 65.
    Zheng W, Lu JJ, Luo F et al (2000) Ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone. Gynecol Oncol 76:80–88PubMedCrossRefGoogle Scholar
  66. 66.
    Hu Y, Ghosh S, Amleh A et al (2005) Modulation of aromatase expression by BRCA1: a possible link to tissue-specific tumor suppression. Oncogene 24:8343–8348PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Dannielle H. Upton
    • 1
  • Emily S. Fuller
    • 2
  • Emily K. Colvin
    • 2
  • Kirsty A. Walters
    • 1
  • Mark Jimenez
    • 1
  • Reena Desai
    • 1
  • David J. Handelsman
    • 1
  • Viive M. Howell
    • 2
  • Charles M. Allan
    • 1
    Email author
  1. 1.ANZAC Research InstituteUniversity of Sydney, Concord HospitalSydneyAustralia
  2. 2.Kolling Institute of Medical ResearchUniversity of SydneySt. LeonardsAustralia

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