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Aberrant granulosa cell-fate related to inactivated p53/Rb signaling contributes to granulosa cell tumors and to FOXL2 downregulation in the mouse ovary

Abstract

Ovarian granulosa cell tumors (GCTs) are indolent tumors of the ovary affecting women at all ages and potentially displaying late recurrence. Even if there is still little information regarding the mechanisms involved in GCT development and progression, FOXL2 would be a major tumor suppressor gene in granulosa cells. We analyzed the mechanisms underlying GCT initiation and progression by using mice with targeted expression of SV40 large T-antigen in granulosa cells (AT mouse), which develop GCTs. Consistent with patients, AT mice with developing GCTs displayed increased levels in circulating anti-Müllerian hormone (AMH), estradiol and androgens, as well as decreased FOXL2 protein abundance. Very few mice developed metastases (1 out of 30). In situ analyses revealed that GCT initiation resulted from both increased granulosa cell survival and proliferation in large antral follicles. Tumorigenesis was associated with the combined inactivation of p53 and Rb pathways, as shown by the impaired expression of respective downstream targets regulating cell apoptosis and proliferation, i.e., Bax, Bak, Gadd45a, Ccna2, Ccne1, E2f1, and Orc1. Importantly, the expression of FOXL2 was still present in newly developed GCTs and its downregulation only started during GCT growth. Collectively, our experiments provide evidence that disrupted p53/Rb signaling can drive tumor initiation and growth. This model challenges the current paradigm that impaired FOXL2 signaling is a major switch of granulosa cell tumorigenesis, albeit possibly contributing to tumor growth.

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References

  1. 1.

    Pectasides D, Pectasides E, Psyrri A. Granulosa cell tumor of the ovary. Cancer Treat Rev. 2008;34:1–12.

    CAS  PubMed  Google Scholar 

  2. 2.

    Rey RA, Lhommé C, Marcillac I, Lahlou N, Duvillard P, Josso N, et al. Antimüllerian hormone as a serum marker of granulosa cell tumorsof the ovary: comparative study with serum alpha-inhibin and estradiol. Am J Obstet Gynecol. 1996;174:958–65.

    CAS  PubMed  Google Scholar 

  3. 3.

    Jamieson S, Fuller PJ. Molecular pathogenesis of granulosa cell tumors of the ovary. Endocr Rev. 2012;33:109–44.

    CAS  PubMed  Google Scholar 

  4. 4.

    McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000;21:200–14.

    CAS  PubMed  Google Scholar 

  5. 5.

    Kalfa N, Sultan C. Juvenile ovarian granulosa cell tumor: a benign or malignant condition? Gynecol Endocrinol J Int Soc Gynecol Endocrinol. 2009;25:299–302.

    Google Scholar 

  6. 6.

    Shah SP, Köbel M, Senz J, Morin RD, Clarke BA, Wiegand KC, et al. Mutation of FOXL2 in granulosa-cell tumors of the ovary. N Engl J Med. 2009;360:2719–29.

    CAS  PubMed  Google Scholar 

  7. 7.

    Gershon R, Aviel-Ronen S, Korach J, Daniel-Carmi V, Avivi C, Bar-Ilan D, et al. FOXL2 C402G mutation detection using MALDI-TOF-MS in DNA extracted from Israeli granulosa cell tumors. Gynecol Oncol. 2011;122:580–4.

    CAS  PubMed  Google Scholar 

  8. 8.

    Goulvent T, Ray-Coquard I, Borel S, Haddad V, Devouassoux-Shisheboran M, Vacher-Lavenu M-C, et al. DICER1 and FOXL2 mutations in ovarian sex cord-stromal tumours: a GINECO Group study. Histopathology. 2016;68:279–85.

    PubMed  Google Scholar 

  9. 9.

    Kalfa N, Philibert P, Patte C, Ecochard A, Duvillard P, Baldet P, et al. Extinction of FOXL2 expression in aggressive ovarian granulosa cell tumors in children. Fertil Steril. 2007;87:896–901.

    CAS  PubMed  Google Scholar 

  10. 10.

    Pannetier M, Chassot A-A, Chaboissier M-C, Pailhoux E. Involvement of FOXL2 and RSPO1 in ovarian determination, development, and maintenance in mammals. Sex Dev Genet Mol Biol Evol Endocrinol Embryol Pathol Sex Determ Differ. 2016;10:167–84.

    CAS  Google Scholar 

  11. 11.

    Georges A, Auguste A, Bessière L, Vanet A, Todeschini A-L, Veitia RA. FOXL2: a central transcription factor of the ovary. J Mol Endocrinol. 2014;52:R17–33.

    CAS  PubMed  Google Scholar 

  12. 12.

    Benayoun BA, Georges AB, L’Hôte D, Andersson N, Dipietromaria A, Todeschini A-L, et al. Transcription factor FOXL2 protects granulosa cells from stress and delays cell cycle: role of its regulation by the SIRT1 deacetylase. Hum Mol Genet. 2011;20:1673–86.

    CAS  PubMed  Google Scholar 

  13. 13.

    Kim J-H, Yoon S, Park M, Park H-O, Ko J-J, Lee K, et al. Differential apoptotic activities of wild-type FOXL2 and the adult-type granulosa cell tumor-associated mutant FOXL2 (C134W). Oncogene. 2011;30:1653–63.

    CAS  PubMed  Google Scholar 

  14. 14.

    Batista F, Vaiman D, Dausset J, Fellous M, Veitia RA. Potential targets of FOXL2, a transcription factor involved in craniofacial and follicular development, identified by transcriptomics. Proc Natl Acad Sci USA. 2007;104:3330–5.

    CAS  PubMed  Google Scholar 

  15. 15.

    Kalfa N, Ecochard A, Patte C, Duvillard P, Audran F, Pienkowski C, et al. Activating mutations of the stimulatory G protein in juvenile ovarian granulosa cell tumors: a new prognostic factor? J Clin Endocrinol Metab. 2006;91:1842–7.

    CAS  PubMed  Google Scholar 

  16. 16.

    Bessière L, Todeschini A-L, Auguste A, Sarnacki S, Flatters D, Legois B, et al. A hot-spot of in-frame duplications activates the oncoprotein AKT1 in juvenile granulosa cell tumors. EBioMedicine. 2015;2:421–31.

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Auguste A, Bessière L, Todeschini A-L, Caburet S, Sarnacki S, Prat J, et al. Molecular analyses of juvenile granulosa cell tumors bearing AKT1 mutations provide insights into tumor biology and therapeutic leads. Hum Mol Genet. 2015;24:6687–98.

    CAS  PubMed  Google Scholar 

  18. 18.

    Arcellana-Panlilio MY, Egeler RM, Ujack E, Magliocco A, Stuart GCE, Robbins SM, et al. Evidence of a role for the INK4 family of cyclin-dependent kinase inhibitors in ovarian granulosa cell tumors. Genes Chromosomes Cancer. 2002;35:176–81.

    CAS  PubMed  Google Scholar 

  19. 19.

    Nogales FF, Musto ML, Sáez AI, Robledo M, Palacios J, Aneiros J. Multifocal intrafollicular granulosa cell tumor of the ovary associated with an unusual germline p53 mutation. Mod Pathol J U S Can Acad Pathol Inc. 2004;17:868–73.

    CAS  Google Scholar 

  20. 20.

    Alexiadis M, Rowley SM, Chu S, Leung DTH, Stewart CJR, Amarasinghe KC, et al. Mutational landscape of ovarian adult granulosa cell tumors from whole exome and targeted TERT promoter sequencing. Mol Cancer Res MCR. 2019;17:177–85.

    CAS  PubMed  Google Scholar 

  21. 21.

    Mayr D, Kaltz-Wittmer C, Arbogast S, Amann G, Aust DE, Diebold J. Characteristic pattern of genetic aberrations in ovarian granulosa cell tumors. Mod Pathol J U S Can Acad Pathol Inc. 2002;15:951–7.

    CAS  Google Scholar 

  22. 22.

    Caburet S, Anttonen M, Todeschini A-L, Unkila-Kallio L, Mestivier D, Butzow R, et al. Combined comparative genomic hybridization and transcriptomic analyses of ovarian granulosa cell tumors point to novel candidate driver genes. BMC Cancer. 2015;10;251.

  23. 23.

    Andreu-Vieyra C, Chen R, Matzuk MM. Conditional deletion of the retinoblastoma (Rb) gene in ovarian granulosa cells leads to premature ovarian failure. Mol Endocrinol. 2008;22:2141–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Makrigiannakis A, Amin K, Coukos G, Tilly JL, Coutifaris C. Regulated expression and potential roles of p53 and Wilms’ tumor suppressor gene (WT1) during follicular development in the human ovary. J Clin Endocrinol Metab. 2000;85:449–59.

    CAS  PubMed  Google Scholar 

  25. 25.

    Tilly KI, Banerjee S, Banerjee PP, Tilly JL. Expression of the p53 and Wilms’ tumor suppressor genes in the rat ovary: gonadotropin repression in vivo and immunohistochemical localization of nuclear p53 protein to apoptotic granulosa cells of atretic follicles. Endocrinology. 1995;136:1394–402.

    CAS  PubMed  Google Scholar 

  26. 26.

    Hafner A, Bulyk ML, Jambhekar A, Lahav G. The multiple mechanisms that regulate p53 activity and cell fate. Nat Rev Mol Cell Biol. 2019;20:199–210.

    CAS  PubMed  Google Scholar 

  27. 27.

    Engelmann D, Pützer BM. The dark side of E2F1: in transit beyond apoptosis. Cancer Res. 2012;72:571–5.

    CAS  PubMed  Google Scholar 

  28. 28.

    Fuller PJ, Leung D, Chu S. Genetics and genomics of ovarian sex cord-stromal tumors. Clin Genet. 2017;91:285–91.

    CAS  PubMed  Google Scholar 

  29. 29.

    Dutertre M, Gouédard L, Xavier F, Long WQ, di Clemente N, Picard JY, et al. Ovarian granulosa cell tumors express a functional membrane receptor for anti-Müllerian hormone in transgenic mice. Endocrinology. 2001;142:4040–6.

    CAS  PubMed  Google Scholar 

  30. 30.

    Anttonen M, Färkkilä A, Tauriala H, Kauppinen M, Maclaughlin DT, Unkila-Kallio L, et al. Anti-Müllerian hormone inhibits growth of AMH type II receptor-positive human ovarian granulosa cell tumor cells by activating apoptosis. Lab Investig J Tech Methods Pathol. 2011;91:1605–14.

    CAS  Google Scholar 

  31. 31.

    Ahuja D, Sáenz-Robles MT, Pipas JM. SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation. Oncogene. 2005;24:7729–45.

    CAS  PubMed  Google Scholar 

  32. 32.

    Fischer M. Census and evaluation of p53 target genes. Oncogene. 2017;36:3943–56.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Ohtani K, DeGregori J, Leone G, Herendeen DR, Kelly TJ, Nevins JR. Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor. Mol Cell Biol. 1996;16:6977–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Ohtani K, DeGregori J, Nevins JR. Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci USA. 1995;92:12146–50.

    CAS  PubMed  Google Scholar 

  35. 35.

    Georges A, L’Hôte D, Todeschini AL, Auguste A, Legois B, Zider A, et al. The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells. eLife. 2014;3. https://doi.org/10.7554/eLife.04207.

  36. 36.

    Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, et al. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell. 2009;139:1130–42.

    CAS  PubMed  Google Scholar 

  37. 37.

    Bukovský A, Caudle MR, Keenan JA, Wimalasena J, Foster JS, Van Meter SE. Quantitative evaluation of the cell cycle-related retinoblastoma protein and localization of Thy-1 differentiation protein and macrophages during follicular development and atresia, and in human corpora lutea. Biol Reprod. 1995;52:776–92.

    PubMed  Google Scholar 

  38. 38.

    Zhao H, Bauzon F, Fu H, Lu Z, Cui J, Nakayama K, et al. Skp2 deletion unmasks a p27 safeguard that blocks tumorigenesis in the absence of pRb and p53 tumor suppressors. Cancer Cell. 2013;24:645–59.

    CAS  PubMed  Google Scholar 

  39. 39.

    Alexander BM, Van Kirk EA, Naughton LMA, Murdoch WJ. Ovarian morphometrics in TP53-deficient mice. Anat Rec (Hoboken NJ). 2007;290:59–64.

    CAS  Google Scholar 

  40. 40.

    Ligtenberg MJ, Siers M, Themmen AP, Hanselaar TG, Willemsen W, Brunner HG. Analysis of mutations in genes of the follicle-stimulating hormone receptor signaling pathway in ovarian granulosa cell tumors. J Clin Endocrinol Metab. 1999;84:2233–4.

    CAS  PubMed  Google Scholar 

  41. 41.

    Fan H-Y, Liu Z, Paquet M, Wang J, Lydon JP, DeMayo FJ, et al. Cell type-specific targeted mutations of Kras and Pten document proliferation arrest in granulosa cells versus oncogenic insult to ovarian surface epithelial cells. Cancer Res. 2009;69:6463–72.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Laguë M-N, Paquet M, Fan H-Y, Kaartinen MJ, Chu S, Jamin SP, et al. Synergistic effects of Pten loss and WNT/CTNNB1 signaling pathway activation in ovarian granulosa cell tumor development and progression. Carcinogenesis. 2008;29:2062–72.

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Chu S, Rushdi S, Zumpe ET, Mamers P, Healy DL, Jobling T, et al. FSH-regulated gene expression profiles in ovarian tumours and normal ovaries. Mol Hum Reprod. 2002;8:426–33.

    CAS  PubMed  Google Scholar 

  44. 44.

    François CM, Petit F, Giton F, Gougeon A, Ravel C, Magre S, et al. A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty. Sci Rep. 2017;7:46222.

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Ogawa T, Yogo K, Ishida N, Takeya T. Synergistic effects of activin and FSH on hyperphosphorylation of Rb and G1/S transition in rat primary granulosa cells. Mol Cell Endocrinol. 2003;210:31–8.

    CAS  PubMed  Google Scholar 

  46. 46.

    Sirotkin AV, Benco A, Tandlmajerova A, Vasícek D, Kotwica J, Darlak K, et al. Transcription factor p53 can regulate proliferation, apoptosis and secretory activity of luteinizing porcine ovarian granulosa cell cultured with and without ghrelin and FSH. Reprod Camb Engl. 2008;136:611–8.

    CAS  Google Scholar 

  47. 47.

    De Cian M-C, Pauper E, Bandiera R, Vidal VPI, Sacco S, Gregoire EP, et al. Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary. Oncogene. 2017;36:208–18.

    PubMed  Google Scholar 

  48. 48.

    Liu Z, Ren YA, Pangas SA, Adams J, Zhou W, Castrillon DH, et al. FOXO1/3 and PTEN depletion in granulosa cells promotes ovarian granulosa cell tumor development. Mol Endocrinol (Balt Md). 2015;29:1006–24.

    CAS  Google Scholar 

  49. 49.

    Benayoun BA, Caburet S, Dipietromaria A, Georges A, D’Haene B, Pandaranayaka PJE, et al. Functional exploration of the adult ovarian granulosa cell tumor-associated somatic FOXL2 mutation p.Cys134Trp (c.402C>G). PLoS ONE. 2010;5:e8789.

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Fleming NI, Knower KC, Lazarus KA, Fuller PJ, Simpson ER, Clyne CD. Aromatase is a direct target of FOXL2: C134W in granulosa cell tumors via a single highly conserved binding site in the ovarian specific promoter. PloS ONE. 2010;5:e14389.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    L’Hôte D, Georges A, Todeschini AL, Kim J-H, Benayoun BA, Bae J, et al. Discovery of novel protein partners of the transcription factor FOXL2 provides insights into its physiopathological roles. Hum Mol Genet. 2012;21:3264–74.

    PubMed  Google Scholar 

  52. 52.

    Merras-Salmio L, Vettenranta K, Möttönen M, Heikinheimo M. Ovarian granulosa cell tumors in childhood. Pediatr Hematol Oncol. 2002;19:145–56.

    PubMed  Google Scholar 

  53. 53.

    Batisse-Lignier M, Sahut-Barnola I, Tissier F, Dumontet T, Mathieu M, Drelon C, et al. P53/Rb inhibition induces metastatic adrenocortical carcinomas in a preclinical transgenic model. Oncogene 2017;36:4445–56.

    CAS  PubMed  Google Scholar 

  54. 54.

    Hillman RT, Celestino J, Terranova C, Beird HC, Gumbs C, Little L, et al. KMT2D/MLL2 inactivation is associated with recurrence in adult-type granulosa cell tumors of the ovary. Nat Commun 2018;9:2496 https://doi.org/10.1038/s41467-018-04950-x

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    François CM, Wargnier R, Petit F, Goulvent T, Rimokh R, Treilleux I, et al. 17β-estradiol inhibits spreading of metastatic cells from granulosa cell tumors through a non-genomic mechanism involving GPER1. Carcinogenesis. 2015;36:564–73.

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Devillers M, Petit F, Cluzet V, François CM, Giton F, Garrel G, et al. FSH inhibits AMH to support ovarian estradiol synthesis in infantile mice. J Endocrinol 2018;240:215–28.

    Google Scholar 

  57. 57.

    Guigon CJ, Fozzatti L, Lu C, Willingham MC, Cheng S-Y. Inhibition of mTORC1 signaling reduces tumor growth but does not prevent cancer progression in a mouse model of thyroid cancer. Carcinogenesis. 2010;31:1284–91.

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We wish to thank Pr. R Veitia, Dr. AL Todeschini (Institut Jacques Monod, Paris, France), and Dr. S Bouret (Institut du Cerveau et de la Moëlle, Paris, France) for valuable discussions regarding this work. They acknowledge the technical assistance of M. Surenaud for gonadotropin assays (IMRB-INSERM U955, Hôpital Henri Mondor, France), and Dr. B. Querat and Dr. D. L’hôte (University Paris 7, BFA unit, France) for providing p53 antibody and Foxl2 primers. They also thank the technical help of Dr. M. Pannetier (INRA, Jouy en Josas, France) for the detection of FOXL2 by Western blots. They thank O. Trassard (UMS-32 INSERM, Hôpital du Kremlin Bicêtre, France) for his kind help in scanning histological slides. They acknowledge the great help of the technicians of the Buffon Animal Facility (University Paris Diderot, France). This work was supported by Ligue contre le Cancer Ile-de-France (attributed to NC), Projet Fondation ARC (PJA 20151203391, attributed to CJG) and GEFLUC Paris-Ile de France (CJG). It was also funded by “Institut National de la Santé & de la Recherche Médicale” (Inserm), “Center National de la Recherche Scientifique” (CNRS), University Paris Diderot, and by fellowships from Ecole Doctorale Bio-SPC (VC, MMD, CMF).

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Cluzet, V., Devillers, M.M., Petit, F. et al. Aberrant granulosa cell-fate related to inactivated p53/Rb signaling contributes to granulosa cell tumors and to FOXL2 downregulation in the mouse ovary. Oncogene 39, 1875–1890 (2020). https://doi.org/10.1038/s41388-019-1109-7

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