Chronology and complexities of ovarian tumorigenesis in FORKO mice: Age-dependent gene alterations and progressive dysregulation of Major Histocompatibility Complex (MHC) Class I and II profiles
Molecular Endocrinology Laboratory, Clinical Research Institute of Montréal, 110 Pine Avenue West, Montréal, Québec H2W 1R7, Canada. Molecular and Cellular Endocrinology
(Impact Factor: 4.41).
11/2010; 329(1-2):37-46. DOI: 10.1016/j.mce.2010.05.015
Among gynecologic malignancies ovarian cancer is the deadliest and most difficult to detect at early stages. As ovarian tumors have long latency and are relatively more frequent in postmenopausal women, revealing chronological changes in model systems might help in the discovery of novel molecular targets and diagnostic biomarkers for disease detection and management. Follitropin receptor knockout (FORKO) mice with early and sustained sex steroid hormone disharmony develop various age-dependent ovarian abnormalities including increased incidence ovarian tumors in complete absence of ovulation. These mutants show various tumor cell types including those related to ovarian surface epithelium around 12-15 months of age. To explore why the FORKO mice develop ovarian tumors later in life, we assessed global gene expression changes during the pre-tumor period (at 8 months). Age-matched wild-type and FORKO mice were compared to gain a comprehensive view of genes that are misregulated, even before overt tumors appear in mutants. Applying a conservative 2-fold change to detect changes, our study identified 476 genes (338 upregulated and 138 downregulated) to be altered between 8-month-old FORKO and wild-type ovaries. Using Ingenuity Pathway Analysis (IPA), we found highly significant alterations in five functional networks in pre-tumor stage FORKO ovaries. Notably, the top network to change in 8-month-old FORKO ovaries was associated with functions implicated in immune system development and function. We selected 9 immune related genes that are reportedly altered in Epithelial Ovarian Cancer (EOC) in women and confirmed their expression and chronology of changes in FORKO ovaries before and after tumor development. Our data indicate that immune surveillance mechanisms are compromised with in a 4-month window of tumorigenic alterations. In addition, expression of previously unrecognized genes misregulated in the dysfunctional FORKO ovaries suggests mechanisms not yet appreciated to date. We propose that a better understanding of genes that change before overt tumors develop could provide useful insights into ovarian carcinogenesis and open the door to additional new targets for treating ovarian cancers.
Available from: Hiren Patel
- "Also the FSHRKO mice exhibit increased incidence of ovarian tumors in complete absence of ovulation. These mutants show various tumor cell types including those related to ovarian surface epithelium around 12–15 months of age . At this juncture it is quite intriguing to find out whether certain compensatory mechanisms or altered cell signaling pathways may result in PF assembly and increased cancer incidence in these animals. "
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ABSTRACT: We have earlier reported that follicle stimulating hormone (FSH) modulates ovarian stem cells which include pluripotent, very small embryonic-like stem cells (VSELs) and their immediate descendants 'progenitors' termed ovarian germ stem cells (OGSCs), lodged in adult mammalian ovarian surface epithelium (OSE). FSH may exert pleiotropic actions through its alternatively spliced receptor isoforms. Four isoforms of FSH receptors (FSHR) are reported in literature of which FSH-R1 and FSH-R3 have biological activity. Present study was undertaken to identify FSHR isoforms mediating FSH action on ovarian stem cells, using sheep OSE cells culture as the study model.
Cultures of sheep OSE cells (a mix of epithelial cells, VSELs, OGSCs and few contaminating red blood cells) were established with and without FSH5IU/ml treatment. Effect of FSH treatment on self-renewal of VSELs and their differentiation into OGSCS was studied after 15 hrs by qRT-PCR using markers specific for VSELs (Oct-4A, Sox-2) and OGSCs (Oct-4). FSH receptors and its specific transcripts (R1 and R3) were studied after 3 and 15 hrs of FSH treatment by immunolocalization, in situ hybridization and qRT-PCR. FSHR and OCT-4 were also immuno-localized on sheep ovarian sections, in vitro matured follicles and early embryos.
FSH treatment resulted in increased stem cells self-renewal and clonal expansion evident by the appearance of stem cell clusters. FSH receptors were expressed on ovarian stem cells whereas the epithelial cells were distinctly negative. An increase in R3 mRNA transcripts was noted after 3 hrs of FSH treatment and was reduced to basal levels by 15 hrs, whereas R1 transcript expression remained unaffected. Both FSHR and OCT-4 were immuno-localized in nuclei of stem cells, showed nuclear or ooplasmic localization in oocytes of primordial follicles and in cytoplasm of granulosa cells in growing follicles.
FSH modulates ovarian stem cells via FSH-R3 to undergo potential self-renewal, clonal expansion as 'cysts' and differentiation into oocytes. OCT-4 and FSHR proteins (required initially to maintain pluripotent state of VSELs and for FSH action respectively) gradually shift from nuclei to cytoplasm of developing oocytes and are later possibly removed by surrounding granulosa cells as the oocyte prepares itself for fertilization.
Available from: Susana Rulli
- "The role of FSH in promoting somatic cell tumors is more complicated. Firstly, most Forko mice older than a year develop both sex-cord and epithelial ovarian tumors including granulosa cell tumors and serous papillary epithelial adenomas [39, 40]. Aging FshrD580H+ mice also develop ovarian surface epithelial hyperplasia  and the presence of FSH accelerates the formation of granulosa cell tumors in inhibin-α-deficient mice  suggesting, that FSH signalling can both suppress and support hyper- and metaplastic growth of several cell types directly or indirectly. "
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ABSTRACT: During the last two decades a large number of genetically modified mouse lines with altered gonadotropin action have been generated. These mouse lines fall into three categories: the lack-of-function mice, gain-of-function mice, and the mice generated by breeding the abovementioned lines with other disease model lines. The mouse strains lacking gonadotropin action have elucidated the necessity of the pituitary hormones in pubertal development and function of gonads, and revealed the processes from the original genetic defect to the pathological phenotype such as hypo- or hypergonadotropic hypogonadism. Conversely, the strains of the second group depict consequences of chronic gonadotropin action. The lines vary from those expressing constitutively active receptors and those secreting follicle-stimulating hormone (FSH) with slowly increasing amounts to those producing human choriogonadotropin (hCG), amount of which corresponds to 2000-fold luteinizing hormone (LH)/hCG biological activity. Accordingly, the phenotypes diverge from mild anomalies and enhanced fertility to disrupted gametogenesis, but eventually chronic, enhanced and non-pulsatile action of both FSH and LH leads to female and male infertility and/or hyper- and neoplasias in most of the gonadotropin gain-of-function mice. Elevated gonadotropin levels also alter the function of several extra-gonadal tissues either directly or indirectly via increased sex steroid production. These effects include promotion of tumorigenesis in tissues such as the pituitary, mammary and adrenal glands. Finally, the crossbreedings of the current mouse strains with other disease models are likely to uncover the contribution of gonadotropins in novel biological systems, as exemplified by the recent crossbreed of LHCG receptor deficient mice with Alzheimer disease mice.
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ABSTRACT: In studying the age dependence and chronology of ovarian tumors in follicle stimulating hormone receptor knockout mice, we identified a novel ovarian tumor associated gene-12 (OTAG-12), which is progressively downregulated and maps to Chr. 8B3.3. OTAG-12 protein overexpression in mouse ovarian and mammary tumor cells suggested powerful anti-proliferative effects. In human epithelial ovarian cancers (OCs) and OC cell lines, OTAG-12 mRNA expression is downregulated in comparison with normal ovaries. Cloning and identification revealed that human OTAG-12 mapping to gene-rich Chr. 19p13.12 is expressed in three spliced forms: hOTAG-12a, hOTAG-12b and hOTAG-12c, of which b is predominant in the normal ovary. Functionally active hOTAG-12b is a simple protein with no disulfide bonds and a nuclear localization signal is present in all variants. Transfection of OTAG-12 variants in OC and tumorigenic HEK293 cells confirmed nuclear localization. hOTAG-12b overexpression in OC and HEK293 cells effectively suppressed cell growth, anchorage-dependent and independent colony formation followed by apoptosis, whereas hOTAG-12a and hOTAG-12c had no such effects. Deletion mutants identified the critical importance of carboxyl terminus for hOTAG-12b function. Doxycycline-inducible growth inhibition of HEK293 cells by hOTAG-12a was associated with effects on G2 cell cycle arrest and apoptosis induction. hOTAG-12b expression rendered tumorigenic cells more sensitive to four apoptotic stimuli including etoposide-a topoisomerase-II inhibitor. Doxycycline-induced hOTAG-12b expression blocked xenograft tumor growth in nude mice, whereas hOTAG-12a was ineffective. Although p53-pathway-dependent apoptotic agents could upregulate endogenous hOTAG-12b and p53 in UCI-101/107 OC cells, hOTAG-12b could also induce apoptosis in p53-null and platinum-resistant SKOV3 OC cells and Doxycycline-induced hOTAG-12b did not alter p53. Further study showed that hOTAG-12b increases mRNAs of pro-apoptotic genes such as BAD, GADD45α and CIEDB, while inhibiting anti-apoptotic NAIP and Akt1 expression, suggesting that hOTAG-12b-induced apoptosis might be p53-independent. These results indicate that hOTAG-12b is a putative ovarian tumor suppressor gene warranting further studies.
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