Adult ovarian granulosa cell tumor transcriptomics: prevalence of FOXL2 target genes misregulation gives insights into the pathogenic mechanism of the p.Cys134Trp somatic mutation.

Ovarian granulosa cell tumors (OGCT) are the most frequent kind of sex cord-stromal tumors, and represent ∼2-5% of all ovarian malignancies. OGCTs exist as two entities, juvenile and adult types, with specific clinical and pathological characteristics. The molecular pathogenesis of these tumors has just begun to be unraveled. Indeed, recent studies have indicated that mutation and/or misregulation of the key ovarian transcription factor FOXL2 has a role in OGCT formation, although the mechanisms remain unclear. To better understand the molecular characteristics of OGCT, we studied the transcriptomic profiles of ten human adult-type OGCT samples, as well as ethnically matched granulosa cell (GC) controls. We find that the OGCT samples analyzed herein exhibit several hallmarks of cancer, including increased expression of genes linked to cell proliferation, but decreased expression of those conferring sensitivity to cell death. Moreover, genes differentially expressed in OGCTs are significantly enriched for known FOXL2 target genes, consistently with the prevalence of FOXL2 somatic mutation in these tumors. Expression of these targets is altered in a way expected to promote malignant transformation, for instance, through induction of genes associated with faster cell cycling and downregulation of genes associated with cell death. Over time, such defects may be responsible at least partly for the malignant transformation of healthy GCs into OGCT. These insights into the molecular pathogenesis of OGCTs may open the way to new efforts in the development of more targeted therapeutic strategies for OGCT patients.

TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary.

Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.

GATA-4 is a granulosa cell factor employed in inhibin-alpha activation by the TGF-beta pathway.

Part of heterodimeric inhibin, inhibin-alpha is crucial for mammalian ovarian function. Regulation of inhibin-alpha expression in granulosa cells is both endocrine, primarily by follicle-stimulating hormone (FSH), and paracrine, primarily by members of the transforming growth factor beta (TGF-beta) superfamily. Smad proteins transmit TGF-beta signals to the nucleus, but the cooperating transcription factors involved in inhibin-alpha promoter activation remain unknown. Transcription factor GATA-4 regulates inhibin-alpha in gonadal cells, and the FSH cascade activates GATA-4. We hypothesized that the TGF-beta signalling cascade and GATA-4 also cooperate to regulate inhibin-alpha expression. In KK-1 granulosa tumour cells, which resemble normal granulosa cells and express inhibin-alpha, we found that TGF-beta upregulated GATA-4 expression. Transient transfection experiments in KK-1 cells demonstrated that dominant negative GATA-4 variants or mutations of GATA-binding sites in the inhibin-alpha promoter attenuated TGF-beta-induced gene activation. In GATA-4-deficient COS-7 cells, TGF-beta enhanced the expression of the inhibin-alpha promoter only in the presence of exogenous GATA-4. Smad3, but not Smad2, cooperated with GATA-4 in the transcriptional activation of the inhibin-alpha promoter, and immunoprecipitation experiments in KK-1 cells revealed a physical Smad3:GATA-4 interaction. Our data suggest that GATA-4, interacting with Smad3, is a cofactor for TGF-beta signalling to activate inhibin-alpha in granulosa cells.

Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4.

The majority of oocytes present in fetal ovaries are depleted before birth, and only about 400 will ovulate during the normal fertile life span. Studies on animals have shown that apoptosis is the mechanism behind oocyte depletion and follicular atresia. In the present study, we investigated the extent and localization of apoptosis in human fetal (aged 13-40 weeks) and adult ovaries. Furthermore, the expression of apoptosis-regulating proteins, bcl-2 and bax, and the relationship of transcription factor GATA-4 were studied. Apoptosis was found in ovarian follicles throughout fetal and adult life. During fetal development, apoptosis was localized mainly to primary oocytes and was highest between weeks 14-28, decreasing thereafter toward term. Expression of bcl-2 was observed only in the youngest fetal ovaries (weeks 13-14), and bax was present in the ovaries throughout the entire fetal period. In adult ovaries, apoptosis was detected in granulosa cells of secondary and antral follicles, and Bcl-2 and bax were expressed from primary follicles onwards. During fetal ovarian development, GATA-4 messenger RNA and protein were localized to the granulosa cells, with expression being highest in the youngest ovaries and decreasing somewhat toward term. The expression pattern of GATA-4 suggests that it may be involved in the mechanisms protecting granulosa cells from apoptosis from fetal to adult life. The results indicate that depletion of ovarian follicles in the human fetus occurs through intrinsic mechanisms of apoptosis in oocytes, and later in adult life the survival of growing follicles may be primarily determined by granulosa cell apoptosis.

Comparison of serous and mucinous ovarian carcinomas: distinct pattern of allelic loss at distal 8p and expression of transcription factor GATA-4.

Using comparative genomic hybridization (CGH), we have previously demonstrated frequent loss of 8p, especially its distal part, in ovarian carcinoma. To compare the deletion map of distal 8p in serous and mucinous ovarian carcinomas, we performed allelic analysis with 18 polymorphic microsatellite markers at 8p21-p23. In serous carcinoma, loss of heterozygosity (LOH) was detected in 67% of the samples, and the majority of the carcinomas showed loss of all or most of the informative markers. In contrast, only 21% of mucinous carcinomas showed allelic loss, with only one or two loci showing LOH in each sample. In serous carcinomas, LOH was associated with higher grade tumors. Three distinct minimal common regions of loss could be defined in serous carcinomas (at 8p21.1, 8p22-p23.1, and 8p23.1). Expression of a transcription factor gene, GATA4, located at one of these regions (8p23.1) was studied in serous and mucinous ovarian carcinomas by Northern blotting and immunohistochemical staining of tumor microarray. Expression was found to be lost in most serous carcinomas but retained in the majority of mucinous carcinomas. Our results suggest distinct pathogenetic pathways in serous and mucinous ovarian carcinomas and the presence of more than one tumor suppressor gene at 8p involved in the tumorigenesis of serous carcinoma.

Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors.

Previous studies have implicated transcription factors GATA-4 and GATA-6 in the regulation of murine ovarian development and function. In rodents, GATA-4 is expressed in granulosa cells of primary and early antral follicles, whereas GATA-6 is expressed in granulosa cells of late antral follicles and luteal glands. Both transcription factors can be detected in lesser amounts in theca cells and interstitial cells. We have now examined the expression of GATA-4 and GATA-6 in human ovaries, human granulosa-luteal (GL) cells and sex cord-derived tumors. We show by in situ hybridization and immunohistochemistry that GATA-4 and GATA-6 messenger RNA (mRNA) and GATA-4 protein are present in granulosa and theca cells in both preantral and antral follicles. Both human ovarian tissue samples and freshly isolated GL cells derived from preovulatory follicles of gonadotropin-treated women express GATA-4, GATA-6, and FOG-2 transcripts, and GATA-6 mRNA expression in GL cell cultures is stimulated by human CG and 8-bromo-cAMP. The vast majority of granulosa and theca cell tumors examined expressed GATA-4 and GATA-6. We also found that mRNA for FOG-2, a recently discovered regulator of GATA-4, is coexpressed with GATA-4 in human ovary samples, normal granulosa cells, and in sex cord-derived tumors. Our results demonstrate that GATA-4, GATA-6, and FOG-2 are expressed in human ovary and in granulosa and theca cell tumors. Our findings support a role for GATA-binding proteins in human ovarian folliculogenesis. Moreover, these data suggest that GATA factors may contribute to the phenotypes of sex cord-derived ovarian tumors.

Transcription factor GATA-4 is expressed in pediatric yolk sac tumors.

Yolk sac tumors (YSTs) are malignant tumors that occur in the gonads of children and young adults, and at extragonadal sites in young children. The histological features of YSTs are variable and can be superimposed on other germ cell tumor histologies. Malignant endodermal cells within YSTs express alpha-fetoprotein, which can be detected in tumor tissue or serum. However, additional markers of endoderm differentiation would be beneficial for the classification of these tumors. Transcription factor GATA-4 regulates the differentiation and function of murine yolk sac endoderm, and its expression correlates with proliferation and cell survival in certain tissues. To see whether GATA-4 plays a role in human YSTs, we surveyed its expression in human germ cell tumors and cell lines. Northern analysis demonstrated expression of GATA-4 mRNA in four human germ cell tumor lines exhibiting yolk sac endoderm differentiation. GATA-4 protein was detected in eight of nine pediatric YSTs by immunohistochemistry. Three of five immature teratomas exhibited GATA-4 in neural blastematous cells and in cylindrical epithelium, whereas all 16 mature teratomas were devoid of GATA-4. We conclude that GATA-4 is a clinically useful marker of human YSTs and speculate that it may play a role in the maintenance of the malignant phenotype.