ERK1/2-SOX9/FOXL2 axis regulates ovarian steroidogenesis and favors the follicular-luteal transition.

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals.

Early gonadal differentiation is associated with the antagonistic action of Foxl2 and Dmrt1l in the Pacific oyster.

As the second largest phylum in the zoological kingdom next to arthropods, the mechanism of gonadal differentiation in mollusca is quite complex. Currently, although much has been carried out on gonadal differentiation in the Pacific oyster, there is still unknown information that needs to be further explored. Here, analysis of the Foxl2 and Dmrt1l expression in samples at different development periods of male and female gonads as well as in annual gonad samples revealed that Log10 (Foxl2/Dmrt1l) values were an effective method for sex identification in oysters. In differentiated gonadal tissue, Log10 (Foxl2/Dmrt1l) values greater than 2 were females and less than 1 for males. Subsequent sequential sampling of the same individuals verified that Log10 (Foxl2/Dmrt1l) values greater than 2 for resting gonads would develop as females and less than 1 would develop as males in the future. Relative expression analysis of Foxl2 and Dmrt1l in the annual samples revealed a negative correlation between Log10 (Foxl2) and Log10 (Dmrt1l). Double fluorescence reporter validation results showed that DMRT1L protein was able to bind the Foxl2 promoter and repress its activity with a weak dosage effect. Antagonism between Dmrt1l and Foxl2 is therefore not restricted to vertebrates, and the competing regulatory networks are of great significance in the maintenance of gonadal sex in oysters after sexual differentiation. This study provides novel ideas and insights into the study of early gonadal differentiation in the adult oyster.

FOXL2 and FOXA1 cooperatively assemble on the TP53 promoter in alternative dimer configurations.

Although both the p53 and forkhead box (FOX) family proteins are key transcription factors associated with cancer progression, their direct relationship is unknown. Here, we found that FOX family proteins bind to the non-canonical homotypic cluster of the p53 promoter region (TP53). Analysis of crystal structures of FOX proteins (FOXL2 and FOXA1) bound to the p53 homotypic cluster indicated that they interact with a 2:1 stoichiometry accommodated by FOX-induced DNA allostery. In particular, FOX proteins exhibited distinct dimerization patterns in recognition of the same p53-DNA; dimer formation of FOXA1 involved protein-protein interaction, but FOXL2 did not. Biochemical and biological functional analyses confirmed the cooperative binding of FOX proteins to the TP53 promoter for the transcriptional activation of TP53. In addition, up-regulation of TP53 was necessary for FOX proteins to exhibit anti-proliferative activity in cancer cells. These analyses reveal the presence of a discrete characteristic within FOX family proteins in which FOX proteins regulate the transcription activity of the p53 tumor suppressor via cooperative binding to the TP53 promoter in alternative dimer configurations.

Identification of SF-1 and FOXL2 and Their Effect on Activating P450 Aromatase Transcription via Specific Binding to the Promoter Motifs in Sex Reversing Cheilinus undulatus.

The giant wrasse Cheilinus undulatus is a protogynous socially hermaphroditic fish. However, the physiological basis of its sex reversal remains largely unknown. cyp19 is a key gender-related gene encoding P450 aromatase, which converts androgens to estrogens. cyp19 transcription regulation is currently unknown in socially sexually reversible fish. We identified NR5A1 by encoding SF-1, and FOXL2 from giant wrasse cDNA and cyp19a1a and cyp19a1b promoter regions were cloned from genomic DNA to determine the function of both genes in cyp19a1 regulation. Structural analysis showed that SF-1 contained a conserved DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD). FOXL2 was comprised of an evolutionarily conserved Forkhead domain. In vitro transfection assays showed that SF-1 could upregulate cyp19a1 promoter activities, but FOXL2 could only enhance cyp19a1b promoter transcriptional activity in the HEK293T cell line. Furthermore, HEK293T and COS-7 cell lines showed that co-transfecting the two transcription factors significantly increased cyp19a1 promoter activity. The -120 to -112 bp (5'-CAAGGGCAC-3') and -890 to -872 bp (5'-AGAGGAGAACAAGGGGAG-3') regions of the cyp19a1a promoter were the core regulatory elements for SF-1 and FOXL2, respectively, to regulate cyp19a1b promoter transcriptional activity. Collectively, these results suggest that both FOXL2 and SF-1 are involved in giant wrasse sex reversal.

miR-133b targets tagln2 and functions in tilapia oogenesis.

microRNAs (miRNAs) are important components of non-coding RNAs that participate in diverse life activities by regulating gene expression at the post transcriptional level through base complementary pairing with 3'UTRs of target mRNAs. miR-133b is a member of the miR-133 family, which play important roles in muscle differentiation and tumorigenesis. Recently, miR-133b was reported to affect estrogen synthesis by targeting foxl2 in mouse, while its role in fish reproduction remains to be elucidated. In the present study, we isolated the complete sequence of miR-133b, which was highly expressed in tilapia ovary at 30 and 90 dah (days after hatching) and subsequently decreased at 120 to 150 dah by qPCR. Interestingly, only a few oogonia were remained in the antagomir-133b treated tilapia ovary, while phase I and II oocytes were observed in the ovaries of the control group. Unexpectedly, the expression of foxl2 and cyp19a1a, as well as estradiol levels in serum were increased in the treated group. Furthermore, tagln2, an important factor for oogenesis, was predicted as the target gene of miR-133b, which was confirmed by dual luciferase reporter vector experiments. miR-133b and tagln2 were co-expressed in tilapia ovaries. Taken together, miR-133b may be involved in the early oogenesis of tilapia by regulating tagln2 expression. This study enriches the understanding of miR-133b function during oogenesis and lays a foundation for further study of the regulatory network during oogenesis.

Identification and functional analysis of a sex-biased transcriptional factor Foxl2 in the bay scallop Argopecten irradians irradians.

Transcription factor Foxl2 is an evolutionarily conserved gene playing pivotal roles in regulation of early ovarian differentiation and maintenance in animals. However, the Foxl2 gene has not been thoroughly studied in hermaphroditic scallops. In this study, we cloned and characterized a Foxl2 (designated as AiFoxl2) from the bay scallop Argopecten irradians irradians. The open reading frame of AiFoxl2 was 1122 bp encoding 373 amino acids residues and contained a conserved forkhead box domain. Quantitative real-time PCR showed that AiFoxl2 was mainly expressed in the ovary. Moreover, the highest expression of AiFoxl2 in the ovary was detected at proliferative stage and growing stage, while the lowest level was found at resting stage. During the embryonic and larval development, expression of AiFoxl2 was found first in fertilized eggs, increased significantly at the blastula stage, and reached peak value at the D-larvae stage. When AiFoxl2 was knocked down, testis development-related genes (Dmrt1, Sox7 and Sox9) were up-regulated significantly while the ovary development-related genes (Vg, HSD14, and GATA-1) were down-regulated manifestly. These findings suggested that AiFoxl2 was a female-related gene in A. i. irradians and may be involved in regulation of ovarian development and differentiation.

Variance in expression and localization of sex-related genes CgDsx, CgBHMG1 and CgFoxl2 during diploid and triploid Pacific oyster Crassostrea gigas gonad differentiation.

Several evolutionarily conserved classes of transcriptional regulators were involved in diverse sex determination and differentiation pathways across taxa, whereas their roles in most mollusks is still limited. The Pacific oyster Crassostrea gigas, a dioecious bivalve with sex reversal, could be an ideal model for this issue because of its complex sexuality and potential disruption of sex differentiation in triploid individuals. Here, two mRNA splicing isoforms of a DM domain gene CgDsx and two isoforms of a novel sex-related CgBHMG1 (ortholog of BHMG1 in mammals) were identified in C. gigas. Real time PCR showed that two isoforms of CgDsx and one isoform of CgBHMG1 displayed male-specific expression in diploid oysters, opposite with the female-specific CgFoxl2 (a potential factor of female gonadic differentiation). Interestingly, the four sex-specific transcripts in diploid oyster were expressed in triploid oysters with opposite sex, triploid hermaphrodites and individuals at stage I that sex could not be determined. Subsequent in situ hybridization analysis on gonads of diploid oysters revealed predominant expression of CgDsx in spermatogonia of testes, CgBHMG1 in spermatocytes of testes and follicle cells of ovaries, and CgFoxl2 in follicle cells of ovaries and some male germ cells in testes. And aberrant co-expression of the three genes in triploid oysters was localized in gonadal tubules of gonads at stage I, ovarian follicle cells and undetermined gonial cells in nontypical hermaphroditic gonads with rare female materials. From the above, temporal and spatial expression of sex-related genes in diploid and triploid gonads indicated that CgDsx and CgFoxl2 might mainly function in C. gigas sex differentiation, and CgBHMG1 appeared as a factor involved in meiosis. This work will help to illuminate the gene network of sex differentiation in bivalves and provides new sight on this issue from comparison between diploid and triploid individuals.

Genomic exploration of the targets of FOXL2 and ESR2 unveils their implication in cell migration, invasion, and adhesion.

FOXL2 and ESR2 are key transcriptional regulators in ovarian granulosa cells. To explore their transcriptional roles and their interplay, we have depleted Foxl2 and Esr2 in mouse primary granulosa cells to assess their ability to bind their targets and/or to modulate gene expression and cellular functions. We show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cell fate. A parallel ChIP-seq analysis showed that FOXL2 mainly binds to sites located in intergenic regions quite far from its targets. A bioinformatic analysis demonstrated that FOXL2-activated genes were enriched in peaks associated with the H3K27ac mark, whereas FOXL2-repressed genes were not, suggesting that FOXL2 can activate transcription through binding to enhancer sites. We also identified about 500 deregulated genes upon Esr2 silencing, of which one third are also targets of FOXL2. We provide evidence showing that both factors modulate, through a coherent feed-forward loop, a number of common targets. Many of the FOXL2/ESR2 targets are involved in cell motility and, consistently, granulosa cells depleted for either Foxl2 or Esr2 exhibit decreased migration, invasion and adhesion. This effect is paralleled by the depletion of their target Phactr1, involved in actin cytoskeleton dynamics. Our analysis expands the number of direct and indirect transcriptional targets of both FOXL2 and ESR2, which deserve investigation in the context of adult-type granulosa cell tumors whose molecular diagnostic hallmark is the presence of the C134W FOXL2 pathogenic variant.

Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia.

Germline sexual fate has long been believed to be determined by the somatic environment, but this idea is challenged by recent studies of foxl3 mutants in medaka. Here, we demonstrate that the sexual fate of tilapia germline is determined by the antagonistic interaction of dmrt1 and foxl3, which are transcriptionally repressed in male and female germ cells, respectively. Loss of dmrt1 rescued the germ cell sex reversal in foxl3Δ7/Δ7 XX fish, and loss of foxl3 partially rescued germ cell sex reversal but not somatic cell fate in dmrt1Δ5/Δ5 XY fish. Interestingly, germ cells lost sexual plasticity in dmrt1Δ5/Δ5 XY and foxl3Δ7/Δ7 XX single mutants, as aromatase inhibitor (AI) and estrogen treatment failed to rescue the respective phenotypes. However, recovery of germ cell sexual plasticity was observed in dmrt1/foxl3 double mutants. Importantly, mutation of somatic cell-specific foxl2 resulted in testicular development in foxl3Δ7/Δ7 or dmrt1Δ5/Δ5 mutants. Our findings demonstrate that sexual plasticity of germ cells relies on the presence of both dmrt1 and foxl3. The existence of dmrt1 and foxl3 allows environmental factors to influence the sex fate decision in vertebrates.

Functional Divergence of Multiple Duplicated Foxl2 Homeologs and Alleles in a Recurrent Polyploid Fish.

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic-pituitary-gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.

An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2.

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.

Production of donor-derived eggs after ovarian germ cell transplantation into the gonads of adult, germ cell-less, triploid hybrid fish†.

In animals, spermatogonial transplantation in sterile adult males is widely developed; however, despite its utility, ovarian germ cell transplantation is not well developed. We previously showed that the interspecific hybrid offspring of sciaenid was a suitable model for germ cell transplantation studies as they have germ cell-less gonads. However, all these gonads have testis-like characteristics. Here, we tested whether triploidization in hybrid embryos could result in germ cell-less ovary development. Gonadal structure dimorphism and sex-specific gene expression patterns were examined in 6-month-old triploid hybrids (3nHybs). Thirty-one percent of 3nHybs had germ cell-less gonads with an ovarian cavity. cyp19a1a and foxl2, ovarian differentiation-related genes, were expressed in these gonads, whereas dmrt1 and vasa were not expressed, suggesting ovary-like germ cell-less gonad development. Some (26%) 3nHybs had testis-like germ cell-less gonads. Ovarian germ cells collected from homozygous green fluorescent protein (GFP) transgenic blue drum (BD) (Nibea mitsukurii) were transplanted into 6-month-old 3nHybs gonads via the urogenital papilla or oviduct. After 9 months, the recipients were crossed with wild type BD. Among the six 3nHyb recipients that survived, one female and one male produced fertile eggs and motile sperm carrying gfp-specific DNA sequences. Progeny tests revealed that all F1 offspring possessed gfp-specific DNA sequences, suggesting that these recipients produced only donor-derived eggs or sperm. Histological observation confirmed donor-derived gametogenesis in the 3nHyb recipients' gonads. Overall, triploidization reduces male-biased sex differentiation in germ cell-less gonads. We report, for the first time, donor-derived egg production in an animal via direct ovarian germ cell transplantation into a germ cell-less ovary.

Aberrant and constitutive expression of FOXL2 impairs ovarian development and functions in mice.

Development and functions of the ovary rely on appropriate signaling and communication between various ovarian cell types. FOXL2, a transcription factor that plays a key role at different stages of ovarian development, is associated with primary ovarian insufficiency and ovarian cancer as a result of its loss-of-function or mutations. In this study, we investigated the impact of aberrant, constitutive expression of FOXL2 in somatic cells of the ovary. Overexpression of FOXL2 that started during fetal life resulted in defects in nest breakdown and consequent formation of polyovular follicles. Granulosa cell differentiation was impaired and recruitment and differentiation of steroidogenic theca cells was compromised. As a consequence, adult ovaries overexpressing FOXL2 exhibited defects in compartmentalization of granulosa and theca cells, significant decreased steroidogenesis and lack of ovulation. These findings demonstrate that fine-tuned expression of FOXL2 is required for proper folliculogenesis and fertility.

Increased FOXL2 expression alters uterine structures and functions†.

The transcription factor forkhead box L2 (FOXL2) regulates sex differentiation and reproductive function. Elevated levels of this transcription factor have been observed in the diseases of the uterus, such as endometriosis. However, the impact of elevated FOXL2 expression on uterine physiology remains unknown. In order to determine the consequences of altered FOXL2 in the female reproductive axis, we generated mice with over-expression of FOXL2 (FOXL2OE) by crossing Foxl2LsL/+ with the Progesterone receptor Pgrcre model. FOXL2OE uterus showed severe morphological abnormality including abnormal epithelial stratification, blunted adenogenesis, increased endometrial fibrosis, and disrupted myometrial morphology. In contrast, increasing FOXL2 levels specifically in uterine epithelium by crossing the Foxl2LsL/+ with the lactoferrin Ltficre mice resulted in the eFOXL2OE mice with uterine epithelial stratification but without defects in endometrial fibrosis and adenogenesis, demonstrating a role of the endometrial stroma in the uterine abnormalities of the FOXL2OE mice. Transcriptomic analysis of 12 weeks old Pgrcre and FOXL2OE uterus at diestrus stage showed multiple signaling pathways related with cellular matrix, wnt/ß-catenin, and altered cell cycle. Furthermore, we found FOXL2OE mice were sterile. The infertility was caused in part by a disruption of the hypophyseal ovarian axis resulting in an anovulatory phenotype. The FOXL2OE mice failed to show decidual responses during artificial decidualization in ovariectomized mice demonstrating the uterine contribution to the infertility phenotype. These data support that aberrantly increased FOXL2 expressions in the female reproductive tract can disrupt ovarian and uterine functions.

Estrogen suppresses SOX9 and activates markers of female development in a human testis-derived cell line.

BACKGROUND: The increasing incidence of reproductive disorders in humans has been attributed to in utero exposure to estrogenic endocrine disruptors. In particular, exposure of the developing testis to exogenous estrogen can negatively impact male reproductive health. To determine how estrogens impact human gonad function, we treated the human testis-derived cell line NT2/D1 with estrogen and examined its impact on SOX9 and the expression of key markers of granulosa (ovarian) and Sertoli (testicular) cell development. RESULTS: Estrogen successfully activated its cognate receptor (estrogen receptor alpha; ESR1) in NT2/D1 cells. We observed a significant increase in cytoplasmic SOX9 following estrogen treatment. After 48 h of estrogen exposure, mRNA levels of the key Sertoli cell genes SOX9, SRY, AMH, FGF9 and PTGDS were significantly reduced. This was followed by a significant increase in mRNA levels for the key granulosa cell genes FOXL2 and WNT4 after 96 h of estrogen exposure. CONCLUSIONS: These results are consistent with estrogen's effects on marsupial gonads and show that estrogen has a highly conserved impact on gonadal cell fate decisions that has existed in mammals for over 160 million years. This effect of estrogen presents as a potential mechanism contributing to the significant decrease in male fertility and reproductive health reported over recent decades. Given our widespread exposure to estrogenic endocrine disruptors, their effects on SOX9 and Sertoli cell determination could have considerable impact on the adult testis.

Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary.

We sequenced more than 52,500 single cells from embryonic day 11.5 (E11.5) postembryonic day 5 (P5) gonads and performed lineage tracing to analyze primordial follicles and wave 1 medullar follicles during mouse fetal and perinatal oogenesis. Germ cells clustered into six meiotic substages, as well as dying/nurse cells. Wnt-expressing bipotential precursors already present at E11.5 are followed at each developmental stage by two groups of ovarian pregranulosa (PG) cells. One PG group, bipotential pregranulosa (BPG) cells, derives directly from bipotential precursors, expresses Foxl2 early, and associates with cysts throughout the ovary by E12.5. A second PG group, epithelial pregranulosa (EPG) cells, arises in the ovarian surface epithelium, ingresses cortically by E12.5 or earlier, expresses Lgr5, but delays robust Foxl2 expression until after birth. By E19.5, EPG cells predominate in the cortex and differentiate into granulosa cells of quiescent primordial follicles. In contrast, medullar BPG cells differentiate along a distinct pathway to become wave 1 granulosa cells. Reflecting their separate somatic cellular lineages, second wave follicles were ablated by diptheria toxin treatment of Lgr5-DTR-EGFP mice at E16.5 while first wave follicles developed normally and supported fertility. These studies provide insights into ovarian somatic cells and a resource to study the development, physiology, and evolutionary conservation of mammalian ovarian follicles.

Phytoestrogen genistein hinders ovarian oxidative damage and apoptotic cell death-induced by ionizing radiation: co-operative role of ER-ß, TGF-ß, and FOXL-2.

Radiotherapy is a well-known cause of premature ovarian failure (POF). Therefore, we investigated the molecular influence of genistein (GEN) on the ovarian reserve of rats exposed to ϒ-radiation. Female Sprague Dawley rats were exposed to a 3.2 Gy γ-radiation to induce POF and/or treated with either GEN (5 mg/kg, i.p.) or Ethinyl estradiol (E2; 0.1 mg/kg, s.c.), once daily for 10 days. GEN was able to conserve primordial follicles stock and population of growing follicles accompanied with reduction in atretic follicles. GEN restored the circulating estradiol and anti-Müllerian hormone levels which were diminished after irradiation. GEN has potent antioxidant activity against radiation-mediated oxidative stress through upregulating endogenous glutathione levels and glutathione peroxidase activity. Mechanistically, GEN inhibited the intrinsic pathway of apoptosis by repressing Bax expression and augmenting Bcl-2 expression resulted in reduced Bax/Bcl-2 ratio with subsequent reduction in cytochrome c and caspase 3 expression. These promising effects of GEN are associated with improving granulosa cells proliferation. On the molecular basis, GEN reversed ovarian apoptosis through up-regulation of ER-ß and FOXL-2 with downregulation of TGF-ß expression, therefore inhibiting transition of primordial follicles to more growing follicles. GEN may constitute a novel therapeutic modality for safeguarding ovarian function of females' cancer survivors.

Ovarian granulosa cell tumor characterization identifies FOXL2 as an immunotherapeutic target.

Granulosa cell tumors (GCT) are rare ovarian malignancies. Due to the lack of effective treatment in late relapse, there is a clear unmet need for novel therapies. Forkhead Box L2 (FOXL2) is a protein mainly expressed in granulosa cells (GC) and therefore is a rational therapeutic target. Since we identified tumor infiltrating lymphocytes (TILs) as the main immune population within GCT, TILs from 11 GCT patients were expanded, and their phenotypes were interrogated to determine that T cells acquired late antigen-experienced phenotypes and lower levels of PD1 expression. Importantly, TILs maintained their functionality after ex vivo expansion as they vigorously reacted against autologous tumors (100% of patients) and against FOXL2 peptides (57.1% of patients). To validate the relevance of FOXL2 as a target for immune therapy, we developed a plasmid DNA vaccine (FoxL2-tetanus toxin; FoxL2-TT) by fusing Foxl2 cDNA with the immune-enhancing domain of TT. Mice immunization with FoxL2-TT controlled growth of FOXL2-expressing ovarian (BR5) and breast (4T1) cancers in a T cell-mediated manner. Combination of anti-PD-L1 with FoxL2-TT vaccination further reduced tumor progression and improved mouse survival without affecting the female reproductive system and pregnancy. Together, our results suggest that FOXL2 immune targeting can produce substantial long-term clinical benefits. Our study can serve as a foundation for trials testing immunotherapeutic approaches in patients with ovarian GCT.

Transcriptome sequencing revealed that knocking down FOXL2 affected cell proliferation, the cell cycle, and DNA replication in chicken pre-ovulatory follicle cells.

Forkhead box L2 (FOXL2) is a single-exon gene encoding a forkhead transcription factor, which is mainly expressed in the ovary, eyelids and the pituitary gland. FOXL2 plays an essential role in ovarian development. To reveal the effects of FOXL2 on the biological process and gene expression of ovarian granulosa cells (GCs), we established stable FOXL2-knockdown GCs and then analysed them using transcriptome sequencing. It was observed that knocking down FOXL2 affected the biological processes of cell proliferation, DNA replication, and apoptosis and affected cell cycle progression. FOXL2 knockdown promoted cell proliferation and DNA replication, decreased cell apoptosis, and promoted mitosis. In addition, by comparing the transcriptome after FOXL2 knockdown, we found a series of DEGs (differentially expressed genes) and related pathways. These results indicated that, through mediating these genes and pathways, the FOXL2 might induce the cell proliferation, cycle, and DNA replication, and play a key role during ovarian development and maintenance.

Mutant FOXL2C134W Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors.

The mutant protein FOXL2C134W is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2C134W contributes to tumorigenesis is not known. Here, we show that mutant FOXL2C134W acquires the ability to bind SMAD4, forming a FOXL2C134W/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2C134W mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2C134W also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2C134W binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFß mitigated the transcriptional effect of FOXL2C134W. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2C134W and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2C134W hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2C134W and the TGFß pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.