The discovery of estrogen receptor-beta (ERbeta) in 1996 stimulated great interest in the physiological roles and molecular mechanisms of ERbeta action. We now know that ERbeta plays a major role in mediating estrogen action in several tissues and organ systems, including the ovary, cardiovascular system, brain, and the immune system, and that ERbeta and ERalpha generally play distinct physiological roles in the body. Although significant progress has been made toward understanding the molecular mechanisms of ERbeta action, particularly in vitro, there remains a large gap in our understanding of the mechanisms by which ERbeta elicits its biological functions in a true physiological context.

Epithelial ovarian cancer (EOC) spreads by direct dissemination of malignant cells and multicellular clusters, known as spheroids, into the peritoneum followed by implantation and growth on abdominal surfaces. Using a spheroid model system of EOC metastasis, we discovered that Liver kinase B1 (LKB1), encoded by the STK11 gene, and its canonical substrate AMP-activated protein kinase (AMPK) are activated in EOC spheroids, yet only LKB1 is required for cell survival. We have now generated STK11-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. STK11KO did not affect growth and viability in adherent culture, but it decreased anchorage-independent growth of EOC cells. EOC spheroids lacking LKB1 had markedly impaired growth and viability, whereas there was no difference in normal FT190 spheroids. To test whether LKB1 loss affects EOC metastasis, we performed intraperitoneal injections of OVCAR8-, HeyA8-, and iOvCa147-STK11KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumour burden and metastatic potential; in particular, OVCAR8-STK11KO tumours had evidence of extensive necrosis, apoptosis and hypoxia. Interestingly, LKB1 loss did not affect AMPKα phosphorylation in EOC spheroids and tumour xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumour growth occurs via other downstream mediators. We identified the dual-specificity phosphatase DUSP4 as a commonly upregulated protein due to LKB1 loss; indeed, DUSP4 knockdown in HeyA8-STK11KO cells restored spheroid formation and viability. Our results strongly indicate that intact LKB1 activity independent of downstream AMPK signaling is required during EOC metastasis.

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.

Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent.

Follicle-stimulating hormone (FSH)-mediated changes in granulosa cell adhesion and morphology are essential for preovulatory follicle development, given the dramatic changes in follicle size and granulosa cell number that occur during this transition. Members of the Eph-ephrin family of cell-positioning and adhesion molecules, a family that consists of ephrin ligands and their Ephrin (Eph) receptors, regulate cell location, adhesion, and migration during embryonic development and tumor growth. However, very little is known about ephrin signaling during folliculogenesis. We have found that FSH increases the expression of several members of the Eph-ephrin family and that this signaling regulates granulosa cell morphology and adhesion. FSH induced increased mRNA levels of the ephrin ligand, ephrin-A5 (Efna5), and its receptors, Eph receptors A3, A5, and A8 (Epha3, Epha5, and Epha8, respectively), in granulosa cells. Immunofluorescence studies indicated that EFNA5 and EPHA5 are located in the membrane of granulosa cells of developing mouse follicles. Eph-ephrin signaling directly affected granulosa cell morphology and adhesion. Recombinant EFNA5 reduced cell spreading and increased cell rounding in mouse primary granulosa cells and in a rat granulosa cell line, whereas EPHA5 reduced granulosa cell adhesion in both model systems. Both FSH and forskolin also increased Efna5 and Epha5 mRNA levels in rat and human granulosa cell lines, indicating that FSH regulates these genes via the cAMP-dependent protein kinase A pathway and that this regulation is conserved across different species. The present study identifies Eph-ephrin signaling as a novel FSH-mediated pathway regulating granulosa cell morphology and adhesion.

Follicle growth and ovulation involve the coordinated expression of many genes, driven by FSH and LH. Reports indicate that Eph receptors and ephrins are expressed in the ovary, suggesting roles in follicle growth and/or ovulation. We previously reported FSH-induced expression of ephrin-A5 (EFNA5) and 4 of its cognate Eph receptors in mouse granulosa cells. We now report that female mice lacking EFNA5 are subfertile, exhibit a compromised response to LH, and display abnormal ovarian histology after superovulation. Efna5(-/-) females litters were 40% smaller than controls, although no difference in litter frequency was detected. The ovarian response to superovulation was also compromised in Efna5(-/-) females, with 37% fewer oocytes ovulated than controls. These results corresponded with a reduction in ovarian mRNA levels of several LH-responsive genes, including Pgr, Ptgs2, Tnfaip6, Ereg, Btc, and Adamts4, suggesting that Efna5(-/-) ovaries exhibit a partially attenuated response to LH. Histopathological analysis indicated that superovulated Efna5(-/-) females exhibited numerous ovarian defects, including intraovarian release of cumulus oocyte complexes, increased incidence of oocytes trapped within luteinized follicles, granulosa cell and follicular fluid emboli, fibrin thrombi, and interstitial hemorrhage. In addition, adult Efna5(-/-) ovaries exhibited a 4-fold increase in multioocyte follicles compared with controls, although no difference was detected in 3-week-old mice, suggesting the possibility of follicle merging. Our observations indicate that loss of EFNA5 in female mice results in subfertility and imply that Eph-ephrin signaling may also play a previously unidentified role in the regulation of fertility in women.

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy often diagnosed at an advanced stage. Although most HGSOC patients respond initially to debulking surgery combined with cytotoxic chemotherapy, many ultimately relapse with platinum-resistant disease. Thus, improving outcomes requires new ways of limiting metastasis and eradicating residual disease. We identified previously that Liver kinase B1 (LKB1) and its substrate NUAK1 are implicated in EOC spheroid cell viability and are required for efficient metastasis in orthotopic mouse models. Here, we sought to identify additional signalling pathways altered in EOC cells due to LKB1 or NUAK1 loss-of-function. Transcriptome analysis revealed that inflammatory signalling mediated by NF-κB transcription factors is hyperactive due to LKB1-NUAK1 loss in HGSOC cells and spheroids. Upregulated NF-κB signalling due to NUAK1 loss suppresses reactive oxygen species (ROS) production and sustains cell survival in spheroids. NF-κB signalling is also activated in HGSOC precursor fallopian tube secretory epithelial cell spheroids, and is further enhanced by NUAK1 loss. Finally, immunohistochemical analysis of OVCAR8 xenograft tumors lacking NUAK1 displayed increased RelB expression and nuclear staining. Our results support the idea that NUAK1 and NF-κB signalling pathways together regulate ROS and inflammatory signalling, supporting cell survival during each step of HGSOC pathogenesis. We propose that their combined inhibition may be efficacious as a novel therapeutic strategy for advanced HGSOC.

Epithelial ovarian cancer (EOC) spreads by direct dissemination of malignant cells and multicellular clusters, known as spheroids, into the peritoneum followed by implantation and growth on abdominal surfaces. Using a spheroid model system of EOC metastasis, we discovered that Liver kinase B1 (LKB1), encoded by the STK11 gene, and its canonical substrate AMP-activated protein kinase (AMPK) are activated in EOC spheroids, yet only LKB1 is required for cell survival. We have now generated STK11-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. STK11KO did not affect growth and viability in adherent culture, but it decreased anchorage-independent growth of EOC cells. EOC spheroids lacking LKB1 had markedly impaired growth and viability, whereas there was no difference in normal FT190 spheroids. To test whether LKB1 loss affects EOC metastasis, we performed intraperitoneal injections of OVCAR8-, HeyA8-, and iOvCa147-STK11KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumour burden and metastatic potential; in particular, OVCAR8-STK11KO tumours had evidence of extensive necrosis, apoptosis and hypoxia. Interestingly, LKB1 loss did not affect AMPKα phosphorylation in EOC spheroids and tumour xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumour growth occurs via other downstream mediators. We identified the dual-specificity phosphatase DUSP4 as a commonly upregulated protein due to LKB1 loss; indeed, DUSP4 knockdown in HeyA8-STK11KO cells restored spheroid formation and viability. Our results strongly indicate that intact LKB1 activity independent of downstream AMPK signaling is required during EOC metastasis.