Investigating the role of ephrins and their receptors in mouse folliculogenesis and ovulation

Follicle stimulating hormone (FSH) promotes granulosa cell (GC) proliferation, differentiation, and steroidogenesis. This series of events is critical for female fertility, and culminates in the formation of mature follicles responsive to the surge of luteinizing hormone (LH) that triggers ovulation. Ephrins (Efn genes) and Eph receptors (Eph genes) are membrane-associated signaling molecules that mediate communication at sites of cell-cell contact, and have been extensively studied in the context of embryonic development. The published literature contains several reports of ovarian Eph and Efn expression, although their precise roles in the ovary are unknown. Dysregulation of Efna5 in GCs of the subfertile Esr2-/- mouse suggests a role for Eph-ephrin signaling in ovarian function. We sought to investigate Eph receptors and ephrins in the mouse ovary using gonadotropin-stimulated animal models and cultured GCs. We identified several Efn and Eph genes for which expression is enhanced in GCs of the gonadotropin-stimulated mouse. Furthermore, we determined that cultured GCs stimulated with recombinant ephrin-A5 or EphA5 exhibit reduced cell spreading or adhesion, respectively, indicating a cell-autonomous response to Eph-ephrin stimulation. In order to ascertain the importance of ephrin-A5 in female fertility, we performed a reproductive assessment of females lacking Efna5, the sole ephrin-encoding gene upregulated by FSH in GCs. Efna5-/- females are subfertile and exhibit an impaired response to LH, displaying attenuated ovulatory potential, reduced ovarian expression of Pgr, Ptgs2, and Adamts4, as well as abnormal follicle rupture. We also found an increased incidence of multi-oocyte follicles in adult, but not juvenile, Efna5-/- females, indicating follicle merging. Finally, we determined that the mouse genomic region upstream of Epha5 is transcriptionally activated by cAMP in a protein kinase A-dependent manner. Transcriptional activation of Efna5, Epha3, Epha5, Epha8, and Ephb2 is reduced in GCs of eCG-treated Esr2-/- females, which exhibit impaired cAMP production. These results suggest that cAMP is a novel transcriptional regulator for several Eph and Efn genes. Our findings establish a place for ephrins and their receptors within the current model of gonadotropin-dependent follicle growth and ovulation, and identify cAMP as a novel transcriptional regulator of Epha5 and possibly other Efn and Eph genes

AMPK-independent LKB1 activity is required for efficient epithelial ovarian cancer metastasis

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 anchorageindependent 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 tumor burden and metastatic potential; in particular, OVCAR8-STK11KO tumors had evidence of extensive necrosis, apoptosis, and hypoxia. Interestingly, LKB1 loss did not affect AMPKα phosphorylation in EOC spheroids and tumor xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumor 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-STK11KOcells partially restored spheroid formation and viability. Implications: LKB1 possesses key tumor-promoting activity independent of downstream AMPK signaling during EOC metastasis

Loss of LKB1-NUAK1 signalling enhances NF-κB activity in a spheroid model of high-grade serous ovarian cancer

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

A novel role for NUAK1 in promoting ovarian cancer metastasis through regulation of fibronectin production in Spheroids

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

BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

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 invivo 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

Minireview: Estrogen Receptor-β: Mechanistic Insights from Recent Studies

The discovery of estrogen receptor-β (ERβ) in 1996 stimulated great interest in the physiological roles and molecular mechanisms of ERβ action. We now know that ERβ 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 ERβ and ER{alpha} generally play distinct physiological roles in the body. Although significant progress has been made toward understanding the molecular mechanisms of ERβ action, particularly in vitro, there remains a large gap in our understanding of the mechanisms by which ERβ elicits its biological functions in a true physiological context