Reduced Gonadotrophin Receptor Expression Is Associated with a More Aggressive Ovarian Cancer Phenotype.

Follicle-stimulating hormone (FSH) and luteinising hormone (LH) play important roles in regulating cell growth and proliferation in the ovary. However, few studies have explored the expression of FSH and LH receptors (FSHR and LHCGR) in ovarian cancer, and their functional roles in cancer progression remain inconclusive. This study investigated the potential impact of both mRNA (FSHR, LHCGR) and protein (FSHR, LHCGR) expression on ovarian cancer progression using publicly available online databases, qRT-PCR (high grade serous ovarian cancers, HGSOC, n = 29 and benign ovarian tumors, n = 17) and immunohistochemistry (HGSOC, n = 144). In addition, we investigated the effect of FSHR and LHCGR siRNA knockdown on the pro-metastatic behavior of serous ovarian cancer cells in vitro. High FSHR or high LHCGR expression in patients with all subtypes of high-grade ovarian cancer was significantly associated with longer progression-free survival (PFS) and overall survival (OS). High FSHR protein expression was associated with increased PFS (p = 0.050) and OS (p = 0.025). HGSOC patients with both high FSHR and high LHCGR protein levels had the best survival outcome, whilst both low FSHR and low LHCGR expression was associated with poorest survival (p = 0.019). Knockdown of FSHR significantly increased the invasion of serous ovarian cancer cells (OVCAR3 and COV362) in vitro. LHCGR knockdown also promoted invasion of COV362 cells. This study highlights that lower FSHR and LHCGR expression is associated with a more aggressive epithelial ovarian cancer phenotype and promotes pro-metastatic behaviour.

Transcriptome analyses of ovarian stroma: tunica albuginea, interstitium and theca interna.

The ovary has specialised stromal compartments, including the tunica albuginea, interstitial stroma and theca interna, which develops concurrently with the follicular antrum. To characterise the molecular determinants of these compartments, stroma adjacent to preantral follicles (pre-theca), interstitium and tunica albuginea were laser microdissected (n = 4 per group) and theca interna was dissected from bovine antral follicles (n = 6). RNA microarray analysis showed minimal differences between interstitial stroma and pre-theca, and these were combined for some analyses and referred to as stroma. Genes significantly upregulated in theca interna compared to stroma included INSL3, LHCGR, HSD3B1, CYP17A1, ALDH1A1, OGN, POSTN and ASPN. Quantitative RT-PCR showed significantly greater expression of OGN and LGALS1 in interstitial stroma and theca interna versus tunica and greater expression of ACD in tunica compared to theca interna. PLN was significantly higher in interstitial stroma compared to tunica and theca. Ingenuity pathway, network and upstream regulator analyses were undertaken. Cell survival was also upregulated in theca interna. The tunica albuginea was associated with GPCR and cAMP signalling, suggesting tunica contractility. It was also associated with TGF-ß signalling and increased fibrous matrix. Western immunoblotting was positive for OGN, LGALS1, ALDH1A1, ACD and PLN with PLN and OGN highly expressed in tunica and interstitial stroma (each n = 6), but not in theca interna from antral follicles (n = 24). Immunohistochemistry localised LGALS1 and POSTN to extracellular matrix and PLN to smooth muscle cells. These results have identified novel differences between the ovarian stromal compartments.

Oxygen-regulated gene expression in murine cumulus cells.

Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

TGF-ß mediates proinflammatory seminal fluid signaling in human cervical epithelial cells.

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-ß1, TGF-ß2, and TGF-ß3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-ß3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-ß isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-ß induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-ß neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-ß isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-ß in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

Engineered CAR-T cells targeting the non-functional P2X purinoceptor 7 (P2X7) receptor as a novel treatment for ovarian cancer.

Objectives: Recent studies have identified expression of the non-functional P2X7 (nfP2X7) receptor on various malignant cells including ovarian cancer, but not on normal cells, which makes it a promising tumour-associated antigen candidate for chimeric antigen receptor (CAR)-T-cell immunotherapies. In this study, we assessed the cytotoxic effects of nfP2X7-CAR-T cells on ovarian cancer using in vitro and in vivo models. Methods: We evaluated the effects of nfP2X7-CAR-T cells on ovarian cancer cell lines (SKOV-3, OVCAR3, OVCAR5), normal peritoneal cells (LP-9) and primary serous ovarian cancer cells derived from patient ascites in vitro using monolayer and 3D spheroid assays. We also evaluated the effects of nfP2X7-CAR-T cells on patient-derived tissue explants, which recapitulate an intact tumour microenvironment. In addition, we investigated the effect of nfP2X7-CAR-T cells in vivo using the OVCAR-3 xenograft model in NOD-scid IL2Rγnull (NSG) mice. Results: Our study found that nfP2X7-CAR-T cells were cytotoxic and significantly inhibited survival of OVCAR3, OVCAR5 and primary serous ovarian cancer cells compared with un-transduced CD3+ T cells in vitro. However, no significant effects of nfP2X7-CAR-T cells were observed for SKOV3 or normal peritoneal cells (LP-9) cells with low P2X7 receptor expression. Treatment with nfP2X7-CAR-T cells increased apoptosis compared with un-transduced T cells in patient-derived explants and correlated with CD3 positivity. Treatment with nfP2X7-CAR-T cells significantly reduced OVCAR3 tumour burden in mice compared with un-transduced CD3 cells for 7-8 weeks. Conclusion: This study demonstrates that nfP2X7-CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer.

ABCA1 is associated with the development of acquired chemotherapy resistance and predicts poor ovarian cancer outcome.

Aim: This study investigated the ATP binding cassette (ABC) transporter (ABCA1, ABCB1, ABCB3, ABCC2 and ABCG2) expression in high grade serous ovarian cancer (HGSOC) tissues, cell lines and primary cells to determine their potential relationship with acquired chemotherapy resistance and patient outcome. Methods: ABC transporter mRNA and protein expression (ABCA1, ABCB1, ABCB3, ABCC2 and ABCG2) was assessed in publicly available datasets and in a tissue microarray (TMA) cohort of HGSOC at diagnosis, respectively. ABC transporter mRNA expression was also assessed in chemosensitive ovarian cancer cell lines (OVCAR-5 and CaOV3) versus matching cell lines with acquired carboplatin resistance and in primary HGSOC cells from patients with chemosensitive disease at diagnosis (n = 10) as well as patients with acquired chemotherapy resistance at relapse (n = 6). The effects of the ABCA1 inhibitor apabetalone in carboplatin-sensitive and -resistant cell lines were also investigated. Results: High ABCA1 mRNA and protein expression was found to be significantly associated with poor patient outcome. ABCA1 mRNA and protein levels were significantly increased in ovarian cancer cell lines (OVCAR-5 CBPR and CaOV3 CBPR) with acquired carboplatin resistance. ABCA1 mRNA was significantly increased in primary HGSOC cells obtained from patients with acquired chemotherapy resistance. Apabetalone treatment reduced ABCA1 protein expression and increased the sensitivity of both parental and carboplatin-resistant ovarian cancer cells to carboplatin. Conclusion: These results suggest that inhibiting ABCA1 transporter may be useful in overcoming acquired chemotherapy resistance and improving outcome for patients with HGSOC.

Epithelial Ovarian Cancer and the Immune System: Biology, Interactions, Challenges and Potential Advances for Immunotherapy.

Recent advances in the understanding of immune function and the interactions with tumour cells have led to the development of various cancer immunotherapies and strategies for specific cancer types. However, despite some stunning successes with some malignancies such as melanomas and lung cancer, most patients receive little or no benefit from immunotherapy, which has been attributed to the tumour microenvironment and immune evasion. Although the US Food and Drug Administration have approved immunotherapies for some cancers, to date, only the anti-angiogenic antibody bevacizumab is approved for the treatment of epithelial ovarian cancer. Immunotherapeutic strategies for ovarian cancer are still under development and being tested in numerous clinical trials. A detailed understanding of the interactions between cancer and the immune system is vital for optimisation of immunotherapies either alone or when combined with chemotherapy and other therapies. This article, in two main parts, provides an overview of: (1) components of the normal immune system and current knowledge regarding tumour immunology, biology and their interactions; (2) strategies, and targets, together with challenges and potential innovative approaches for cancer immunotherapy, with attention given to epithelial ovarian cancer.

Stress response genes are suppressed in mouse preimplantation embryos by granulocyte-macrophage colony-stimulating factor (GM-CSF).

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to promote the development and survival of human and mouse preimplantation embryos; however, the mechanism of action of GM-CSF in embryos is not defined. METHODS: Mouse blastocysts were cultured from zygote stage in vitro with and without recombinant mouse GM-CSF (rmGM-CSF), and in vivo developed blastocysts were flushed from Csf2 null mutant and wild-type mice. The effect of GM-CSF on blastocyst expression of stress response and apoptosis genes was evaluated by microarray, qPCR and immunochemistry. RESULTS: Microarray analysis of the gene transcription profile showed suppression of stress response and apoptosis gene pathways in blastocysts exposed to rmGM-CSF in vitro. qPCR analysis confirmed that rmGM-CSF inhibited expression of heat shock protein (HSP) and apoptosis pathway genes Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5 in in vitro blastocysts. Immunocytochemical analysis of HSP 1 (HSPA1A/1B; HSP70), BAX, BCL2 and TRP53 (p53) in in vitro blastocysts showed that HSPA1A/1B and BCL2 proteins were less abundant when embryos were cultured with rmGM-CSF. BAX and TRP53 were unchanged at the protein level, but Bax mRNA expression was reduced after GM-CSF treatment. In in vivo developed blastocysts, Csf2 null mutation caused elevated expression of Hsph1 but not other stress response genes. CONCLUSIONS: We conclude that GM-CSF inhibits the cellular stress response and apoptosis pathways to facilitate embryo growth and survival, and the protective effects of GM-CSF are particularly evident in in vitro culture media, whereas in vivo other cytokines can partly compensate for absence of GM-CSF.

4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer.

We have recently shown that the extracellular matrix molecule hyaluronan (HA) plays a role in the development of ovarian cancer chemoresistance. This present study determined if HA production is increased in chemotherapy-resistant ovarian cancers and if the HA inhibitor 4-methylubelliferone (4-MU) can overcome chemoresistance to the chemotherapeutic drug carboplatin (CBP) and inhibit spheroid formation and the expression of cancer stem cell (CSC) markers. We additionally assessed whether 4-MU could inhibit in vivo invasion of chemoresistant primary ovarian cancer cells in the chicken embryo chorioallantoic membrane (CAM) assay. The expression of the HA synthases HAS2 and HAS3 was significantly increased in chemoresistant compared to chemosensitive primary ovarian cancer cells isolated from patient ascites. 4-MU significantly inhibited HA production, cell survival, and spheroid formation of chemoresistant serous ovarian cancer cells. In combination with CBP, 4-MU treatment significantly decreased ovarian cancer cell survival and increased apoptosis of chemoresistant primary cells compared to CBP alone. 4-MU significantly reduced spheroid formation, expression of CSC markers ALDH1A1 and ABCG2 in primary cell spheroid cultures, and ALDH1 immunostaining in patient-derived tissue explant assays following treatment with CBP. Furthermore, 4-MU was very effective at inhibiting in vivo invasion of chemoresistant primary cells in CAM assays. Inhibition of HA is therefore a promising new strategy to overcome chemoresistance and to improve ovarian cancer survival.

Novel ex vivo ovarian cancer tissue explant assay for prediction of chemosensitivity and response to novel therapeutics.

The majority of ovarian cancer patients present with advanced disease and despite aggressive treatment, prognosis remains poor. Response to first-line carboplatin-containing chemotherapy is usually good, however, recurrence rates and subsequent chemoresistance are very high and ultimately responsible for the fatal outcome of the disease. To improve treatment outcomes pre-clinical models that can predict individual patient response to 1st line chemotherapy and novel therapeutics are urgently required. In this study, we employed an ex vivo ovarian cancer tissue explant assay to assess response to carboplatin and an inhibitor of the extracellular matrix molecule, hyaluronan (4-methylubelliferone, 4-MU), shown to inhibit cancer metastasis. Cryopreserved ovarian cancer tissues were cultured on gelatine sponges for 48-120 h with increasing concentrations of carboplatin (0-400 µM) or 4-MU (1 mM) alone or the combination of both drugs. Effects on apoptosis and proliferation were assessed by immunohistochemistry using antibodies to cleaved caspase 3 or Ki67, respectively. The ex vivo tissue explant assay maintained viable tumor cells in an intact tumor microenvironment similar to the in vivo situation over the 120 h culture period. Carboplatin treatment promoted apoptosis in chemosensitive (P = 0.0047) but not chemoresistant cancer tissues. The combination of 4-MU (1 mM) and carboplatin (100 µM) significantly increased apoptosis (P = 0.0111) and reduced proliferation (P = 0.0064) in chemoresistant tissues. Overall, our results show that the ex vivo explant assay is a robust and cost effective model to assess chemosensitivity and the effect of novel therapeutics in ovarian cancer.

Keratin 5 overexpression is associated with serous ovarian cancer recurrence and chemotherapy resistance.

This study investigated the clinical significance of keratin 5 and 6 expression in serous ovarian cancer progression and chemotherapy resistance. KRT5 and KRT6 (KRT6A, KRT6B & KRT6C) gene expression was assessed in publically available serous ovarian cancer data sets, ovarian cancer cell lines and primary serous ovarian cancer cells. Monoclonal antibodies which detect both K5/6 or only K5 were used to assess protein expression in ovarian cancer cell lines and a cohort of high grade serous ovarian carcinomas at surgery (n = 117) and after neoadjuvant chemotherapy (n = 21). Survival analyses showed that high KRT5 mRNA in stage III/IV serous ovarian cancers was significantly associated with reduced progression-free (HR 1.38, P < 0.0001) and overall survival (HR 1.28, P = 0.013) whilst high KRT6 mRNA was only associated with reduced progression-free survival (HR 1.2, P = 0.031). Both high K5/6 (≥ 10%, HR 1.78 95% CI; 1.03-2.65, P = 0.017) and high K5 (≥ 10%, HR 1.90, 95% CI; 1.12-3.19, P = 0.017) were associated with an increased risk of disease recurrence. KRT5 but not KRT6C mRNA expression was increased in chemotherapy resistant primary serous ovarian cancer cells compared to chemotherapy sensitive cells. The proportion of serous ovarian carcinomas with high K5/6 or high K5 immunostaining was significantly increased following neoadjuvant chemotherapy. K5 can be used to predict serous ovarian cancer prognosis and identify cancer cells that are resistant to chemotherapy. Developing strategies to target K5 may therefore improve serous ovarian cancer survival.

Csf2 null mutation alters placental gene expression and trophoblast glycogen cell and giant cell abundance in mice.

Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31(+) glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C(+), P57(Kip2+)) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.

Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells.

Exposure to semen elicits an inflammatory response in the female reproductive tract of rodents and other animals. The nature and regulation of any similar response in humans is poorly understood. This study investigated seminal plasma induction of inflammatory cytokine and chemokine gene regulation in human cervical and vaginal epithelial cells in vitro. Affymetrix microarray gene profiling revealed that inflammatory cytokine genes were prevalent among 317 known genes differentially expressed in immortalized ectocervical epithelial (Ect1) cells after incubation with pooled human seminal plasma. A dose- and time-dependent induction by seminal plasma of IL8, IL6, CSF2 and CCL2 mRNA expression in Ect1 cells was verified by quantitative RT-PCR. This was accompanied by increases in Ect1 secretion of immunoactive gene products IL-8, IL-6, GM-CSF and MCP-1. Similar cytokine responses were elicited in primary ectocervical epithelial cells. Endocervical epithelial (End1) and vaginal epithelial (Vk2) cells were less responsive to seminal fluid, with induction of IL-8 and MCP-1, but not GM-CSF or IL-6. In a panel of 10 seminal plasma samples, considerable variation in inflammatory cytokine-inducing activity was evident. These experiments show that seminal plasma can elicit expression of a range of inflammatory cytokines and chemokines in reproductive tract epithelia, and implicate the ectocervix as the primary site of responsiveness, with gene-specific differences in the kinetics and site-restrictedness of the response. Seminal factor regulation of inflammatory cytokines in the cervical epithelium is implicated in controlling the immune response to seminal antigens, and defence against infectious agents introduced at intercourse.