Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer.

BACKGROUND: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. METHODS: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2'-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. RESULTS: Approximately 70-90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. CONCLUSIONS: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer.

DNA repair in primordial follicle oocytes following cisplatin treatment.

PURPOSE: Genotoxic chemotherapy and radiotherapy can cause DNA double stranded breaks (DSBs) in primordial follicle (PMF) oocytes, which then undergo apoptosis. The development of effective new fertility preservation agents has been hampered, in part, by a limited understanding of DNA repair in PMF oocytes. This study investigated the induction of classical DSB repair pathways in the follicles of wild type (WT) and apoptosis-deficient Puma-/- mice in response to DSBs caused by the chemotherapy agent cisplatin. METHODS: Adult C57BL/6 WT and Puma-/- mice were injected i.p. with saline or cisplatin (5 mg/kg); ovaries were harvested at 8 or 24 h. Follicles were counted, and H2A histone family member (γH2AX) immunofluorescence used to demonstrate DSBs. DNA repair protein RAD51 homolog 1 (RAD51) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) immunofluorescence were used to identify DNA repair pathways utilised. RESULTS: Puma-/- mice retained 100% of follicles 24 h after cisplatin treatment. Eight hours post-treatment, γH2AX immunofluorescence showed DSBs across follicular stages in Puma-/- mice; staining returned to control levels in PMFs within 5 days, suggesting repair of PMF oocytes in this window. RAD51 immunofluorescence eight hours post-cisplatin was positive in damaged cell types in both WT and Puma-/- mice, demonstrating induction of the homologous recombination pathway. In contrast, DNA-PKcs staining were rarely observed in PMFs, indicating non-homologous end joining plays an insignificant role. CONCLUSION: PMF oocytes are able to conduct high-fidelity repair of DNA damage accumulated during chemotherapy. Therefore, apoptosis inhibition presents a viable strategy for fertility preservation in women undergoing treatment.

Expression of TIMPs and MMPs in Ovarian Tumors, Ascites, Ascites-Derived Cells, and Cancer Cell Lines: Characteristic Modulatory Response Before and After Chemotherapy Treatment.

BACKGROUND: The tissue inhibitors of metalloproteinase (TIMPs) and their associated metalloproteinase (MMPs) are essential regulators of tissue homeostasis and are essential for cancer progression. This study analyzed the expression of TIMP-1,-2,-3 and the associated MMPs (MMP-2,-9,-11,-14) in different Stages, Grades and World Health Organization (WHO) classifications of serous ovarian tumors, ascites, ascites-derived cells from chemo-naïve (CN) and relapsed (CR) patients, and in ovarian cancer cell lines. The status of TIMPs and associated MMPs in response to chemotherapy treatment was assessed in cancer cell lines; TCGA data was interrogated to gauge TIMPs and associated MMPs as prognostic and platinum-response indicators. METHODS: The levels of TIMP-1, -2 and -3 were assessed by immunohistochemistry. The mRNA expression of TIMPs and MMPs was quantified by real time PCR (qRT-PCR). The chemosensitivity (IC50 values) to Cisplatin or Paclitaxel in cell lines was evaluated by MTT assay. The levels of TIMPs in ascites and cell lysates were analyzed by an ELISA assay. RESULTS: The expression of TIMP-2 was significantly upregulated in Type 2 compared to Type 1 tumors and normal/benign ovarian tissues. TIMP-3 expression was significantly enhanced in Stage III, Grade 3 and Type 2 tumors compared to normal/benign ovarian tissues. The mRNA expression of MMP-9,-11 and -14 was significantly upregulated in Stage IV compared to normal/benign ovarian tissues. The expression of TIMP-1 was highest, followed by TIMP-2 and then TIMP-3 in CN ascites. At the cellular level, TIMP-2 mRNA expression was significantly higher in CN compared to CR epithelial cells in patients. The expression of TIMP-1 and -2, MMPs and cancer stem cells (CSCs) were upregulated in response to chemotherapy treatments in cancer cell lines. Interrogation of the TCGA dataset suggests shifts in platinum responses in patients consistent with genetic alterations in TIMP-2, -3 and MMP-2, -11 genes in tumors; and decreased overall survival (OS) and progression-free survival (PFS) in patients with altered MMP-14 genes. CONCLUSIONS: TIMPs and related MMPs are differentially expressed in serous ovarian tumors, ascites, ascites-derived cells and ovarian cancer cell lines. Chemotherapy treatment modulates expression of TIMPs and MMPs in association with increased expression of genes related to cancer stem cells.

TIMP-2 regulates proliferation, invasion and STAT3-mediated cancer stem cell-dependent chemoresistance in ovarian cancer cells.

BACKGROUND: The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. METHODS: FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. RESULTS: Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. CONCLUSIONS: The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.

Reproductive science and the future of the planet.

Reproductive sciences have made major contributions to human health, livestock production and environmental management in the past and will continue to do so in future. In collaboration with other disciplines, reproductive scientists can provide scientifically valid information that will allow the rational development of policies on topics such as declining fertility in men and women, livestock breeding efficiencies, climate change, pest animal control, wildlife management and environmental influences. It is imperative that the reproductive sciences are recognised by the community and policy makers as important contributors to future health and welfare of animals, humans and the planet if these potential benefits are to be captured and utilised. Reproductive Health Australia (RHA) was launched recently to advocate for reproductive biology as a national health, economic and social priority. This short review provides a snapshot of why it is imperative that reproductive science receives the recognition that is due to it and provides examples of how it can contribute to the future of the planet.

Oocytes from stem cells.

Folliculogenesis describes the process of activating an oocyte-containing primordial follicle from the ovarian reserve and its development to the mature ovulatory stage. This process is highly complex and is controlled by extra- and intra-ovarian signaling events. Oocyte competence and capacity for fertilization to support a viable pregnancy are acquired during folliculogenesis. Cancer and cancer-based therapies can negatively affect this process, compromising fertility. Currently, preservation of fertility in these patients remains limited to surrogacy, oocyte freezing, oocyte donation, or in vitro maturation (IVM). Recent reports of stem cells being used to produce fully competent oocytes and subsequently healthy offspring in mice have opened up a novel avenue for fertility preservation. However, translating these findings into human health first relies on enhancing our understanding of follicle growth and mimicking its intricacies in vitro. Indeed, the future of oocytes from stem cells in humans comes with many possibilities but currently faces several technical and ethical obstacles.

Loss of PUMA protects the ovarian reserve during DNA-damaging chemotherapy and preserves fertility.

Female gametes are stored in the ovary in structures called primordial follicles, the supply of which is non-renewable. It is well established that DNA-damaging cancer treatments can deplete the ovarian reserve of primordial follicles, causing premature ovarian failure and infertility. The precise mechanisms underlying this chemotherapy-driven follicle loss are unclear, and this has limited the development of targeted ovarian-protective agents. To address this fundamental knowledge gap, we used gene deletion mouse models to examine the role of the DNA damage-induced pro-apoptotic protein, PUMA, and its transcriptional activator TAp63, in primordial follicle depletion caused by treatment with cyclophosphamide or cisplatin. Cyclophosphamide caused almost complete destruction of the primordial follicle pool in adult wild-type (WT) mice, and a significant destructive effect was also observed for cisplatin. In striking contrast, Puma-/- mice retained 100% of their primordial follicles following either genotoxic treatment. Furthermore, elimination of PUMA alone completely preserved fertility in cyclophosphamide-treated mice, indicating that oocytes rescued from DNA damage-induced death can repair themselves sufficiently to support reproductive function and offspring health. Primordial follicles were also protected in TAp63-/- mice following cisplatin treatment, but not cyclophosphamide, suggesting mechanistic differences in the induction of apoptosis and depletion of the ovarian reserve in response to these different chemotherapies. These studies identify PUMA as a crucial effector of apoptosis responsible for depletion of primordial follicles following exposure to cyclophosphamide or cisplatin, and this indicates that inhibition of PUMA may be an effective ovarian-protective strategy during cancer treatment in women.

Momelotinib decreased cancer stem cell associated tumor burden and prolonged disease-free remission period in a mouse model of human ovarian cancer.

Despite a good initial response to front-line chemotherapy, majority of the ovarian cancer patients relapse with consecutive phases of recurrences; and nearly 60% die within 5 years due to the development of a chemoresistant disease. This study investigated whether inhibition of the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway by momelotinib is sufficient in suppressing tumor burden and prolonging the disease-free survival period in a mouse model of ovarian cancer. We demonstrate that paclitaxel treatment enhanced JAK2/STAT3 activation which resulted in the enrichment of cancer stem cell (CSC)-like phenotype in the surviving ovarian cancer cells in vitro and in in vivo mouse xenografts. Combined treatment with paclitaxel and momelotinib inhibited paclitaxel-induced JAK2/STAT3 activation and CSC-like development in mice xenografts, and consequently reduced the tumor burden significantly greater than that achieved by paclitaxel-treatment alone. However, robust recurrent tumor growth with enhanced JAK2/STAT3 activation and CSC-like phenotype was observed in all mice groups after termination of treatments, but was delayed significantly in the paclitaxel and momelotinib treated group compared to other treatment groups. Daily oral gavage of momelotinib after termination of paclitaxel treatment showed sustained inhibition of tumor growth and a prolonged disease-free survival period in 50% of the mice. The other 50% of mice that developed tumors with ongoing momelotinib treatment also showed significantly increased survival benefit and a smaller tumor burden. These preliminary findings may have a profound clinical impact in developing an effective momelotinib-based 'maintenance-therapy' in ovarian cancer patients' post-chemotherapy treatment.

The Many Facets of Metzincins and Their Endogenous Inhibitors: Perspectives on Ovarian Cancer Progression.

Approximately sixty per cent of ovarian cancer patients die within the first five years of diagnosis due to recurrence associated with chemoresistance. The metzincin family of metalloproteinases is enzymes involved in matrix remodeling in response to normal physiological changes and diseased states. Recently, there has been a mounting awareness of these proteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), as superb modulators of cellular communication and signaling regulating key biological processes in cancer progression. This review investigates the role of metzincins and their inhibitors in ovarian cancer. We propose that understanding the metzincins and TIMP biology in ovarian cancer may provide valuable insights in combating ovarian cancer progression and chemoresistance-mediated recurrence in patients.

The ovarian reserve is depleted during puberty in a hormonally driven process dependent on the pro-apoptotic protein BMF.

In females, germ cells are maintained in ovarian structures called primordial follicles. The number of primordial follicles in the ovarian reserve is a critical determinant of the length of the fertile lifespan. Despite this significance, knowledge of the precise physiological mechanisms that regulate primordial follicle number is lacking. In this study we show that a wave of primordial follicle depletion occurs during the transition to adulthood in mice. We demonstrate that this sudden and dramatic loss of primordial follicles is hormonally triggered and identify the pro-apoptotic BH3-only protein, BCL-2 modifying factor (BMF), as essential for this process, implicating the intrinsic apoptotic pathway as a key mechanism. The elimination of primordial follicles during puberty is not only a striking developmental event, it is also physiologically important because it ultimately reduces the availability of primordial follicles and determines the duration of fertility. Collectively, these findings show that puberty is a critical developmental window for the regulation of the size of ovarian reserve, impacting on female fertility and reproductive longevity.

Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling.

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas.

Coalition of Oct4A and ß1 integrins in facilitating metastasis in ovarian cancer.

BACKGROUND: Ovarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis. METHODS: The expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice. RESULTS: We report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin ß1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34 and CD31 compared to vector control cell-derived xenografts. CONCLUSION: The expression of Oct4A may be crucial to promote and sustain integrin-mediated extracellular matrix (ECM) remodeling requisite for tumor metastasis in ovarian cancer patients.

Endogenous inhibins regulate steroidogenesis in mouse TM3 Leydig cells by altering SMAD2 signalling.

This study tested the hypothesis that inhibins act in an autocrine manner on Leydig cells using a pre-pubertal Leydig cell line, TM3, as a model of immature Leydig cells. The expression of Inha, Inhba, and Inhbb in TM3 cells was determined by RT-PCR and the production of the inhibin-alpha subunit was confirmed by western blot. Knockdown of Inha expression resulted in significant decreases in the expression of Leydig cell markers Cyp17a1, Cyp11a1, Nr5a1, and Insl3. Western blot showed that activin A, TGFß1 and TGFß2 activated SMAD2, and that knockdown of Inha expression in TM3 cells enhanced both activin A- and TGFß-induced SMAD2 activation. SB431542, a chemical inhibitor of the TGFß/activin type I receptors, blocked ligand-induced SMAD2 activation and the downregulation of Cyp17a1 expression. Our findings demonstrate that TGFßs and activin A negatively regulate steroidogenic gene expression in TM3 cells via ALK4/5 and SMAD2 and endogenous inhibins can counter this regulation.

Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells.

Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission after initial surgery and chemotherapy. However, most patients die within <5 years due to episodes of recurrences resulting from the growth of residual chemoresistant cells. In an effort to identify mechanisms associated with chemoresistance and recurrence, we compared the expression of proteins in ascites-derived tumor cells isolated from advanced-stage ovarian cancer patients obtained at diagnosis (chemonaive, CN) and after chemotherapy treatments (chemoresistant/at recurrence, CR) by using in-depth, high-resolution label-free quantitative proteomic profiling. A total of 2,999 proteins were identified. Using a stringent selection criterion to define only significantly differentially expressed proteins, we report identification of 353 proteins. There were significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells. In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cells from CN and CR ovarian cancer patients.

BCL2-modifying factor promotes germ cell loss during murine oogenesis.

Apoptosis plays a prominent role during ovarian development by eliminating large numbers of germ cells from the female germ line. However, the precise mechanisms and regulatory proteins involved in germ cell death are yet to be determined. In this study, we characterised the role of the pro-apoptotic BH3-only protein, BCL2-modifying factor (BMF), in germ cell apoptosis in embryonic and neonatal mouse ovaries. BMF protein was immunohistochemically localised to germ cells at embryonic days 15.5 (E15.5) and E17.5 and postnatal day 1 (PN1), coincident with entry into the meiotic prophase, but was undetectable at E13.5, and only present at low levels at PN3 and PN5. Consistent with this expression pattern, loss of BMF in female mice was associated with a decrease in apoptosis at E15.5 and E17.5. Furthermore, increased numbers of germ cells were found in ovaries from Bmf(-/-) mice compared with WT animals at E15.5 and PN1. However, germ cell numbers were comparable between Bmf(-/-) and WT ovaries at PN3, PN5 and PN10. Collectively, these data indicate that BMF mediates foetal oocyte loss and its action limits the maximal number of germ cells attained in the developing ovary, but does not influence the number of primordial follicles initially established in ovarian reserve.

A critical role of Oct4A in mediating metastasis and disease-free survival in a mouse model of ovarian cancer.

BACKGROUND: High grade epithelial ovarian cancer (EOC) is commonly characterised by widespread peritoneal dissemination and ascites. Metastatic EOC tumour cells can attach directly to neighbouring organs or alternatively, maintain long term tumourigenicity and chemoresistance by forming cellular aggregates (spheroids). Cancer stem-like cells are proposed to facilitate this mechanism. This study aimed to investigate the role of Oct4A, an embryonic stem cell factor and known master regulator of pluripotency in EOC progression, metastasis and chemoresistance. METHODS: To investigate the expression of Oct4A in primary EOC tumours, IHC and qRT-PCR analyses were used. The expression of Oct4A in chemonaive and recurrent EOC patient ascites-derived tumour cells samples was investigated by qRT-PCR. The functional role of Oct4A in EOC was evaluated by generating stable knockdown Oct4A clones in the established EOC cell line HEY using shRNA-mediated silencing technology. Cellular proliferation, spheroid forming ability, migration and chemosensitivty following loss of Oct4A in HEY cells was measured by in vitro functional assays. These observations were further validated in an in vivo mouse model using intraperitoneal (IP) injection of established Oct4A KD clones into Balb/c nu/nu mice. RESULTS: We demonstrate that, compared to normal ovaries Oct4A expression significantly increases with tumour dedifferentiation. Oct4A expression was also significantly high in the ascites-derived tumour cells of recurrent EOC patients compared to chemonaive patients. Silencing of Oct4A in HEY cells resulted in decreased cellular proliferation, migration, spheroid formation and increased chemosensitivity to cisplatin in vitro. IP injection of Oct4A knockdown cells in vivo produced significantly reduced tumour burden, tumour size and invasiveness in mice, which overall resulted in significantly increased mouse survival rates compared to mice injected with control cells. CONCLUSIONS: This data highlights a crucial role for Oct4A in the progression and metastasis of EOC. Targeting Oct4A may prove to be an effective strategy in the treatment and management of epithelial ovarian tumours.

Betaglycan blocks metastatic behaviors in human granulosa cell tumors by suppressing NFκB-mediated induction of MMP2.

Metastatic ovarian granulosa cell tumors (GCT) exhibit loss of betaglycan. Here we test the hypothesis that betaglycan blocks GCT metastasis by suppressing NFκB/TGFß2-induced matrix metalloprotinease-2 (MMP2). Human GCT and a human GCT cell model demonstrated prominent MMP2 expression, which was dependent on NFκB activity and stimulated by TGFß2 in an NFκB-dependent manner. Betaglycan suppressed both basal and TGFß2-induced MMP2 expression and countered metastatic behaviors of GCT cells in non-adherent spheroid culture and in vivo xenograft models of metastasis. These data suggest that NFκB/TGFß2 promotes, and betaglycan impedes, the early stages of GCT metastasis, when tumor cells first invade the peritoneum.

Inhibition of the JAK2/STAT3 pathway in ovarian cancer results in the loss of cancer stem cell-like characteristics and a reduced tumor burden.

BACKGROUND: Current treatment of ovarian cancer patients with chemotherapy leaves behind a residual tumor which results in recurrent ovarian cancer within a short time frame. We have previously demonstrated that a single short-term treatment of ovarian cancer cells with chemotherapy in vitro resulted in a cancer stem cell (CSC)-like enriched residual population which generated significantly greater tumor burden compared to the tumor burden generated by control untreated cells. In this report we looked at the mechanisms of the enrichment of CSC-like residual cells in response to paclitaxel treatment. METHODS: The mechanism of survival of paclitaxel-treated residual cells at a growth inhibitory concentration of 50% (GI50) was determined on isolated tumor cells from the ascites of recurrent ovarian cancer patients and HEY ovarian cancer cell line by in vitro assays and in a mouse xenograft model. RESULTS: Treatment of isolated tumor cells from the ascites of ovarian cancer patients and HEY ovarian cancer cell line with paclitaxel resulted in a CSC-like residual population which coincided with the activation of Janus activated kinase 2 (JAK2) and signal transducer and activation of transcription 3 (STAT3) pathway in paclitaxel surviving cells. Both paclitaxel-induced JAK2/STAT3 activation and CSC-like characteristics were inhibited by a low dose JAK2-specific small molecule inhibitor CYT387 (1 µM) in vitro. Subsequent, in vivo transplantation of paclitaxel and CYT387-treated HEY cells in mice resulted in a significantly reduced tumor burden compared to that seen with paclitaxel only-treated transplanted cells. In vitro analysis of tumor xenografts at protein and mRNA levels demonstrated a loss of CSC-like markers and CA125 expression in paclitaxel and CYT387-treated cell-derived xenografts, compared to paclitaxel only-treated cell-derived xenografts. These results were consistent with significantly reduced activation of JAK2 and STAT3 in paclitaxel and CYT387-treated cell-derived xenografts compared to paclitaxel only-treated cell derived xenografts. CONCLUSIONS: This proof of principle study demonstrates that inhibition of the JAK2/STAT3 pathway by the addition of CYT387 suppresses the 'stemness' profile in chemotherapy-treated residual cells in vitro, which is replicated in vivo, leading to a reduced tumor burden. These findings have important implications for ovarian cancer patients who are treated with taxane and/or platinum-based therapies.

PUMA regulates germ cell loss and primordial follicle endowment in mice.

The number of primordial follicles initially established within the ovary is influenced by the extent of germ cell death during foetal ovarian development, but the mechanisms that mediate this death have not been fully uncovered. In this study, we identified BBC3 (PUMA) (p53 upregulated modulator of apoptosis, also known as BCL2-binding component 3), a pro-apoptotic BH3-only protein belonging to the BCL2 family, as a critical determinant of the number of germ cells during ovarian development. Targeted disruption of the Bbc3 gene revealed a significant increase in the number of germ cells as early as embryonic day 13.5. The number of germ cells remained elevated in Bbc3(-/-) female mice compared with WT female mice throughout the remainder of embryonic and early postnatal life, resulting in a 1.9-fold increase in the number of primordial follicles in the ovary on postnatal day 10. The increase in the number of germ cells observed in the ovaries of Bbc3(-/-) mice could not be attributed to the altered proliferative activity of germ cells within the ovaries. Furthermore, BBC3 was found to be not required for the massive germ cell loss that occurs during germ cell nest breakdown. Our data indicate that BBC3 is a critical regulator of germ cell death that acts during the migratory phase of oogenesis or very soon after the arrival of germ cells in the gonad and that BBC3-mediated cell death limits the number of primordial follicles established in the initial ovarian reserve.

Targeted Disruption of the JAK2/STAT3 Pathway in Combination with Systemic Administration of Paclitaxel Inhibits the Priming of Ovarian Cancer Stem Cells Leading to a Reduced Tumor Burden.

Chemotherapy resistance associated with recurrent disease is the major cause of poor survival of ovarian cancer patients. We have recently demonstrated activation of the JAK2/STAT3 pathway and the enhancement of a cancer stem cell (CSC)-like phenotype in ovarian cancer cells treated in vitro with chemotherapeutic agents. To elucidate further these mechanisms in vivo, we used a two-tiered paclitaxel treatment approach in nude mice inoculated with ovarian cancer cells. In the first approach, we demonstrate that a single intraperitoneal administration of paclitaxel in mice 7 days after subcutaneous transplantation of the HEY ovarian cancer cell line resulted in a significant increase in the expression of CA125, Oct4, and CD117 in mice xenografts compared to control mice xenografts which did not receive paclitaxel. In the second approach, mice were administered once weekly with paclitaxel and/or a daily dose of the JAK2-specific inhibitor, CYT387, over 4 weeks. Mice receiving paclitaxel only demonstrated a significant decrease in tumor volume compared to control mice. At the molecular level, mouse tumors remaining after paclitaxel administration showed a significant increase in the expression of Oct4 and CD117 coinciding with a significant activation of the JAK2/STAT3 pathway compared to control tumors. The addition of CYT387 with paclitaxel resulted in the suppression of JAK2/STAT3 activation and abrogation of Oct4 and CD117 expression in mouse xenografts. This coincided with significantly smaller tumors in mice administered CYT387 in addition to paclitaxel, compared to the control group and the group of mice receiving paclitaxel only. These data suggest that the systemic administration of paclitaxel enhances Oct4- and CD117-associated CSC-like marker expression in surviving cancer cells in vivo, which can be suppressed by the addition of the JAK2-specific inhibitor CYT387, leading to a significantly smaller tumor burden. These novel findings have the potential for the development of CSC-targeted therapy to improve the treatment outcomes of ovarian cancer patients.