Peter C Leung

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

View record

Placental insufficiency disorders, including preeclampsia (PET), intrauterine growth restriction (IUGR) and preterm labor (PTL), are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes share a common phenomenon of poor placental trophoblast cell invasion into the uterine tissue. To date there are no effective treatments for these illnesses. Placental invasion is controlled by many hormones and growth factors, among which the transforming growth factor (TGF)-β superfamily is a well-known regulator. Myostatin (MSTN) is a TGF-β superfamily member recognized for its important role in muscle growth control. Recently, MSTN was shown to be secreted and functioning in the placenta. In addition, MSTN serum and/or placental levels were found to be upregulated in common pregnancy complications including PET and IUGR. Yet, the role of MSTN in placental biology is poorly understood. In this study, I confirmed the positive effect of MSTN treatment on human trophoblast invasion and performed mRNA sequencing on control and MSTN-treated primary trophoblast cells (n=5). This analysis identified 610 differentially expressed genes (DEGs) with a false discovery rate
View record

Many pregnancy disorders, including pregnancy loss, preeclampsia, preterm birth and intrauterine growth restriction are associated with defects in placental development, including abnormal placental trophoblast invasion and differentiation. Bone morphogenetic protein 2 (BMP2) belongs to the transforming growth factor-β (TGF-β) superfamily and controls various physiological and developmental processes. Previous studies have demonstrated the pro-invasive action of BMP2 signaling in human trophoblasts in vitro. However, the expression of BMP2 in the placenta and underlying molecular mechanisms of how BMP2 regulates trophoblast function remain unclear. In this study, we analyzed several publicly available microarray datasets and revealed that BMP2 was expressed in the placenta across all three trimesters. Importantly, BMP2 levels were significantly reduced in placenta samples from early-onset preeclampsia pregnancies compared with gestational age-matched normal pregnancies. RNAscope in situ hybridization showed that in the first trimester BMP2 was localized in all subtypes of trophoblasts as well as in decidua. To further investigate downstream transcriptional targets of BMP2 signaling, we performed mRNA-sequencing on control and BMP2-treated primary human trophoblasts (n=5). This analysis identified 431 differentially expressed genes, of which 253 were upregulated and 178 were downregulated, with a false discovery rate
View record

During placentation, extravillous cytotrophoblasts (EVTs) derived from villous cytotrophoblasts invade into the uterine wall for proper placentation and successful establishment of human pregnancy. Insufficient trophoblast invasion contributes to several pregnancy complications including preeclampsia, which is a leading cause of maternal mortality and affects 2-8% of pregnancies worldwide. As an important member of the transforming growth factor β (TGF-β) superfamily, bone morphogenetic protein 2 (BMP2) is abundantly produced at the maternal-fetal interface and its expression is spatiotemporally correlated with embryo placentation. BMP2 is crucial for endometrial decidualization in humans and normal fertility in mice. In addition, BMP2 exerts pro-invasive effects in a variety of cancer cells. However, whether BMP2 can promote trophoblast cell invasion during placentation remains unknown. BMPs increase mesenchymal adhesion molecule N-cadherin expression, activin A production, an inhibitor of DNA-binding protein 1 (ID1) expression, and WNT/β-catenin signaling in different cell types. All of the above mentioned molecules and signals have been shown to positively regulate human trophoblast or cancer cell invasion, thus we hypothesized that BMP2 could promote human trophoblast cell invasion by regulating the expression of N-cadherin, activin A and ID1 as well as the activation of canonical WNT/β-catenin signaling. Primary and immortalized (HTR8/SVneo) cultures of human EVT cells were used as study models. Activin receptor-likekinase 2/3 (ALK2/3) inhibitor DMH1 and ALK4/5/7 inhibitor SB431542 were used to block receptor-mediated signaling. Small interfering RNA (siRNA) was used to study the involvement of key signaling molecules. Cell invasiveness was examined using the Matrigel-coated transwell invasion assay. Overall, our results demonstrate that BMP2 promotes trophoblast cell invasion via the following mechanisms: 1) Up-regulating N-cadherin via non-canonical ALK2/3/4-SMAD2/3-SMAD4 signaling; 2) Up-regulating inhibin βA and activin A production via ALK3-BMPR2/ACVR2A-SMAD1/5/8-SMAD4 signaling; 3) Inducing ID1-mediated up-regulation of insulin-like growth factor binding protein 3 (IGFBP3); and 4) Inducing WNT/β-catenin signaling activation mediated by bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI). These findings deepen our understanding of the roles of BMP2 in placentation and provide insights into the molecular mechanisms of human trophoblast invasion.

View record

Epithelial ovarian cancer is the most lethal gynecological malignancy in developed countries. Although extensive studies have been done in the past decades to develop early detection methods and novel therapeutic strategies for epithelial ovarian cancer, 5-year survival rate of patients diagnosed in late stages remains lower than 30%. The epidermal growth factor (EGF) family of ligands and receptors is well-known for its roles in oncogenesis and cancer progression. As a unique member in this family, betacellulin has been shown to stimulate cell proliferation in several types of human tumors, and has also been suggested to associate with poor clinical outcome in breast cancer. However, little is known about the role and mechanism of betacellulin in epithelial ovarian cancer cell activities. In the current study, we hypothesized that betacellulin enhances epithelial ovarian cancer migration and proliferation by modulating several key target genes. Epithelial ovarian cancer cell lines OVCAR3, OVCAR4, OVCAR5 and SKOV3 were used as study models. Cancer cell migration was assessed by transwell assays; and cell proliferation was measured by MTT assays. Our results showed that betacellulin induced epithelial ovarian cancer cell migration by down-regulating E-cadherin and up-regulating Connexin43 expression; and these effects were mainly mediated by MEK/ERK and PI3K/Akt signaling via EGFR. Moreover, the potential role of ERBB4 in betacellulin signaling and ovarian cancer cell migration has also been suggested. On the other hand, we also demonstrated that betacellulin stimulated cell proliferation by up-regulating CCN1 expression. Taken together, our findings provide important insight into ovarian cancer biology and fill in the gaps of our knowledge of betacellulin, which may lead to the development of novel therapeutic tools for epithelial ovarian cancer.

View record

Endometrial cancer is the fourth most common female cancer and the most common gynecological malignancy. Although it comprises only ~10% of all endometrial cancers, the serous histological subtype accounts for ~40% of deaths due to its aggressive behavior and propensity to metastasize. Moreover, the number of endometrial cancer related deaths keeps rising, which can be attributed to the increased incidence of advanced-stage tumor and high risk histologies. Histopathological studies suggest that in non-endometrioid endometrial cancers (type II, mostly serous), elevated expression of activin/inhibin βB subunit is associated with reduced survival and TGFβ signalings are closely associated with the neoplastic transformation of human endometrium and the initiation of invasion of endometrial cancer. However, little is known about the specific roles and mechanisms of activin B (βB dimer) and TGFβ1 in type II endometrial cancer cell progression. We hypothesized that integrin αvβ3, E-cadherin, and PTEN play critical roles in activin B or TGFβ1 induced endometria cancer cell adhesion or migration. Type II endometrial cancer cell lines KLE, HEC-1B and HEC-50 were used as study models. Cancer cell adhesion was assessed by extracellular matrix coated 96 well adhesion assays. Cancer cell migration or invasiveness was assessed by transwell assays without or with coated matrigel following exposure to recombinant human activin B or TGFβ1. Small interfering RNA (siRNA)-mediated knockdown or vector-mediated overexpression approaches were used to investigate the molecular determinants of activin B or TGFβ1-mediated functions. In summary, our results demonstrate that SMAD-mediated integrin β3 up-regulation by activin B promotes type II endometrial cancer cell adhesion and migration while ERK1/2-SNAIL-mediated E-cadherin down-regulation by activin B plays important roles in cancer migration. Moreover, TGFβ1 induces type II endometrial cancer cell migration via ERK1/2 mediated-up-regulation of integrin αvβ3. TGFβ1 also promotes cancer cell migration by down-regulating PTEN via both SMAD-dependent and independent pathways. Our findings provide important insights into the molecular mechanisms underlying the effects of activin and TGFβ on endometrial cancer cell migration and suggest novel therapeutic targets for treating type II endometrial cancer.

View record

The overexpression of epidermal growth factor receptor (EGFR) has been shown in ovarian cancer and is associated with poor prognosis of this malignant disease. Thus, exploring the EGFR-mediated cell signaling in ovarian cancer may deepen our understanding of this disease. The down-regulation of E-cadherin may promote cell proliferation, motility and invasiveness, leading to the cancer progression. We have previously demonstrated that epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor-α (TGF-α), all of which bind exclusively to EGFR, down-regulate E-cadherin expression and induce ovarian cancer cell invasion. In this study, we showed that, as was the case for the effect of EGF, the TGF-α- and AREG-induced down-regulation of E-cadherin expression involved both EGFR and HER2. However, in contrast to the cases of EGF and AREG, the transcription factor Snail was not required for the TGF-α-induced down-regulation of E-cadherin expression. This study showed that TGF-α uses common and divergent molecular mediators to regulate E-cadherin expression and cell invasion.Cyclooxygenase-2 (COX-2) has been shown to participate in cancer metastasis by down-regulating E-cadherin expression, and elevated expression of COX-2 has been reported in ovarian cancer. We have previously demonstrated that COX-2-derived prostaglandin E2 (PGE2) promotes cell invasion in human ovarian cancer. In this study, we showed that EGF/EGFR-induced cell invasion was mediated by the elevation of COX-2 expression and PGE2 production in an E-cadherin-independent manner. Aside from the pro-invasive effect, EGF may strongly promote the cell proliferation. Connexin 43 (Cx43) has been shown to regulate cell proliferation, and this gap junction protein can be regulated by EGF. To date, the functional role of EGF in regulating Cx43 expression in human ovarian cancer has never been investigated. Interestingly, we demonstrated that EGF/EGFR up-regulated Cx43 expression through the activation of Akt1. Functionally, Cx43 may act as a negative regulator of EGF/EGFR-induced cell proliferation in human ovarian cancer, in a gap junction-independent manner. Overall, our studies provide important insights into the molecular mechanisms regulating EGF-stimulated human ovarian cancer cell invasion and proliferation.

View record

During implantation, extravillous cytotrophoblast (EVT) differentiation follows invasive interstitial and endovascular routes. Aberrant placentation due to restricted trophoblast invasion and spiral artery remodeling is responsible for the pathogenesis of common complications during pregnancy, including miscarriage, preeclampsia and IUGR. Activin A, which is abundantly produced at the maternal-fetal interface, exerts its stimulatory effect in trophoblast invasion. Whether two additional activin isoforms, activin B and AB exert similar effects as activin A in trophoblast invasion remains unknown. We hypothesized that mesenchymal adhesion molecule neural cadherin (N-cadherin) and matrix metalloproteinase 2 (MMP2) play essential roles in activin-induced trophoblast invasion. In addition, the effect of activin A on trophoblast endovascular differentiation has never been studied. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation and activin A has been shown to stimulate VEGF-A expression in human hepatocellular carcinoma cells. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation has yet to be determined. An established immortalized EVT cell line HTR8/SVneo and primary cultures of human EVT cells were used as study models. Trophoblast invasiveness and endovascular differentiation were assessed by matrigel-coated transwell assays and endothelial-like tube formation assays following exposure to recombinant human activin. Small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin-mediated functions. In summary, our results demonstrate that activin A, B and AB produce comparable increases in human trophoblast cell invasion by up-regulating N-cadherin expression. Activin A induces human trophoblast cell invasion by up-regulating SNAIL-mediated MMP2 expression through ALK4 in a SMAD2/3-SMAD4-dependent manner. In addition, activin A induces human trophoblast cells endothelial-like tube formation by up-regulating VEGF-A expression in a SMAD2/3-SMAD4-dependent manner. Our findings provide important insights into the molecular mechanisms underlying the dual effects of activin on human EVTs, and enhance our understanding of the establishment of pregnancy under normal or pathological conditions.

View record

Ovulation is a complex and finely-tuned process whereby a series of closely-regulated events happen, including cumulus-oocyte complex (COC) expansion, resumption of oocyte meiosis and follicle rupture. These events must occur before luteinization; otherwise the oocytes are trapped within follicles and destined to degenerate. Appropriate timing requires specific changes in granulosa cell functions that are governed by gonadotropins and regulated by intrafollicular molecular regulators. In animal models, bone morphogenetic proteins (BMPs) are essential for successful ovulation by inhibiting early luteinization. However, the detailed functions and underlying mechanisms of BMPs on the ovulation/luteinization process have not been fully established. In the present research, immortalized (SVOG) and primary human granulosa cells were used to investigate the effects of recombinant human BMPs on steroid hormones and COC expansion-related protein production. Receptor-mediated signaling was investigated using activin receptor-like kinase (ALK) inhibitors and small interfering RNAs targeting ALKs and SMADs. In human granulosa cells, BMP4 and BMP7 suppress progesterone production and steroidogenic acute regulatory protein (StAR) expression while increasing estradiol production and aromatase expression. This fact suggests that BMPs could inhibit premature luteinization by decreasing the progesterone/estradiol ratio. BMPs (BMP4, BMP6, BMP7 and BMP15) up-regulate hyaluronan synthases 2 (HAS2) expressions and hyaluronan production as well as the expression of the hyaluronan binding protein versican. BMP4 also induced A Disintegrin and Metalloproteinase with TromboSPondin Repeats-1 (ADAMTS-1) protein levels and activity of versican proteolytic cleavage. It suggests that BMPs could promote COC mass formation while simultaneously loosing the matrix by increasing versican cleavage. In addition, the findings that the effects of BMPs on different gene expressions are differentially mediated by ALK inhibitors suggest that human granulosa cells have the capacity to signal via multiple BMP type I receptors in response to these factors, but that the receptor(s) are differentially coupled to specific target genes or cellular responses. Moreover, BMPs regulate different biological processes by activating different SMAD signaling: down-regulate progesterone production through canonical SMAD1/5/8 signaling, while up-regulate hyaluronan production through noncanonical SMAD2/3 signaling. These findings deepen knowledge of the physiological roles of BMPs during periovulatory interval and may lead to applying to in vitro fertilization (IVF) protocol improvement.

View record

To date, the pro-invasive role of the GnRH-GnRHR system has been demonstrated in several cell types including carcinoma cells. Placental expression of GnRH I, GnRH II, and their mutual receptor (GnRHR) is indicative of a potential mechanism(s) that occurs during the dynamic process of human placenta formation and differentiation, particularly during the development of an invasive phenotype of extravillous trophoblasts (EVTs). However, current studies haven’t completely revealed the role of GnRH in regulating invasive EVT function and the underlying mechanism(s) is not yet well-established. Dynamic reprogramming of cell adhesion and proteolytic machinery is frequently accompanied with cell invasive and angiogenic phenotypes. I hypothesized that GnRH could regulate trophoblast invasion and vascular remodeling via modulation of mesenchymal cadherins and matrix matelloproteinases (MMPs). In these studies, I have found that both GnRH forms could regulate N-cadherin and cadherin-11 expression distinctly by activating of transcription factors TWIST and c-FOS/c-JUN, respectively. Furthermore, I have demonstrated that GnRH I and GnRH II are capable of increasing MMP-2 and MMP-9 expression in EVT cells via up-regulation of the transcription factor RUNX2. Specific inhibition of TWIST/N-cadherin, c-FOS/c-JUN/cadherin-11 and RUNX2/MMP-2/MMP-9 in EVT cells attenuates both basal and GnRH-induced trophoblast invasion. Additionally, both forms of GnRH stimulate matrigel-mediated capillary-like network formation of trophoblastic cells and this phenomenon is also mediated by GnRH induced N-cadherin, cadherin-11, MMP-2 and MMP-9 expression. Collectively, our observations strengthen our hypothesis that GnRH is an important regulator of EVT cell behavior during implantation and placentation. These studies systemically described the underlying molecular mechanisms involved in GnRH induced adhesion molecule and proteolysis reprogramming.

View record

HOX genes are transcription factors which act as morphological regulators during development and in adult tissues. HOX paralogs often function cooperatively or synergistically in a specific tissue both in development and carcinogenesis. We have previously shown that HOXA4 enhances cell-cell adhesion and inhibits cell migration in epithelial ovarian cancer (EOC) cells. HOXB4, a close paralog of HOXA4, is more highly expressed in EOC cells than ovarian surface epithelium (OSE), a putative source of EOC. We therefore hypothesized that HOXB4 might have functions similar or complementary to those of HOXA4 in EOC.In our present study, we found variable expression levels of HOXB4 in EOC cell lines. Treatment with a DNA hypo-methylation reagent (5-Aza-dC) significantly induced HOXB4 expression in EOC cells. Functionally, neither siRNA-mediated down-regulation nor retroviral overexpression of HOXB4 affected EOC cell proliferation, as assessed by MTT and cell counting assays. Down-regulation of endogenous HOXB4 significantly enhanced trans-well Matrigel invasion of EOC cells and immortalized fallopian tube epithelial cells (OE-E6/E7), whereas forced-expression of HOXB4 suppressed EOC cell invasiveness. Gene expression microarray analysis indicated that expression of the extracellular matrix receptor CD44 was positively regulated by HOXB4, and down-regulation of CD44 reversed the suppression of cell invasion in HOXB4 over-expressing EOC cells. Immunohistochemical analysis of HOXB4 expression was performed on two ovarian carcinoma tissue microarrays. Strong nuclear immuno-reactivity for HOXB4 was detected in the fimbrial epithelium of fallopian tube, a putative source of high-grade serous carcinoma (HGSC), whereas negative staining was observed in OSE, moreiiithan 85% of HGSCs and all mucinous ovarian carcinomas in both arrays. Indeed, HOXB4 staining was significantly correlated with subtype (p
View record

Exerting a broad range of biological effects in various tissues, bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor β superfamily. Accumulating evidence indicates that ovarian BMPs are critical regulators of ovarian function and play important roles in the female reproductive system. Mutual communication between oocytes and the surrounding somatic cells is mandatory for normal follicle development, as these locally expressed growth factors function mainly as paracrine/autocrine effectors in granulosa cells. Previous studies have shown that oocytes may secrete an anti-luteinization factor and theca cells may have luteinization-inhibiting activities. We therefore hypothesized that during the late follicular stage, oocyte-derived and theca cell-derived BMPs may prevent premature luteinization by down-regulating several ovulation-related genes. This study aims to investigate the anti-luteinization effects of oocyte- and theca cell-derived BMPs in human granulosa cells. An established immortalized human granulosa cell line (SVOG), granulosa cell tumor cell line (KGN) and primary granulosa-lutein cells were used as study models. Several parameters of luteinization were investigated following exposure to recombinant human BMP4, BMP7 or BMP15. Dual pharmacological and siRNA-based approaches were used to examine the underlying mechanisms and verify the specificity of the effects. Our studies demonstrate that oocyte-derived BMP15 functions as a paracrine factor to decrease progesterone production and that theca cell-derived BMP4 and BMP7 down-regulate PTX3 expression in human granulosa cells. In addition, all three growth factors decrease intercellular communication by down-regulating Cx43-coupled gap junction formation. Furthermore, both BMP4 and BMP7 increase the production of a luteinization inhibitor activin A by up-regulating the expression of inhibin βA subunit and furin. Interestingly, different BMPs act through differential subsets of type I receptor-driven SMAD-dependent pathways. These results suggest that oocytes and theca cells may play important roles in the prevention of premature luteinization, a process that is essential for normal ovarian function and fertility. Our findings provide important insight into ovarian biology, and may lead to the development of novel therapeutic tools for fertility regulation.

View record

E-cadherin is a membrane glycoprotein located at cell adherens junctions. A switch from E-cadherin to N-cadherin expression has been considered a hallmark of the epithelial-mesenchymal transition (EMT), which is primarily due to the up-regulation of the transcription factors Snail, Slug, Twist and ZEB1. Epithelial ovarian cancer cells with low E-cadherin expression are more invasive, and the absence of E-cadherin expression in ovarian cancer is associated with poor prognosis and survival. Serous borderline ovarian tumors (SBOT) are slow-growing, non-invasive ovarian epithelial neoplasms. SBOT are considered distinct entities that give rise to invasive low-grade serous carcinomas (LGSC), which have a relatively poor prognosis and are unrelated to high-grade serous carcinomas (HGSC). The mechanisms underlying the progression of non-invasive SBOT to invasive LGSC are not understood. We have established short-term cultures of SBOT cells from tumor biopsies and have shown that inactivation of p53, Rb and/or PP2A by the SV40 large T (LT) and small T (ST) antigens allows SBOT cells to acquire characteristics associated with neoplastic progression, including increased cell motility, invasion and EMT. However, the overexpression of N-cadherin does not induce cell invasion in SBOT cells. In this study, using loss- and gain-of-function approaches, we show that p53 acts as a tumor suppressor in the regulation of SBOT and LGSC cell invasion by regulating E-cadherin expression through PI3K/Akt-mediated transcriptional and epigenetic machineries. In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) and transforming growth factor-beta (TGF-β) induce cell invasion by activating the EMT. However, the effects of EGF and TGF-β on SBOT and LGSC cell invasion remain unknown. We show that EGF induces SBOT cell invasion by activating the EMT. In addition, our results suggest that there are EMT-independent mechanisms that mediate EGF-induced LGSC cell invasion. Interestingly, we show a dual function for TGF-β in which it induces invasion in SBOT cells by activating the EMT and promotes apoptosis in LGSC cells. Overall, this study demonstrates that the loss of E-cadherin expression in SBOT may play an important role in the transition to invasive LGSC.

View record

E-cadherin is a cell-cell adhesion protein and tumor suppressor that is silenced in many malignancies. However, the role of E-cadherin in ovarian cancer progression is still controversial. In an attempt to define the regulation of E-cadherin in ovarian cancer, we found that local growth factors, FGF2 and IGF1, suppress E-cadherin expression in ovarian cancer cells. To elucidate the role of E-cadherin in ovarian cancer progression, we found that stable knockdown of E-cadherin significantly enhances, whereas overexpression of E-cadherin reduces tumor cell growth and invasion. Loss of E-cadherin results in constitutive activation of phosphoinositide 3-kinase (PI3K)/Akt signaling by inhibition of PTEN transcription through downregulation of Egr1. In addition, immunofluorescence microscopy and TCF promoter/luciferase reporter assays showed that E-cadherin loss was associated with enhanced nuclear beta-catenin signaling. Constitutive activation of PI3K/Akt signaling reinforced nuclear beta-catenin signaling by inactivating glycogen synthase kinase-3beta indicating cross talk between the PI3K/Akt and beta-catenin signaling pathways. Furthermore, we found that E-cadherin negatively regulates tumor cell growth, in part, by positively regulating PTEN expression via beta-catenin-mediated Egr1 regulation, thus influencing PI3K/Akt signaling. Finally, the constitutive activation of PI3K/Akt signaling activates its downstream mammalian target of rapamycin (mTOR) signaling pathway. The pharmacological inhibition of PI3K and mTOR suggests that PI3K/Akt/mTOR is required for E-cadherin-depletion-induced tumor cell motility. Moreover, loss of E-cadherin induces tumor cell invasion, in part, by activation of Rho GTPase, Cdc42 and Rac1. In summary, endogenous E-cadherin inhibits PI3K/Akt signaling and Rho GTPase activation. Thus, the loss of E-cadherin itself may contribute to dysregulate PI3K/Akt signaling and Rho GTPase activation to promote tumor proliferation and invasion in human ovarian cancer cells.

View record

Aberrant epidermal growth factor receptor (EGFR) activity contributes to the development of epithelial ovarian cancer (EOC), a common and lethal female malignancy. Elucidating the regulation of EGFR function will improve treatments for EOC and the survival of patients. This study aims to elucidate the role of Sprouty (SPRY) proteins, which are EGFR regulators, in EOC. The investigation began with demonstrating the downregulation of mRNA levels of two SPRY members, SPRY2 and SPRY4, in EOC tissues and/or cell lines. Deletion of the SPRY2 gene was found to cause reduced SPRY2 mRNA. Loss of the SPRY2 gene and thus its expression are particularly common in high-grade serous tumors, suggesting that SPRY2 deficiency may be involved in the pathogenesis of this prevailing subtype of EOC. The regulatory mechanisms of SPRY level are incompletely understood. The EGFR ligand EGF strongly upregulates SPRY4 protein level primarily through the ERK pathway. In addition, the PI3K/AKT pathway and hypoxia-inducible factor-1 (HIF-1α) have been shown to be involved in SPRY4 regulation, allowing the possibility that SPRY4 is regulated by micro-environmental (hypoxia) and genetic (PI3K mutation) abnormalities. Functionally, SPRY2 and SPRY4 counteract various aspects of EGFR activity and generally have tumor suppressor functions. First, in contrast to the EGFR, SPRY2 and SPRY4 prevent loss of cell adhesion by E-cadherin and therefore suppress cancer cell invasion. Second, SPRY4 inhibits PI3K/AKT signalling activated by EGF, as AKT activation is enhanced in the absence of SPRY4. Finally, the HIF-1α oncogene has been identified as a novel SPRY4 target. In ovarian cancer cell lines, SPRY4 suppresses the basal and EGF-stimulated expression of HIF-1α. The negative effects of SPRY4 on HIF-1α are also reflected by modulation of HIF-1 activity and target gene expression. SPRY4 has also been shown to destabilise HIF-1α protein, independent of the classic HIF-1α degradation pathway. The current study investigated the expression, regulation and function of SPRY in ovarian cancer. Understanding the tumor suppressor role of SPRY will not only enhance our knowledge about the pathophysiology of ovarian cancer but also identifies a possible therapeutic intervention against this lethal malignancy.

View record

It is increasingly apparent that GnRH-II acts as an autocrine/paracrine regulator in non-pituitary tissues, in addition to its role in the regulation of gonadotropin synthesis, and is an important player in cancer cell biology. High levels of GnRH-II and GnRHR in malignant ovarian tumors as compared with benign ovarian tissues underlies the importance of understanding GnRH-II function in ovarian cancer. In an attempt to define the regulation of GnRH-II in these tissues, we found that a cyclic-AMP responsive element (CRE) is critical for GnRH-II promoter transcription. In this scenario, the transcription factors p-CREB, C/EBPβ and CBP are recruited to this region in a temporarily-defined manner in response to cAMP/PKA signaling, thereby enhancing GnRH-II transcription and increasing GnRH-II mRNA levels in cancer cells of reproductive tissues. We also verified that EGF/EGFR-activated p-CREB/C/EBPβ interactions target the CRE region within GnRH-II promoter to enhance GnRH-II production in ovarian cancer. Importantly, EGF-stimulated GnRH-II expression constitutes a specific autocrine loop that contributes to ovarian cancer motility. In an attempt to define the downstream mechanisms responsible for this autocrine action of GnRH-II, we identified that MMP-2 and MT1-MMP are critical mediators of GnRH-II-enhanced ovarian cancer cell invasion. Specifically, GnRH-II acts via GnRHR to up-regulate 37kDa laminin receptor precusor expression which dimerizes to yield the non-integrin 67kDa laminin receptor (67LR). This leads to an increase interaction between 67LR and laminin in the extracellular matrix, and increases MMP-2 production in ovarian cancer cells. In parellel, GnRH-II/GnRHR-activated PI3K/Akt/β-catenin signaling to up-regulate MT1-MMP production which is known to be an activator for MMP-2 zymogen. Lastly, we attempted to define the implication of 67LR in high grade serous ovarian carcinoma due to its critical role in enhancing ovarian cancer progression in our in vitro model. However 67LR did not correlate with the overall survival of stage III & IV high-grade serous ovarian cancer patients. Overall, this study contributes to our understanding of the impact of GnRH-II/GnRHR in ovarian cancer invasive potential and provides insights into the progression of ovarian cancer and the development of new therapeutic strategies.

View record

Nuclear receptors including estrogen receptors (ERs) and progesterone receptors (PRs) areactivated by their ligands as well as by signaling pathways in response to peptide hormones and growth factors. In gonadotrophs, gonadotropin releasing hormones (GnRHs) act via the GnRH receptor (GnRHR). Both GnRH-I and GnRH-II activate an estrogen response element (ERE)-driven luciferase reporter gene in LβT2 mouse pituitary cells, and GnRH-I is more potent in this regard. The ERα is phosphorylated at Ser¹¹⁸ in the nucleus and at Ser¹⁶⁷ in both nucleus and cytoplasm after GnRI-I treatments, and this coincides with increased ERct binding to its co-activator, the P300/CBP-associated factor (PCAF). Most importantly, both GnRH subtypes robustly up-regulate expression of the immediate early response gene, Fosb, while co-treatment with ERα siRNA or PCAF siRNA attenuates this effect. This appears to occur at the transcriptional level because co-recruitment of ERα and PCAF to an ERE within the endogenous Fosb promoter is increased by GnRH treatments, as shown by chromatinimmunoprecipitation assays. Furthermore, cross-talk between GnRH-I and PR accentuatesgonadotropin production. GnRH-I activates a progesterone response element (PRE)-drivenluciferase reporter gene and gonadotropin a subunit (Gsua) gene expression in two mousegonadotroph cell lines, αT3-1 and LβT2. Up-regulation of the PRE-luciferase reporter geneby GnRH-I is attenuated by pre-treatment with protein kinase A (H89) and protein kinase C(GF109203X) inhibitors, while only GF109203X inhibits GnRH-1-induced Gsua mRNA levels. In both cell lines within the same time-frame, knockdown of PR levels by siRNA reduces GnRH-I activation of Gsua mRNA levels by approximately 40%. Both GnRH-I and GnRH-II also increase mouse Gnrhr-luciferase promoter activity and this is significantlyreduced by knockdown of PR in LβT2 cells. We conclude that the effects of GnRH-I on Fosband Gsua expression, as well as mouse Gnrhr promoter activity in mouse gonadotrophs aremediated by ligand-independent activation of ERα and PR. These ligand-independent effectsof GnRHs on steroid hormone receptor function may influence the magnitude of changes inthe expression of specific genes in the pituitary during the mouse estrous cycle, which in thiscontext may serve as a model in the human menstrual cycle.

View record

Gonadotropin-releasing hormone (GnRH) is the key regulator of hypothalamus-pituitary gonadal function. In the anterior pituitary, GnRH stimulates the synthesis and the release of the gonadotropins. GnRH and its receptors have also been found in extra pituitary tissues such as the ovary, breast, and placenta. GnRH has been reported to have antiproliferative effects on granulosa cells from rats, pigs, and humans; however, the underlying mechanisms are unclear.We have successfully established two immortalized human granulosa cell lines by SV40 large T antigen transfection. In the process, we have characterized multiple cellular and molecular features of these immortalized cells and compared these characteristics with those of primary cultured granulosa cells. The features examined include the expression patterns of cytoskeletal proteins (vimentin, cytokeratin 5/6 and desmin), adhesion molecules (Connexin 43 and E-cadherin), steroidogenic enzymes (StAR, p450scc and 3β-HSD, steroid receptors (ER-α,-β and PR-α,-β and gonadotropin receptors (LH and FSH), as well as cell morphology, anchorage independent growth, growth potential, and progesterone production.In addition to its well-established actions on the pituitary–gonadal axis, GnRH-I or II suppressed proliferation and directly induced apoptosis in human granulosa cells. The intracellular signals of apoptosis induced by GnRH I or II, in turn, are mediated by activation of the proteolyic caspase cascade, involving caspase 8,3 and 7. In addition, FSH protected human granulosa cells from undergoing apoptosis induced by GnRH I or II.GnRH I or II also significantly attenuated the stimulatory effect of IGF I on granulosa cell proliferation. Correspondingly, IGF I decreased while GnRH I or II increased cleaved caspases 3 in our granulosa cell line; furthermore, IGF I significantly attenuated the expression of cleaved caspase 3 induced by GnRH I or II. These interactions of IGF I and GnRH I or II in regulating granulosa cell proliferation or apoptosis are mediated through Akt in human granulosa cells.In conclusion, we have established two granulosa cell lines and performed integrated investigations of multiple characteristics to elucidate more clearly the characteristicsof human ovarian granulosa cells. Additionally, the present study demonstrated that GnRH I or II mediates antiproliferate effects via stimulation of granulosa cell apoptosis and inhibition of IGF I activities through Akt signaling pathway in human granulosa cells.

View record

Activin A (homodimer of inhibin βA-subunit) is known to increase inhibin βB-subunit and inhibin B (heterodimer of inhibin α- and βB-subunit) levels and decrease progesterone accumulation in human granulosa cells. Growth differentiation factor 9 (GDF9) is a potent paracrine regulator of ovarian function, but its overall effects, particularly relating to activin A actions, are unknown.We examined the potential crosstalk between activin A and GDF9 in primary cultures of human granulosa-lutein (hGL) cells. Pretreatment of hGL cells with GDF9 for 24 h resulted in an increased expression of activin receptors and Smad2/3, and decreased inhibitory Smad7 activity. These effects were attenuated by BMP type II receptor ectodomain (BMPR2 ECD), a GDF9 antagonist. These GDF9-induced changes, in turn, increased the cellular response to activin A stimulation and resulted in significantly greater production of βB-subunit mRNA and inhibin B compared to activin A treatment alone.Interestingly, endogenous GDF9 mRNA and protein were detected in hGL cells. Reduction of endogenous GDF9 by GDF9 siRNA resulted in decreased levels of activin receptors and Smad2/3/4, but increased expression of Smad7. Consequently, GDF9 siRNA treatment significantly attenuated the stimulation of activin A on βB-subunit mRNA and inhibin B levels.Additionally, GDF9 suppressed the expression of follistatin (FST) and follistatin-like 3 (FSTL3), which are extracellular inhibitors of activin A. These effects were attenuated by BMPR2 ECD and GDF9 siRNA. Treatment with FST or FSTL3 siRNA augmented activin A-induced βB-subunit mRNA levels. Conversely, GDF9 enhancement of activin A-induced βB-subunit mRNA was attenuated by FST.Activin A decreased expression of StAR but not P450scc and 3βHSD, this effect lead to reduced basal and FSH-induced progesterone accumulation. GDF9 reversed these effects of activin A on StAR and progesterone; these GDF9 effects were attenuated by inhibin α-subunit siRNA.Together, these findings support a novel hypothesis that GDF9 exerts both paracrine and autocrine control of key components in the activin receptor-signaling pathway and the extracellular inhibition of activin A in hGL cells. As a result, GDF9 may serve to enhance activin A-induced accumulation of inhibin B, which in turn acts to reverse activin A suppression of progesterone accumulation during granulosa cell luteinization.

View record

Benign ovarian tumors and majority of epithelial ovarian cancers possess steroid receptors including estrogen receptors (ERs). However, the estrogen-ER signaling in ovarian carcinomas is not completely understood. Tumorigenesis is a multiple-step process involving dysregulated cell growth and metastasis. Tumor cells acquire the capacity of migration and invasion by temporal phenotypical and genotypical changes termed epithelial-mesenchymal transition (EMT). Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In this study, I have focused on the role of 17β-estradiol (E2) in ovarian tumorigenesis. EMT related genes including E-cadherin, Snail, Slug, and Twist were examined. E2 treatment led to clear morphological changes and an enhanced cell migratory propensity. These morphologic and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin was strikingly suppressed, whereas EMT-associated transcription factors Snail and Slug were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of the endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated control in E-cadherin. In addition, the E2-induced cell migration was neutralized by Snail and Slug siRNAs, implying that both transcription factors are indispensable for the pro-metastatic actions of E2. Importantly, by using selective ER agonists as well as over-expression and siRNA approaches, it was identified that E2 triggered the metastatic behaviors exclusively through an ER⍺-dependent pathway. In contrast, overexpression of ERβ opposed the phenotypic changes and down-regulation of E-cadherin induced by ER⍺. In addition, microarray analysis was performed to characterize more putative downstream mediators of E2. Expression levels of 486 genes were found to be altered by at least 50% upon E2 treatment, and included several genes involved in oncogenesis, cell cycle control, apoptosis, signal transduction and the gene expression machinery. These candidate genes may be valuable for better delineating the ER pathways and functions. In summary, this study provides compelling arguments that estrogen can potentiate tumor progression by EMT induction, and highlight the crucial role of ER⍺ in ovarian tumorigenesis.

View record