Wai-Kin So

Fibroblast Growth Factor 2 Induces E-Cadherin Down-Regulation via PI3K/Akt/mTOR and MAPK/ERK Signaling in Ovarian Cancer Cells

Fibroblast growth factor 2 (FGF2) is produced by ovarian cancer cells and it has been suggested to play an important role in tumor progression. In this study, we report that FGF2 treatment down-regulated E-cadherin by up-regulating its transcriptional repressors, Slug and ZEB1, in human ovarian cancer cells. The pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K), mammalian target of rapamycin (mTOR), and MEK suggests that both PI3K/Akt/mTOR and MAPK/ERK signaling are required for FGF2-induced E-cadherin down-regulation. Moreover, FGF2 up-regulated Slug and ZEB1 expression via the PI3K/Akt/mTOR and MAPK/ERK signaling pathways, respectively. Finally, FGF2-induced cell invasion was abolished by the inhibition of the PI3K/Akt/mTOR and MAPK/ERK pathways, and the forced expression of E-cadherin diminished the intrinsic invasiveness of ovarian cancer cells as well as the FGF2-induced cell invasion. This study demonstrates a novel mechanism in which FGF2 down-regulates E-cadherin expression through the activation of PI3K/Akt/mTOR and MAPK/ERK signaling, and the up-regulation of Slug and ZEB1 in human ovarian cancer cells.

Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview

The hypothalamic decapeptide gonadotropin‐releasing hormone (GnRH) is well known for its role in the control of pituitary gonadotropin secretion, but the hormone and receptor are also expressed in extrapituitary tissues and tumor cells, including epithelial ovarian cancers. It is hypothesized that they may function as a local autocrine regulatory system in nonpituitary contexts. Numerous studies have demonstrated a direct antiproliferative effect on ovarian cancer cell lines of GnRH and its synthetic analogs. This effect appears to be attributable to multiple steps in the GnRH signaling cascade, such as cell cycle arrest at G0/G1. In contrast to GnRH signaling in pituitary gonadotropes, the involvement of Gαq, protein kinase C and mitogen‐activated protein kinases is less apparent in neoplastic cells. Instead, in ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin‐sensitive protein Gαi, leading to the activation of protein phosphatase, which in turn interferes with growth factor‐induced mitogenic signals. Apoptotic involvement is still controversial, although GnRH analogs have been shown to protect cancer cells from doxorubicin‐induced apoptosis. Recently, data supporting a regulatory role of GnRH analogs in ovarian cancer cell migration/invasion have started to emerge. In this minireview, we summarize the current understanding of the antiproliferative actions of GnRH analogs, as well as the recent observations of GnRH effects on ovarian cancer cell apoptosis and motogenesis. The molecular mechanisms that mediate GnRH actions and the clinical applications of GnRH analogs in ovarian cancer patients are also discussed.

Amphiregulin induces human ovarian cancer cell invasion by down-regulating E-cadherin expression

Aberrant epidermal growth factor receptor (EGFR) activation is associated with ovarian cancer progression. In this study, we report that the EGFR ligand amphiregulin (AREG) stimulates cell invasion and down‐regulates E‐cadherin expression in two human ovarian cancer cell lines, SKOV3 and OVCAR5. In addition, AREG increases the expression of transcriptional repressors of E‐cadherin including SNAIL, SLUG and ZEB1. siRNA targeting SNAIL or SLUG abolishes AREG‐induced cell invasion. Moreover, ERK1/2 and AKT pathways are involved in AREG‐induced E‐cadherin down‐regulation and cell invasion. Finally, we show that three EGFR ligands, AREG, epidermal growth factor (EGF) and transforming growth factor‐α (TGF‐α), exhibit comparable effects in down‐regulating E‐cadherin and promoting cell invasion. This study demonstrates that AREG induces ovarian cancer cell invasion by down‐regulating E‐cadherin expression.

Integrin β1 mediates epithelial growth factor-induced invasion in human ovarian cancer cells

Integrins function as cell-extracellular matrix adhesion proteins and have been implicated in tumor progression. In ovarian tumors, elevated integrin β1 expression correlates with high clinical stage and poor patient survival. In this study, we report that EGF treatment up-regulated integrin β1 mRNA and protein levels in ovarian cancer cells. Moreover, pharmacological inhibition of MEK totally abolished EGF-induced integrin β1 up-regulation and cell invasion suggesting that MAPK/ERK signaling is required for EGF-induced integrin β1 up-regulation and cell invasion. Furthermore, we found that knockdown of integrin β1 expression reduced the intrinsic invasiveness of ovarian cancer cells and the EGF-induced cell invasion. Finally, we found that overexpression of integrin β1 was sufficient to promote ovarian cancer cell invasion. This study demonstrates that integrin β1 mediates EGF-induced cell invasion in ovarian cancer.

Rapid Effect of GNRH1 on Follicle-Stimulating Hormone Beta Gene Expression in LbetaT2 Mouse Pituitary Cells Requires the Progesterone Receptor

Abstract Gonadotropin-releasing hormone (GNRH) activates the progesterone receptor (PGR) in pituitary cells and accentuates gonadotropin expression. We show that GNRH1 increases Fshb mRNA levels in LbetaT2 mouse pituitary cells within 8 h and is three times more effective than GNRH2. By contrast, GNRH1 and GNRH2 do not affect Lhb gene expression in these cells. Within the same time frame, small interfering RNA (siRNA) knockdown of the PGR in LbetaT2 cells reduced GNRH1 activation of a PGR response element (PRE)-driven luciferase reporter gene and Fshb mRNA levels by >50%. Chromatin immunoprecipitation (ChIP) assays also demonstrated that PGR loading on the PRE within the Fshb gene promoter in LbetaT2 cells occurred within 8 h after GNRH1 treatment and was lost by 24 h. While the GNRH1-induced upregulation of the PRE reporter gene and Fshb mRNA levels was attenuated by cotreatment with protein kinase A (H-89) and protein kinase C (GF109203X) inhibitors, only GF109203X inhibited PGR phosphorylation at Ser249 in LbetaT2 cells. Immunoprecipitation assays also showed a progressive increase in the interaction between the PGR and its coactivator NCOA3 that peaked at 8 h coincident with the increase in Fshb mRNA after GNRH1 treatment. The siRNA-mediated knockdown of NCOA3 in LbetaT2 cells also reduced Fshb mRNA levels after GNRH1 treatment and loading of NCOA3 on the Fshb promoter PRE in a ChIP assay. We conclude that the rapid effect of GNRH1 on Fshb expression in LbetaT2 cells is mediated by PGR phosphorylation and loading at the PRE within the Fshb promoter together with NCOA3.

Transforming growth factor-α induces human ovarian cancer cell invasion by down-regulating E-cadherin in a Snail-independent manner

Transforming growth factor-α (TGF-α), like epidermal growth factor (EGF) and amphiregulin (AREG) binds exclusively to EGF receptor (EGFR). We have previously demonstrated that EGF, AREG and TGF-α down-regulate E-cadherin and induce ovarian cancer cell invasion, though whether these ligands use the same molecular mediators remains unknown. We now show that, like EGF, TGF-α- and AREG-induced E-cadherin down-regulation involves both EGFR and HER2. However, in contrast to EGF and AREG, the transcription factor Snail is not required for TGF-α-induced E-cadherin down-regulation. This study shows that TGF-α uses common and divergent molecular mediators to regulate E-cadherin expression and cell invasion.

Gonadotropin-Releasing Hormone-I-Mediated Activation of Progesterone Receptor Contributes to Gonadotropin α-Subunit Expression in Mouse Gonadotrophs

In pituitary cells, cross talk between GnRH-I and the progesterone receptor accentuates gonadotropin production. We show that GnRH-I activates a progesterone response element (PRE)-driven luciferase reporter gene at 8 h and gonadotropin alpha-subunit (gsu alpha) gene expression at 24 h in two mouse gonadotrope cell lines, alpha T3-1 and L beta T2. In alpha T3-1 cells, progesterone had an additive effect on GnRH-I-induced PRE-luciferase reporter gene activity but not on GSU alpha mRNA levels. However, progesterone had no synergistic effect on the GnRH-I-induced expression of these genes in L beta T2 cells. Up-regulation of the PRE-luciferase reporter gene by GnRH-I was attenuated by pretreatment with protein kinase A (H89) and protein kinase C (GF109203X) inhibitors in both cell lines, whereas only GF109203X inhibited GnRH-I-induced GSU alpha mRNA levels. Most important, in both cell lines within the same time frame, knockdown of progesterone receptor levels by small interfering RNA reduced GnRH-I activation of GSU alpha mRNA levels by approximately 40%. We conclude that the effect of GnRH-I on gsu alpha expression in both alpha T3-1 and L beta T2 cells is mediated by ligand-independent activation of progesterone receptor and that this contributes to the self-priming effect of GnRH-I in the pituitary.

Temporal Recruitment of Transcription Factors at the 3′,5′-Cyclic Adenosine 5′-Monophosphate-Response Element of the Human GnRH-II Promoter

GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including alphaT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-beta coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPbeta levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPbeta occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPbeta at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2-4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPbeta, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.

Loss of Sprouty2 in human high‐grade serous ovarian carcinomas promotes EGF‐induced E‐cadherin down‐regulation and cell invasion

Sprouty (SPRY) proteins are well‐characterized factors that inhibit receptor tyrosine kinase signaling. Our Human Exonic Evidence‐Based Oligonucleotide (HEEBO) microarray results showed that the mRNA levels of SPRY2, but not of SPRY1 or SPRY4, are down‐regulated in high‐grade serous ovarian carcinoma (HGSC) tissues and epithelial ovarian cancer (EOC) cell lines. Molecular inversion probe (MIP) copy number analysis showed the deletion of the SPRY2 locus in HGSC. Overexpression of SPRY2 reduced EGF‐induced cell invasion by attenuating EGF‐induced E‐cadherin down‐regulation. Moreover, a positive correlation between SPRY2 and E‐cadherin protein levels was observed in HGSC tissues. This study reveals the loss of SPRY2 in HGSC and indicates an important tumor‐suppressive role for SPRY2 in mediating the stimulatory effect of EGF on human EOC progression.

Sprouty4 mediates amphiregulin-induced down-regulation of E-cadherin and cell invasion in human ovarian cancer cells.

Sprouty (SPRY) proteins are well-characterized factors that inhibit receptor tyrosine kinase (RTK)-mediated activation of cellular signaling pathways. The down-regulation of SPRY4 expression has been reported in human ovarian cancer. However, the specific roles and mechanisms by which SPRY4 affects ovarian cancer progression are completely unknown. Amphiregulin (AREG) binds exclusively to the epidermal growth factor receptor (EGFR) and has been considered to be a dominant autocrine/paracrine EGFR ligand in ovarian cancer. In the present study, we first examined the effects of AREG on SPRY4 expression and the possible underlying molecular mechanisms involved in this process in two human ovarian cancer cell lines. Our results demonstrated that treatment with AREG up-regulated SPRY4 expression by activating the ERK1/2 signaling pathway. In addition, we showed that small interfering RNA (siRNA)-mediated knockdown of SPRY4 attenuated the AREG-induced down-regulation of E-cadherin by inhibiting the expression of SNAIL but not SLUG. In contrast, overexpression of SPRY4 enhanced AREG-induced down-regulation of E-cadherin by increasing the expression of SNAIL. Moreover, SPRY4 knockdown attenuated AREG-induced cell migration and invasion. Overexpression of SPRY4 enhanced AREG-induced cell invasion. This study reveals that SPRY4 is involved in EGFR-mediated human ovarian cancer progression.