-
[show abstract]
[hide abstract]
ABSTRACT: Overexpression of HER2 is correlated with a poor prognosis in many types of human cancers. Due to the interaction between HER2 and other ErbB receptors, HER2 is implicated in the EGF family of ligands-regulated tumor progression. In ovarian cancer, although the relationships between HER2 amplification and patient prognosis remain controversial, the underlying molecular mechanisms of HER2-mediated tumor progression are not fully understood. Our previous studies demonstrated that EGF induces ovarian cancer cell invasion by down-regulating E-cadherin expression through the up-regulation of its transcriptional repressors, Snail and Slug. It has been shown that overexpression of HER2 down-regulates E-cadherin expression in human mammary epithelial cells. However, whether HER2 mediates EGF-induced down-regulation of E-cadherin remains unknown. In this study, we examined the potential role of HER2 in EGF-induced down-regulation of E-cadherin and increased cell invasion. We show that EGF treatment induces the interaction of EGFR with HER2 and increases the activation of HER2 in human ovarian cancer cells; we also show that these effects are diminished by knockdown of EGFR. Importantly, treatment with HER2-specific tyrosine kinase inhibitor, AG825, and HER2 siRNA diminished the up-regulation of Snail and Slug as well as the down-regulation of E-cadherin by EGF. Finally, we also show that EGF-induced cell invasion was attenuated by treatment with HER2 siRNA. This study demonstrates an important role for HER2 in mediating the effects of EGF on Snail, Slug and E-cadherin expression as well as invasiveness in human ovarian cancer cells.
Biochemical and Biophysical Research Communications 03/2013; · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Context:Connexin43 (Cx43)-coupled gap junctions in granulosa cells play important roles in follicular and oocyte development and may be modulated by theca cell-derived bone morphogenic protein (BMP) 4 and BMP7.Objective:The aim of this study was to examine the effects of BMP4 and BMP7 on Cx43 expression in human granulosa cells and its potential mediation by the Smad-dependent pathway.Design:An immortalized human granulosa (SVOG) cell was used to investigate Cx43 expression and gap junction intercellular communication (GJIC) activity after exposure to BMP4 and BMP7. A BMP type I inhibitor, dorsomorphin, and small interfering RNAs targeting Smad4 were used to verify the specificity of the effects.Setting:The study was conducted in an academic center.Main Outcome Measures:Extracts were prepared from cultured cells, the Cx43 mRNA levels were examined using RT-quantitative real-time PCR, and the levels of Cx43 protein and phosphorylated Smad1/5/8 were assayed using Western blot analyses. GJIC activities between SVOG cells were evaluated using a scrape loading and dye transfer assay.Results:Treatment with BMP4 and BMP7 significantly decreased Cx43 mRNA and protein levels, as well as GJIC activities. These suppressive effects were attenuated by cotreatment with the BMP type I receptor inhibitor dorsomorphin. Furthermore, Smad4 knockdown reversed the effects of BMP4 and BMP7 on Cx43 expression.Conclusion:Theca cell-derived BMP4 and BMP7 down-regulate Cx43 expression and decrease GJIC activity in human granulosa cells. Our findings indicate that this biological effect is most likely mediated by a Smad-dependent pathway.
The Journal of clinical endocrinology and metabolism 02/2013; · 6.50 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The etiology of granulosa cell tumors (GCTs) is largely unknown. The primary mode of treatment is surgical, however not all women are cured by surgery alone. Thus, it is important to develop improved treatments through a greater understanding of the molecular mechanisms that contribute to this disease. Recently, it has been shown that a FOXL2 402C>G (C134W) mutation is present in 97% of human adult-type GCTs, suggesting an important role for this mutation in the development of GCTs. We have shown previously that gonadotropin-releasing hormone (GnRH)-I and -II induce apoptosis in cultured normal human granulosa cells. Moreover, it has been reported that FOXL2 can bind to the promoter of the mouse GnRH receptor gene and regulate its transcription. Thus, we hypothesized that C134W mutant FOXL2 could modulate the pro-apoptotic effects of GnRH via aberrant regulation of GnRH receptor levels. Using KGN cells, a human GCT-derived cell line which harbors the FOXL2 402C>G mutation, we show that treatment with GnRH-I and -II induces cell apoptosis, and that small interfering RNA-mediated depletion of GnRH receptor abolishes these effects. Overexpression of wild-type FOXL2 increases both mRNA and protein levels of GnRH receptor and consequently enhances GnRH-induced apoptosis. Importantly, neither the expression levels of GnRH receptor nor GnRH-induced apoptosis were affected by overexpression of the C134W mutant FOXL2. Interestingly, knockdown of endogenous FOXL2 down-regulates GnRHR expression in normal human granulosa cells with wild-type FOXL2, but not in KGN cells. These results suggest that the FOXL2 402C>G mutation may contribute to the development of human adult-type GCTs by reducing the expression of GnRH receptor, thus conferring resistance to GnRH-induced cell apoptosis.
PLoS ONE 01/2013; 8(1):e55099. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hypoxia-inducible factor 1α (HIF-1α) regulates the transcription of a number of genes under hypoxia and other extracellular stimulations. It has been shown that E-cadherin is down-regulated by epidermal growth factor receptor (EGF) stimulation, and that cells with low E-cadherin expression are more invasive. Our recent study demonstrated a novel mechanism by which EGF down-regulates E-cadherin expression through production of hydrogen peroxide (H(2)O(2)) and the activation of p38 MAPK in human ovarian cancer cells. In this study, we were interested in examining the potential role of HIF-1 in cell invasion under normoxic conditions, specifically when cells are treated with EGF, which is known to down-regulate E-cadherin and increase invasiveness. We show that EGF treatment induces HIF-1α expression in two human ovarian cancer cell lines (SKOV3 and OVCAR5), and that this effect is diminished by treatment with a membrane-permeable H(2)O(2) scavenger, PEG-catalase. However, the induction of HIF-1α by EGF did not require the activation of p38 MAPK. Treatment with siRNA targeting HIF-1α reduces both basal and EGF-induced HIF-1α levels. Importantly, treatment with HIF-1α siRNA diminishes the up-regulation of Snail and Slug as well as the down-regulation of E-cadherin by EGF. The involvement of HIF-1α in the down-regulation of E-cadherin was confirmed with cobalt chloride (CoCl(2)), a hypoxia-mimetic reagent. Finally, we also show that EGF-induced cell invasion is attenuated by treatment with HIF-1α siRNA. This study demonstrates an important role for HIF-1α in mediating the effects of EGF on Snail, Slug and E-cadherin expression as well as invasiveness in human ovarian cancer cells.
Cancer letters 11/2012; · 4.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The loss of E-cadherin enhances cell invasiveness. There is increasing evidence that high-grade serous ovarian cancer may arise from oviductal epithelial cells rather than the ovarian surface epithelium. Despite the controversy over the cellular origins of this disease, the roles of epidermal growth factor (EGF) in human oviductal epithelial cells are largely unknown.
We examined whether EGF could induce oviductal epithelial cell invasion by its down-regulation of E-cadherin.
Matrigel-coated transwells were used for the invasion assay. Small interfering RNA was used to knock down the expression of EGF receptor (EGFR). Specific mRNA and protein levels were examined by quantitative RT-PCR and Western blot, respectively.
The expression of Pax8 confirmed the secretory type of the cultured human oviductal epithelial cell line OE-E6/E7. EGFR was expressed in OE-E6/E7 cells, and treatment with EGF down-regulated E-cadherin expression. The effect of EGF on the down-regulation of E-cadherin was abolished by small interfering RNA-mediated depletion of EGFR. EGF treatment led to the activation of ERK1/2, p38, and Akt. Snail and Slug are transcriptional repressors of E-cadherin. Interestingly, our results show that EGF induced Slug but not Snail expression. Moreover, the inhibition of EGF-induced ERK1/2, p38, and Akt activation by pharmacological inhibitors attenuated EGF-induced Slug expression and the down-regulation of E-cadherin, as well as subsequent cell invasion.
EGF induces human oviductal epithelial cell invasion through the activation of ERK1/2, p38, and Akt, the up-regulation of Slug, and the down-regulation of E-cadherin.
The Journal of clinical endocrinology and metabolism 05/2012; 97(8):E1380-9. · 6.50 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) induces cell invasion by activating an epithelial-mesenchymal transition (EMT). However, the effect of EGF on serous borderline ovarian tumors (SBOT) and low-grade serous carcinomas (LGC) cell invasion remains unknown. Here, we show that EGF receptor (EGFR) was expressed, that EGF treatment increased cell migration and invasion in two cultured SBOT cell lines, SBOT3.1 and SV40 large T antigen-infected SBOT cells (SBOT4-LT), and in two cultured LGC cell lines, MPSC1 and SV40 LT/ST-immortalized LGC cells (ILGC). However, EGF induced down-regulation of E-cadherin and concurrent up-regulation of N-cadherin in SBOT cells but not in LGC cells. In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment. Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt. The MEK1 inhibitor PD98059 diminished the EGF-induced cadherin switch and the up-regulation of Snail, Slug and ZEB1 and the EGF-mediated increase in SBOT cell migration and invasion. The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1. This study demonstrates that EGF induces SBOT cell migration and invasion by activating EMT, which involves the activation of the ERK1/2 and PI3K/Akt pathways and, subsequently, Snail, Slug and ZEB1 expression. Moreover, our results suggest that there are EMT-independent mechanisms that mediate the EGF-induced LGC cell migration and invasion.
PLoS ONE 01/2012; 7(3):e34071. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Apoptosis in ovarian surface epithelial (OSE) cells is induced by transforming growth factor-beta (TGF-β). However, high-grade serous ovarian carcinomas (HGC) are refractory to the inhibitory functions of TGF-β; their invasiveness is up-regulated by TGF-β through epithelial-mesenchymal transition (EMT) activation. Serous borderline ovarian tumors (SBOT) have been recognized as distinct entities that give rise to invasive low-grade serous carcinomas (LGC), which have a relatively poor prognosis and are unrelated to HGC. While it is not fully understood how TGF-β plays disparate roles in OSE cells and its malignant derivative HGC, its role in SBOT and LGC remains unknown. Here we demonstrate the effects of TGF-β on cultured SBOT3.1 and LGC-derived MPSC1 cells, which express TGF-β type I and type II receptors. TGF-β treatment induced the invasiveness of SBOT3.1 cells but reduced the invasiveness of MPSC1 cells. The analysis of apoptosis, which was assessed by cleaved caspase-3 and trypan blue exclusion assay, revealed TGF-β-induced apoptosis in MPSC1, but not SBOT3.1 cells. The pro-apoptotic effect of TGF-β on LGC cells was confirmed in another immortalized LGC cell line ILGC. TGF-β treatment led to the activation of Smad3 but not Smad2. The specific TβRI inhibitor SB431542 and TβRI siRNA abolished the SBOT3.1 invasion induced by TGF-β, and it prevented TGF-β-induced apoptosis in MPSC1 cells. In SBOT3.1 cells, TGF-β down-regulated E-cadherin and concurrently up-regulated N-cadherin. TGF-β up-regulated the expression of the transcriptional repressors of E-cadherin, Snail, Slug, Twist and ZEB1. In contrast, co-treatment with SB431542 and TβRI depletion by siRNA abolished the effects of TGF-β on the relative cadherin expression levels and that of Snail, Slug, Twist and ZEB1 as well. This study demonstrates dual TGF-β functions: the induction of SBOT cell invasion by EMT activation and apoptosis promotion in LGC cells.
PLoS ONE 01/2012; 7(8):e42436. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A major function of the p53 tumor suppressor is the regulation of the cell cycle and apoptosis. In addition to its well-documented functions in malignant cancer cells, p53 can also regulate cell migration and invasion, which contribute to metastasis. Growth differentiation factor-15 (GDF-15), a member of the TGF-β superfamily, has been shown to be a downstream target of p53 and is associated with diverse human diseases and cancer progression. In this study, we examined the potential role of GDF-15 in p53-regulated cancer cell motility. We show that overexpression of wild-type p53 in two highly invasive p53-null human cancer cell lines, SKOV3 and PC3, attenuated cell migration and the movement through Matrigel. Using wild-type p53 and DNA-binding-deficient p53 mutants, we found that the transcriptional activity of p53 is required in the induction of GDF-15 expression. Cell movement through uncoated and Matrigel-coated transwell decreased in response to treatment with recombinant GDF-15, whereas the cell proliferation was not affected by GDF-15 treatment. Moreover, the induction of GDF-15 expression and secretion by p53 and the reduction in cell movement through Matrigel were diminished by treatment with GDF-15 small interfering RNA. This study demonstrates a mechanism by which p53 attenuates cancer cell motility through GDF-15 expression. In addition, our results indicate that GDF-15 mediates the functions of p53 by autocrine/paracrine action.
Endocrinology 05/2011; 152(8):2987-95. · 4.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The extracellular matrix (ECM) has been shown to induce the epithelial-mesenchymal transition (EMT), which is characterized by the loss of E-cadherin. However, little is known about the mechanisms by which collagen I, the most abundant component of ECM, induces this process. Here, we show that E-cadherin was down-regulated in response to collagen I in SKOV3 ovarian cancer cells and PC3 prostate cancer cells. Furthermore, collagen I induced the up-regulation of PIK3CA, which in turn contributed to the collagen I-induced down-regulation of E-cadherin by up-regulating its transcriptional repressors, Snail and Slug. Our data demonstrate that PIK3CA is a mediator of collagen I-induced down-regulation of E-cadherin.
Cancer letters 03/2011; 304(2):107-16. · 4.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The growth hormone-releasing hormone (GHRH) antagonists have been shown to inhibit growth of human cancer cells, but the underlying molecular mechanisms and their actions have not been fully investigated. In this study, we first showed that GHRH-R splice variant 1 (SV1) was expressed in two human endometrial cancer cell lines, Ishikawa and ECC-1. By using MTT assay, immunoblotting for cleaved caspase-3 and TUNEL assays, we found that cell growth inhibition and apoptosis were induced in GHRH antagonist, JMR-132-treated cells by activating PKCδ and could be inhibited by treatment with PKC inhibitor, GF109203X. In addition, activation and protein expression of p53 as well as the expression of its downstream effector, p21, were increased by JMR-132 treatment. Moreover, JMR-132-induced p53 and p21 expression were diminished by treatment with PKC inhibitor. Knockdown of endogenous p53 and p21 by siRNAs abolished the JMR-132-induced cell growth inhibition and apoptosis. This study demonstrates a novel mechanism in which GHRH antagonist-induced cell growth inhibition and apoptosis through PKCδ-mediated activation of p53/p21 in human endometrial cancer cells. These findings may suggest the feasibility of GHRH antagonists as a therapeutic approach for human cancer.
Cancer letters 12/2010; 298(1):16-25. · 4.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In ovarian cancer, it has been shown that E-cadherin is down-regulated by epidermal growth factor (EGF) receptor (EGFR) activation, and that cells with low E-cadherin expression are particularly invasive. Although it is generally believed that reactive oxygen species play important roles in intracellular signal transduction, the role of reactive oxygen species in EGF-mediated reductions in E-cadherin remains to be elucidated. In this study, we show that EGF treatment down-regulated E-cadherin by up-regulating its transcriptional repressors, Snail and Slug, in human ovarian cancer cells. Using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester staining, we found that intracellular hydrogen peroxide (H(2)O(2)) production was increased in EGF-treated cells and could be inhibited by treatment with an EGFR inhibitor, AG1478, or an H(2)O(2) scavenger, polyethylene glycol (PEG)-catalase. In addition, PEG-catalase diminished EGF-induced p38 MAPK, but not ERK1/2 or c-Jun N-terminal kinase, phosphorylation. PEG-catalase and the p38 MAPK inhibitor SB203580 abolished EGF-induced Snail, but not Slug, expression and E-cadherin down-regulation. Furthermore, the involvement of p38 MAPK in the down-regulation of E-cadherin was confirmed using specific p38alpha MAPK small interfering RNA. Finally, we also show that EGF-induced cell invasion was abolished by treatment with PEG-catalase and SB203580, as well as p38alpha MAPK small interfering RNA, and that forced expression of E-cadherin diminished intrinsic invasiveness as well as EGF-induced cell invasion. This study demonstrates a novel mechanism in which EGF down-regulates E-cadherin expression through production of H(2)O(2), activation of p38 MAPK, and up-regulation of Snail in human ovarian cancer cells.
Molecular Endocrinology 08/2010; 24(8):1569-80. · 4.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Gonadotropin-releasing hormone type II (GnRH-II) has an antiproliferative effect on human endometrial cancer cells. Apoptosis in cancer cells may play a critical role in regulating cell proliferation. However, more studies are necessary to elucidate the underlying molecular mechanisms and develop potential applications of GnRH-II. Therefore, we explored the mechanisms of GnRH-II-induced apoptosis and the effects of GnRH-II on GADD45alpha activation in human endometrial cancer cell lines. GnRH-II decreased cell viability in a dose- and time-dependent manner. Apoptosis was induced with increased terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling apoptotic cells after GnRH-II treatment. Knockdown of the endogenous GnRH-I receptor with small interfering RNA (siRNA) rescued the cells from GnRH-II-mediated cell growth inhibition and abolished the induction of apoptosis. GnRH-II activated extracellular signal-regulated kinase (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) in a time-dependent manner, and the activation was abolished by GnRH-I receptor siRNA and MAPK inhibitors. Cells pretreated with MAPK inhibitors were rescued from GnRH-II-mediated cell growth inhibition. Moreover, both inhibitors abolished GnRH-II-induced apoptosis. GnRH-II induced GADD45alpha expression, which was abolished by knockdown of endogenous GnRH-I receptors and MAPK inhibitors. GnRH-II-stimulated cell growth inhibition was rescued by knockdown of endogenous GADD45alpha with siRNA. Cells treated with GADD45alpha siRNA were refractory to GnRH-II-induced apoptosis. Thus, GnRH-II inhibits cell growth by inducing apoptosis through binding of the GnRH-I receptor, activation of the ERK1/2 and p38 MAPK pathways, and induction of GADD45alpha signaling. This finding may provide a new concept relating to the mechanism of GnRH-II-induced antiproliferation and apoptosis in endometrial cancer cells, indicating the possibility of GnRH-II as a promising therapeutic intervention for human endometrial cancer.
Cancer Research 05/2009; 69(10):4202-8. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 G(0)/G(1). In contrast to GnRH signaling in pituitary gonadotropes, the involvement of G(alpha q), 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 G(alpha i), 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.
FEBS Journal 12/2008; 275(22):5496-511. · 3.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ovarian epithelial carcinomas (OECs) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In our study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.
International Journal of Cancer 10/2008; 123(8):1761-9. · 5.44 Impact Factor
