FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

Downregulation of ASPP2 in choriocarcinoma contributes to increased migratory potential through Src signaling pathway activation.

Gestational choriocarcinoma is a malignant tumor derived from placental trophoblast and the most aggressive member of gestational trophoblastic disease (GTD). Apoptosis-stimulating protein of p53-2 (ASPP2) is a member of ASPP family that transactivates p53 and thereby functions as a tumor suppressor. In this study, the expression profile of ASPP2 in choriocarcinoma was examined in comparison with normal placentas and hydatidiform moles, the latter being a type of GTD that carries malignant potential. Downregulation of ASPP2 messenger RNA and protein was demonstrated in choriocarcinoma by quantitative PCR and immunohistochemistry. ASPP2-transfected choriocarcinoma cells (JEG-3 and JAR) showed an increase in apoptosis and a decrease in cell migration as detected by TdT-mediated dUTP nick end labeling and wound healing assays, respectively, illustrating the complex action of ASPP2 on cell functions other than programmed cell death. Activated Src is known to be important in tumor progression. Transfection of ASPP2 but not ASPP1, another tumor-suppressive ASPP, was found to be related to subsequent decreased Src-pY416 phosphorylation, suggesting an inactivating effect of ASPP2 on Src. Moreover, this ASPP2-mediated inactivation of Src could be abolished by RNA interference with C-terminal Src kinase (Csk), a kinase that can inhibit Src activation. Our findings suggested that the ability of ASPP2 to attenuate Src activation was specific to ASPP2 in a Csk-dependent manner. Taken together, we demonstrated a loss of tumor-suppressive ASPP2 in choriocarcinoma with effects on cell migration and apoptosis. We also unveiled a possible mechanistic link between ASPP2 and Csk/Src signaling pathway, implicating the multiple cellular functions of ASPP2.

p21-activated kinase 4 regulates ovarian cancer cell proliferation, migration, and invasion and contributes to poor prognosis in patients.

Ovarian cancer is a lethal gynecological malignancy, and to improve survival, it is important to identify novel prognostic and therapeutic targets. In this study, we present a role for p21-activated kinase 4 (Pak4) in ovarian cancer progression. We show a significant association between increased expression of Pak4 and its activated form, phosphorylated (p)-Pak4 Ser(474), with metastasis of ovarian cancers, shorter overall and disease-free survival, advanced stage and high-grade cancers, serous/clear cell histological subtypes, and reduced chemosensitivity. Pak4 overexpression was also observed in ovarian cancer cell lines. Pak4 and p-Pak4 expression were detected both in the nucleus and cytoplasm of ovarian cancer cells, in vitro as well as in vivo. Stable knockdown of Pak4 in ovarian cancer cell lines led to reduced cell migration, invasion, and proliferation, along with reduced c-Src, ERK1/2, and epidermal growth factor receptor (EGFR) activation and decreased matrix metalloproteinase 2 (MMP2) expression. Conversely, Pak4 overexpression promoted ovarian cancer cell migration and invasion in a c-Src, MEK-1, MMP2, and kinase-dependent manner, and induced cell proliferation through the Pak4/c-Src/EGFR pathway that controls cyclin D1 and CDC25A expression. Stable knockdown of Pak4 also impeded tumor growth and dissemination in nude mice. This report reveals the association between Pak4 and important clinicopathologic parameters, suggesting Pak4 to be a significant prognostic marker and potential therapeutic molecular target in ovarian cancer. The implied possible cross-talk between Pak4 and EGFR suggests the potential of dual targeting of EGFR and Pak4 as a unique therapeutic approach for cancer therapy.

Downregulation of ASPP1 in gestational trophoblastic disease: correlation with hypermethylation, apoptotic activity and clinical outcome.

Gestational trophoblastic disease encompasses a spectrum of trophoblastic lesions including true neoplasms such as choriocarcinomas and the potentially malignant hydatidiform moles, which may develop persistent disease requiring chemotherapy. ASPP1, a member of apoptosis-stimulating proteins of p53 (ASPPs), is a proapoptotic protein that can stimulate apoptosis through its interaction with p53. We evaluated the promoter methylation and expression profiles of ASPP1 in different trophoblastic tissues and its in vitro functional effect on two choriocarcinoma cell lines, namely JEG-3 and JAR. Significant downregulation of ASPP1 mRNA and protein levels was demonstrated in hydatidiform moles and choriocarcinomas, when compared with normal placentas by quantitative-PCR and immunohistochemistry. The ASPP1 mRNA level was significantly correlated with its hypermethylation status, evaluated with methylation-specific PCR, in placenta and gestational trophoblastic disease samples (P=0.024). Moreover, lower ASPP1 immunoreactivity was shown in hydatidiform moles that progressed to persistent gestational trophoblastic neoplasms than in those that regressed (P=0.045). A significant correlation was also found between expression of ASPP1 and proliferative indices (assessed by Ki67 and MCM7), apoptotic activity (M30 CytoDeath antibody), p53 and caspase-8 immunoreactivities. An in vitro study showed that ectopic expression of ASPP1 could trigger apoptosis through intrinsic and extrinsic pathways as indicated by an increase in cleaved caspase-9 and Fas ligand protein expression. The latter suggests a hitherto unreported novel link between ASPP1 and the extrinsic pathway of apoptosis. Our findings suggest that downregulation of ASPP1 by hypermethylation may be involved in the pathogenesis and progress of gestational trophoblastic disease, probably through its effect on apoptosis.

Overexpression of dedicator of cytokinesis I (Dock180) in ovarian cancer correlated with aggressive phenotype and poor patient survival.

AIMS: Dedicator of cytokinesis I (Dock180) is a novel guanine nucleotide exchange factor for Rho guanosine triphosphates (GTPases) important for cell migration. The aim of this study was to evaluate the role of Dock180 in ovarian carcinogenesis. METHODS AND RESULTS: Using immunohistochemistry, real-time polymerase chain reaction and Western blotting, overexpression of Dock180 RNA and protein was demonstrated in the nucleus and cytoplasm of ovarian cancer cell lines (n = 5) and clinical samples of ovarian borderline tumours (n = 21) and invasive cancers (n = 108) when compared with ovarian epithelial cell lines (n = 3) and benign cystadenomas (n = 10) (P < 0.05). High Dock180 cytoplasmic expression in ovarian cancer (n = 108) was associated significantly with serous histological type, high-grade cancer and advanced stage (P < 0.05), as well as poor overall and disease-free survival (P = 0.004). Using multivariate progression analysis, high Dock180 cytoplasmic expression and advanced cancer stage were found to be independent prognostic factors for short overall survival and disease-free survival (P < 0.05). Exogenous expression of Dock180 by transient transfection enhanced cancer cell migration and invasion, whereas knockdown of Dock180 by an siRNA approach retarded cancer cell migration and invasion in association with down-regulation of matrix metalloproteinase 2. CONCLUSIONS: Our findings suggest that Dock180 contributes to ovarian carcinogenesis and dissemination and is a potential prognostic marker and therapeutic target.

Overexpression of proto-oncogene FBI-1 activates membrane type 1-matrix metalloproteinase in association with adverse outcome in ovarian cancers.

BACKGROUND: FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) is a member of the POK (POZ and Kruppel) family of transcription factors and play important roles in cellular differentiation and oncogenesis. Recent evidence suggests that FBI-1 is expressed at high levels in a subset of human lymphomas and some epithelial solid tumors. However, the function of FBI-1 in human ovarian cancers remains elusive. RESULTS: In this study, we investigated the role of FBI-1 in human ovarian cancers, in particularly, its function in cancer cell invasion via modulating membrane type 1-matrix metalloproteinase (MT1-MMP). Significantly higher FBI-1 protein and mRNA expression levels were demonstrated in ovarian cancers samples and cell lines compared with borderline tumors and benign cystadenomas. Increased FBI-1 mRNA expression was correlated significantly with gene amplification (P = 0.037). Moreover, higher FBI-1 expression was found in metastatic foci (P = 0.036) and malignant ascites (P = 0.021), and was significantly associated with advanced stage (P = 0.012), shorter overall survival (P = 0.032) and disease-free survival (P = 0.016). In vitro, overexpressed FBI-1 significantly enhanced cell migration and invasion both in OVCA 420 and SKOV-3 ovarian carcinoma cells, irrespective of p53 status, accompanied with elevated expression of MT1-MMP, but not MMP-2 or TIMP-2. Moreover, knockdown of MT1-MMP abolished FBI-1-mediated cell migration and invasion. Conversely, stable knockdown of FBI-1 remarkably reduced the motility of these cells with decreased expression of MT1-MMP. Promoter assay and chromatin immunoprecipitation study indicated that FBI-1 could directly interact with the promoter spanning ~600 bp of the 5'-flanking sequence of MT1-MMP and enhanced its expression in a dose-dependent manner. Furthermore, stable knockdown and ectopic expression of FBI-1 decreased and increased cell proliferation respectively in OVCA 420, but not in the p53 null SKOV-3 cells. CONCLUSIONS: Our results suggested an important role of FBI-1 in ovarian cancer cell proliferation, cell mobility, and invasiveness, and that FBI-1 can be a potential target of chemotherapy.

Differential expression and phosphorylation of Pak1 and Pak2 in ovarian cancer: effects on prognosis and cell invasion.

Ovarian cancer is a gynecological malignancy with high mortality. Therefore, the identification of novel prognostic and therapeutic targets is important. p21-activated kinases (Paks) are involved in cytoskeleton reorganization. This study investigated the clinical significance of total and phosphorylated (p) Pak1 and Pak2 as well as their functional roles in ovarian cancer. Expressions of Pak1, p-Pak1 Thr(212), Pak2 and p-Pak2 Ser(20) in ovarian normal and cancerous cell lines as well as in clinical samples of ovarian tumors were evaluated. The effects of Pak1 and Pak2 on ovarian cancer cell functions were determined. Pak1, p-Pak1 and p-Pak2 were overexpressed in ovarian cancer cell lines, and clinical samples of ovarian cancers were compared with benign ovarian lesions/inclusion cysts. Similar Pak2 expression levels were observed among normal and cancerous cell lines and clinical samples. After multiple testing correction, high Pak1 and nuclear p-Pak1 expression in ovarian cancers was significantly associated with histological type and tumor grade, respectively. Pak1 and p-Pak1 expression was associated with poor overall and disease-free survival. Pak1 was an independent prognostic factor. Knockdown of Pak1 and Pak2 in ovarian cancer cell lines reduced cell migration and invasion but did not affect cell proliferation and apoptosis. Knockdown of Pak1 also reduced p38 activation and downregulated vascular endothelial growth factor. Conversely, ectopic Pak1 overexpression enhanced ovarian cancer cell migration and invasion in a kinase-dependent manner, along with increased p38 activation. Our findings suggest that Pak1, p-Pak1 and p-Pak2 play important roles in ovarian carcinogenesis. Pak1 and p-Pak1 may be potential prognostic markers and therapeutic molecular targets in ovarian cancer.

Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation.

Aberrant activation of hedgehog (HH) pathway has been implicated in the development of human malignancies. This study aimed at investigating the role of HH molecules in human ovarian carcinogenesis. The expression profiles of HH molecules were examined in ovarian tumor samples and ovarian cancer cell lines and the in vitro effects of HH molecules on cell proliferation, apoptosis, migration, invasion and cell differentiation as well as related downstream target genes were assessed. Overexpression of Patched and Gli1 protein in ovarian cancers correlated with poor survival of the patients (P = 0.008; P = 0.004). Significantly elevated expression of Sonic hedgehog messenger RNA was observed in ovarian cancers compared with normal tissues and benign ovarian tumors and such differential expression was specific to histological types (P < 0.05). Ectopic Gli1 overexpression in ovarian cancer cells conferred increased cell proliferation, cell mobility, invasiveness and change in differentiation in association with increased expression of E-cadherin, vimentin, Bcl-2, caspases as well as beta1 integrin, membrane type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF). Treatment with 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)-cyclopamine induced cancer cell apoptosis, suppressed cell growth, mobility and invasiveness and induced cancer cell dedifferentiation with decreased expression of E-cadherin, cytokeratin 7, Snail, calretinin, vimentin, Bcl-2, caspases, beta1 integrin, MT1-MMP and VEGF. Our data suggested that abnormal HH signaling activation plays important roles in the development and progression of ovarian cancers. Gli1 expression is an independent prognostic marker. Inhibition of the HH pathway molecules might be a valid therapeutic strategy for ovarian cancers.

Aberrant activation of hedgehog signaling pathway contributes to endometrial carcinogenesis through beta-catenin.

The hedgehog and Wnt signaling pathways play important roles in human cancers with possible interaction. This study aimed at analysis and correlation of the expression of Gli1, a transcriptional factor and target gene of hedgehog signaling pathway, with clinicopathological parameters and expression of beta-catenin, an important member of the Wnt pathway, in normal, hyperplastic and malignant endometrium. Immunohistochemical study on 15 normal endometrium, 14 simple and complex hyperplasia without atypia, 37 atypical complex hyperplasia and 80 endometrial cancers showed significant Gli1 overexpression and beta-catenin nuclear immunoreactivity in endometrial cancers and atypical endometrial hyperplasia when compared with normal endometrium (P<0.05). Overexpression of Gli1 in endometrial cancers correlated with well-differentiated histological grade (P<0.001), non-myometrial invasion (P=0.004) and superficial myometrial invasion (P=0.041). beta-Catenin nuclear immunoreactivity was also associated with well-differentiated histology (P=0.013). Gli1 overexpression positively correlated with beta-catenin nuclear immunoreactivity in atypical complex hyperplasia (P=0.013) and endometrial carcinoma (P=0.017). Similar Gli1 and beta-catenin protein expression pattern was observed in normal and endometrial cancer cell lines by western blotting. We further showed a complex formation between Gli1 and beta-catenin protein in endometrial cancer cell lines in an immunoprecipitation study. Ectopic overexpression of Gli1 into endometrial cancer cells led to reduced expression of beta-catenin in cell cytoplasm and increased expression of beta-catenin in the nuclei. In summary, overexpression of Gli1 was an early event in endometrial carcinogenesis. Aberrant activation of hedgehog pathway may play important roles in endometrial cancer through beta-catenin nuclear accumulation.

Overexpression of NANOG in gestational trophoblastic diseases: effect on apoptosis, cell invasion, and clinical outcome.

Gestational trophoblastic disease includes choriocarcinoma, a frankly malignant tumor, and hydatidiform mole (HM), which often leads to the development of persistent gestational trophoblastic neoplasia and requires chemotherapy. NANOG is an important transcription factor that is crucial for maintaining embryonic stem cell self-renewal and pluripotency. We postulated that NANOG is involved in the pathogenesis of gestational trophoblastic disease. In this study, significantly higher NANOG mRNA and protein expression levels, by quantitative PCR and immunoblotting, respectively, were demonstrated in HMs, particularly those that developed persistent disease, when compared with normal placentas. In addition, significantly increased nuclear NANOG immunoreactivity was found by immunohistochemistry in HMs (P < 0.001) and choriocarcinoma (P = 0.002). Higher NANOG expression levels were demonstrated in HMs that developed persistent disease, as compared with those that regressed (P = 0.025). Nuclear localization of NANOG was confirmed by confocal microscopy and immunoblotting in choriocarcinoma cell lines. There was a significant inverse correlation between NANOG immunoreactivity and apoptotic index assessed by M30 CytoDeath antibody (P = 0.012). After stable knockdown of NANOG in the choriocarcinoma cell line JEG-3 by an shRNA approach, increased apoptosis was observed in relation to with enhanced caspases and poly(ADP-ribose) polymerase activities. NANOG knockdown was also associated with decreased mobility and invasion of JEG-3 and down-regulation of matrix metalloproteases 2 and 9. These findings suggest that NANOG is involved in the pathogenesis and clinical progress of gestational trophoblastic disease, likely through its effect on apoptosis, cell migration, and invasion.

Deregulated Gab2 phosphorylation mediates aberrant AKT and STAT3 signaling upon PIK3R1 loss in ovarian cancer.

Copy number loss of PIK3R1 (p85α) most commonly occurs in ovarian cancer among all cancer types. Here we report that ovarian cancer cells manifest a spectrum of tumorigenic phenotypes upon knockdown of PIK3R1. PIK3R1 loss activates AKT and p110-independent JAK2/STAT3 signaling through inducing changes in the phosphorylation of the docking protein Gab2, thereby relieving the negative inhibition on AKT and promoting the assembly of JAK2/STAT3 signalosome, respectively. Additional mechanisms leading to AKT activation include enhanced p110α kinase activity and a decrease in PTEN level. PIK3R1 loss renders ovarian cancer cells vulnerable to inhibition of AKT or JAK2/STAT3. The combination of AKT and STAT3 inhibitors significantly increases the anti-tumor effect compared to single-agent treatments. Together, our findings provide a rationale for mechanism-based therapeutic approach that targets tumors with loss of PIK3R1.

p21-Activated Kinases 1, 2 and 4 in Endometrial Cancers: Effects on Clinical Outcomes and Cell Proliferation.

p21-activated kinases (Paks) are serine/threonine protein kinases involved in biological events linked to malignant tumor progression. In this study, expression of Pak1, p-Pak2 Ser20, Pak4, pPak4 Ser474 in 21 normal endometrium, 16 hyperplastic endometrium without atypia, 17 atypical complex hyperplasia and 67 endometrial cancers was assessed by immunohistochemistry and correlated with clinicopathological parameters. We also accessed the proliferative role and downstream targets of Pak1 in endometrial cancer. Pak1 was expressed in cytoplasm whereas Pak4 and p-Pak4 were expressed in both cytoplasm and nucleus of endometrial tissues. In normal endometrium, significantly higher Pak1 (P = 0.028) and cytoplasmic p-Pak2 (P = 0.048) expression was detected in proliferative endometrium than secretory endometrium. Pak1, cytoplasmic and nuclear Pak4 and nuclear p-Pak4 was significantly overexpressed in endometrial cancer when compared to atrophic endometrium (all P<0.05). Moreover, type I endometrioid carcinomas showed significantly higher Pak1 expression than type II non-endometrioid carcinomas (P<0.001). On the other hand, Pak1, Pak4 and p-Pak4 expression negatively correlated with histological grade (all P<0.05) while p-Pak2 and cytoplasmic Pak4 expression inversely correlated with myometrial invasion (all P<0.05). Furthermore, patients with endometrial cancers with lower cytoplasmic Pak4 expression showed poorer survival (P = 0.026). Multivariate analysis showed cytoplasmic Pak4 is an independent prognostic factor. Functionally, knockdown of Pak1, but not Pak4, in endometrial cancer cell line led to reduced cell proliferation along with reduced cyclin D1, estrogen receptor (ERα) and progestogen receptor (PR) expression. Significant correlation between Pak1 and PR expression was also detected in clinical samples. Our findings suggest that Pak1 and cytoplasmic p-Pak2 may promote cell proliferation in normal endometrium during menstral cycle. Pak1, cytoplasmic and nuclear Pak4 and nuclear p-Pak4 are involved in the pathogenesis of endometrial cancer especially in postmenopausal women. Pak1 promote endometrial cancer cell proliferation, particular in type I endometrioid carcinoma. Cytoplasmic Pak4 can be potential prognostic marker in endometrial cancer.

Overexpression of forkhead box protein M1 (FOXM1) in ovarian cancer correlates with poor patient survival and contributes to paclitaxel resistance.

AIM: Deregulation of FOXM1 has been documented in various cancers. The aim of this study was to evaluate the role of FOXM1 in ovarian cancer tumorigenesis and paclitaxel resistance. EXPERIMENTAL DESIGN: Expression of FOXM1 was examined in 119 clinical samples by immunohistochemistry and correlated with clinicopathological parameters. Effects of FOXM1 knockdown on ovarian cancer cell migration, invasion and mitotic catastrophe were also studied. qPCR and ChIP-qPCR were used to establish KIF2C as a novel FOXM1 target gene implicated in chemoresistance. RESULTS: High nuclear FOXM1 expression in ovarian cancer patient samples was significantly associated with advanced stages (P = 0.035), shorter overall (P = 0.019) and disease-free (P = 0.014) survival. Multivariate analysis confirmed FOXM1 expression as an independent prognostic factor for ovarian cancer. FOXM1 knockdown significantly inhibited migration and invasion of ovarian cancer cells and enhanced paclitaxel-mediated cell death and mitotic catastrophe in a p53-independent manner. Bioinformatics analysis suggested a number of potential transcription targets of FOXM1. One of the potential targets, KIF2C, exhibited similar expression pattern to FOXM1 in chemosensitive and chemoresistant cells in response to paclitaxel treatment. FOXM1 could be detected at the promoter of KIF2C and FOXM1 silencing significantly down-regulated KIF2C. CONCLUSION: Our findings suggest that FOXM1 is associated with poor patient outcome and contributes to paclitaxel resistance by blocking mitotic catastrophe. KIF2C is identified as a novel FOXM1 transcriptional target that may be implicated in the acquisition of chemoresistance. FOXM1 should be further investigated as a potential prognostic marker and therapeutic target for ovarian cancer.

Paradoxical impact of two folate receptors, FRα and RFC, in ovarian cancer: effect on cell proliferation, invasion and clinical outcome.

Despite being an essential vitamin, folate has been implicated to enhance tumor growth, as evidenced by reports on overexpression of folate receptor alpha (FRα) in carcinomas. The role of another folate transporter, reduced folate carrier (RFC), is largely unknown. This study investigated the roles of folate, FRα and RFC in ovarian cancers. We demonstrated FRα mRNA and protein overexpression and reduced RFC expression in association with FRα gene amplification and RFC promoter hypermethylation, respectively. FRα overexpression was associated with tumor progression while RFC expression incurred a favorable clinical outcome. Such reciprocal expression pattern was also observed in ovarian cancer cell lines. Folate was shown to promote cancer cell proliferation, migration and invasion in vitro, and down-regulate E-cadherin expression. This effect was blocked after either stable knockdown of FRα or ectopic overexpression of RFC. This hitherto unreported phenomenon suggests that, RFC can serve as a balancing partner of FRα and confer a protective effect in patients with high FRα-expressing ovarian carcinomas, as evidenced by their prolonged overall and disease-free survivals. In conclusion, we report on the paradoxical impact of FRα (putative oncogenic) and RFC (putative tumor suppressive) in human malignancies. FRα and RFC may potentially be explored as therapeutic target or prognostic marker respectively. We recommend caution and additional research on folate supplements in cancer patients.

iASPP and chemoresistance in ovarian cancers: effects on paclitaxel-mediated mitotic catastrophe.

PURPOSE: iASPP is a specific regulator of p53-mediated apoptosis. Herein, we provided the first report on the expression profile of iASPP in ovarian epithelial tumor and its effect on paclitaxel chemosensitivity. EXPERIMENTAL DESIGN: Expression and amplification status of iASPP was examined in 203 clinical samples and 17 cell lines using immunohistochemistry, quantitative real-time PCR, and immunoblotting, and correlated with clinicopathologic parameters. Changes in proliferation, mitotic catastrophe, apoptosis, and underlying mechanism in ovarian cancer cells of different p53 status following paclitaxel exposure were also analyzed. RESULTS: The protein and mRNA expression of iASPP was found to be significantly increased in ovarian cancer samples and cell lines. High iASPP expression was significantly associated with clear cell carcinoma subtype (P = 0.003), carboplatin and paclitaxel chemoresistance (P = 0.04), shorter overall (P = 0.003), and disease-free (P = 0.001) survival. Multivariate analysis confirmed iASPP expression as an independent prognostic factor. Increased iASPP mRNA expression was significantly correlated with gene amplification (P = 0.023). iASPP overexpression in ovarian cancer cells conferred resistance to paclitaxel by reducing mitotic catastrophe in a p53-independent manner via activation of separase, whereas knockdown of iASPP enhanced paclitaxel-mediated mitotic catastrophe through inactivating separase. Both securin and cyclin B1/CDK1 complex were involved in regulating separase by iASPP. Conversely, overexpressed iASPP inhibited apoptosis in a p53-dependent mode. CONCLUSIONS: Our data show an association of iASPP overexpression with gene amplification in ovarian cancer and suggest a role of iASPP in poor patient outcome and chemoresistance, through blocking mitotic catastrophe. iASPP should be explored further as a potential prognostic marker and target for chemotherapy.

Tyrosine kinase B receptor and BDNF expression in ovarian cancers - Effect on cell migration, angiogenesis and clinical outcome.

In this report, we demonstrated that overexpression of tropomyosin-related kinase B (TrkB) was associated with shorter survival in ovarian cancer patients. Brain-derived neurotrophic factor (BDNF), the TrkB ligand, induced activation (phosphorylation) of TrkB in a dose dependent manner. Besides demonstrating the effect of BDNF/TrkB pathway in enhancing cancer cell migration and invasion but inhibiting apoptosis, we also report for the first time that exogenous hepatocyte growth factor induced TrkB expression at both mRNA and protein levels as well as phosphorylation. Our findings suggest that BDNF/TrkB pathway is important in ovarian carcinogenesis and TrkB may be a potential therapeutic target for ovarian cancer.

Hypermethylation of SOX2 gene in hydatidiform mole and choriocarcinoma.

This study investigated the expression and methylation profiles of SOX2, a stem cell-related transcription factor, in placentas and gestational trophoblastic disease. The methylation status of SOX2 promoter region in 55 hydatidiform moles, 4 choriocarcinoma, 23 first trimester, and 15 term placentas was evaluated by methylation-specific polymerase chain reaction. The methylated allele was found in 4.4% (1/23) of first trimester placentas, 26.7% (4/15) term placentas, and 56.4% (31/55) of hydatidiform moles and all choriocarcinoma samples and cell lines. A significant reduction in SOX2 messenger RNA expression was found in the hydatidiform moles (P = .027) when compared with that in the placentas. SOX2 messenger RNA expression was significantly correlated with SOX2 hypermethylation (P < .001). SOX2 expression was restored in choriocarcinoma cell lines following treatment to 5-Aza-2(')-deoxycytidine and/or Trichostatin A, demethylation and histone deacetylase inhibitors, respectively, and the response was synergistic. Epigenetic mechanisms may play important role on the transcriptional regulation of SOX2 and contribute to pathogenesis of gestational trophoblastic disease.