Oscar Gee-Wan Wong

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 Ser474, 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.

Ascorbic acid inhibits ROS production, NF-κB activation and prevents ethanol-induced growth retardation and microencephaly

In this study, we established an embryo model to study the effects of ethanol on fetal development. When embryos of Xenopus laevis (the African clawed frog) were exposed to ethanol, the resultant tadpoles had significantly reduced brain sizes (microencephaly) and retarded growth rates. These effects, similar to those observed in human fetal alcohol syndrome (FAS), were dose- and time-dependent. We further showed that the antioxidant ascorbic acid (vitamin C) could inhibit the ethanol-induced reactive oxygen species (ROS) production and NF-kappaB activation and protect the ethanol-treated embryos against microencephaly and growth retardation. These results suggest the involvement of NF-kappaB and oxidative stress in ethanol-mediated developmental defects, and the potential use of ascorbic acid as a new and effective protective agent for FAS.

Overexpressed PAK4 promotes proliferation, migration and invasion of choriocarcinoma

Gestational trophoblastic disease (GTD) includes frankly malignant choriocarcinoma (CCA) and placental site trophoblastic tumor and potentially malignant hydatidiform mole. p21-Activated kinase (PAK) 4 promotes cell motility. This study investigated the role of PAK4 in the pathogenesis of GTD. PAK4 messenger RNA and protein expressions in clinical samples and cell lines of normal placentas and GTD were determined by quantitative real-time polymerase chain reaction and western blot, respectively. The effects of human chorionic gonadotropin (hCG) and phosphoinositide 3 kinase (PI3K) on the expression and activation of PAK4 were investigated by treating CCA JEG3 and JAR cells with anti-hCG antibody and PI3K inhibitor, respectively. The effects of PAK4 on CCA cell proliferation, migration and invasion were assessed by corresponding functional assays. We demonstrated overexpression of PAK4 in GTD and CCA cell lines at both RNA and protein level. hCG is one of the upstream regulators of PAK4 expression, whereas activation of PAK4 is PI3K/PKB dependent in JEG3 and JAR cells. Significant correlation was found between PAK4 expression and proliferation index minichromosome maintenance complex component 7 (P = 0.007). In JEG3 and JAR cells, stably transfected PAK4 increased proliferation, migration and invasion, whereas small interfering RNA knockdown of PAK4 decreased proliferation, migration and invasion along with downregulated CDK6 and membrane-type 1 matrix metalloproteinase (MT1-MMP) and upregulated p16. We further found PAK4-mediated transcription of MT1-MMP in CCA cells by luciferase reporter assay. Our results demonstrated for the first time that overexpressed PAK4 was involved in the pathogenesis of GTD, promoting proliferation and enhancing cell migration and invasion in CCA cells.According to JAMAs retraction statement, the first author of the article admitted to data fabrication following an internal investigation.² The source article does not provide subgroup analysis to determine how much of an effect the fabricated data may have had on the final reported outcome.

AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

Recent evidence has suggested that AMPK activators may be applied as therapeutic drugs in suppressing cancer cell growth. However, the molecular mechanism of their suppressive function in cancer cells is still unclear. Here we show that AMPK activators impair cervical cancer cell growth through the reduction of DVL3, a positive regulator in Wnt/β-catenin signaling and an oncogenic player in cervical cancer tumorigenesis. By western blot and immunohistochemical analyses, we demonstrated that DVL3 was frequently upregulated and significantly associated with elevated β-catenin (P = 0.009) and CyclinD1 (P = 0.009) expressions in cervical cancer. Enforced expression of DVL3 elevated β-catenin and augmented cervical cancer cell growth, verifying that DVL3-mediated Wnt/β-catenin activation is involved in cervical cancer oncogenesis. On the other aspect, we noted that the cervical cancer cell growth was remarkably suppressed by AMPK activators and such cell growth inhibition was in concomitant with the reduction of DVL3 protein level in dose- and time-dependent manners. Besides, impaired mTOR signaling activity also reduced DVL3 expression. In contrast, co-treatment with Compound C (AMPK inhibitor) could significantly abrogate metformin induced DVL3 reduction. In addition, co-treatment with AM114 or MG132 (proteosomal inhibitors) could partially restore DVL3 expression under the treatment of metformin. Further in vivo ubiquitination assay revealed that metformin could reduce DVL3 by ubiquitin/proteasomal degradation. To our knowledge, this is the first report showing the probable molecular mechanisms of that the AMPK activators suppress cervical cancer cell growth by impairing DVL3 protein synthesis via AMPK/mTOR signaling and/or partially promoting the proteasomal degradation of DVL3.

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. Clin Cancer Res; 17(21); 6924–33. ©2011 AACR.

A critical role of Pax6 in alcohol-induced fetal microcephaly

Maternal alcohol abuse during pregnancy is one of the leading causes of birth defects in humans. Despite extensive studies, the molecular basis is still not clear. Here we transiently exposed Xenopus embryos to alcohol and showed that alcohol dose-dependently produced microcephaly and growth retardation. Moreover, it reduced the expression of several key neural genes (xPax6, xOtx2, xSox3, xSox2, and xNCAM), of which xPax6 was most vulnerable. An alcohol concentration as low as 0.3% could produce more than 90% reduction of xPax6 expression. Consistently, microinjection of xPax6 expression plasmid to Xenopus embryos dose-dependently rescued alcohol-induced microcephaly and restored the expression of xOtx2, xSox3, xSox2, and xNCAM. To test whether reactive oxygen species (ROS) is the upstream signal for alcohol-induced microcephaly and xPax6 suppression, we overexpressed catalase in Xenopus embryos and found that catalase not only decreased alcohol-induced H(2)O(2) formation, but also fully restored Pax6 expression and reversed microcephaly. In contrast, xPax6 and catalase could only provide partial protection against growth retardation. Results from this study illustrate for the first time the critical role of H(2)O(2)-mediated Pax6 suppression in alcohol-induced microcephaly and suggest the presence of additional mechanisms for alcohol-induced fetal growth retardation.

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.

Dysregulated stemness-related genes in gynecological malignancies

In recent years, much attention has been paid to the concept of cancer stem cells (CSC) and self-renewal related pathways in cancer biology. This review outlines the dysregulated stemness-related genes or transcription factors in gynecological cancers. Hedgehog (Hh) and Notch signaling are important pathways in tissue pattern programming and cell fate determination during embryonic development. Hyperactivation of these two pathways was frequently observed in gynecological malignancies such as ovarian, endometrial and cervical cancers. In contrast, the expression profiles of pluripotency-regulating core transcriptional circuitry: Nanog, Oct4 and Sox2 appear heterogeneous. Among these transcription factors, overexpression of Nanog was found to exert a prominent effect in gynecological tumorigenesis, while dysregulations of Oct4 and Sox2 may vary in a context dependent manner. On the other hand, the isolation of putative CSC illustrates a hierarchy model of tumor heterogeneity, in which only a subset of cells among biologically distinct populations can initiate tumor growth. Re-activation of these pluripotent transcription factors (Nanog, Oct4 and/or Sox2) in association with distinct tumorigenic properties could be found in clones isolated from gynecological tumors using various approaches. Recent understanding on the roles of Hh and Notch signaling in enhancing CSC survival may help to better understand the mechanism of carcinogenesis and identify new pharmaceutical targets for gynecological malignancies.

Efficacy of Abbott RealTime High Risk HPV test in evaluation of atypical squamous cells of undetermined significance from an Asian screening population.

BACKGROUND Abbott RealTime High Risk HPV test is a new qualitative real-time PCR assay for the detection of 14 high risk HPV (HR-HPV) types and specific identification of HPV16 and HPV18. For each new HPV DNA test, it is important to validate its clinical performance using established tests as benchmarks. Hybrid Capture 2 (HC2) is the first USA FDA-approved HR-HPV DNA test. OBJECTIVES To compare the performance of Abbott RealTime High Risk HPV test with that of Hybrid Capture 2 in detecting cytology samples with varying prognosis. STUDY DESIGN 250 liquid-based cervical cytology samples diagnosed of Atypical Squamous cells of Undetermined Significance (ASC-US) collected from an Asian Screening Population were independently tested with both Abbott RealTime High Risk HPV test and HC2. Their utility in predicting disease progression was evaluated in 82 of the samples for which follow up cytology or colposcropic histology data was available. RESULTS Good to excellent agreement between the two tests was demonstrated (Kappa=0.800, 95% CI: 0.726-0.874). The sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of the two tests in detecting cases with underlying HSIL/CIN2+ were evaluated (Abbott: 100%, 20.83%, 14.93% and 100% respectively; HC2: 100%, 12.50%, 13.70% and 100% respectively). HPV16/18 genotyping provided by the Abbott test enhanced specific identification of cases with LSIL/CIN1+ (specificity 91.30%, PPV 84.62%) and HSIL/CIN2+ (specificity 86.11%, PPV 23.08%) at follow-up. CONCLUSIONS The Abbott test performed similarly to HC2 and is unlikely to be affected by ethnicity. Abbott combined HPV detection and HPV 16/18 genotyping is found to provide enhanced sensitivity and specificity for triage of ASC-US.

Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis

This paper aimed at investigating the expression and methylation profiles of SOX2, a gene coding for the stem cell-related transcription factor SOX2, in endometrial carcinomas. By methylation-specific polymerase chain reaction (MS-PCR), the methylation status of SOX2 promoter region in 72 endometrial carcinomas and 12 normal endometrial samples was examined. Methylated allele was found in 37.5% (27/72) of endometrial carcinomas but only in 8.3% (1/12) of normal endometrial, significantly more frequent in cancers (P = .0472). SOX2 mRNA level was significantly reduced in endometrial carcinoma compared with nonneoplastic endometrium (P = .045). A significant correlation between SOX2 mRNA expression and hypermethylation of SOX2 was found (P = .024). Hypermethylation of SOX2 tended to be more frequently found in type II serous or clear cell adenocarcinoma. SOX2 methylation was also significantly correlated with shorter survival of patients (P = .046). In conclusion, epigenetic mechanisms may play a crucial role on the transcriptional regulation of SOX2 and loss of SOX2 expression may be related to endometrial carcinogenesis.