Zehua Wang

miR-335 represents an invasion suppressor gene in ovarian cancer by targeting Bcl-w

microRNAs (miRNAs) are a class of non-coding small RNAs that bind to target mRNAs, usually resulting in post-transcriptional repression by translational inhibition or target degradation. mRNAs can function as tumor suppressors or oncogenes (also referred to as oncomirs) in human tumors. Although aberrant expression of miR-335 has been reported in ovarian cancer, whether it is an active participant or a mere bystander remains unknown. To clarify its role in ovarian carcinogenesis, we first examined the relative expression of miR-335 in 17xa0normal ovarian tissues and 4 ovarian cancer cell lines using qPCR. We found that miR-335 was downregulated in the ovarian cancer cell lines relative to normal ovarian epithelium tissues. Inxa0vitro, overexpression of miR-335 suppressed cell migration and invasion and resulted in depolymerization of F-actin in ovarian cancer cell lines, but exhibited a negligible effect on cell proliferation. B-cell CLL/lymphomaxa02 likexa02 (Bcl-w or BCL2L2), a pro-survival member of the Bcl-2 protein family, was identified as a potential target of miR-335 according to the results of bioinformatic analysis, and the expression of Bcl-w and its effector matrix metalloproteinase-2 (MMP‑2) was downregulated after transfection with miR-335 mimics. In addition, ectopic Bcl-w could almost fully nullify the effect of miR-335 overexpression on ovarian cancer cell migration and invasion. These findings indicate that the tiny genome product, miR-335, whose lack of expression brings about the abnormal accumulation of Bcl-w and subsequent unchecked cell invasion in ovarian cancer, may help us to understand one of the many steps ovarian cells take on their way toward the acquisition of malignant phenotypes and miR-335 may be a promising predictor of survival.

miR-335 Represents an Independent Prognostic Marker in Epithelial Ovarian Cancer

OBJECTIVESnTo investigate the clinical significance of miR-335 expression in epithelial ovarian cancer (EOC).nnnMETHODSnExpression of miR-335 in normal ovaries, EOC, and omental metastases was determined by quantitative realtime polymerase chain reaction. Overall survival (OS) and relapse-free survival (RFS) were examined using KaplanMeier curves and the Cox proportional hazards regression model.nnnRESULTSnmiR-335 expression level was reduced in malignant tissue samples, especially in omental metastases. Low miR-335 levels in EOC were associated with shorter OS (P < .001) and RFS (P = .002). In the multivariate analyses, low miR-335 levels emerged as an independent prognostic factor for poor OS (P = .005) and RFS (P = .016). miR-335 was downregulated in the recurrence group compared with the nonrecurrence group. The area under the receiver operating characteristic curve was 0.818 (95% confidence interval, 0.675-0.961).nnnCONCLUSIONSnOur results indicate that miR-335 relates to the prognosis of patients with EOC and is a promising predictor of EOC recurrence.

Prognostic significance of several biomarkers in epithelial ovarian cancer: a meta-analysis of published studies

ObjectiveAbnormal expression of several biomarkers might predict disease prognosis and response to chemotherapy in patients with epithelial ovarian cancer (EOC). However, the published data are inconsistent.MethodsEligible studies that investigated the association between survival or response to platinum-based chemotherapy in EOC and the expression status of Bcl-2, EGFR, GST, LRP, p16, p21, P-gp and TNF-α were identified by an electronic search of PubMed and Embase. The measures of interest were hazard ratio (HR) for survival or risk ratio for chemotherapy response. A meta-analysis was performed using the fixed-effect or random-effect models.ResultsThe number of eligible studies analyzed was 27 for Bcl-2, 22 for EGFR, 29 for GST, 12 for LRP, 16 for p16, 22 for p21, 27 for P-gp and three for TNF-α. A meta-analysis showed that high EGFR and P-gp expression was associated with poor overall survival (OS) (pooled adjusted HRxa0=xa01.826 and HRxa0=xa01.822). Only high GST expression was associated with improved OS (HRxa0=xa00.780). Furthermore, high p16 and P-gp expression was associated with poor progression-free survival (PFS) (HRxa0=xa01.550 and HRxa0=xa02.136). High GST expression was associated with improved PFS (HRxa0=xa00.689). Among these factors, only LRP, P-gp and TNF-α were associated with response to platinum-based chemotherapy.ConclusionsThe markers we analyzed are unlikely to be useful as predictors of prognosis and response to platinum-based chemotherapy in EOC patients in clinical practice.

Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer.

Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression.

The Use of Laser Microdissection in the Identification of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Human FFPE Epithelial Ovarian Tissue Samples

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

The Role of the PTEN/PI3K/Akt Pathway on Prognosis in Epithelial Ovarian Cancer: A Meta-Analysis

The PTEN/PI3K/Akt signaling pathway, a key player in mediating apoptosis, metabolism, cell proliferation, and cell growth, is frequently dysregulated in many cancers. However, the pathways prognostic impact in epithelial ovarian cancer (EOC) is still inconsistent. We performed a meta-analysis based on individual study outcomes to more precisely evaluate its clinical significance in EOC patients. Methods. We searched all potentially relevant studies published between January 1, 1990, and March 1, 2013, that assessed the association between PTEN, PI3K, and Akt status and survival in EOC. Meta-analysis was performed using a fixed-effect or random-effects model as appropriate. We investigated the possibility of publication bias through a funnel plot and identified the heterogeneity by I(2) statistics. Results. Eleven eligible studies were analyzed for PTEN, 5 for PI3K, and 11 for pAkt. High PI3K and pAkt expression was associated with poor overall survival (OS; pooled adjusted hazard ratio [HR] = 1.44, 95% CI, 1.08-1.91 for PI3K; HR = 1.60, 95% CI, 1.26-2.04 for pAkt). In addition, both the meta-analyses of univariate and multivariate estimates showed that only high pAkt expression was significantly associated with poor progression-free survival (PFS; pooled unadjusted HR = 1.24, 95% CI, 1.10-1.39; pooled adjusted HR = 1.65, 95% CI, 1.07-2.55). Conclusion. Published studies suggest that high pAkt expression is significantly associated with poor OS and PFS in EOC patients, but currently available evidence is insufficient to recommend that PTEN, PI3K, or Akt be used as prognostic predictors in EOC in clinical practice.

Tumor-associated macrophages induce lymphangiogenesis in cervical cancer via interaction with tumor cells.

Our studies were conducted to investigate the clinical and functional significance of tumor‐associated macrophages (TAMs) in cervical tumor lymphatic metastasis. We found that the increase in macrophages in tumor stroma is significantly associated with lymphatic metastasis (p = 0.017), through performing immunohistochemical staining in 111 cervical samples (55 invasive squamous carcinomas of uterine cervix, 27 cervical intraepithelial neoplasms III, and 29 normal cervix). The human lymphatic endothelial cells (HLEC), which were cultured in conditioned medium of cervical cancer cell‐macrophage coculture, formed more tube‐like structures in vitro, when compared with those in conditioned mediums of LEC, normal cervical epithelium, single macrophage, and single cervical cancer cell (all p < 0.001). The mRNA expressions of IL‐1β and IL‐8 in cervical cancer cells cocultured with macrophages were increased, compared with those in cervical cancer cell cultured alone (pIL‐1β < 0.05 and pIL‐8 < 0.01). Meanwhile, the mRNA expression of VEGF‐C and VEGF‐A was increased in macrophages cocultured with cervical cancer cells, compared with the expression in those macrophages cultured alone (both p < 0.05). Taken together, the results suggest that TAMs promote lymphangiogenesis mainly through interaction with surrounding cervical cancer cells.

EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

Enhancer of zeste homolog 2 (EZH2) is often increased in malignant tumors and is involved in metastasis. EZH2 silences gene expression by tri-methylating the lysine 27 residue of histone H3 (H3K27me3). However, the mechanism underlying EZH2 promotion of ovarian cancer metastasis remains elusive. Here, we showed that EZH2 is up-regulated in ovarian cancer and is associated with tumor metastasis and poor survival by mRNA sequencing and microarray results from databases. Tissue microarray and immunohistochemistry results revealed that EZH2 was negatively correlated with the expression of tissue inhibitor of metalloproteinases 2 (TIMP2). EZH2 overexpression inhibited TIMP2 expression and promoted proteolytic activities of matrix metalloproteinases 2 and 9 and vice versa. EZH2 promoted ovarian cancer invasion and migration, which could be largely reversed by TIMP2 down-regulation in vitro and in vivo. Both H3K27me3 inhibition and demethylation could reduce methylation of the TIMP2 promoter and finally reactivate TIMP2 transcription. The presence of EZH2 and H3K27me3 at the TIMP2 promoter was confirmed by chromatin immunoprecipitation. H3K27me3 and DNA methyltransferases at the promoter were significantly increased by EZH2 overexpression. These results suggest that EZH2 inhibits TIMP2 expression via H3K27me3 and DNA methylation, which relieve the repression of MMP and facilitate ovarian cancer invasion and migration.

Biodegradable polymer–platinum drug conjugates to overcome platinum drug resistance

Drug resistance and severe dose-dependent side effects are the major obstacles to the further use of platinum agents. Two well-acknowledged mechanisms of resistance are the decreased intracellular accumulation and the increased sulfur-binding-detoxification of platinum agents. The polymer–drug conjugate approach can remarkably enhance the uptake of substances by cancer cells, therefore can achieve better anticancer effects with lower dosage, and thus reduce the dose-dependent side effects. Here we synthesized a biodegradable polymer containing pendant 1,2-bidentate carboxyl groups to chelate platinum without introducing sulfur atoms to potentially detoxify the platinum drugs. The polymer–platinum conjugate formed nanomicelles showed significantly increased intracellular accumulation and could partially overcome drug resistance in ovarian cancer cells.

Role of EZH2 in cancer stem cells: from biological insight to a therapeutic target

Epigenetic modifications in cancer stem cells largely result in phenotypic and functional heterogeneity in many solid tumors. Increasing evidence indicates that enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb repressor complex 2, is highly expressed in cancer stem cells of numerous malignant tumors and has a critical function in cancer stem cell expansion and maintenance. Here, we review up-to-date information regarding EZH2 expression patterns, functions, and molecular mechanisms in cancer stem cells in various malignant tumors and discuss the therapeutic potential of targeting EZH2 in tumors.