Expression of PTEN in ovarian epithelial tumors and its relation to tumor behavior and growth

OBJECTIVE: To evaluate the expression of tumor suppressor gene phosphatase and tensin homologue on chromosome 10 (PTEN) in ovarian epithelial tumors and its correlation with tumor growth and clinicopathologic features in ovarian adenocarcinomas. STUDY DESIGN: Immunohistochemical staining with anti-PTEN antibody was performed in 54 adenocarcinomas and 23 borderline tumors of the ovary. The apoptotic cells were visualized by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling, and proliferative cells were visualized by staining with Ki-67 antibody. RESULTS: Reduced PTEN expression was significantly higher among the adenocarcinomas than the borderline tumors (p < 0.001). Reduced PTEN expression in adenocarcinomas did not correlate with International Federation of Obstetrics and Gynecology (FIGO) stage. The Ki-67 index (KI) and apoptotic index were significantly higher in adenocarcinomas as compared with borderline tumors (p < 0.001). Tumors with reduced PTEN expression in ovarian adenocarcinomas had a significantly higher KI than those with normal PTEN expression (p < 0.01). By univariate analysis, FIGO stage and histologic type correlated with survival. However, FIGO stage was the only independent prognostic factor by multivariate analysis. CONCLUSION: Our results suggest that alteration of the PTEN gene may be associated with malignant transformation of ovarian epithelial tumors. The PTEN gene seems to be a negative regulator of cell proliferation in ovarian adenocarcinomas.     

Inhibition of Ovarian Epithelial Carcinoma Tumorigenesis and Progression by microRNA 106b Mediated through the RhoC Pathway

Epithelial ovarian cancer (EOC) is the most lethal of the gynecological malignancies. Exploring the molecular mechanisms and major factors of invasion and metastasis could have great significance for the treatment and prognosis of EOC. Studies have demonstrated that microRNA 106b (miR-106b) may be a promising therapeutic target for inhibiting breast cancer bone metastasis, but the role of miR-106b in EOC is largely unknown. In this work, miRNA-106b expression was quantified in various ovarian tissues and tumors. Ovarian carcinoma cell lines were transfected with miR-106b, after which, cell phenotype and expression of relevant molecules was assayed. Dual-luciferase reporter assays and xenograft mouse models were also used to investigate miR-106b and its target gene. MiR-106b mRNA expression was found to be significantly higher in normal ovarian tissues and benign tumors than in ovarian carcinomas and borderline tumors (p < 0.01), and was negatively associated with differentiation (Well vs. Por & Mod) and the International Federation of Gynecology and Obstetrics (FIGO) staging (stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05). MiR-106b transfection reduced cell proliferation; promoted G1 or S arrest and apoptosis (p < 0.05); suppressed cell migration and invasion (p < 0.05); reduced Ras homolog gene family member C (RhoC), P70 ribosomal S6 kinase (P70S6K), Bcl-xL, Matrix metallopeptidase 2 (MMP2), MMP9 mRNA and protein expression; and induced p53 expression (p < 0.05). Dual-luciferase reporter assays indicated that miR-106b directly targets RhoC by binding its 3’UTR. MiR-106b transfection also suppressed tumor development and RhoC expression in vivo in xenograft mouse models. This is the first demonstration that miR-106b may inhibit tumorigenesis and progression of EOC by targeting RhoC. The involvement of miR-106b-mediated RhoC downregulation in EOC aggression may give extended insights into molecular mechanisms underlying cancer aggression. Approaches aimed at overexpressing miR-106b may serve as promising therapeutic strategies for treating EOC patients.     

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades ^1,2. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages ^3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren''t specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.     

Copper content in ascitic fluid is associated with angiogenesis and progression in ovarian cancer

BackgroundAscites is associated with the poor prognosis of malignant tumors. The biological importance of the changes in the content of trace elements in the ascitic fluid is unknown. Herein, we analyzed trace elements in the ascitic fluid of patients with ovarian tumors and used cultured cells to determine the copper (Cu)-induced changes in gene expression in ovarian cancer.MethodsInductively coupled plasma mass spectrometry (ICP-MS) was used to compare ascitic fluid trace element levels in patients with benign ovarian tumors (n = 22) and borderline/malignant tumors (n = 5) for primary screening. Cu levels were validated using atomic absorption spectrometry (AAS) in 88 benign, 11 borderline, and 25 malignant ovarian tumor patients. To confirm Cu-induced gene expression changes, microarray analysis was performed for Cu-treated OVCAR3, A2780, and Met5A cells. The vascular endothelial growth factor (VEGF) concentration in the cell supernatant or ascitic fluid (ovarian cancer samples) was measured using ELISA.ResultsICP-MS showed that Co, Ni, Cu, Zn, As, Se, and Mo levels significantly increased in patients with malignant/borderline ovarian tumors compared to those in patients with benign ovarian tumors. AAS showed that malignant ovarian tumors were independently associated with elevated levels of Cu in ascites adjusted for age, body mass index, alcohol, smoking, and supplement use (p < 0.001). Microarray analysis of both Cu-treated ovarian cancer cell lines OVCAR3 and A2780 and the mesothelial cell line Met-5A revealed the upregulation of the angiogenesis biological process. Real-time polymerase chain reaction and ELISA demonstrated that an increased Cu content significantly enhanced VEGF mRNA expression and protein secretion in OVCAR3, A2780, and Met-5A cells. VEGF levels and clinical stages of the tumors correlated with the ascitic fluid Cu content in patients with malignant ovarian tumors (correlation coefficient 0.445, 95 % confidence interval [CI]: 0.069–0.710, p = 0.023 and correlation coefficient 0.406, 95 % CI: 0.022–0.686, p = 0.040, respectively).ConclusionCu levels significantly increased in patients with malignant ovarian cancer. Cu induced angiogenic effects in ovarian cancer and mesothelial cells, which affected ascites fluid production. This study clarifies the link between elevated Cu in ascites and malignant ovarian tumor progression. Strategies to decrease Cu levels in the ascitic fluid may help downregulate VEGF expression, thereby improving the prognosis of ovarian malignancies.     

Genome-Wide Maps of Mononucleosomes and Dinucleosomes Containing Hyperacetylated Histones of Aspergillus fumigatus

It is suggested that histone modifications and/or histone variants influence the nucleosomal DNA length. We sequenced both ends of mononucleosomal and dinucleosomal DNA fragments of the filamentous fungus Aspergillus fumigatus, after treatment with the histone deacetylase inhibitor trichostatin A (TSA). After mapping the DNA fragments to the genome, we identified >7 million mononucleosome positions and >7 million dinucleosome positions. We showed that the distributions of the lengths of the mononucleosomal DNA fragments after 15-min and 30-min treatments with micrococcal nuclease (MNase) showed a single peak at 168 nt and 160 nt, respectively. The distributions of the lengths of the dinucleosomal DNA fragments after 15-min- and 30-min-treatment with MNase showed a single peak at 321 nt and 306 nt, respectively. The nucleosomal DNA fragments obtained from the TSA-treated cells were significantly longer than those obtained from the untreated cells. On the other hand, most of the genes did not undergo any change after treatment. Between the TSA-treated and untreated cells, only 77 genes had ≥2-fold change in expression levels. In addition, our results showed that the locations where mononucleosomes were frequently detected were conserved between the TSA-treated cells and untreated cells in the gene promoters (lower density of the nucleosomes). However, these locations were less conserved in the bodies (higher density of the nucleosomes) of genes with ≥2-fold changes. Our findings indicate that TSA influences the nucleosome positions, especially of the regions with high density of the nucleosomes by elongation of the nucleosomal DNA. However, most of the nucleosome positions are conserved in the gene promoters, even after treatment with TSA, because of the low density of nucleosomes in the gene promoters.     

Activation of the RhoB Signaling Pathway by Thyroid Hormone Receptor β in Thyroid Cancer Cells

Thyroid hormone receptor (TR) mediates the crucial effects of the thyroid hormone (T3) on cellular growth, development, and differentiation. Decreased expression or inactivating somatic mutations of TRs have been found in human cancers of the liver, breast, lung, and thyroid. The mechanisms of TR-associated carcinogenesis are still not clear. To establish the function of TRβ in thyroid cancer cell proliferation, we constructed a recombinant adenovirus vector, AdTRβ, which expresses human TRβ1 cDNA. Thyroid cancer cell lines in which TRβ protein levels were significantly decreased as compared to intact thyroid tissues were infected with AdTRβ and the function of TRβ on cell proliferation and migration was analyzed. Ligand-bound TRβ induced HDAC1 and HDAC3 dissociation from, and histone acetylation associated with the RhoB promoter and enhanced the expression of RhoB mRNA and protein. In AdTRβ-infected cells, T3 and farnesyl transferase inhibitor (FTI)-treatment induced the distribution of RhoB on the cell membrane and enhanced the abundance of active GTP-bound RhoB. This RhoB protein led to p21-associated cell-cycle arrest in the G0/G1 phase, following inhibition of cell proliferation and invasion. Conversely, lowering cellular RhoB by small interfering RNA knockdown in AdTRβ-infected cells led to downregulation of p21 and inhibited cell-cycle arrest. The growth of BHP18-21v tumor xenografts in vivo was significantly inhibited by AdTRβ injection with FTIs-treatment, as compared to control virus-injected tumors. This novel signaling pathway triggered by ligand-bound TRβ provides insight into possible mechanisms of proliferation and invasion of thyroid cancer and may provide new therapeutic targets for thyroid cancers.     

Epigenetic inactivation of the novel candidate tumor suppressor gene ITIH5 in colon cancer predicts unfavorable overall survival in the CpG island methylator phenotype

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression as well as its potential clinical and functional impact in colon cancer. Based on 30 tumor and 30 adjacent normal tissues we examined ITIH5 mRNA expression and promoter methylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA) platform. In addition, ITIH5 protein expression was evaluated using immunohistochemistry. ITIH5 mRNA expression loss was significantly associated (P < 0.001) with hypermethylation of the ITIH5 promoter in primary colon tumors. In addition, treatment of tumor cell lines with demethylating (DAC) and histone acetylating (TSA) agents induced ITIH5 expression. In line, independent TCGA data revealed a significant expression loss of ITIH5, particularly in the MSI-high and CIMP-positive phenotype concordant with an increased ITIH5 hypermethylation in CIMP-positive colon tumors (P < 0.001). In proximal, i.e., right-sided tumors, abundant ITIH5 expression was associated with longer overall survival (OS, P = 0.049) and the CIMP-positive (P = 0.032) subgroup. Functionally, ITIH5 re-expression mediated a reduced proliferation in HCT116 and CaCo2 cells. In conclusion, our results indicate that ITIH5 is a novel putative tumor suppressor gene in colon cancer with a potential impact in the CIMP-related pathway. ITIH5 may serve as a novel epigenetic-based diagnostic biomarker with further clinical impact for risk stratification of CIMP-positive colon cancer patients.     

Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

Ovarian cancer is a highly lethal disease with poor prognosis and especially in high-grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers still remains unclear. In this study, we found that GRB7 (P<0.0001), ERK phosphorylation (P<0.0001) and FOXM1 (P = 0.001) were frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer by immunohistochemical analysis. Intriguingly, the expressions of GRB7 (P<0.0001), ERK phosphorylation (P<0.001) and FOXM1 (P<0.001) showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancers. Biochemical studies using western blot analysis demonstrated that enforced expression or knockdown of GRB7 showed GRB7 could elevate the levels of ERK phosphorylation and FOXM1, whereas enforced expression of FOXM1 could not alter levels of GRB7 and ERK phosphorylation. But inhibition of ERK signaling by U0126 or PD98059 could reduce the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or PD98059, or decreased FOXM1 expression by Thiostrepton significantly inhibited cell migration/invasion, tumor growth in vitro and in vivo. Collectively, our findings confer that targeting GRB7/ERK/FOXM1 signaling cascade may be a promising molecular therapeutic choice in combating ovarian cancer.     

Legumain: a biomarker for diagnosis and prognosis of human ovarian cancer

Legumain is a member of the asparaginyl endopeptidase family that is over‐expressed in response to hypoxic stress on mammary adenocarcinoma, colorectal cancer, proliferating endothelial cells, and tumor‐associated macrophages (TAMs). Here, we demonstrate that elevated expression of legumain in ovarian cancer by a proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) followed by liquid chromatography–mass spectrometry (LC–MS/MS). To investigate the relationship between legumain expression and ovarian cancer development, we tested legumain expression in malignant human ovarian tumors (n = 60), borderline ovarian tumors (n = 20), benign ovarian tumors (n = 20), and normal ovary samples (n = 20) using immunohistochemical assay (IHC). A correlation between legumain expression, and clinocopathologic and biological variables was also established. Importantly, increased legumain expression was validated by real‐time PCR and Western blots, correlated positively with an increased malignancy of ovarian tumors (P < 0.01). In fact, patients with strong legumain expression had a worse prognosis (P = 0.03). In addition, results of in vitro experiments revealed that over‐expression of legumain correlates with increased cell migration and invasion of ovarian cancer cells. Although legumain's functional role and clinical utility remain to be established, our results indicated that a sensitive assay for early expression of legumain may serve as both a potential biomarker and a molecular target for treatment of ovarian cancer. J. Cell. Biochem. 113: 2679–2686, 2012. © 2012 Wiley Periodicals, Inc.     

Trichostatin A-treated eight-cell bovine embryos had increased histone acetylation and gene expression, with increased cell numbers at the blastocyst stage

The objective was to determine the effects of trichostatin A (TSA), a potent histone deacetylase inhibitor, on eight-cell bovine embryos. That treatment increased histone acetylation was confirmed by immunostaining with anti-AcH4K5 and AcH4K8 antibodies. Embryos treated with TSA (100 nM) for various intervals (4, 8, and 12 h) developed to the blastocyst stage as frequently as untreated embryos (average development rate, 49.5%). Treatment with TSA for 12 h increased (P < 0.05) the numbers of inner cell mass (ICM) cells and total cells (TC), as well as the ICM/TC ratio in the blastocyst, but the number of cells in the trophectoderm decreased (P < 0.05). Treated embryos had increased relative abundance (RA) of OCT3/4 and E-CADHERIN mRNA relative to controls at the morula stage (P < 0.05), however, the RA of CDX2 mRNA was unchanged. In conclusion, TSA-treated eight-cell stage embryos had increased histone acetylation and gene expression, which increased ICM and TC numbers and the ICM/TC ratio, but significantly decreased the number of cells in the trophectoderm of resulting blastocysts.     

The GADD45A (1506T>C) Polymorphism Is Associated with Ovarian Cancer Susceptibility and Prognosis

GADD45A (growth arrest and DNA damage 45 A) is the first stress-inducible gene identified to be a target of p53. However, no studies to date have assessed variants of the GADD45 gene and their potential relationship to tumor susceptibility. We investigated the association of the GADD45A (1506T>C) polymorphism with ovarian cancer development in 258 ovarian cancer patients and 332 age-matched healthy women as controls using sequence analysis. We found a statistically significant difference in the GADD45A (1506T>C) genotype distributions between the case and control groups (TT vs. TC vs. CC, P = 0.0021) and found that variant 1506T>C was significantly associated with an increased risk of ovarian cancer (P<0.001, OR = 1.71, 95% CI [1.28–2.29]). We observed a statistically significant effect between tumor histology (P = 0.032) and CA125 status (P = 0.021). Carrying the C allele (TC+CC) was associated with an increased risk of positive CA125 (OR = 3.20, 95% CI [1.15–8.71). Carrying the T allele (TT+TC) showed a significant correlation with both higher GADD45A mRNA expression and longer ovarian cancer RFS (relapse-free survival) and OS (overall survival). We are the first group to demonstrate that the GADD45A (1506T>C) polymorphism is associated with ovarian cancer susceptibility and prognosis. These data suggest that GADD45A (1506T>C) is a new tumor susceptibility gene and could be a useful molecular marker for assessing ovarian cancer risk and for predicting ovarian cancer patient prognosis.     

Clinicopathological study of metallothionein immunohistochemical expression, in benign, borderline and malignant ovarian epithelial tumors

Metallothioneins (MTs) are a family of cystein-rich metal-binding proteins, which are expressed in normal cells during fetal and postnatal life but also in a variety of human neoplasms. MT expression in human tumors has been linked to resistance to anticancer drugs and differentiation and progression in some types of tumors. This study examined the immunohistochemical expression of MTs in benign, borderline and malignant tumors of ovarian surface epithelium and the possible correlations with clinicopathological parameters and survival. A total of 87 cases with diagnosis of ovarian surface epithelial tumors were included. Specifically, 21 cases of benign cystadenomas (11 serous and 10 mucinous), 14 borderline (low malignant potential tumors, 8 mucinous and 6 serous) and 52 cases of ovarian cancer were analysed. Immunohistochemical expression of MT (cut-off level > 10% of tumor cells) was clearly associated with malignancy. A statistically significant correlation was found between the expression of MT in cancer cases and benign tumors (p < 0.0001) and cancer cases and borderline tumors p = 0.003. In cancer cases a difference was observed between grade I and III (p = 0.002). There was no correlation of MT overexpression with survival in the small number of ovarian carcinoma patients where it was analysed. MT constitutes a marker that characterizes aggressiveness and a high malignant potential in ovarian epithelial tumors. In diagnostic problems MT may help distinguish between benign, borderline and malignant tumors.     

An Outlook on Ovarian Cancer and Borderline Ovarian Tumors: Focus on Genomic and Proteomic Findings

Among the gynaecological malignancies, ovarian cancer is one of the neoplastic forms with the poorest prognosis and with the bad overall and disease-free survival rates than other gynaecological cancers. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian tumors display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline, and invasive tumors. Several studies on ovarian tumors, have focused on the identification of both diagnostic and prognostic markers for applications in clinical practice. High-throughput technologies have accelerated the process of biomolecular study and genomic discovery; unfortunately, validity of these should be still demonstrated by extensive researches on sensibility and sensitivity of ovarian cancer novel biomarkers, determining whether gene profiling and proteomics could help differentiate between patients with metastatic ovarian cancer and primary ovarian carcinomas, and their potential impact on management. Therefore, considerable interest lies in identifying molecular and protein biomarkers and indicators to guide treatment decisions and clinical follow up. In this review, the current state of knowledge about the genoproteomic and potential clinical value of gene expression profiling in ovarian cancer and ovarian borderline tumors is discussed, focusing on three main areas: distinguishing normal ovarian tissue from ovarian cancers and borderline tumors, identifying different genotypes of ovarian tissue and identifying proteins linked to cancer or tumor development. By these targets, authors focus on the use of novel molecules, developed on the proteomics and genomics researches, as potential protein biomarkers in the management of ovarian cancer or borderline tumor, overlooking on current state of the art and on future perspectives of researches.Key Words: Ovarian cancer, borderline ovarian tumors, markers, genomics, proteomics, oncogenes.     

Co-Expression of Lewis y Antigen with Human Epididymis Protein 4 in Ovarian Epithelial Carcinoma

Objective

The main aims of this study were to explore the molecular structural relationship between Human epididymis protein 4 (HE4) and Lewis y antigen by determining their expression patterns and clinical significance in ovarian epithelial carcinoma.

Methods

The structural relationship between HE4 and Lewis y antigen was examined using immunoprecipitation and confocal laser scanning microscopy. HE4 and Lewis y were detected in tissues from malignant (53 cases), borderline (27 cases), benign (15 cases) and normal ovarian tissues (15 cases) using immunohistochemical analysis.

Results

HE4 was present in ovarian cancer, benign tumor tissues, ovarian carcinoma cells, and culture medium, and contained Lewis y antigen. Moreover, expression of Lewis y antigen in HE4 from ovarian cancer was higher than that from benign tumor (P<0.05). HE4 possibly exists as two protein isoforms, both containing Lewis y antigen. Our immunohistochemistry data revealed significantly higher positive expression rates of HE4 in malignant ovarian tissues, compared to benign tumor and normal tissue (P<0.05), similar to Lewis y antigen levels in ovarian cancer (P<0.05). Notably, tissues displaying marked expression of HE4 simultaneously expressed high levels of Lewis y antigen. A linear correlation between the expression patterns of HE4 and Lewis y antigen was evident. Consistently, double-labeling immunofluorescence experiments illustrated co-localization of HE4 and Lewis y antigen within the same area.

Conclusions

HE4 contains Lewis y antigen. Our results further demonstrate a close correlation between the expression levels of the two antigens, which are significantly high in ovarian cancer.