The RUNX1 transcription factor is expressed in serous epithelial ovarian carcinoma and contributes to cell proliferation,migration and invasion

Previously, we have identified the RUNX1 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared with primary cultures derived from matched primary (prior to CT) tumors. Here we show that RUNX1 displays a trend of hypomethylation, although not significant, in omental metastases compared with primary EOC tumors. Surprisingly, RUNX1 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. The RUNX1 expression levels were almost identical in primary tumors and omental metastases, suggesting that RUNX1 hypomethylation might have a limited impact on its overexpression in advanced (metastatic) stage of the disease.

Knockdown of the RUNX1 expression in EOC cells led to sharp decrease of cell proliferation and induced G₁ cell cycle arrest. Moreover, RUNX1 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX1 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced.

Taken together, our data are indicative for a strong oncogenic potential of the RUNX1 gene in EOC progression and suggest that RUNX1 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX1 and other members of the RUNX gene family in ovarian tumorigenesis.     

Expression Profiling of Primary and Metastatic Ovarian Tumors Reveals Differences Indicative of Aggressive Disease

The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.     

Enhancer of zeste homolog 2 promotes the proliferation and invasion of epithelial ovarian cancer cells

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes noncatalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared with primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12, and EED were expressed at higher levels in all 8 human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared with pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n = 134) when compared with normal ovarian surface epithelium (n = 46; P < 0.001). EZH2 expression positively correlated with expression of Ki67 (P < 0.001; a marker of cell proliferation) and tumor grade (P = 0.034) but not tumor stage (P = 0.908) in EOC. There was no correlation of EZH2 expression with overall (P = 0.3) or disease-free survival (P = 0.2) in high-grade serous histotype EOC patients (n = 98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics.     

NuMA overexpression in epithelial ovarian cancer

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon.NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis.Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008).NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.     

Effect of Ovarian Cancer Ascites on Cell Migration and Gene Expression in an Epithelial Ovarian Cancer In Vitro Model

A third of patients with epithelial ovarian cancer (EOC) present ascites. The cellular fraction of ascites often consists of EOC cells, lymphocytes, and mesothelial cells, whereas the acellular fraction contains cytokines and angiogenic factors. Clinically, the presence of ascites correlates with intraperitoneal and retroperitoneal tumor spread. We have used OV-90, a tumorigenic EOC cell line derived from the malignant ascites of a chemonaive ovarian cancer patient, as a model to assess the effect of ascites on migration potential using an in vitro wound-healing assay. A recent report of an invasion assay described the effect of ascites on the invasion potential of the OV-90 cell line. Ascites sampled from 31 ovarian cancer patients were tested and compared with either 5% fetal bovine serum or no serum for their nonstimulatory or stimulatory effect on the migration potential of the OV-90 cell line. A supervised analysis of data generated by the Affymetrix HG-U133A GeneChip identified differentially expressed genes from OV-90 cells exposed to ascites that had either a nonstimulatory or a stimulatory effect on migration. Ten genes (IRS2, CTSD, NRAS, MLXIP, HMGCR, LAMP1, ETS2, NID1, SMARCD1, and CD44) were upregulated in OV-90 cells exposed to ascites, allowing a nonstimulatory effect on cell migration. These findings were validated by quantitative polymerase chain reaction. In addition, the gene expression of IRS2 and MLXIP each correlated with prognosis when their expression was assessed in an independent set of primary cultures established from ovarian ascites. This study revealed novel candidates that may play a role in ovarian cancer cell migration.     

Subtype specific elevated expression of hyaluronidase-1 (HYAL-1) in epithelial ovarian cancer

Background

Epithelial ovarian cancer (EOC) is morphologically heterogeneous being classified as serous, endometrioid, clear cell, or mucinous. Molecular genetic analysis has suggested a role for tumor suppressor genes located at chromosome 3p in serous EOC pathogenesis. Our objective was to evaluate the expression of HYAL1, located at chromosome 3p21.3, in these EOC subtypes, and to investigate its correlation with the expression of steroid hormone receptors.

Methodology/Principal Findings

We determined the mRNA expression of HYAL1, estrogen receptor (ER)-α, ERβ and progesterone receptor (PR) in EOC tumor samples and cell lines using quantitative RT-PCR. We also examined the expression of these genes in a publicly available microarray dataset. HYAL-1 enzyme activity was measured in EOC cell lines and in plasma samples from patients. We found that HYAL1 mRNA expression was elevated in clear cell and mucinous EOC tissue samples, but not in serous and endometrioid samples, normal ovaries or benign tumors. Similar results were obtained by two different techniques and with tissue sample cohorts from two independent institutions. Concordantly, HYAL1 mRNA levels and enzymatic activity were elevated only in EOC cell lines derived from clear cell and mucinous subtypes. We also showed that HYAL1 mRNA was inversely correlated to that of ERα specifically in clear cell and mucinous EOCs. Additionally, ectopic expression of ERα in a clear cell EOC cell line (ER- and PR-negative) induced 50% reduction of HYAL1 mRNA expression, supporting a role of ERα in HYAL1 gene regulation. Significantly, HYAL-1 activity was also high in the plasma of patients with these EOC subtypes.

Conclusions/Significance

This is the first report showing high HYAL-1 levels in EOC and demonstrating HYAL1 gene repression by ERα. Our results identify Hyaluronidase-1 as a potential target/biomarker for clear cell and mucinous EOCs and especially in tumors with low ERα levels.     

Long non-coding RNA LINC01215 promotes epithelial-mesenchymal transition and lymph node metastasis in epithelial ovarian cancer through RUNX3 promoter methylation

Epithelial ovarian cancer (EOC) still remains the most lethal gynaecological malignancy in women, despite the recent progress in the management, including surgery and chemotherapy. According to the microarray data of the {"type":"entrez-geo","attrs":{"text":"GSE18520","term_id":"18520"}}GSE18520 and {"type":"entrez-geo","attrs":{"text":"GSE54388","term_id":"54388"}}GSE54388 datasets, LINC01215 was identified as an upregulated long noncoding RNA (lncRNA) in EOC. Therefore, this study aimed to figure out the involvement of LINC01215 in the progression of EOC. RT-qPCR was conducted to select the EOC cell line with the highest expression of LINC01215. Methylation of RUNX3 was then examined in EOC cells by MS-PCR. Furthermore, the interaction between LINC01215 and methylation-related proteins was revealed according to the results of RIP and RNA pull down assays. Subsequently, the involvement of LINC01215 and RUNX3 in regulating biological behaviors of EOC cells was investigated. Finally, the effects of the ectopic expression of LINC01215 and RUNX3 on the tumor formation and lymph node metastasis (LNM) of EOC cells were assessed in the xenograft tumors of nude mice. Overexpressing LINC01215 contributed to downregulated levels of RUNX3, as demonstrated by the recruitment of methylation-related proteins. Silencing of LINC01215 elevated the expression of RUNX3, thus suppressing cell proliferation, migration, invasion and EMT and decreasing the expressions of MMP-2, MMP-9 and Vimentin, but increased the expression of E-cadherin. The tumor growth and LNM were suppressed by downregulated levels of LINC01215 through inducing the expression of RUNX3. Collectively, the down-regulating LINC01215 could upregulate the expression of RUNX3 by promoting its methylation, thus suppressing EOC cell proliferation, migration and invasion, EMT, tumor growth and LNM.     

MicroRNA-7 Inhibits Tumor Metastasis and Reverses Epithelial-Mesenchymal Transition through AKT/ERK1/2 Inactivation by Targeting EGFR in Epithelial Ovarian Cancer

Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.     

Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion

Background

Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC.

Methodology/Principal Findings

A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration.

Conclusion/Significance

Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.     

Identification of tumor suppressors and oncogenes from genomic and epigenetic features in ovarian cancer

The identification of genetic and epigenetic alterations from primary tumor cells has become a common method to identify genes critical to the development and progression of cancer. We seek to identify those genetic and epigenetic aberrations that have the most impact on gene function within the tumor. First, we perform a bioinformatic analysis of copy number variation (CNV) and DNA methylation covering the genetic landscape of ovarian cancer tumor cells. We separately examined CNV and DNA methylation for 42 primary serous ovarian cancer samples using MOMA-ROMA assays and 379 tumor samples analyzed by The Cancer Genome Atlas. We have identified 346 genes with significant deletions or amplifications among the tumor samples. Utilizing associated gene expression data we predict 156 genes with altered copy number and correlated changes in expression. Among these genes CCNE1, POP4, UQCRB, PHF20L1 and C19orf2 were identified within both data sets. We were specifically interested in copy number variation as our base genomic property in the prediction of tumor suppressors and oncogenes in the altered ovarian tumor. We therefore identify changes in DNA methylation and expression for all amplified and deleted genes. We statistically define tumor suppressor and oncogenic features for these modalities and perform a correlation analysis with expression. We predicted 611 potential oncogenes and tumor suppressors candidates by integrating these data types. Genes with a strong correlation for methylation dependent expression changes exhibited at varying copy number aberrations include CDCA8, ATAD2, CDKN2A, RAB25, AURKA, BOP1 and EIF2C3. We provide copy number variation and DNA methylation analysis for over 11,500 individual genes covering the genetic landscape of ovarian cancer tumors. We show the extent of genomic and epigenetic alterations for known tumor suppressors and oncogenes and also use these defined features to identify potential ovarian cancer gene candidates.     

High neuropilin and tolloid‐like 1 expression associated with metastasis and poor survival in epithelial ovarian cancer via regulation of actin cytoskeleton

Abnormal expression of neuropilin and tolloid‐like 1 (NETO1) has been detected in some human carcinomas. However, the expression of NETO1 and the underlying mechanism in epithelial ovarian cancer (EOC) remain unknown. In this study, we found that a higher NETO1 expression in EOC tissue samples compared to normal ovarian tissue samples was significantly correlated with worse overall survival. Additionally, Cox regression analysis suggested that NETO 1 was independently associated with overall survival. NETO1 overexpression enhanced the EOC cells’ migration and invasion capability in vitro via regulation of actin cytoskeleton. Mechanistically, silencing NETO1 reduced the expression of β‐tubulin, F‐actin and KIF2A. In conclusion, our results demonstrated the critical role of NETO1 in EOC invasion, and therapies aimed at inhibiting its expression or activity might significantly control EOC growth, invasion and metastatic dissemination.     

β-hCG promotes epithelial ovarian cancer metastasis through ERK/MMP2 signaling pathway

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, with typically extensive intraperitoneal implantation leading to poor prognosis. Our previous study preliminarily demonstrated β-hCG can promote tumorigenesis in immortalized nontumorigenic ovarian epithelial cells. In this study, the roles and mechanisms of β-hCG in regulating EOC proliferation and metastasis were thoroughly explored. First, histologically, β-hCG was aberrantly overexpressed in human EOC metastatic tissues, and significantly correlated with FIGO stage, tumor size, differentiation, histologic grade and high grade serous ovarian carcinoma (HGSOC) (P < 0.05). However, serologically, β-hCG expression showed no significant difference between EOC and nonmalignant ovarian patients. Second, β-hCG was confirmed to have no significant effects on EOC proliferation in vitro and in vivo, while β-hCG upregulation was proven to promote migration and invasion ability in ES-2 and OVCAR-3 cells in vitro (P < 0.05), and β-hCG downregulation in SKOV3 cells had the opposite effect. Moreover, more invadopodia protrusions, mitochondria accumulations and cytoskeletal rearrangements were observed in β-hCG-overexpressing ES-2 cells, while β-hCG-depleted SKOV3 cells produced the opposite effect. Furthermore, β-hCG was confirmed to clearly facilitate intraperitoneal metastasis in nude mouse orthotopic ovarian xenograft models. Importantly, these effects of β-hCG were mediated by activation of the ERK/MMP2 signaling pathway, independently of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) presence, and inhibition the pathway with the p-ERK1/2 inhibitor SCH772984 significantly impaired the tumor-promoting effects induced by β-hCG. Collectively, these data provide new insight into the roles and mechanisms of β-hCG in regulating EOC metastasis through ERK/MMP2 signaling pathway and may become a new target for therapeutic intervention.     

Identification of Ovarian Cancer Metastatic miRNAs

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2–4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.