Ovarian Epithelial Cells Research Articles

Abstract C216: Small molecule antitubulin drugs that evade multiple drug resistance mechanisms.

Abstract Background: A new library of small molecules with structural features resembling combretastatin analogs was synthesized and two analogues were chosen by the NCI for screening. Both agents exhibited activity in all NCI-60 panel cell lines with GI50 values <10–100 nM. Based on structural characteristics of similar drugs we hypothesized that the cytotoxic activity was due to interaction with tubulin. Furthermore, these compounds appeared to overcome P-glycoprotein (P-gp) mediated resistance as they were equipotent in OVCAR8 and NCI/ADR-Res cells. Given that antitubulin drugs are among the most effective agents for the treatment of advanced prostate cancer we sought to validate the the NCI-60 panel results using prostate cancer cell lines and to explore the molecular mechanism of their cytotoxic action. Methods: Biochemical assays to assess the interaction with tubulin were carried out using purified tubulin. Immunofluorescence staining for beta-tubulin was also evaluated in PC3 cells following drug treatment. Vinblastine and docetaxel were used to demonstrate tubulin destabilizing and stabilizing modes of action, respectively. Cytotoxicity assays (72 hour GI50) were carried out in PC3, DU145, LNCaP and C4/2 prostate cancer cells and in PC3-DR cells (docetaxel resistant PC3 cells). To assess the effect of P-gp, we used NCI/ADR-Res ovarian cancer cells and L-MDR1 porcine epithelial cells and their non-expressing parent lines. Assays were performed in 96-well plates using AlamarBlue cell viability dye. Cell-cycle analysis was performed using propidium iodide staining and flow activated cell sorting. Western blot analyses were used to assess the effect of drug treatment on apoptotic pathway and on cell cycle proteins. Results: The GI50 in all cell lines was in the 1–10 nM range with no apparent resistance in P-gp expressing cells or in docetaxel resistant PC3-DR cells. The lead combretastatin analog showed a concentration dependent antitubulin interaction with a mode of action similar to vinblastine. This suggests that these compounds act by interfering with the microtubule polymerization process. Immunofluorescence staining for beta-tubulin showed diffuse staining patterns with drug increased concentration suggesting the lack of microtubule formation. Cell-cycle analysis demonstrated a G2/M cell cycle accumulation after 24-hr treatment with drug. Interestingly, there was no drug dependent effect on the number of cells in the sub-G1 fraction suggesting that the mode of cell death may be different than apoptosis. This was corroborated with the lack of caspase activation as well as the lack of cleaved-PARP. A concentration dependent p-Bcl2 was noted, however, suggesting that the drug interferes with the anti-apoptotic role of Bcl-2. Nuclear staining with Hoechst 33342 and DAPI demonstrated the presence of multinucleated cells suggesting that mitotic catastrophe could be the primary mode of cell death. This was further corroborated by the concentration dependent centrosome overduplication and presence of multiple spindle poles. Conclusion: These exquisitely potent antitubulin agents appear to overcome multiple modes of drug resistance including P-glycoprotein, p-53 deregulation, and Bcl-2. Further studies are ongoing to verify the exact molecular mechanism mediating cell death and to determine the in-vivo activity of these novel compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C216.

Melatonin synergistically enhances cisplatin‐induced apoptosis via the dephosphorylation of ERK/p90 ribosomal S6 kinase/heat shock protein 27 in SK‐OV‐3 cells

To evaluate melatonin's ability to enhance ovarian cancer cells to cisplatin treatment for ovarian cancer, this study was performed. Melatonin by itself had no significant cytotoxicity against SK-OV-3 cells, while cisplatin suppressed the cell viability in a dose-dependent manner. Combined treatment with cisplatin and melatonin synergistically inhibited the viability of SK-OV-3 cells with the synergism between two drugs (1 > combination index). In contrast, melatonin revealed the protective effect against cisplatin-induced cytotoxicity in OSEN normal ovarian epithelial cells. Cotreatment with cisplatin and melatonin increased the sub-G1 DNA contents and TdT-mediated dUTP nick end-labeling (TUNEL)-positive cells compared with cisplatin control in SK-OV-3 cells, suggesting that melatonin augments cisplatin-induced apoptosis. Consistently, combined treatment of cisplatin and melatonin increased the cleavage of caspase-3 and poly-(ADP-ribose) polymerase (PARP). Importantly, melatonin synergistically inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) along with dephosphorylation of 90-kDa ribosomal S6 kinase (p90RSK) and heat shock protein 27 (HSP27) induced by cisplatin. Furthermore, melatonin remarkably blocked the expression and colocalization of p90RSK and HSP27 by combination treatment with cisplatin. Taken together, our findings demonstrate that melatonin enhances cisplatin-induced apoptosis via the inactivation of ERK/p90RSK/HSP27 cascade in SK-OV-3 cells as a potent synergist to cisplatin treatment.

Ginkgo may prevent genetic-associated ovarian cancer risk

Women carrying BRCA1 mutations have a higher risk of developing ovarian cancers. Options to reduce this risk are largely limited to prophylactic surgery, which leads to a decrease in the quality of life and permanently damages fertility. There is a obvious and an urgent need to identify a noninvasive approach to effectively prevent the ovarian cancer risk, specifically for those women of reproductive age. Our previous studies showed that the use of the herbal remedy Ginkgo biloba may reduce the risk for nonmucinous ovarian cancer. Here, we explored whether Ginkgo biloba might also be an effective agent to reduce BRCA1-associated ovarian cancer (always serous-type) risk. A human ovarian surface epithelial cell line-636 was developed from a BRCA1-mutant carrier. Cells were treated with ginkgolide B (GB) or dimethyl sulfoxide, and protein lysates from the cells were applied to antibody microarrays to determine upregulated or downregulated protein expression patterns. Anticancer activities and the associated networking pathways with the altered proteins were analyzed by using the Pathway Studio software. After GB treatment, 28 proteins were shown to be consistently upregulated (1.5-15.5-fold), and 22 proteins were downregulated (1.5-28.3-fold). Bioinformatics software analysis indicated that multiple mechanisms and signal pathways are involved in anticancer activities in BRCA1-mutant cells induced by GB treatment. These pathways include cell proliferation, tumor suppression, and DNA damage repair. Our study suggested that GB found in the herbal Ginkgo biloba may have cancer-preventive activities in BRCA1-mutant ovarian epithelial cells.

5-AZA-2'-deoxycytidine induced demethylation influences N-glycosylation of secreted glycoproteins in ovarian cancer

Glycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stably transmitted to daughter cells without the requirement for genetic sequence alterations. Aberrant regulation of both epigenetic programming and glycosylation patterning are integral aspects of carcinogenesis. The objective of this study was to determine the interplay between these two complex cellular processes. We demonstrate that global DNA methylation changes in ovarian cancer epithelial cells (OVCAR3) resulted in significant alterations in the glycosylation of secreted glycoproteins. These changes included a reduction in core fucosylation, increased branching and increased sialylation. We further show that the change in core fucose levels was mirrored by altered expression of GMDS and FX, key enzymes in fucose biosynthesis. Alterations in the expression of key glycosyltransferase enzymes such as MGAT5 reflect the changes seen in the branching and sialylation of secreted glycans. Overall, our results highlight that modifications to the epigenetic machinery have a profound effect on the glycan structures generated by cells, which may be a key step in understanding metastasis and drug resistance during cancer progression.

The In Vitro Antioxidant and Anti-Proliferative Effect and Induction of Phase II Enzymes by a Mistletoe (Viscum Album ) Extract

The increasing interest for phytochemical antioxidants, found in many medicinal plants, is demonstrated experimentally by epidemiological and in vitro studies which show their positive effects on human health. Mistletoe contains therapeutically active ingredients which incubated with several human cancer cell lines revealed cytotoxicity for the cancer cells, but no evidence is available for their chemoprotective properties. We investigated in vitro the potential of extracts from Viscum album growing on Mallus domestica (VAM) to modulate the activity of phase II detoxifying enzymes using the gluthatione S-transferase (GST) and quinone oxidoreductase (QR) assays and to inhibition of adherent epithelial human ovarian tumor cells A2780 proliferation using the MTT assay. We found that different concentrations of VaM extract had inhibitory effects on the growth of adherent epithelial human ovarian tumor cells A2780 (IC50 = 120.62 µg/ml, after 48 h treatment). Also, this extract is a good antioxidant source (total phenol content of 91.51 mg gallic acid equivalents /g dry weight), having a radical scavenging ability and a reducing potential proportional to the dose used (from 0.312 to 5 mg/ml) in vitro. At 120 µg/ml, this extract increased, significantly, the activity of GST and QR, as well the level of GSH.

The Role of HE4 in Ovarian Cancer: Inhibiting Tumour Cell Proliferation and Metastasis

As a promising biomarker, human epididymis protein 4 (HE4) has been widely used for the early detection and differential diagnosis of ovarian cancer. This study evaluated the function of HE4 in the carcinogenesis and progression of ovarian cancer. An enzyme immunometric assay, used to detect HE4 in the serum of ovarian cancer patients, showed that the protein could discriminate between malignant and benign ovarian tumours with high specificity. An exogenous HE4 gene was transfected into ovarian cancer cell lines and an immortalized ovarian epithelial cell line. Compared with the controls, HE4 overexpression significantly promoted cell apoptosis and adhesion. Overexpression of HE4 also led to significant inhibition of cell proliferation, migration and invasiveness in vitro, as well as xenograft tumour formation in vivo. This is the first report to demonstrate the functional importance of HE4 in multiple cellular processes and indicates that HE4 may play a protective role in the progression of ovarian cancer.

Transcription Factor DLX5 As a New Target for Promising Antitumor Agents

The crystal structure of the human transcription factor DLX5 has been used for the screening of a library consisting of 10(6 )compounds by the molecular docking technique.In vitro testsof the 14 top-rated ligands showed that compound Q12 displays the best ability to inhibit the proliferation ofDlx5 positive mouse lymphoma cells, which correlates with the down-regulation ofc-mycexpression. Compound Q12 has low toxicity on normal human ovarian epithelial cells and mouse lymphoma cells with absent expression ofDlx5, and can be used for further chemical optimization and for the development of novel, highly efficient cancer treatments.

Ovarian clear cell carcinoma—bad endometriosis or bad endometrium?

It has become increasingly clear that the four main histological subtypes of epithelial ovarian cancer (EOC), high-grade serous, endometrioid, clear cell and mucinous, are entities with different epidemiologies, clinical presentations, responses to treatment, and ultimate outcomes. In fact, for all intents and purposes, they can be considered different diseases, their only common denominator being that they frequently involve the ovary and pelvic organs. However, clinical practice has not caught up with these insights and the treatment of EOC is that of a single disease entity. In part, this is because we lack detailed knowledge of the molecular mechanisms driving the pathogenesis of each disease, which is vital in order to develop therapeutic approaches against common driver events. In the last few years, mutations in ARID1A and PIK3CA have been described in a substantial fraction of cases of ovarian clear cell carcinoma, yet the paper by Yamamoto et al in this issue of The Journal of Pathology reveals that PIK3CA mutations can be detected in precursor endometriosis tissues. These and other recent observations underscore the importance of investigating whether mutations in the eutopic endometrium actually predispose to endometriosis and eventually to malignancy.

Modelling genetic and clinical heterogeneity in epithelial ovarian cancers

The biology underlying early-stage epithelial ovarian cancer (EOC) development is poorly understood. Identifying biomarkers associated with early-stage disease could have a significant impact on reducing mortality. Here, we describe establishment of a three-dimensional (3D) in vitro genetic model of EOC initiation and early-stage neoplastic progression. Normal primary ovarian epithelial (POE) cells, immortalized using hTERT (immortalised ovarian epithelial [IOE] cells), were partially transformed by overexpressing the CMYC oncogene (IOE(CMYC) cells). Subsequent expression of mutant alleles of KRAS (KRAS(G12V)) or BRAF (BRAF(V600E)) created double-mutant lines (IOE(CMYC.KRAS) and IOE(CMYC.BRAF)). The transformed phenotype of IOE(CMYC) cells was further enhanced in concert with KRAS(G12V)/BRAF(V600E) expression, as in vitro analyses indicated that IOE(CMYC) cells had undergone morphological and phenotypic changes characteristic of neoplastic progression. When cultured as 3D spheroids, IOE cells underwent growth arrest, reminiscent of nonproliferative, unstimulated POE in vivo. In contrast, IOSE(CMYC+BRAF/KRAS) cells formed highly proliferative, poly-aggregate spheroid structures, showing increased expression of the Wilms tumour 1 tumourigenic marker and MIB1 proliferation marker. Transcriptomic analyses identified different gene expression profiles between the different cell lines and novel candidate genes (e.g. RGS4, CTGF and THBS1) that are somatically altered in EOCs. Gene expression signatures were compared with signatures from primary EOCs; tumours with IOE(CMYC) 'like' signatures were more likely to be high grade (P = 0.018); tumours with BRAF signatures were associated with improved relapse-free survival (P = 0.003). In conclusion, we have established in vitro 3D models of early-stage EOCs, which reflect genetic and phenotypic heterogeneity of the disease. Molecular genetic characteristics of these models correlated with molecular and clinical features of primary EOCs.

Downregulation of Filamin A Interacting Protein 1-Like is Associated with Promoter Methylation and Induces an Invasive Phenotype in Ovarian Cancer

Ovarian cancer is the most lethal gynecologic malignancy with a five-year survival rate below 25% for patients with stages III and IV disease. Identifying key mediators of ovarian cancer invasion and metastasis is critical to the development of more effective therapeutic interventions. We previously identified Filamin A interacting protein 1-like (FILIP1L) as an important mediator of cell proliferation and migration. In addition, targeted expression of FILIP1L in tumors inhibited tumor growth in vivo. In our present study, we confirmed that both mRNA and protein expression of FILIP1L were downregulated in ovarian cancer cells compared with normal ovarian epithelial cells. FILIP1L expression was inversely correlated with the invasive potential of ovarian cancer cell lines and clinical ovarian cancer specimens. We also provide evidence that DNA methylation is a mechanism by which FILIP1L is downregulated in ovarian cancer. The CpG island in the FILIP1L promoter was heavily methylated in ovarian cancer cells. Methylation status of the FILIP1L promoter was inversely correlated with FILIP1L expression in ovarian cell lines and clinical ovarian specimens. Reduced methylation in the FILIP1L promoter following treatment with a DNA demethylating agent was associated with restoration of FILIP1L expression in ovarian cancer cells. A transcription activator, cAMP-responsive element binding protein (CREB) was shown to bind to the CREB/ATF site in the CpG island of the FILIP1L promoter. Overall, these findings suggest that downregulation of FILIP1L associated with DNA methylation is related with the invasive phenotype in ovarian cancer and that modulation of FILIP1L expression has the potential to be a target for ovarian cancer therapy.

Wnt and Hedgehog Gene Pathway Expression in Serous Ovarian Cancer

Ovarian cancer has very heterogeneous histological classification, and response to therapy of the same grade and type varies. We studied genes in the Wnt and hedgehog (Hh) pathways, which are essential for embryonic development and which play critical roles in proliferation in a variety of human cancers. Variations in these pathway genes causing proliferation could play a role in the variation in tumor progression and response to therapy. Using real-time polymerase chain reaction, we studied 16 primary grade 3 International Federation of Gynecology and Obstetrics stage III serous ovarian cancer samples for expression of the Wnt pathway gene AXIN2, fibroblast growth factor 9, and Hh pathway gene expressions of glioma-associated oncogene 1, glioma-associated oncogene 2, patched homolog 1, patched homolog 2, Indian Hedgehog (HH), sonic HH, and Smoothened, a G protein-coupled receptor protein. Normal ovary epithelial cell line was used as control. We found wide variation of up-regulation of pathway component and target genes in the primary tumor samples and apparent cross talk between the pathways. AXIN2, a Wnt target gene, showed increased expression in all serous ovarian cancer samples. Fibroblast growth factor 9 was also overexpressed in all tumors with greater than 1000-fold increase in gene expression in 4 tumors. Expression of Hh pathway genes varied greatly. More than half of the tumor samples showed involvement of Hh signaling or pathway activation either by expression of transcription factors and Hh ligands or by overexpression of Indian HH/sonic HH and the receptor-encoding patched homolog 1/patched homolog 2. We found a wide variation in fold expression of genes involved in the Wnt and Hh pathway between patient samples.

Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

Trypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated by T. cruzi surface trans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used by T. cruzi to enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant to T. cruzi became highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore, trkC transfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive to T. cruzi after transfection with the trkC gene. Additionally, NT-3 specifically blocked T. cruzi infection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blocked T. cruzi infection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected by T. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broad T. cruzi infection both in vitro and in vivo.

Dual inhibition of SRC and Aurora kinases induces postmitotic attachment defects and cell death

Increased activity of SRC family kinases promotes tumor invasion and metastasis, and overexpression of the mitotic regulator Aurora kinase A (AURKA) drives tumor aneuploidy and chromosomal instability. These functions nominate SRC and AURKA as valuable therapeutic targets for cancer, and inhibitors for SRC and Aurora kinases are now being used in the clinic. In this study, we demonstrate potent synergy between multiple inhibitors of Aurora and SRC kinases in ovarian and colorectal cancer cell lines, but not in normal ovarian epithelial cell lines. Combination of Aurora and SRC inhibitors selectively killed cells that have undergone a preceding aberrant mitosis, and was associated with a postmitotic reattachment defect, and selective removal of aneuploid cell populations. Combined inhibition of Aurora kinase and SRC potentiated dasatinib-dependent loss of activated (Y(416)-phosphorylated) SRC. SRC and AURKA share a common interaction partner, NEDD9, which serves as a scaffolding protein with activities in cell attachment and mitotic control, suggesting SRC and AURKA might interact directly. In vitro, we observed physical interaction and mutual cross-phosphorylation between SRC and AURKA that enhanced SRC kinase activity. Together, these findings suggest that combination of SRC and Aurora-targeting inhibitors in the clinic may be a productive strategy.

Evidence for the Complexity of MicroRNA-Mediated Regulation in Ovarian Cancer: A Systems Approach

MicroRNAs (miRNAs) are short (∼22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. Previous studies have shown that miRNAs inhibit the translation and facilitate the degradation of their targeted messenger RNAs (mRNAs) making them attractive candidates for use in cancer therapy. However, the potential clinical utility of miRNAs in cancer therapy rests heavily upon our ability to understand and accurately predict the consequences of fluctuations in levels of miRNAs within the context of complex tumor cells. To evaluate the predictive power of current models, levels of miRNAs and their targeted mRNAs were measured in laser captured micro-dissected (LCM) ovarian cancer epithelial cells (CEPI) and compared with levels present in ovarian surface epithelial cells (OSE). We found that the predicted inverse correlation between changes in levels of miRNAs and levels of their mRNA targets held for only ∼11% of predicted target mRNAs. We demonstrate that this low inverse correlation between changes in levels of miRNAs and their target mRNAs in vivo is not merely an artifact of inaccurate miRNA target predictions but the likely consequence of indirect cellular processes that modulate the regulatory effects of miRNAs in vivo. Our findings underscore the complexities of miRNA-mediated regulation in vivo and the necessity of understanding the basis of these complexities in cancer cells before the therapeutic potential of miRNAs can be fully realized.

Overexpression of the β Subunit of Human Chorionic Gonadotropin Promotes the Transformation of Human Ovarian Epithelial Cells and Ovarian Tumorigenesis

Ovarian carcinoma is the most lethal gynecologic malignancy, however underlying molecular events remain elusive. Expression of human chorionic gonadotropin β subunit (β-hCG) is clinically significant for both trophoblastic and nontrophoblastic cancers; however, whether β-hCG facilitates ovarian epithelial cell tumorigenic potential remains uncharacterized. Immortalized nontumorigenic ovarian epithelial T29 and T80 cells stably overexpressing β-hCG were examined for alterations in cell cycle and apoptotic status by flow cytometry, expression of proteins regulating cell cycle and apoptosis by Western blot, proliferation status by MTT assay, anchorage-independent colony formation, and mouse tumor formation. Immunoreactivity for β-hCG was evaluated using mouse xenografts and on human normal ovarian, fallopian tube, endometrium, and ovarian carcinoma tissues. T29 and T80 cells overexpressing β-hCG demonstrated significantly increased proliferation, anchorage-independent colony formation, prosurvival Bcl-X(L) protein expression, G2-checkpoint progression, elevated cyclins E/D1 and Cdk 2/4/6, and decreased apoptosis. Collectively, these transformational alterations in phenotype facilitated increased xenograft tumorigenesis (P < 0.05). Furthermore, β-hCG immunoreactivity was elevated in malignant ovarian tumors, compared with normal epithelial expression in ovaries, fallopian tube, and endometrium (P < 0.001). Our data indicate that elevated β-hCG transforms ovarian surface epithelial cells, facilitating proliferation, cell cycle progression, and attenuated apoptosis to promote tumorigenesis. Our results further decipher the functional role and molecular mechanism of β-hCG in ovarian carcinoma. β-hCG may contribute to ovarian cancer etiology, which introduces a new therapeutic intervention target for ovarian cancer.

Pituitary tumor transforming gene induces epithelial to mesenchymal transition by regulation of Twist, Snail, Slug, and E-cadherin

Pituitary tumor-transforming gene (PTTG) is an oncogene with its expression levels correlating with tumor development and metastasis. Epithelial to mesenchymal transition (EMT) is a crucial step in tumor progression and metastasis. Using ovarian epithelial tumor cell line (A2780) for loss-of-function or gain-of-function of PTTG in our experiments, we observed up regulation of TGF-β, Twist, Snail, Slug, vimentin and down regulation of E-cadherin on infection of cells with Ad-PTTG cDNA. In contrast reverse phenomena was observed on depletion of PTTG on infection of cells with Ad-PTTG siRNA, suggesting an important role of PTTG in induction of EMT in ovarian cancer cells.

Novel antagonists of growth hormone‐releasing hormone inhibit growth and vascularization of human experimental ovarian cancers

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancer cell lines and experimental tumors by mechanisms that include direct action on GHRH receptors in cancer cells. In this study, the effects of newly synthesized GHRH antagonists, MIA-313, MIA-602, MIA-604, and MIA-610, were investigated in 2 human ovarian epithelial adenocarcinoma cell lines, OVCAR-3 and SKOV-3, in vitro and in vivo. The expression of receptors for GHRH was demonstrated by Western blot analysis and ligand competition methods in the OVCAR-3 and SKOV-3 cell lines and in tumors from those cells grown in athymic nude mice. The effects of GHRH antagonists on the secretion of vascular endothelial growth factor (VEGF) by OVCAR-3 cells and on the vascularization of OVCAR-3 xenografts also were evaluated. Both the pituitary and the splice variant type 1 (SV1) GHRH receptors were detected in the 2 cell lines and in tumor xenografts, and SV1 was expressed at higher levels. Cell viability assays revealed the antiproliferative effect of all GHRH antagonists that were. Maximal tumor growth inhibition was approximately 75% in both models. MIA-313 and MIA-602 decreased VEGF secretion of OVCAR-3 cells, as measured by enzyme-linked immunosorbent assay, and reduced tumor vascularization in a Matrigel plug assay, but caused no change in the expression of VEGF or VEGF receptor in the terminal ileum of mice with OVCAR-3 tumors. Results from the current study indicated that a he novel approach based on GHRH antagonists may offer more effective therapeutic alternatives for patients with advanced ovarian cancer and who do not tolerate conventional anti-VEGF therapy.

Identification of the Chemokine CX3CL1 as a New Regulator of Malignant Cell Proliferation in Epithelial Ovarian Cancer

BackgroundLittle is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX3CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX3CL1 in EOC.ResultsEpithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX3CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX3CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX3CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX3CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX3CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX3CL1 products. Conversely, CX3CL1 promoted through its binding to CX3CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX3CL1 and faster tumor growth.ConclusionOur findings highlight the previously unappreciated constitutive expression of CX3CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients.

RhoGDI2 antagonizes ovarian carcinoma growth, invasion and metastasis

Previous studies described functional roles for Rho GDP dissociation inhibitor 2 (RhoGDI2) in bladder, gastric and breast cancers. However, only limited expression and no functional analyses have been done for RhoGDI2 in ovarian cancer. We determined RhoGDI2 protein expression and function in ovarian cancer. First, western blot analysis was performed to determine the expression levels of RhoGDI2 in ovarian cells lines. RhoGDI2 but not RhoGDI1 protein expression levels varied widely in ovarian carcinoma cell lines, with elevated levels seen in Ras-transformed ovarian epithelial cells. Next, immunohistochemistry was performed to detect RhoGDI2 expression in patient samples of ovarian cysts and ovarian cancer with known histological subtype, stage, grade and outcome. RhoGDI2 protein was significantly overexpressed in high-grade compared to low-grade ovarian cancers, correlated with histological subtype, and did not correlate with stage of ovarian cancer nor between carcinomas and benign cysts. Unexpectedly, stable suppression of RhoGDI2 protein expression in HeyA8 ovarian cancer cells increased anchorage-independent growth and Matrigel invasion in vitro and in tail-vein lung colony metastatic growth in vivo. Finally, we found that RhoGDI2 stably-associated preferentially with Rac1 and suppression of RhoGDI2 expression resulted in decreased Rac1 activity and Rac-associated JNK and p38 mitogen-activated protein kinase signaling. RhoGDI2 antagonizes the invasive and metastatic phenotype of HeyA8 ovarian cancer cells. In summary, our results suggest significant cell context differences in RhoGDI2 function in cancer cell growth.

WNT7A Regulates Tumor Growth and Development Through the WNT/CTNNB1 Pathway in Ovarian Cancer.

The canonical WNT signaling pathway regulates embryonic ovarian development as well as normal ovarian function and folliculogenesis by governing cellular proliferation and differentiation. Alterations in the WNT system have been implicated in ovarian cancer, however, the contribution of specific ligand partners associated with WNT signaling to ovarian tumorigenesis have not been elucidated. The goal of the current study was to examine the role and functional mechanisms of WNT7A in ovarian cancer. WNT7A was expressed in 40-60 % of high grade serous ovarian malignant tumors, and 20-30 % of mucinous and clear cell adenocarcinomas, but was not detected in borderline/benign tumors, normal ovary or endometrioid carcinomas. Further, WNT7A was detected in only the highly invasive SKOV3.ip1 cell line in a cohort of normal and malignant ovarian epithelial cells. Using lentiviral transfection of shRNA, WNT7A was silenced in SKOV3.ip cells and two subclones, ip11 and ip12, were selected for further study after confirmation of WNT7A mRNA and protein suppression. To characterize the role of WNT7A in advanced ovarian cancer, nude mice were injected with SKOV3.ip1, ip11, and ip12 intraperitoneally or subcutaneously. In the i.p. group, mice receiving ip11 and ip12 cells had significantly lower tumor burden and implanted tumor number. While mice injected with SKOV3.ip1 parental cells failed to gain weight during the experiment (a sign of advanced illness), those with ip11 and ip12 cells displayed increased body weight throughout the study. Gross and histological examination further revealed greatly reduced invasion of ip11 and ip12 cells into intestinal mesentery and serosa compared to SKOV3.ip parental cells. Tumor growth of ip11 and ip12 was significantly decreased compared to SKOV3.ip controls in mice given s.c. injection as well, and western blot analysis of homogenates verified sustained reduction of WNT7A expression in ip11 and ip12 tumors. Immunohistochemical staining of s.c. tumors showed decreased expression of cyclin D1, a WNT7A transcriptional target and regulator of cell cycle and proliferation, in ip11 and ip12 tumors compared to SKOV3.ip1. In vitro analysis of cell function revealed significantly reduced proliferation, adhesion, and migration, as well as cell cycle arrest in ip11 and ip12 cells compared to SKOV3.ip1 parental cells. Further, expression of MMP7, a transcriptional target of WNT7A involved in tumor invasion, was reduced in ip11 and ip12 cells compared to SKOV3.ip1 cells. These results were verified by stable overexpression of WNT7A in ovarian cancer cells which lack endogenous WNT7A. Increased proliferation, adhesion, and migration were observed in these cells. Activation of the TopFlash (TCF/LEF) reporter confirmed that transfection of WNT7A activates the canonical WNT/CTNNB1 pathway. Further, co-transfection with FZD5 (WNT receptor) produced an even greater increase in TopFlash reporter activity, and this effect was inhibited by co-transfection with the WNT receptor antagonist SFRP2. These results indicate that WNT7A is a critical regulator of ovarian cancer tumorigenesis and disease progression mediated by intracellular signaling through the WNT/CTNNB1 pathway. Supported by American Cancer Society Illinois Division #139038. (platform)