Progression Of Human Ovarian Cancer Research Articles

Targeting Gonadotropin-releasing Hormone Receptor Inhibits the Early Step of Ovarian Cancer Metastasis by Modulating Tumor-mesothelial Adhesion

Ovarian cancer has a clear predilection to metastasize to the peritoneum, which represents one of the most important prognostic factors of poor clinical outcome. Gonadotropin-releasing hormone (GnRH) receptor is significantly overexpressed during the malignant progression of human ovarian cancer. Here, using lentiviral-based small interfering RNA (siRNA) technology to downregulate GnRH receptor in metastatic ovarian cancer cells, we show that GnRH receptor is an important mediator of ovarian cancer peritoneal metastasis. GnRH receptor downregulation dramatically attenuated their adhesion to the peritoneal mesothelium. By inhibiting the expression of GnRH receptor, we showed decreased expression of α2β1 and α5β1 integrin and adhesion to specific extracellular matrix (ECM) proteins. This was also associated with a reduction of P-cadherin. Furthermore, adhesion of ovarian cancer cells to different ECMs and the mesothelium were abrogated in response to β1 integrin and P-cadherin reduction, confirming that the effects were β1 integrin- and P-cadherin-specific. Using a mouse model of human ovarian cancer metastasis, we found that the inhibition of GnRH receptor, β1 integrin, and P-cadherin significantly attenuated tumor growth, ascites formation, and the number of metastatic implants. These results define a new role for GnRH receptor in early metastasis and offer the possibility of novel therapeutic targets.

Discovery of serum biomarkers implicated in the onset and progression of serous ovarian cancer in a rat model using iTRAQ technique

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, and early tumor detection is the most promising approach for improving the EOC survival rate. The goal of this study was to identify the biomarkers underlying ovarian carcinogenesis. To mimic the onset and progression of human ovarian cancer, we established a rat model of ovarian neoplasm by implanting 7,12-dimethylbenz(a)anthracene (DMBA)-coated silk cloth strips onto the ovaries. Sera collected from rats bearing serous ovarian carcinoma (SOC) at baseline, 12 and 24 weeks after DMBA treatment and from controls were analyzed using iTRAQ combined with two-dimensional liquid chromatography and tandem mass spectrometry. The data were analyzed with ProteinPilot software for peptide matching, protein identification, and protein quantitation. Ingenuity pathway analysis software was used to identify the canonical pathways and biological interaction networks of differentially expressed proteins. The cumulative ovarian tumor incidence rate reached 75% at 32 weeks after DMBA treatment. Out of all tumors, 94% were EOC, and 51% of the EOC cases were SOC. A total of 225 unique, non-redundant proteins were identified with 95% confidence. Twenty-seven differentially expressed proteins were significantly up- or down-regulated during the early or advanced carcinogenesis of SOC. Fifteen proteins were previously reported to be involved in ovarian cancer, and 12 proteins, including MMRN1, SERPINC1, TLN1, AHSG, PLG, APOA2, HPX, APOC1, APOC2, FERMT3, FETUB and HBB, were identified for the first time in our study. The discovery of these differentially expressed proteins provides valuable clues for understanding the molecular mechanism underlying the dynamic carcinogenic process of ovarian cancer. These proteins could be used as diagnostic biomarkers for early detection, disease monitoring and therapeutic targets.

AT-406, an orally active antagonist of multiple inhibitor of apoptosis proteins, inhibits progression of human ovarian cancer

Ovarian carcinoma is the most deadly gynecological malignancy. Current chemotherapeutic drugs are only transiently effective and patients with advance disease often develop resistance despite significant initial responses. Mounting evidence suggests that anti-apoptotic proteins, including those of the inhibitor of apoptosis protein (IAP) family, play important roles in the chemoresistance. There has been a recent emergence of compounds that block the IAP functions. Here, we evaluated AT-406, a novel and orally active antagonist of multiple IAP proteins, in ovarian cancer cells as a single agent and in the combination with carboplatin for therapeutic efficacy and mechanism of action. We demonstrate that AT-406 has significant single agent activity in 60% of human ovarian cancer cell lines examined in vitro and inhibits ovarian cancer progression in vivo and that 3 out of 5 carboplatin-resistant cell lines are sensitive to AT-406, highlighting the therapeutic potential of AT-406 for patients with inherent or acquired platinum resistance. Additionally, our in vivo studies show that AT-406 enhances the carboplatin-induced ovarian cancer cell death and increases survival of the experimental mice, suggesting that AT-406 sensitizes the response of these cells to carboplatin. Mechanistically, we demonstrate that AT-406 induced apoptosis is correlated with its ability to down-regulate XIAP whereas AT-406 induces cIAP1 degradation in both AT-406 sensitive and resistance cell lines. Together, these results demonstrate, for the first time, the anti-ovarian cancer efficacy of AT-406 as a single agent and in the combination with carboplatin, suggesting that AT-406 has potential as a novel therapy for ovarian cancer patients, especially for patients exhibiting resistance to the platinum-based therapies.

P16 Gene Alterations in Human Ovarian Cancers

To determine the alterations of tumor suppressor gene p16INK4A in human ovarian cancers to explore the possibilities of identifying potential minimally invasive markers in blood of the patients, which could help in the clinical practice as a diagnostic and prognostic marker. Ovarian cancer tissue and corresponding blood samples were collected from patients (n = 50). The promoter methylation and mutation status of p16 gene in blood and ovarian tissue DNA was then assessed using methylation-specific polymerase chain reaction and denaturing high-performance liquid chromatography along with DNA sequencing method. In addition, the protein expression in ovarian cancer tissue samples was detected by immunostaining method using monoclonal antibodies against p16. Methylation of p16 was observed in 56% (28/50) of the cases. The data showed concordance in promoter methylation status of p16 gene between the tumor tissue and the corresponding blood DNA samples of the patients with ovarian cancer. There was a weak statistical agreement (Kendall tau b = +0.31), and a perfect correspondence was observed in 50% of the cases. The p16 mutations were comparatively low, revealing only 2 variations among the samples analyzed. The percentage of protein expression was inversely correlated with the p16 gene promoter methylation. This study demonstrates that the p16 gene plays a role in the progression of human ovarian cancers and the blood DNA methylation of p16 gene promoter region is a weak predictor of tumor tissue methylation status.

Abstract 4613: A role of integrin-associated CD151 in human ovarian cancer

Abstract Despite being implicated in multiple types of human epithelia-origin cancer, laminin-binding (LB) integrins (e.g., α3α1 and α6β4) have rarely been investigated for their roles in human ovarian tumors. More recently, we and others have shown that CD151, a member of the tetraspanin family, not only forms tight surface complexes with these adhesion receptors, but also mediates the malignancy of human carcinomas largely in a LB integrin-dependent manner. Here we report our studies on the role of CD151 in human ovarian cancer. Initially, we stained human ovarian tumor tissue microarrays with CD151-specific antibody. Our data showed that the majority of ovarian tumors exhibited a reduced expression of CD151 protein, compared to the fallopian tube, implying a putative suppressing role of this tetraspanin in ovarian cancer. With this hint we next evaluated the impact of CD151 ablation on the behaviors and proliferation of cultured human ovarian cancer cells. While CD151 removal had a minimal impact on cell motility or invasion, it markedly enhanced cell proliferation. Also, CD151-deficeint cells appeared to have reduced cell-cell contacts, suggestive of epithelial-mesenchymal transition (EMT)-like changes. However, there was no dramatic difference in the surface expression of E-cadherin protein, according to our FACS analyses. To substantiate these observations, we also injected ovarian cancer cells with or without CD151 knockdown into immuno-deficient nude mice subcutaneously or orthotopically (intra-peritoneal). Results from these ex vivo analyses showed that disruption of CD151 accelerated ovarian tumor growth and production of ascites fluid in mice, further supporting a role of CD151 in ovarian tumor progression. While tumors in the control group had homogenous epithelial-like appearance, those derived from CD151-ablated cells exhibited mucin-related vacuoles and diverse morphologies. Our subsequent DNA array and protein analyses indicated that there was indeed a marked increase in the expression of a mucin family member in CD151-deficient tumor cells. Collectively, these results provide strong evidence that CD151 suppresses human ovarian cancer progression by regulating tumor cell proliferation and extracellular microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4613. doi:1538-7445.AM2012-4613

Under-expression of the opioid growth factor receptor promotes progression of human ovarian cancer

The opioid growth factor (OGF) and its receptor, OGFr, serve as a tonically active inhibitory axis regulating the proliferation of human ovarian cancer cells. In the present study, we have investigated the repercussion on the progression of this deadly neoplasia when cells are engineered to molecularly under-express OGFr. shRNA constructs were used to knockdown OGFr in SKOV-3 cells; two clonal cell lines were examined. OGFr protein expression was decreased up to 73% in clones compared with wild-type (WT) and empty vector (EV) controls. OGFr-binding assays of clones revealed 50-55% decreases in binding capacity compared with control cells; binding affinity was comparable in all groups. Cell number in clones was increased 33-132%, and doubling times decreased 29-35%, compared with WT and EV cultures. Addition of exogenous OGF or naltrexone did not affect cell number in cultures with silenced OGFr. DNA synthesis of clonal cell lines was increased 136-146% from the WT and EV groups; no changes were noted in cell survival. Nude mice injected subcutaneously with cells under-expressing OGFr had an increased tumor incidence, decreased latency to tumor formation, increased tumor volume and decreased OGFr expression in tumors compared with WT and EV controls. OGF treatment in mice with WT or EV tumors, but not OGFr under-expressing tumors, inhibited tumor volume and weight. Collectively, these data demonstrate the critical nature of the OGF-OGFr axis as a determinant of the progression of human ovarian cancer, and suggest that attenuation of this system has an important bearing on the survival of these patients.

The expression of human epididymis protein 4 and cyclindependent kinase inhibitor p27Kip1 in human ovarian carcinoma

Background: Cyclin-dependent kinase inhibitor p27Kip1 is a new class of tumor suppressor in a dosage dependent manner to control cell cycle progression. Human epididymis protein 4 (HE4) is a potentially useful biomarker for ovarian carcinoma, comparable with cancer antigen 125 in identifying women with ovarian cancer, both localized and advanced. However, the prognostic significance of p27Kip1 and HE4 in ovarian cancer is unclear. Objective: Investigate the expression of p27Kip1 and HE4 in the progression of human ovarian cancer. Material and method: Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 61 specimens. The association between HE4 and p27Kip1expression and clinicopathological features was analyzed using ?2-test. For analysis of survival data, Kaplan-Meier curves were constructed, and the log-rank test was performed. Results: The expression of p27Kip1 negatively related with HE4 expression, but it correlated significantly with lymph nodes. On the other hand, HE4 expression correlated significantly with disease stage and lymph nodes. Kaplan-Meier analysis of the survival curves of p27Kip1 and HE4 revealed a highly significant separation between low vs. high expressers in ovarian carcinoma. Conclusion: Expression of HE4 was up-regulated significantly in human ovarian carcinoma. Overexpression of HE4 might be responsible for oncogenesis and development of ovarian carcinoma. HE4 and p27Kip1 may be of prognostic significance in human ovarian cancer. Keywords: HE4 (WFDC2), human ovarian cancer; p27Kip1, immunohistochemistry, prognosis

Expression of the Transient Receptor Potential Channel C3 Correlates with a Favorable Prognosis in Patients with Adenocarcinoma of the Lung

The cation channels of the transient receptor potential (TRP) superfamily are implicated in cancer formation; in particular, TRPC3 has been shown to contribute to the progression of human ovarian cancer. However, the relationship between TRP expression and the clinicopathological characteristics of lung cancer and patient prognosis is not well understood. Therefore, we investigated the relationship between TRP expression and the prognosis of patients with adenocarcinoma of the lung. We used semiquantitative real-time reverse transcription polymerase chain reaction to assess the expression of TRP mRNA in tumor samples from 95 patients with adenocarcinoma of the lung. We then correlated the TRP mRNA levels with clinicopathological factors. We also used immunohistochemical staining to determine the localization of expressed TRP. The 5-year overall and disease-free survival rates among patients expressing higher levels of TRPC3 mRNA were significantly better than the corresponding rates among patients expressing lower levels (P=0.004, P=0.002, respectively, by log-rank test). Multivariate Cox proportional hazard analyses revealed that tumor size (hazard ratio, 2.46; 95% confidence interval [CI], 1.06 to 5.79; P=0.036), n2 (hazard ratio, 3.81; 95% CI, 1.29 to 11.77; P=0.015) and TRPC3 (hazard ratio, 2.71; 95% CI, 1.33 to 5.59; P=0.006) were independent factors affecting the 5-year overall survival rate. Immunohistochemistry showed that the cytoplasm of tumor cells were stained positively for TRPC3. Higher levels of TRPC3 expression in tumor cells are an independent predictor of a better prognosis in patients with adenocarcinoma of the lung.

Abstract 4540: Follicle-stimulating hormone (FSH) enhances the proliferation of epithelial ovarian cancer cells by activating transient receptor potential channel C3 (TRPC3)

Abstract At present, epithelial ovarian cancer is the leading killer among all gynecological malignancies. Despite much research, development of this neoplasm is still not fully understood. The five-year survival rate for this disease still remains about 30%. Recent studies suggest that follicle-stimulating hormone (FSH) may play a major role in ovarian epithelial carcinogenesis. Our group has demonstrated that FSH stimulates the proliferation and invasion of ovarian cancer cells, inhibits apoptosis, and facilitates neovascularization by increasing vascular endothelial growth factor (VEGF) expression. To understand the mechanisms by which FSH stimulates ovarian cancer growth, we have performed gene expression arrays and confirmed changes in protein expression by Western blots. Interestingly, we found that FSH stimulation enhances expression of transient receptor potential channel C3 (TRPC3) at both mRNA and protein levels. Silencing TRPC3 expression by siRNA inhibited the ability of FSH to stimulate proliferation of ovarian cancer cell lines and to block apoptosis. TRPC channels are nonselective cation channels with permeability to Ca2+. Increases in cytosolic free Ca2+ can promote different physiological changes including cell growth, differentiation and death. TRPC3 is one of 7 TRPC family members. Our previous work has shown that TRPC3 contributes to the progression of human ovarian cancer. TRPC3 protein levels in human ovarian cancer specimens were greatly increased relative to those in normal ovarian specimens. Downregulating TRPC3 expression led to reduced proliferation, suppression of epidermal growth factor-induced Ca2+ influx, and decreased growth of xenografts. In this study, we further explored the interaction of FSH-TRPC3. FSH stimulation was associated with upregulation of TRPC3 levels and translocation of TRPC3 from the cytoplasm to cell membrane, facilitating the influx of Ca2+ after treatment with a TRPC3 specific agonist. Knocking down TRPC3 abrogated Akt/PKB phosphorylation by FSH stimulation, leading to decreased expression of down-stream effectors including survivin, HIF1α and VEGF. Our findings indicate that TRPC3 plays a significant role in the tumor-stimulating activity of FSH and could be a potential therapeutic target of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2011-4540

Abstract 4306: KLF8 promotes human ovarian tumorigenesis in athymic mouse models

Abstract KLF8 is a dual transcription factor that plays a role in oncogenic transformation in mouse fibroblast cells and is highly over-expressed in human ovarian cancer. To test if KLF8 alone or together with each of the ovarian oncoprotein c-Myc, Stat3c or Akt plays a role in ovarian cell transformation, we used the immortalized normal human ovarian epithelial cell line T80 to generate stable cell lines that overexpress KLF8 alone or its combination with one of the other three oncoproteins. The Soft agar assay showed that T80/KLF8 formed significantly more colonies compared with the mock control cells, suggesting that KLF8 may play a role to transform T80 cells. Interestingly the cells expressing both KLF8 and c-Myc formed the largest amounts of colonies that were even greater than the sum of colonies formed by the cells expressing either KLF8 or c-Myc alone, suggesting a potential synergistic role of KLF8 with c-Myc. Surprisingly, however, overexpression of KLF8 alone was sufficient to induce tumorigenesis in athymic mice when the T80/KLF8 cells were injected subcutaneously (s.c.), intraperitoneally (i.p.) or orthotopically into the ovarian bursa. HE and IHC staining identified the tumors’ epithelial origin characteristic of human ovarian cancer. The s.c. growth rate of the T80/KLF8 tumors was greater than the T80/Stat3c tumors and similar to T80/Akt tumors although slower than the tumors formed by SKOV3ip1 and T80/c-Myc cells. The average survival time for the mice with the i.p. implantations was about six months for T80/KLF8 as compared to approximately two months for SKOV3ip1. These novel findings suggest that KLF8 plays a critical oncogenic role in synergy with c-Myc in the ovarian cancer progression. The mouse tumor models generated in this work provide important tools for the studies of mechanisms underlying human ovarian cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4306. doi:10.1158/1538-7445.AM2011-4306

B7-H4 Expression Promotes Tumorigenesis in Ovarian Cancer

It has been previously shown that B7-H4, one of the B7 family members that serve as negative regulators of T cell function, has altered expression levels in a variety of cancers, overexpression of B7-H4 promotes cellular transformation. However, there is still lack of adequate evidence to establish a direct connection between B7-H4 expression and malignant transformation. Herein, we constructed pE-green fluorescent protein-N1/B7-H4 mammalian expression vector and transfected into B7-H4-negative human ovarian cancer cell line SKOV3. Cellular proliferation, apoptosis, adhesion, motility, and invasion were examined in vitro. Cells injected subcutaneously into severe combined immunodeficient mouse were analyzed for the possible functions of B7-H4 in ovarian tumorigenesis in vivo. Fluorescence microscopy studies confirmed that the B7-H4-green fluorescent protein localizes in the cytoplasm of SKOV3/B7-H4 cells, whereas green fluorescent protein is uniformly distributed throughout the cell. B7-H4 promoted cellular proliferation rate and increased cell adhesion, migration, and invasion. In addition, SKOV3 cells expressing B7-H4 gained growth advantage in the xenograft model in vivo. These studies demonstrate that B7-H4 directly promotes malignant transformation of ovarian cancer cell line, and provides a potential therapeutic strategy for targeting B7-H4 to inhibit progression of human ovarian cancers.

Roles of clusterin in progression, chemoresistance and metastasis of human ovarian cancer

Clusterin (CLU) is a multivalent glycoprotein with ubiquitous tissue distribution. To address the possible differential functional roles assumed by different isoforms of CLU in the progression of human ovarian cancer, we constructed 2 human ovarian cancer cell models that represent examples of contradistinctive CLU expression levels. One is constitutively overexpressing different clusterin isoforms in SKOV3 cells by transfection of the 3 different expression vectors, another is silencing the intrinsically expressing clusterin in cisplatin-resistant human A2780-cis(CP70) tumor cells with the usage of shRNA-mediated CLU gene silencing. Then, the different cellular localization, biological effects, and functional roles played in tumor progression and drug resistances were studied. We found that (i) in the distinct cellular contexts of human ovarian carcinoma SKOV3 and CP70 cells assayed, sCLU is a central molecule in cell homeostasis that functions as a cytoprotective protein, whereas nCLU is proapoptotic; (ii) In SKOV3 cells, nuclear localization of the truncated CLU is NLS dependent, without which the pnCLU protein was sequestrated in cytoplasm to prevent cytotoxicity. (iii) sCLU plays a significant role in the development of the chemoresistance phenotype in ovarian cancer cells. Moreover, with the CLU-specific shRNA oligonucleotides, we successfully sensitized cells for chemotherapy, and inhibited cells' proliferation, migration and invasion. Collectively, our results reveal that, CLU gene expression might play a crucial role in ovarian cancer progression, adaptation and eventual resistance to chemotherapy through differential processing of CLU isoforms. Specifically, sCLU as an antiapoptotic protein, upregulated in an adaptive cell-survival manner by chemotherapy, confers resistance to various cell-death triggers.

Transient receptor potential channel C3 contributes to the progression of human ovarian cancer

Ovarian cancer (OC) is the leading cause of death from gynecological malignancy. However, the mechanism by which OC develops remains largely unknown. Increases in cytosolic free Ca(2+) ([Ca(2+)](i)) can result in different physiological changes including cell growth, differentiation and death. The transient receptor potential (TRP) C channels are nonselective cation channels with permeability to Ca(2+). Here we report that TRPC3 channels promote human OC growth. The TRPC3 protein levels in human OC specimens were greatly increased than those in normal ovarian specimens. Downregulating TRPC3 expression in SKOV3 cells, a human OC cell line, led to reduction of proliferation, suppression in epidermal growth factor-induced Ca(2+) influx, dephosphorylation of Cdc2 and CaMKIIalpha and prolonged progression through M phase of these cells. Further, decreased the expression of TRPC3 suppressed the tumor formation generated by injecting SKOV3 cells in nude mice. Together, our results suggest that increased activity of TRPC3 channels is necessary for the development of OCs.

The lysophosphatidic acid (LPA) receptors their expression and significance in epithelial ovarian neoplasms

To investigate the lysophosphatidic acid (LPA) receptors expression situation and their biological significance in human ovarian cancer cell lines and in human epithelial ovarian neoplasms. The reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were employed to measure the expression levels of LPA(1), LPA(2) and LPA(3) mRNA, LPA(2) and LPA(3) protein expression in cultured human ovarian cancer cell lines (3AO, SKOV3 and OVCAR3) and in human epithelial ovarian neoplasms. The immunocytochemical method was used to detect LPA(2) and LPA(3) protein expression in cultured human ovarian cancer cell lines. RT-PCR revealed that all ovarian cancer cell lines expressed LPA(1), LPA(2) and LPA(3) mRNA. The positive rates (100%; 86.4%; 88.2%) of LPA(1) mRNA in normal ovarian tissue, benign tumor and ovarian cancer were no significant difference (p>0.05). The expression level of LPA(1) mRNA was significantly higher in normal ovarian tissue compared with that in benign tumor and in ovarian cancer tissue (p<0.01). LPA(1) expression level was no significant difference in both benign tumor and ovarian cancer tissue (p>0.05). LPA(2) mRNA-positive rates (92.6%) and expression level were significantly higher in ovarian cancer compared with that in benign tumor (31.8%) and in normal ovarian tissue (31.3%) (p<0.01); LPA(2) mRNA-positive rates and expression level were no significant difference in both benign tumor and normal ovarian tissue (p>0.05). LPA(3) mRNA-positive rates (92.6%) and expression level were significantly higher in ovarian cancer compared with that in benign tumor (31.8%) and in normal ovarian tissue (31.3%) (p<0.01), LPA(3) mRNA-positive rates and expression level were no significant difference in both benign tumor and normal ovarian tissue (p>0.05). LPA(1) mRNA expression level was significantly decreased compared with that of LPA(2) and LPA(3) in ovarian cancer (p<0.01); Western blotting clearly revealed that all ovarian cancer cell lines showed LPA(2) and LPA(3) protein. The positive rates and expression level of LPA(2) and LPA(3) protein were significantly increased in ovarian cancer (92.6%; 92.6%) compared with that in benign tumor (45.5%; 45.5%) and that in normal ovarian tissue (43.8%; 43.8%) (p<0.01); LPA(2) and LPA(3) protein-positive rates and expression level were no significant difference in both benign tumor and normal ovarian tissue (p>0.05). Correlation of clinicopathological parameters showed that LPA receptors mRNA and protein expression were associated with FIGO stage and histological grade, except pathologic types and age. The mRNA and protein expression of LPA(2) and LPA(3) in stages III and IV was significantly higher than that in stages I and II epithelial ovarian cancer (p<0.05). The mRNA and protein expression of pathologic grade G(3) was significantly higher compared with grade G(1) (p<0.05). LPA(1), LPA(2) and LPA(3) mRNA and protein expressed widely in human epithelial ovarian neoplasms. LPA(2) and LPA(3) may be involved in the development and progression of human ovarian cancer. There was a significant correlation between LPA(2), LPA(3) and invasion and metastasis of epithelial ovarian cancer. LPA(2) and LPA(3) may be a prognostic indicator in patients with epithelial ovarian cancer.

Disruption of the retinoblastoma pathway by small interfering RNA and ectopic expression of the catalytic subunit of telomerase lead to immortalization of human ovarian surface epithelial cells

The risk of developing ovarian cancer is about 1% over a lifetime, but it is the most deadly gynecologic cancer, in part due to lack of diagnostic markers for early-stage disease and cell model system for studying early neoplastic changes. Most existing immortal human ovarian surface epithelial cells were achieved by using viral protein such as SV40 T/t antigen or E6/E7, which inactivate multiple cellular pathways. In the current study, we used a small interfering RNA (siRNA) against the retinoblastoma gene (pRb) and ectopic expression of human telomerase reverse transcriptase (hTERT) to immortalize the primary ovarian epithelial cell line OSE137 and two additional human ovarian surface epithelial cells. The immortalized OSE137 showed increased telomerase activity, lengthened telomeres, increased G2/M phase, altered cell-cycle regulatory proteins but nontumorigenic. As both Rb and hTERT pathways are commonly altered in human ovarian cancer and these genetic changes are faithfully modeled in these cells without using viral protein, these immortal cells represent an authentic in vitro model system with which to study the initiation and progression of human ovarian cancer.

Microarray Comparative Genomic Hybridization Profile of a Murine Model for Epithelial Ovarian Cancer Reveals Genomic Imbalances Resembling Human Ovarian Carcinomas

Microarray comparative genomic hybridization (mCGH) is emerging as a high-resolution technology to detect gene dosage alterations in numerous pathologies, including cancer. We optimized cDNA microarrays to identify genome-wide imbalances in spontaneously transformed mouse ovarian surface epithelial cell lines, an in vitro murine model for ovarian cancer. Amplification of chromosome 19 and a more variable gain pattern of chromosomes 15 and 5 were detected and independently validated using conventional metaphase CGH. In addition, cryptic aberrations in segments of chromosomes 4, 7, 8, 9, 11, 17, and X, allowed identification of 2 related genomic variants among six cell lines studied. Mouse-human synteny revealed an overall early transformation stage with approximately 80% conservation relative to human ovarian malignancies of epithelial origin including low malignant potential tumors, serous carcinoma, and carcinoma cell lines. Importantly, three of the cells bear gained segments 13 and 41 Mbp length of chromosomes 5 and 15, respectively, which are syntenic to human 22q11–13, 8q24 and 12p11–q24, the two latter chromosomal regions thought to define one pathway of karyotypic changes in the development of human ovarian tumors. Our findings support the utility of mouse ovarian surface epithelial (MOSE) cells in studying initiation and progression of human ovarian cancer and as a suitable model to evaluate therapeutic approaches.

Epigenetic silencing of the imprinted gene ZAC by DNA methylation is an early event in the progression of human ovarian cancer

ZAC is a paternally expressed, imprinted gene located on chromosome 6q24, within a region known to harbor a tumor suppressor gene for several types of neoplasia, including human ovarian cancer (HOC). We have failed to identify genetic mutations in the ZAC gene in tumor material. Many imprinted genes contain differentially allele-specific-methylated regions (DMR) and harbor promoter activity that is regulated by the DNA methylation. Aberrant DNA methylation is a common feature of neoplasia and changes in DNA methylation at the ZAC locus have been reported in some cases of HOC. We investigated the DNA methylation and ZAC mRNA expression levels in a larger sample of primary HOC material, obtained by laser capture microdissection. ZAC mRNA expression was reduced in the majority of samples and this correlated with hypermethylation of the ZAC-DMR. Treatment of hypermethylated cells lines with a demethylating agent restored ZAC expression. Our studies indicate that transcriptional silencing of ZAC is likely to be caused by DNA methylation in HOC. Forced expression of ZAC resulted in a reduction in proliferation and marked induction of apoptotic cell death. The ZAC-mediated apoptosis signal is p53-independent and eliminated by inhibitors of caspase 3, 8 and 9. Reduced expression of ZAC would therefore favor tumor progression. As there were no significant differences in either DNA methylation or expression of ZAC mRNA between localized and advanced tumors, our data indicates that loss of ZAC is a relatively early event in HOC. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.)

Trans-3,4,5'-Trihydroxystibene inhibits hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in human ovarian cancer cells.

trans-3,4,5'-Trihydroxystibene (resveratrol) is a natural product commonly found in the human diet and has been shown recently to have anticancer effects on various human cancer cells. However, the molecular basis for its anticancer action remains to be elucidated. In this study, we investigated the effect of resveratrol on hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression in human ovarian cancer cells A2780/CP70 and OVCAR-3. We found that although resveratrol did not affect HIF-1alpha mRNA levels, it did dramatically inhibit both basal-level and growth factor-induced HIF-1alpha protein expression in the cells. Resveratrol also greatly inhibited VEGF expression. Mechanistically, we demonstrated that resveratrol inhibited HIF-1alpha and VEGF expression through multiple mechanisms. First, resveratrol inhibited AKT and mitogen-activated protein kinase activation, which played a partial role in the down-regulation of HIF-1alpha expression. Second, resveratrol inhibited insulin-like growth factor 1-induced HIF-1alpha expression through the inhibition of protein translational regulators, including M(r) 70,000 ribosomal protein S6 kinase 1, S6 ribosomal protein, eukaryotic initiation factor 4E-binding protein 1, and eukaryotic initiation factor 4E. Finally, we showed that resveratrol substantially induced HIF-1alpha protein degradation through the proteasome pathway. Our data suggested that resveratrol may inhibit human ovarian cancer progression and angiogenesis by inhibiting HIF-1alpha and VEGF expression and thus provide a novel potential mechanism for the anticancer action of resveratrol.

Animal models of ovarian cancer

Ovarian cancer is the most lethal of all of the gynecological cancers and can arise from any cell type of the ovary, including germ cells, granulosa or stromal cells. However, the majority of ovarian cancers arise from the surface epithelium, a single layer of cells that covers the surface of the ovary. The lack of a reliable and specific method for the early detection of epithelial ovarian cancer results in diagnosis occurring most commonly at late clinical stages, when treatment is less effective. In part, the deficiency in diagnostic tools is due to the lack of markers for the detection of preneoplastic or early neoplastic changes in the epithelial cells, which reflects our rather poor understanding of this process. Animal models which accurately represent the cellular and molecular changes associated with the initiation and progression of human ovarian cancer have significant potential to facilitate the development of better methods for the early detection and treatment of ovarian cancer. This review describes some of the experimental animal models of ovarian tumorigenesis that have been reported, including those involving specific reproductive factors and environmental toxins. Consideration has also been given to the recent progress in modeling ovarian cancer using genetically engineered mice.

CD44 Interaction with c-Src Kinase Promotes Cortactin-mediated Cytoskeleton Function and Hyaluronic Acid-dependent Ovarian Tumor Cell Migration

In this study we have demonstrated that both CD44 (the hyaluronan (HA) receptor) and c-Src kinase are expressed in human ovarian tumor cells (SK-OV-3.ipl cell line), and that these two proteins are physically associated as a complex in vivo. Using a recombinant cytoplasmic domain of CD44 and an in vitro binding assay, we have detected a specific interaction between CD44 and c-Src kinase. Furthermore, the binding of HA to SK-OV-3.ipl cells promotes c-Src kinase recruitment to CD44 and stimulates c-Src kinase activity, which, in turn, increases tyrosine phosphorylation of the cytoskeletal protein, cortactin. Subsequently, tyrosine phosphorylation of cortactin attenuates its ability to cross-link filamentous actin in vitro. In addition, transfection of SK-OV-3.ipl cells with a dominant active form of c-Src (Y527F)cDNA promotes CD44 and c-Src association with cortactin in membrane projections, and stimulates HA-dependent/CD44-specific ovarian tumor cell migration. Finally, overexpression of a dominant-negative mutant of c-Src kinase (K295R) in SK-OV-3.ipl cells impairs the tumor cell-specific phenotype. Taken together, these findings strongly suggest that CD44 interaction with c-Src kinase plays a pivotal role in initiating cortactin-regulated cytoskeleton function and HA-dependent tumor cell migration, which may be required for human ovarian cancer progression.