Normal Human Ovarian Surface Epithelium Research Articles

Targeting Aurora A Kinase (AAK) in Platinum-Resistant High Grade Serous Ovarian Cancer.

Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.

Abstract 1774: Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC)

Abstract Resistance to platinum-based chemotherapy is a major problem in the clinical management of recurrent HGSOC. While the use of poly ADP-ribose polymerase inhibitors (PARPi) have made an impact in BRCA1/2 mutated and homologous recombinant-deficient HGSOC, resistance and limited response-rates to PARPi persist. Alisertib (AL) an inhibitor of AAK can inhibit entry and progression of cells through mitosis as well as the interaction with N-myc and its degradation (Front. Oncol. doi:10.3389/fonc.2015.00189). In models of HGSOC from patients with recurrent platinum-resistant disease, cisplatin (CP) treatment does not lead to cell cycle traverse perturbations and significant accumulation of cells in G2-M. Since a defect in the G2-M DNA damage checkpoint is a potential mechanism of resistance to cisplatin, we sought to test the hypothesis that inhibition of AAK that is involved in centrosome maturation and entry into mitosis would enhance the anti-tumor activity of cisplatin in platinum-resistant HGSOC. To test this hypothesis, we treated HGSOC cell lines from patients with recurrent platinum-refractory disease with increasing doses of CP (1- 2.5 μM), of AL (0.05 μM) or the combination and evaluated effects on cell proliferation, apoptosis and generation of reactive oxygen species (ROS). The sequential treatment of four different HGSOC cell lines with CP for 3 hours followed by AL for 48 hours and recovery in drug-free medium for 96-120 hours led to a synergistic increase in apoptosis (p&amp;lt;0.01) and a synergistic decrease in cell survival (p&amp;lt;0.001) compared to treatment with either drug alone. The enhanced apoptosis and decreased cell survival with (CP→AL) treatment was accompanied by a significant increase (p&amp;lt;0.05) in ROS. In contrast, sequential treatment with AL followed by CP (AL→CP) led to an antagonistic response. The synergistic effect of (CP→AL) was accompanied by a modest increase in mRNA and protein levels of AAK. However, protein levels of N-myc protein, which interacts with AAK and is degraded, were not altered. Interestingly, (CP→AL) treatment did not elicit the synergistic cytotoxic response or increased ROS in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. In platinum-refractory HGSOC cells that are wild-type for BRCA1/2 and resistant to olaparib, treatment with the combination of 0.01 μM AL and 1 μM olaparib for 48 hours resulted in an additive cytotoxic response. Based on the mutation landscape of the HGSOC cell panel evaluated, the synergistic cytotoxicity with the CP→AL treatment was independent of BRCA1/2 or TP53 mutation status. In summary, CP resistance in HGSOC due to a defective G2-M DNA damage response, can be potentially circumvented by sequential treatment with CP followed by AL. Citation Format: Ram N. Ganapathi, Eric J. Norris, Ashley P. Sutker, Hala Soliman, Mahrukh K. Ganapathi. Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1774.

Duodenal stent placement for the preparation of systemic chemotherapy in patients with advanced pancreatic cancer and gastric outlet obstruction

Objective: Determine the effects of factors secreted by normal human ovarian stroma on the proliferation of benign and malignant ovarian epithelia, in vitro. Methods: Primary cultures of normal human ovarian surface epithelium (HOSE), human ovarian stromal tissue (HOST), and epithelial ovarian carcinomas (CSOC) were established from surgical specimens and characterized immunohistochemically using anti-cytokeratin, vimentin, and Factor VIII antibodies. Stroma-conditioned media (SCM) were collected over 3 days from confluent HOST cultures. The SCM were dialyzed, lyophilized, resuspended, and added to HOSE, CSOC, SKOV-3, and Caov-3 ovarian cancer cell cultures and growth inhibitory effects were assayed by MTS and [3H]thymidine uptake. Results: SCM inhibited the growth and DNA synthesis of normal HOSE cells and cancer cells by 79-99% in >10-cell lines studied to date. The inhibitory effect was rapid in onset with 31-82% reduction in DNA synthesis at 1 hr and approximately 50% return of activity by 23 hr following a 1-hr SCM pulse treatment. The SCM inhibitory activity was not abolished by boiling or by absorption with heparin-agarose. Size exclusion filtration places the molecular weight of the inhibitory substance between 1 and 3 kDa. Neither trypsin nor proteinase K treatments altered the inhibitory activity of SCM, while a Bligh-Dyer organic extraction placed the activity in the aqueous phase. Conclusion: A heat-stable, nonheparin-binding, low-molecular-weight, water-soluble substance secreted by normal ovarian stroma significantly inhibits HOSE and ovarian cancer cell proliferation. Derangements in normal ovarian stroma-epithelial interactions may contribute to growth dysregulation of the surface epithelia and result in ovarian carcinogenesis.

Local estrogen metabolism in epithelial ovarian cancer suggests novel targets for therapy

Epithelial ovarian cancer (EOC) accounts for about 90% of malignant ovarian tumors, and estrogen is often implicated in disease progression. We therefore compared the potential for gating of estrogen action via pre-receptor metabolism in normal human ovarian surface epithelium (OSE), EOC and selected EOC cell lines (SKOV3 and PEO1). Steroid sulphatase (STS), estrogen sulfotransferase (EST), 17β-hydroxysteroid dehydrogenases 2 (17BHSD2) and 5 (17BHSD5) mRNAs, proteins and enzymatic activities were all detectable in primary cell cultures of OSE and EOC, whereas aromatase and 17BHSD1 expression was negligible. qRT-PCR assay on total mRNA revealed significantly higher EST mRNA expression in OSE compared to EOC (P<0.05). Radioenzymatic measurements confirmed reduced sulfoconjugation (neutralization) of free estrogen in EOC relative to OSE. OSE cells were more effective at converting free [3H]-E1 to [3H]-E1S or [3H]-E2S, while EOC cell lines mainly converted [3H]-E1 to [3H]-E2 with minimal formation of [3H]-E1S or [3H]-E2S. IL1α treatment suppressed EST (P<0.01) and 17BHSD2 (P<0.001) mRNA levels in OSE and stimulated STS mRNA levels (P<0.001) in cancer (SKOV3) cells. These results show that estrogen is differentially metabolized in OSE and EOC cells, with E2 ‘activation’ from conjugated estrogen predominating in EOC. Inflammatory cytokines may further augment the local production of E2 by stimulating STS and suppressing EST. We conclude that local estrogen metabolism may be a target for EOC treatment.

Citrate synthase expression affects tumor phenotype and drug resistance in human ovarian carcinoma.

Citrate synthase (CS), one of the key enzymes in the tricarboxylic acid (TCA) cycle, catalyzes the reaction between oxaloacetic acid and acetyl coenzyme A to generate citrate. Increased CS has been observed in pancreatic cancer. In this study, we found higher CS expression in malignant ovarian tumors and ovarian cancer cell lines compared to benign ovarian tumors and normal human ovarian surface epithelium, respectively. CS knockdown by RNAi could result in the reduction of cell proliferation, and inhibition of invasion and migration of ovarian cancer cells in vitro. The drug resistance was also inhibited possibly through an excision repair cross complementing 1 (ERCC1)-dependent mechanism. Finally, upon CS knockdown we observed significant increase expression of multiple genes, including ISG15, IRF7, CASP7, and DDX58 in SKOV3 and A2780 cells by microarray analysis and real-time PCR. Taken together, these results suggested that CS might represent a potential therapeutic target for ovarian carcinoma.

SUZ12 Promotes Human Epithelial Ovarian Cancer by Suppressing Apoptosis via Silencing HRK

Epithelial ovarian cancer (EOC) ranks first as the cause of death for gynecological cancers in the United States. SUZ12 is a component of the polycomb repressive complex 2 (PRC2) and is essential for PRC2-mediated gene silencing by generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3). The role of SUZ12 in EOC has never been investigated. Here, we show that SUZ12 is expressed at significantly higher levels in human EOC (n = 117) compared with either normal human ovarian surface epithelium (n = 35, P < 0.001) or fallopian tube epithelium (n = 15, P < 0.001). There is a positive correlation between expression of SUZ12 and EZH2 in human EOC (P < 0.001). In addition, expression of SUZ12 positively correlates with Ki67, a marker of cell proliferation (P < 0.001), and predicts shorter overall survival (P = 0.0078). Notably, knockdown of SUZ12 suppresses the growth of human EOC cells in vitro and in vivo in both orthotopic and subcutaneous xenograft EOC models. In addition, SUZ12 knockdown decreases the levels of H3K27Me3 and triggers apoptosis of human EOC cells. Mechanistically, we identified Harakiri (HRK), a proapoptotic gene, as a novel SUZ12 target gene, and showed that HRK upregulation mediates apoptosis induced by SUZ12 knockdown in human EOC cells. In summary, we show that SUZ12 promotes the proliferation of human EOC cells by inhibiting apoptosis and HRK is a novel SUZ12 target gene whose upregulation contributes to apoptosis induced by SUZ12 knockdown.

Primary cilia and aberrant cell signaling in epithelial ovarian cancer

BackgroundOvarian cancer is the fourth leading cause of cancer-related deaths among women in Denmark, largely due to the advanced stage at diagnosis in most patients. Approximately 90% of ovarian cancers originate from the single-layered ovarian surface epithelium (OSE). Defects in the primary cilium, a solitary sensory organelle in most cells types including OSE, were recently implicated in tumorigenesis, mainly due to deregulation of ciliary signaling pathways such as Hedgehog (Hh) signaling. However, a possible link between primary cilia and epithelial ovarian cancer has not previously been investigated.MethodsThe presence of primary cilia was analyzed in sections of fixed human ovarian tissue as well as in cultures of normal human ovarian surface epithelium (OSE) cells and two human OSE-derived cancer cell lines. We also used immunofluorescence microscopy, western blotting, RT-PCR and siRNA to investigate ciliary signaling pathways in these cells.ResultsWe show that ovarian cancer cells display significantly reduced numbers of primary cilia. The reduction in ciliation frequency in these cells was not due to a failure to enter growth arrest, and correlated with persistent centrosomal localization of aurora A kinase (AURA). Further, we demonstrate that ovarian cancer cells have deregulated Hh signaling and platelet-derived growth factor receptor alpha (PDGFRα) expression and that promotion of ciliary formation/stability by AURA siRNA depletion decreases Hh signaling in ovarian cancer cells. Lastly, we show that the tumor suppressor protein and negative regulator of AURA, checkpoint with forkhead-associated and ring finger domains (CHFR), localizes to the centrosome/primary cilium axis.ConclusionsOur results suggest that primary cilia play a role in maintaining OSE homeostasis and that the low frequency of primary cilia in cancer OSE cells may result in part from over-expression of AURA, leading to aberrant Hh signaling and ovarian tumorigenesis.

Abstract 2030: A novel role for the 185delAG BRCA1 mutant protein, BRAT, in ovarian cancer pathology

Abstract Objective: Familial history is the strongest risk factor for developing ovarian cancer (OC) and is associated with BRCA1 truncation mutations, such as the founder mutation 185delAG. Studying these risk-associated mutations may reveal signaling pathways important in OC etiology. Normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT, exhibit phenotypic changes due, in part, to up-regulation of maspin, a tumor suppressor important in apoptosis, invasion, and metastasis that exhibits unique up-regulation in some cancer types. In the current study, we determined BRAT's impact on expression of Interleukin-1beta (IL1β), another key player in invasion and metastasis. Methods: HOSE cells transiently and stably expressing the 185delAG mutation and HOSE cells with endogenous 185delAG were analyzed for differential target gene expression by semi-quantitative, real time PCR, western blot; as well as, IL1β bioactivity. Western blot was used to identify cell signaling targets of BRAT mediated IL1β expression via MAPK pathways. Breast epithelial and breast cancer cells transiently and stably expressing BRAT were evaluated by real time PCR to determine whether BRAT induced IL1β is tissue specific. Results: BRAT-expressing HOSE cells exhibited enhanced IL1β mRNA expression. Higher levels of pro-IL1β protein were detected by Western blotting of cell lysate and conditioned media. Increased phosphorylation of MAPK pathway constituents were seen in BRAT-expressing HOSE cells as detected by Western blotting of cell lysate. However, expression of BRAT in normal breast and breast cancer cell lines failed to significantly alter IL1β expression levels. Conclusion: Normal HOSE cells expressing the BRAT mutation exhibit increased expression of IL1β. Further investigation is warranted to elucidate the mechanism of tissue-specific BRAT-mediated IL1β expression since increased IL1β expression may represent an early step of malignant transformation contributing to 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 2030. doi:10.1158/1538-7445.AM2011-2030

Abstract 4969: RNA interference screens to identify genes essential for ovarian tumor growth and maintenance

Abstract BACKGROUND: Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the U.S. Although most women afflicted with EOC respond to primary therapy, the relapse rate is 85%. It is essential that we better understand the genetic and epigenetic factors that contribute to EOC in order to improve prevailing conventional therapies. We have used a functional genomic approach to help identify potential molecular targets that represent key points of therapeutic intervention. METHODS: Building on the concept of “oncogenic addiction” we employed a small interfering RNA (siRNA) screen against 6022 druggable genes using high-throughput cell-based, one-well/one-gene screening approach and tested for effects on growth and/or survival of A1847 EOC cells. From primary lethality screens, we identified 300 genes that were considered essential for tumor cell growth and/or survival. We derived a custom siRNA library consisting of 1,200 siRNAs (pooled 4 siRNAs/gene) targeting these 300 genes. We optimized high-throughput transfection conditions for six additional EOC cell lines (OVCAR5, SKOV3, A2780, C30, CP70 and UPN275) and three non-tumorigenic, immortalized human ovarian surface epithelial (HOSE) cell lines (HIO-80, HIO-117, HIO-120) and screened our custom library. We identified lead candidates and eliminated potential off-target effects by rescreening individual siRNAs following deconvolution of siRNAs pools and by quantitative RT-PCR (qRT-PCR). We examined gene expression of the validated hits in all the EOC cell lines under two different growth conditions (monolayer and 3D spheroids) by qRT-PCR (Fluidigm instrument) and in primary serous adenocarcinoma tumors by microarrays (Affymetrix). RESULTS: A hit was defined as a gene that reduced cell viability by &amp;gt;15% with a false discovery rate of ≤5%. In the 300-gene library screen, each of the ten cell lines exhibited a unique number of hits ranging from 27-94% of the total siRNAs screened. We validated the top priority candidates and ultimately identified four genes (HSPA5, NDC80, NUF2 and PTN) that when suppressed, significantly reduced the viability of all the EOC cell lines tested but none of the non-tumorigenic HOSE lines. Expression of two of the genes was significantly (p=0.1) down-regulated (&amp;gt;1.5-fold) in cells grown as 3D spheroids versus monolayers. Expression of all four genes was significantly higher in primary serous tumors (n=53) compared to normal HOSE samples (n=10) (p&amp;lt; 0.005). CONCLUSION: We identified four druggable candidates that could contribute to ovarian tumorigenesis and form the basis of targeted combination therapy to block the multiple oncogenic pathways activated in EOC. This work has the potential to contribute to the next-generation approach for cancer therapies: combining targeted drugs in coherently designed multi-agent combinations useful in the clinical treatment of EOC to improve patient survival. 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 4969. doi:10.1158/1538-7445.AM2011-4969

Immunohistochemical analysis of 17β-hydroxysteroid dehydrogenase isozymes in human ovarian surface epithelium and epithelial ovarian carcinoma

Epidemiological studies have indicated a relationship between gonadal steroid hormones, primarily estrogens, and epithelial ovarian carcinoma. In situ estrogen metabolism and synthesis have been considered to play important roles in the development of the progression of epithelial ovarian carcinoma. 17β-Hydroxysteroid dehydrogenases (17β-HSDs) are a group of intracellular isozymes catalyzing interconversions between estradiol (E2) and estrone (E1). In the last step of steroidogenesis, 17β-HSD type 1 catalyzes the 17β-reduction and produces E2 from E1. The oxidative enzymes known as types 2, 4, and 8 are potent estrogen-inactivating enzymes that convert E2 to E1. Here we report the immunoexpression of 17β-HSD types 1, 2, 4, and 8 in normal human ovarian surface epithelium (OSE) and epithelial ovarian carcinoma. For this study, novel polyclonal antibodies were generated against each type of 17β-HSD. Of the six normal OSE cases investigated, 17β-HSD types 1, 4, and 8, but not type 2, were found in the cytoplasm of epithelial cells. In 58 cases of epithelial ovarian carcinoma (45 serous, 4 endometrioid, 4 mucinous, and 5 clear cell), estrogen-inactivating 17β-HSDs were commonly found (type 2, 84.5%; type 4, 82.8%; type 8, 86.2%), whereas type 1 was detected in only 10 cases (17.2%). These results indicate that 17β-HSDs may be involved in the protective and/or suppressive effects against the estrogen-dependent proliferation of epithelial ovarian carcinoma.

Highlights of This Issue

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy in the United States. Thus, there is an urgent need to identify new targets for developing novel therapeutics for EOC. EZH2 is an epigenetic regulator that plays an important role in gene silencing. Li and colleagues showed that EZH2 is significantly overexpressed in primary human EOCs when compared with normal human ovarian surface epithelium. Moreover, loss of function analysis revealed a pivotal role of EZH2 in suppressing apoptosis and promoting proliferation and invasion of human EOC cells. There results suggest that EZH2 is a potential target for developing EOC therapeutics.Integrin α6β4 is upregulated in invasive human cancers and has been implicated in poor prognosis for cancer patients. α6β4 has been shown to enhance cancer cell function via activation of translation initiation factor eIF4E. In this study, Korneeva and colleagues found that α6β4 regulates the activity of eIF4E through Mnk1. α6β4 signals through MEK and p38 MAPK to increase in the phosphorylation and activity of Mnk1. Inhibition of Mnk1 by CGP57380 or shRNA blocks α6β4-dependent eIF4E phosphorylation and translation of VEGF mRNA. This suggests that Mnk1 could be a therapeutic target in cancers in which the α6β4 levels are high.The cellular readout of TGF-β signal is highly contextual and differs between normal and cancer cells. Using mammary epithelial cells transformed by the HER2 oncogene and a panel of breast cancer cell lines, Yu and colleagues found a contextdependent, bidirectional regulation of the DNA mismatch repair protein MSH2 as a function of TGF-β. TGF-β activated MSH2 promoter in a p53-dependent manner in nontransformed cells, whereas in transformed cells, it downregulated MSH2 expression through a miR-21-mediated mechanism. Reduced MSH2 level further resulted in tolerance to DNA-damaging chemotherapy agents in cancer cells, suggesting a role of TGF-β in inducing chemoresistance.The role of nonsteroidal anti-inflammatory drug activated Gene-1 (Nag-1) in prostate cancer is not fully determined and remains an area of interest. The data show that Nag-1 protein expression is induced by the NSAIDs R-flurbiprofen and ibuprofen downstream of NSAID induction of p75NTR, which is an established tumor suppressor and metastasis suppressor of prostate cancer cells. Induction of p75NTR decreases both survival and migration of PC-3 prostate cancer cells; however, Nag-1 induction only inhibits cell migration and not cell survival. These results suggest that Nag-1 contributes to metastasis suppressor activity but not tumor suppressor activity downstream of p75NTR.

Phenotypic Plasticity of the Ovarian Surface Epithelium: TGF-β1 Induction of Epithelial to Mesenchymal Transition (EMT) in Vitro

Ovarian surface epithelium (OSE) is the most conceivable cell origin of epithelial ovarian carcinomas. Unlike many other epithelial tumors, the precancerous lesion acquires expression of epithelial markers, e.g. E-cadherin and claudins, suggesting that OSE cells undergo mesenchymal to epithelial transition (MET) during transformation. Recent findings indicate that TGF-β1, a prototypic stimulus of epithelial to mesenchymal transition (EMT), i.e. reverse to MET, is produced at significant amounts in the intact ovary. In the present study, we therefore investigated whether TGF-β1 changes the OSE phenotype accordingly, focusing on epithelial junction proteins and transcriptional EMT regulators quantified by real-time RT-PCR and Western blotting in cultured normal human OSE. Early OSE passages were found to paradoxically express de novo E-cadherin and also establish tight junctions exhibiting claudin-1 (but not claudin-3 and -4) and occludin. Stimulation with TGF-β1 (100 ng/ml) for 3-5 d down-regulated all these epithelial markers including Crumbs3 and also prevented the formation of an epithelial barrier This was accompanied by sustained expression of Snail and N-cadherin and transient expression of Slug, whereas Zeb1 (zinc finger E-box binding homeobox 1) and Twist mRNA levels were not significantly changed. In conclusion, TGF-β1 enforces the mesenchymal phenotype of OSE cells in vitro by an EMT-like process, leading to an altered molecular composition of the epithelial junction complex that partly coincides with the expression pattern of the native OSE. This suggests a potential role of TGF-β1-induced EMT in OSE under physiological conditions and possibly also in epithelial ovarian tumorigenesis.

Abstract 1852: A novel role for the 185delAG BRCA1 mutant protein, BRAT, in ovarian cancer pathology

Abstract Objective: Familial history is the strongest risk factor for developing ovarian cancer (OC). Studying risk-associated BRCA1 truncation mutations, such as the founder mutation 185delAG, may reveal signaling pathways important in OC etiology. Normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT, exhibit phenotypic changes due possibly in part to up-regulation of maspin, a tumor suppressor important in apoptosis, invasion, and metastasis that exhibits unique up-regulation in some cancer types. In the current study, we wish to determine BRAT's impact on expression and activity of matrix metalloprotease 1 (MMP1), another key player in invasion and metastasis, and the mechanism of this modulation. Methods: HOSE, breast epithelial cells, and breast cancer cells transiently and stably expressing the 185delAG mutation were analyzed for differential target gene expression by semi-quantitative and real time PCR. SiRNA transfection was performed to achieve knockdown of c-Jun. Western blotting was used to evaluate MMP1 and c-Jun protein levels. FRET ELISA was performed on conditioned media to evaluate levels and functionality of secreted MMP1. Cells were treated with cisplatin and subjected to MTS assay to evaluate chemosensitivity. Results: BRAT-expressing HOSE cells exhibited enhanced MMP1 mRNA expression. Higher levels of pro-MMP1 protein were detected by Western blotting of conditioned media. FRET ELISA revealed total MMP1 (pro and active forms) was also higher in BRAT-expressing cells, and that MMP1 in the conditioned media was functional. c-Jun knockdown diminished MMP-1 expression and secretion in HOSE. Expression of BRAT in normal breast and breast cancer cell lines failed to significantly alter chemosensitivity or MMP1 expression levels. Conclusion: Normal HOSE cells expressing the BRAT mutation exhibit c-Jun-dependent changes in expression of MMP1. Further investigation is warranted to elucidate the mechanism of this change since increased expression of this gene may represent an initial step forward on the continuum of malignant transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1852.

Abstract 4789: Functional characterization and clinical correlation of the tumor antigen casein kinase 1-epsilon in ovarian cancer

Abstract OBJECTIVES: Prior studies have identified antibodies to casein kinase I epsilon (CKI-ε) in the serum of patients with ovarian cancer. We investigated the expression pattern, functional roles, and clinical correlation of CKI-ε in ovarian cancer. METHODS: The expression of CKI-ε in 8 healthy ovaries and 68 ovarian tumor samples was determined by IHC and scored on intensity of antibody expression (scale 0-3) and the percentage of the area stained (scale 0-3). The average from both scores was assigned to each patient. Clinical characteristics for each patient were retrieved from electronic medical records. Statistical significance was determined using the Fisher exact test. Univariate analysis was performed by constructing probability curves according to the Kaplan-Meier method. Immortalized normal human ovarian surface epithelial (HOSE) cell lines with ectopic expression of CKI-ε were established by transfection with a CKI-ε expression construct. CKI-ε-targeting shRNAs were used to generate ovarian cancer cell lines that have stably suppressed CKI-ε expression. Protein expression was determined by Western blot. Growth rate was determined by cell counting. Three-dimensional spheroid culture was performed on Matrigel. The growth of ovarian cancer cell lines harboring either a control shRNA or a CKI-ε-shRNA was evaluated in a mouse xenograft model. The effects of IC261, an inhibitor of CKI-ε, on ovarian cancer cells were evaluated by MTT assay. RESULTS: CKI-ε was overexpressed in 67% of invasive ovarian tumors (P-value=0.001) and in 14 of 17 ovarian cancer cell lines. Normal HOSE cells that have ectopic expression of CKI-ε have accelerated growth rate and formed spheroids in Matrigel that were two to four times larger than those formed by control HOSE. In contrast, cancer cell lines harboring specific CKI-ε shRNA had reduced growth rate and 6-fold reduced tumor weight in the xenograft model (P-value=0.007). The cancer cell lines also showed CKI-ε-dependent sensitivity to IC261. The median CKI-ε expression level among the ovarian cancer tissue samples was 3. For patients whose score was ≤ 3 and &amp;gt; 3, the mean overall survival was 91 months and 48 months, respectively. Similarly, the mean disease free survival interval was 43 months and 22 months for patients whose score was ≤ 3 and &amp;gt; 3, respectively. Patients who scored &amp;gt; 3 tended to have late stage disease (61%) compared to patients who scored ≤ 3 (39%), (p=0.06). CONCLUSIONS: CKI-ε is highly expressed in ovarian tumors and cancer cell lines. Studies of cell lines with alterations in CKI-ε expression showed that this signaling protein regulates cell growth and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4789.

Overexpression of Epithelial Cell Adhesion Molecule in Primary, Metastatic, and Recurrent/Chemotherapy-Resistant Epithelial Ovarian Cancer

To evaluate the potential of epithelial cell adhesion molecule (Ep-CAM/TROP-1)-specific immunotherapy against epithelial ovarian carcinomas (EOCs), we have analyzed the expression of Ep-CAM at RNA and protein level in patients harboring primary, metastatic, and chemotherapy-resistant/recurrent EOC. Epithelial cell adhesion molecule expression was evaluated by real-time polymerase chain reaction and immunohistochemistry in 168 fresh-frozen biopsies and paraffin-embedded tissues. In addition, Ep-CAM surface expression was evaluated by flow cytometry in several freshly established ovarian carcinoma cell lines derived from patients harboring tumors resistant to chemotherapy in vivo as well as in vitro. Epithelial cell adhesion molecule transcript was found significantly overexpressed in primary, metastatic, and recurrent EOC when compared with normal human ovarian surface epithelium cell lines and fresh-frozen normal ovarian tissue (P < 0.001). Similarly, by immunohistochemistry, Ep-CAM protein expression was found significantly higher in primary, metastatic, and recurrent EOC when compared with normal ovarian tissues. Of interest, metastatic/recurrent tumors were found to express significantly higher levels of Ep-CAM protein when compared with primary ovarian carcinomas (P < 0.001). Finally, a high surface expression of Ep-CAM was found in 100% (5/5) of the chemotherapy-resistant ovarian carcinoma cell lines studied by flow cytometry. These results demonstrate high Ep-CAM overexpression in ovarian carcinoma, especially in metastatic and recurrent/chemotherapy-resistant ovarian disease. The lack of Ep-CAM expression on the chelomic epithelium in the peritoneal cavity, combined with the recent development of fully human monoclonal antibodies against this surface molecule, suggest Ep-CAM as a promising target for antibody-mediated therapies in ovarian carcinoma patients harboring tumors refractory to standard treatment modalities.

Inflammatory Cytokine Tumor Necrosis Factor α Confers Precancerous Phenotype in an Organoid Model of Normal Human Ovarian Surface Epithelial Cells

In this study, we established an in vitro organoid model of normal human ovarian surface epithelial (HOSE) cells. The spheroids of these normal HOSE cells resembled epithelial inclusion cysts in human ovarian cortex, which are the cells of origin of ovarian epithelial tumor. Because there are strong correlations between chronic inflammation and the incidence of ovarian cancer, we used the organoid model to test whether protumor inflammatory cytokine tumor necrosis factor alpha would induce malignant phenotype in normal HOSE cells. Prolonged treatment of tumor necrosis factor alpha induced phenotypic changes of the HOSE spheroids, which exhibited the characteristics of precancerous lesions of ovarian epithelial tumors, including reinitiation of cell proliferation, structural disorganization, epithelial stratification, loss of epithelial polarity, degradation of basement membrane, cell invasion, and overexpression of ovarian cancer markers. The result of this study provides not only an evidence supporting the link between chronic inflammation and ovarian cancer formation but also a relevant and novel in vitro model for studying of early events of ovarian cancer.

Repertoire of microRNAs in Epithelial Ovarian Cancer as Determined by Next Generation Sequencing of Small RNA cDNA Libraries

BackgroundMicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells.MethodologyWe used massively parallel pyrosequencing (i.e., “454 sequencing”) to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.ConclusionsThis report expands the body of miRNAs known to be expressed in epithelial ovarian cancer and provides a useful resource for future studies of the role of miRNAs in the pathogenesis and early detection of ovarian cancer.

Oncogenic transformation of human ovarian surface epithelial cells with defined cellular oncogenes

Ovarian surface epithelium (OSE) is considered to give rise to epithelial ovarian carcinomas (EOCs). To elucidate early processes contributing to the development of EOCs from the OSE, two batches of primary human OSE cells were transduced with non-viral human genes (mutant Cdk4, cyclinD1 and hTERT) so as to efficiently establish normal diploid OSE cells without chromosomal instability. Then defined genetic alterations frequently observed in EOCs were transduced into the OSE cells. A combination of p53 inactivation and oncogenic Kras transduction did not confer tumor-forming ability in immunodeficient mice, though additional transduction of Akt or combined transduction of c-myc with bcl-2 did result in tumor formation. In the latter case, tumors demonstrated phenotypes reminiscent of human EOCs, including cytokeratin expression, a highly aggressive phenotype, metastatic behavior and formation of ascites. These results indicate that inactivation of p53 and activation of the Ras pathway play critical roles in ovarian carcinogenesis in co-operation with the Akt or c-myc pathways. This first in vitro model system faithfully recapitulating the development of EOCs using normal human OSE cells should greatly facilitate further studies of EOCs.

Overexpression of CEACAM6 in borderline and invasive mucinous ovarian neoplasms

Objective Identifying markers specific for mucinous ovarian neoplasms (MON) is important for cancer diagnosis and surveillance, and will help improve our general understanding of the pathobiology of these tumors. CEACAM6 overexpression appears to be an early molecular event with prognostic significance in gastrointestinal carcinomas. Microarray analysis previously demonstrated high CEACAM6 overexpression in MON's and this study sought to validate this finding. Methods Western blot compared CEACAM6 expression in normal human ovarian surface epithelium (HOSE) and ovarian cancer cell lines. Quantitative RT-PCR (qRT-PCR) was performed on 75 laser-microdissected HOSE and ovarian cancer tissue samples. Immunohistochemistry (IHC) was performed and slides were analyzed in a semi-quantitative manner. Results CEACAM6 was expressed in 2 of 3 mucinous cancer cell lines. Expression was absent in all 2 HOSE, 7 serous cancer, and 2 clear cell cancer cell lines. 100-fold CEACAM6 overexpression (qRT-PCR) was demonstrated in 13/16 (81%) borderline, low-grade, and high-grade invasive MON's, compared to 5/50 (10%) serous and 1/5 (20%) benign mucinous samples. CEACAM6 expression was not different between borderline and invasive MON's ( p = 0.55) or across tumor stage ( p = 0.76). CEACAM6 staining was present in 24/28 (86%) borderline, low-grade, and high-grade invasive MON's; 13/28 (46%) exhibited moderate-strong staining. Neither CEACAM6 expression ( p = 0.36) nor staining intensity ( p = 0.51) was different between borderline and invasive MON's. None of the serous or benign mucinous tumors exhibited CEACAM6 staining. Conclusions CEACAM6 is overexpressed in borderline and invasive MON's.

Claudin-7 expression in human epithelial ovarian cancer

Claudin-7 (CLDN-7) is a tight junction protein recently found highly differentially expressed in ovarian carcinoma. To evaluate its potential as a novel biomarker, in this study, we quantified and compared claudin-7 expression at messenger RNA and protein level in 110 patients harboring various histologic types of epithelial ovarian carcinomas (EOC). CLDN-7 transcript was found significantly overexpressed in both primary and metastatic EOCs compared to normal human ovarian surface epithelium cell lines (fold change = 111.4, P < 0.001) by reverse transcription-polymerase chain reaction. At the protein level, CLDN-7 expression was found significantly higher in tumors of primary and metastatic origin when compared to normal ovaries (P < 0.001), regardless of the histologic type, the grade of differentiation, and the pathologic stage of the disease (P = 0.12). Moreover, a strong immunoreactivity for CLDN-7 was detected in EOC cells present in ascites fluids, whereas ascites-derived inflammatory cells, histiocytes, and reactive mesothelial cells were negative. Finally, immunohistochemical expression of CLDN-7 was observed in several human normal epithelial control tissues analyzed. CLDN-7 is significantly overexpressed in all main histologic types of EOC and in single neoplastic cells disseminated in peritoneal cavity and pleural effusions, suggesting its potential role as novel diagnostic marker in ovarian cancer. Despite widespread expression of CLDN-7 in several human normal tissues, the high density of CLDN-7 molecules, their membranous localization on EOC cells, and their lack of expression on the celomic epithelium in the peritoneal cavity suggest that this target could be potentially suitable for antibody-mediated localized therapies of ovarian adenocarcinoma.