Normal Ovarian Epithelium Research Articles

Stearoyl-CoA desaturase 1 inhibition induces ER stress-mediated apoptosis in ovarian cancer cells

Ovarian cancer is a leading cause of death among gynecologic tumors, often detected at advanced stages. Metabolic reprogramming and increased lipid biosynthesis are key factors driving cancer cell growth. Stearoyl-CoA desaturase 1 (SCD1) is a crucial enzyme involved in de novo lipid synthesis, producing mono-unsaturated fatty acids (MUFAs). Here, we aimed to investigate the expression and significance of SCD1 in epithelial ovarian cancer (EOC). Comparative analysis of normal ovarian surface epithelial (NOSE) tissues and cell lines revealed elevated SCD1 expression in EOC tissues and cells. Inhibition of SCD1 significantly reduced the proliferation of EOC cells and patient-derived organoids and induced apoptotic cell death. Interestingly, SCD1 inhibition did not affect the viability of non-cancer cells, indicating selective cytotoxicity against EOC cells. SCD1 inhibition on EOC cells induced endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR) sensors and resulted in apoptosis. The addition of exogenous oleic acid, a product of SCD1, rescued EOC cells from ER stress-mediated apoptosis induced by SCD1 inhibition, underscoring the importance of lipid desaturation for cancer cell survival. Taken together, our findings suggest that the inhibition of SCD1 is a promising biomarker as well as a novel therapeutic target for ovarian cancer by regulating ER stress and inducing cancer cell apoptosis.

Abstract B095: Tenascin C promotes ovarian cancer stem cells interactions with the niche

Abstract Background: Effective treatment of ovarian cancer (OC) is greatly impacted by advanced, late-stage diagnosis which usually presents with intra-peritoneal metastases, chemoresistance and high recurrence. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids disseminated in the peritoneal fluid. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components that stiffen the tumor stroma and promote the aberrant activation of oncogenic processes, including survival, invasion, and stemness. Tenascin C (TNC) is one of the molecules secreted in the ECM and correlates with migration and metastatic progression in a variety of carcinomas. However, the functional role of TNC in spheroid formation and in the maintenance of the platinum resistant ovarian cancer stem phenotype has not been investigated yet. We tested the hypothesis that the increased expression of TNC and its secretion in the ECM compartment promotes survival of OCSCs, supports OC spheroid formation and tumor progression. Methods: We compared TNC expression in OC cells grown as spheroids and monolayers and in chemoresistant vs sensitive OC cells by realtime-PCR and Western Blot (WB). TNC blockade by siRNA knockdown (KD) was evaluated in combination with carboplatin on spheroid assay, colony formation, and by qPCR of stemness related gene expression. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the TNC promoter. Results: OC cells grown as spheroids showed a significant increase in TNC expression compared to monolayers. Our results also demonstrated an increase in TNC expression levels in platinum resistant OC cell lines when compared to the platinum sensitive counterpart. A comparison of normal adjacent ovarian epithelium and primary OC tumors on a multitissue array revealed that TNC expression coincided with a pro-tumorigenic phenotype (n=88, P<0.001), correlating with poor outcome. TNC-KD in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. Mechanistically, b-catenin-KD decreased TNC expression levels and β-catenin coimmunoprecipitated with the TNC promoter, suggesting that TNC is a direct β-catenin target. Additionally, OC spheroids treated with the Wnt ligand WNT-3A showed increased TNC expression compared to control untreated cells, as demonstrated by WB. Importantly, the enhanced expression of TNC was followed by its secretion in the ECM compartment, as observed via immunofluorescence imaging, implying a direct role of TNC in the ECM-rearrangement which is responsible for CSC maintenance and spheroid formation. Conclusions: TNC is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that strategies aimed at interfering with TNC expression and/or secretion along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Amber Rogers, Maya Shebeshi, Morganna Gordon, Rula Atwani, Virginie Lazar, Salvatore Condello. Tenascin C promotes ovarian cancer stem cells interactions with the niche [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B095.

Tripartite-motif 3 represses ovarian cancer progression by downregulating lactate dehydrogenase A and inhibiting AKT signaling.

The E3 ubiquitin ligase Tripartite-motif 3 (TRIM3) is known to play a crucial role in tumor suppression in various tumors through different mechanisms. However, its function and mechanism in ovarian cancer have yet to be elucidated. Our study aims to investigate the expression of TRIM3 in ovarian cancer and evaluate its role in the development of the disease. Our findings revealed a significant decrease in TRIM3 mRNA and protein levels in ovarian cancer tissues and cells when compared to normal ovarian epithelial tissues and cells. Furthermore, we observed a negative correlation between the protein level of TRIM3 and the FIGO stage, as well as a positive correlation with the survival of ovarian cancer patients. Using gain and loss of function experiments, we demonstrated that TRIM3 can inhibit cell proliferation, migration and invasion of the ovarian cancer cells in vitro, as well as suppress tumor growth in vivo. Mechanistic studies showed that TRIM3 interacts with lactate dehydrogenase A, a key enzyme in the glycolytic pathway, through its B-box and coiled-coil domains and induces its ubiquitination and proteasomal degradation, leading to the inhibition of glycolytic ability in ovarian cancer cells. RNA-sequencing analysis revealed significant alterations in the phosphatidylinositol signaling pathways upon TRIM3 overexpression. Additionally, overexpression of TRIM3 inhibited the phosphorylation of AKT. In conclusion, our study demonstrated that TRIM3 exerts a tumor-suppressive effect in ovarian cancer, at least partially, by downregulating LDHA and inhibiting the AKT signaling pathway, and thus leading to the inhibition of glycolysis and limiting the growth of ovarian cancer cells.

CircAGFG1 Promotes Ovarian Cancer Progression Through the miR-409-3 p/ZEB1 Axis.

Circular RNAs (circRNAs) serve a crucial regulatory role in ovarian cancer (OC). Circular RNA ArfGAP with FG repeats 1 (circAGFG1) has been shown to be involved in promoting the progression of several cancers, containing triple-negative breast cancer, esophageal cancer and colorectal cancer. However, the function of circAGFG1 in OC is unclear. Quantitative real-time reverse transcription PCR (RT-qPCR) was conducted to determine the expression levels of circAGFG1 and miR-409-3p. The proliferation and metastasis of cells were determined by colony formation assays, EdU assays, transwell assays and wound healing assays. In addition, a dual-luciferase reporter assay was performed to validate the mechanism between circAGFG1, miR-409-3p, and ZEB1. Our data suggested that circAGFG1 was significantly overexpressed in OC tissues compared to normal ovarian epithelial tissues. Overexpression of circAGFG1 was correlated with intraperitoneal metastasis, tumor recurrence and advanced stage. Additionally, circAGFG1 overexpression revealed a poor prognosis in OC patients. Knockdown of circAGFG1 suppressed the proliferation, invasion and migration of OC cells. Mechanistically, circAGFG1 acted as a sponge of miR-409-3p to enhance the expression level of zinc finger E-box binding homeobox 1 (ZEB1), thereby conferring OC cell proliferation, invasion and migration. Importantly, re-expression of ZEB1 effectively reversed the effects of circAGFG1 knockdown on OC cells. In summary, our study indicated that circAGFG1 may act as a prognostic biomarker and potential therapeutic target for patients with OC.

Development of a machine learning-based signature utilizing inflammatory response genes for predicting prognosis and immune microenvironment in ovarian cancer.

Ovarian cancer (OC) represents a significant health challenge, characterized by a particularly unfavorable prognosis for affected women. Accumulating evidence supports the notion that inflammation-related factors impacting the normal ovarian epithelium may contribute to the development of OC. However, the precise role of inflammatory response-related genes (IRRGs) in OC remains largely unknown. To address this gap, we performed an integration of mRNA expression profiles from 7 cohorts and conducted univariate Cox regression analysis to screen 26 IRRGs. By utilizing these IRRGs, we categorized patients into subtypes exhibiting diverse inflammatory responses, with subtype B displaying the most prominent immune infiltration. Notably, the elevated abundance of Treg cells within subtype B contributed to immune suppression, resulting in an unfavorable prognosis for these patients. Furthermore, we validated the distribution ratios of stromal cells, inflammatory cells, and tumor cells using whole-slide digitized histological slides. We also elucidated differences in the activation of biological pathways among subtypes. In addition, machine learning algorithms were employed to predict the likelihood of survival in OC patients based on the expression of prognostic IRRGs. Through rigorous testing of over 100 combinations, we identified CXCL10 as a crucial IRRG. Single-cell analysis and vitro experiments further confirmed the potential secretion of CXCL10 by macrophages and its involvement in lymphangiogenesis within the tumor microenvironment. Overall, the study provides new insights into the role of IRRGs in OC and may have important implications for the development of novel therapeutic approaches.

Loss of RBPMS in ovarian cancer compromises the efficacy of EGFR inhibitor gefitinib through activating HER2/AKT/mTOR/P70S6K signaling

RBPMS may be a tumor suppressor in cancer, but its impact in modulation of drug sensitivity is unclear. This study aimed to investigate the regulatory role of RBPMS in cellular response to EGFR inhibitor gefitinib in ovarian cancer (OC). By western blotting assay, we revealed RBPMS was down-regulated in epithelial ovarian cancer tissues compared to normal control ovarian epithelial tissues. Overexpression of RBPMS inhibited cell viability and proliferation, and conferred gefitinib sensitivity, accompanied by reduced expression of p-EGFR, and vice versa. Proteomic analysis and flow cytometry experiments showed that RBPMS induced S-stage cell cycle arrest in gefitinib-treated OC cells. Co-IP assay suggested that HER2 was a downstream target of RBPMS, and RBPMS negatively regulated HER2 expression. HER2 counteracted the stimulation of RBPMS to cell growth blocking, gefitinib sensitivity and cell cycle arrest. We further demonstrated that RBPMS overexpression suppressed the activation of p-AKT, p-mTOR and p-P70S6K, which was rescued by up-regulation of HER2. The combination of AKT inhibitor MK2206 and gefitinib had a synergistic effect on OC cells with high level of RBPMS. In conclusion, through the direct inhibition of HER2/AKT/mTOR/P70S6K pathway, RBPMS may be a potential therapeutic target for improving gefitinib sensitivity in OC.

TRAF4 promotes the malignant progression of high-grade serous ovarian cancer by activating YAP pathway

High-grade serous ovarian cancer (HGSOC) accounts for the majority of deaths caused by epithelial ovarian cancer. The specific molecular changes attributable to the pathogenesis of HGSOC are still largely unknown. TRAF4 has been identified to be up-regulated in certain cancers. However, the role and mechanism of TRAF4 in HGSOC remain unclear. In this study, we aim to explore the prognostic value and function of TRAF4 in HGSOC. Immunohistochemical staining and prognostic analysis were used to estimate the prognosis value of TRAF4 in HGSOC. Cell counting assays, colony formation assays, sphere formation assays and tumorigenic assays were used to explore the function of TRAF4 in ovarian cancer cells. Furthermore, RNA-seq, qPCR and western blotting were performed to investigate the molecular mechanism of TRAF4 in ovarian cancer cells. The results showed that TRAF4 was significantly higher expressed in ovarian cancer than normal ovarian epithelium. Moreover, high expression of TRAF4 was significantly associated with shorter overall survival and recurrence-free survival in HGSOC. Knockdown of TRAF4 significantly inhibited the proliferation and tumorigenicity of ovarian cancer cells, whereas overexpression of TRAF4 promoted the proliferation and tumorigenicity of ovarian cancer cells both in vitro and in vivo. Mechanistically, our study demonstrated that TRAF4 expression was positively correlated with the YAP pathway gene signatures, and the malignant progression induced by TRAF4 was inhibited after silencing YAP signaling by its selective inhibitor. In conclusion, our findings suggested that TRAF4 promoted the malignant progression of ovarian cancer cells by activating YAP pathway and might serve as a prognostic biomarker for HGSOC.

Abstract 906: Targeting CD166 to overcome platinum resistance in ovarian cancer

Abstract Background: Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids by OC cells disseminated in the peritoneal fluid. Within a spheroid, OC cells undergo an epithelial-to-mesenchymal transition (EMT), which involves changes in the expression of cell adhesion molecules (CAMs) and acquisition of cancer stem cell (CSC) characteristics, therefore protecting OC cells during chemotherapy. Cluster of differentiation 166 (CD166) is one of the CAMs involved in tumor progression and its modulation impacts cell-cell adhesion, leading to impaired ability of tumor cells to metastasize. However, limited number of studies are available on the functional role of CD166 in the initiation, progression, and chemoresistance of OC. Our goal was to investigate the contribution of CD166 in supporting chemoresistance and the tumorigenic OCSC phenotype, thus proposing this molecule as a potential new therapeutic target in OC. Methods: We compared CD166 expression in primary OC cells grown as spheroids and monolayers and in chemoresistant versus sensitive OC cell lines by q-PCR and Western Blot (WB). CD166 blockade by shRNA knockdown (KD) or anti-CD166 inhibitory antibody (clone AZN-L50) was evaluated on spheroid assay and colony formation. Combinatorial carboplatin and AZN-L50 treatment was assessed on spheroid assays and by WB of pro-apoptotic proteins. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the CD166 promoter. Results: OC cells grown as spheroids showed a significant increase in CD166 expression compared to monolayers. Our results also demonstrated an increase in CD166 expression levels in chemoresistant OC cell lines when compared to the chemosensitive counterpart. A comparison of normal adjacent ovarian epithelium, localized, and metastatic OC tissues on a multitissue array revealed that CD166 expression coincided with an advanced promigratory phenotype (n=88, P<0.001), correlating with poor outcome. CD166 blockade by either shRNA-mediated KD or AZN-L50 treatment in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. The combination of carboplatin and CD166 blockade increased cleaved-PARP and -caspase-3 levels compared to single agent alone, indicating sustained DNA damage. Mechanistically, β-catenin-KD decreased CD166 expression levels and β-catenin coimmunoprecipitated with the CD166 promoter, suggesting that CD166 is a direct β-catenin target. Conclusions: CD166 is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that inhibition of CD166 along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Rula Atwani, Mayuri Prasad, George E Sandusky, Salvatore Condello. Targeting CD166 to overcome platinum resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 906.

Abstract 4185: Targeting variant of CEACAM5 and CEACAM6 using NEO-201 and IL-15 in gynecologic cancers

Abstract Background: Uterine and ovarian cancers express glycosylated forms of CEACAMs 5 and 6 that regulate interactions with the tumor microenvironment. NEO-201 is a novel monoclonal antibody that binds a glycosylated variant of these CEACAMs that is upregulated in cancer compared to normal uterine or ovarian epithelia. Previous work has shown that NEO-201 induced cancer cell death predominantly by antibody-dependent cellular cytotoxicity (ADCC) that can be enhanced by the addition of IL-15. Methods: NEO-201 target expression was assessed on uterine and ovarian cancer cell lines, as well as a tissue microarray of primary uterine cancer samples. Cell lines were profiled with DNA and RNA sequencing. Chromium-release assays were performed to quantify ADCC without and with varying concentrations of IL-15 and varying target to effector ratios. In vivomodeling was performed using ovarian cancer cell line OV90, and mice were treated with NEO-201, with PBMC or NK cells, with or without IL-15. Results: NEO-201 target antigen was highly expressed in uterine cancer cell line ACI158 and ovarian cancer cell line OV90. The antigen was expressed in primary uterine cancer samples as well. IL-15 consistently increased the in vitro killing of both cell lines and was further enhanced with the use of NK cells compared to PBMC. These findings were replicated in the in vivo mouse model, where mice survived longest with the combination of NEO-201, IL-15 and NK cells. Conclusions: The ADCC activity of NEO-201 monoclonal antibody was enhanced with the addition of IL-15, especially in the presence of purified NK cells. NEO-201 with IL-15 shows promising results as a potential treatment for biomarker-selected gynecologic cancers. Citation Format: Maria Pia Morelli, Massimo Fantini, Kwong Y. Tsang, Phillip Arlen, Paulette Fauceglia, Lidia Hernandez, Soumya Korrapati, Elijah Edmonson, Christopher Cole, Christina M. Annunziata. Targeting variant of CEACAM5 and CEACAM6 using NEO-201 and IL-15 in gynecologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4185.

Peptide PDHPS1 Inhibits Ovarian Cancer Growth through Disrupting YAP Signaling.

The lives of patients with ovarian cancer are threatened largely due to metastasis and drug resistance. Endogenous peptides attract increasing attention in oncologic therapeutic area, a few antitumor peptides have been approved by the FDA for clinical use over the past decades. However, only few peptides or peptide-derived drugs with antiovarian cancer effects have been identified. Here we focused on the biological roles and mechanism of a peptide named PDHPS1 in ovarian cancer development. Our results indicated that PDHPS1 reduced the proliferation ability of ovarian cancer cells in vitro and inhibited the ovarian cancer growth in vivo. Peptide pull down and following mass spectrometry, Western blot and qRT-PCR revealed that PDHPS1 could bind to protein phosphatase 2 phosphatase activator (PTPA), an essential activator of protein phosphatase 2A (PP2A), which resulted in increase of phosphorylated YAP, further inactivated YAP, and suppressed the expression of its downstream target genes. Flow cytometry, cell membrane permeability test, and IHC staining study demonstrated that there were no observable side effects of PDHPS1 on normal ovarian epithelium and hepatorenal function. Besides, modification of membrane penetration could improve the physicochemical properties and biological activity of PDHPS1. In conclusion, our study demonstrated that the endogenous peptide PDHPS1 serves as an antitumor peptide to inhibit YAP signaling pathway though interacting with PTPA in ovarian cancer.

TRIM21 deficiency promotes cell proliferation and tumorigenesis via regulating p21 expression in ovarian cancer

ABSTRACT Tripartite motif-containing 21 (TRIM21) has been reported to have a cancer-promoting or anticancer effect in various tumors; however, its role in ovarian cancer (OC) remains to be elucidated. In this study, we explored the biological function of TRIM21 in OC progression and investigated the potential mechanisms. We found that TRIM21 was remarkably decreased in OC tissues and cell lines compared with adjacent-cancerous tissues and normal ovarian epithelium cell. Decreased expression of TRIM21 in OC patients was significantly correlated with shorter overall and disease-specific survival by The Cancer Genome Atlas database (TCGA) analysis. Functional assays revealed that TRIM21 inhibited the migration and invasion of OC cells; and that TRIM21 also obviously impaired cell proliferation by inhibiting cell cycle progression in vitro and in vivo. Taken together, our results suggest that TRIM21 may be a promising biomarker and target for OC diagnosis and treatment.

TMED9 Expression Level as a Biomarker of Epithelial Ovarian Cancer Progression and Prognosis.

Transmembrane emp24 domain-containing protein 9 (TMED9) belongs to the TMED/p24 family that transports, modifies, and packs proteins and lipids into vesicles for delivery to specific locations and is important in innate immune signaling via the endoplasmic reticulum-Golgi cargo pathway. TMED9 has been implicated in various cancer types; however, its role in epithelial ovarian cancer (EOC) is unclear. In this study, we aimed to elucidate the role and clinical significance of TMED9 in EOC. mRNA and protein levels of TMED9 and their associations with clinicopathological features in EOCs were evaluated using RNA-sequencing and immunohistochemistry data. Functional studies assessing the tumorigenic role of TMED9 in EOC cell lines were also performed. The mRNA expression of TMED9 was up-regulated in EOC compared to that in normal ovarian epithelium. TMED9 protein expression increased in progression from normal ovarian epithelium to EOC (p<0.001). Moreover, high expression of TMED9 was associated with advanced stage, serous cell type and poor histological grade in EOC and demonstrated independent prognostic significance for both disease-free and overall survival. Further functional studies showed that TMED9 knockdown reduced migration, invasion, cell proliferation, and colony formation of EOC cells. Overall, our results support the use of TMED9 as a valuable prognostic biomarker and provide evidence for targeting of TMED9 as a novel strategy for EOC treatment.

Reference Module-Based Analysis of Ovarian Cancer Transcriptome Identifies Important Modules and Potential Drugs.

Ovarian cancer (OVC) is often diagnosed at the advanced stage resulting in a poor overall outcome for the patient. The disease mechanisms, prognosis, and treatment require imperative elucidation. A rank-based module-centric framework was proposed to analyze the key modules related to the development, prognosis, and treatment of OVC. The ovarian cancer cell line microarray dataset GSE43765 from the Gene Expression Omnibus database was used to construct the reference modules by weighted gene correlation network analysis. Twenty-three reference modules were tested for stability and functionally annotated. Furthermore, to demonstrate the utility of reference modules, two more OVC datasets were collected, and their gene expression profiles were projected to the reference modules to generate a module-level expression. An epithelial-mesenchymal transition module was activated in OVC compared to the normal epithelium, and a pluripotency module was activated in ovarian cancer stroma compared to ovarian cancer epithelium. Seven differentially expressed modules were identified in OVC compared to the normal ovarian epithelium, with five up-regulated, and two down-regulated. One module was identified to be predictive of patient overall survival. Four modules were enriched with SNP signals. Based on differentially expressed modules and hub genes, five candidate drugs were screened. The hub genes of those modules merit further investigation. We firstly propose the reference module-based analysis of OVC. The utility of the analysis framework can be extended to transcriptome data of other kinds of diseases.

How Does Endometriosis Lead to Ovarian Cancer? The Molecular Mechanism of Endometriosis-Associated Ovarian Cancer Development.

Simple SummaryDriver gene mutations have been identified in not only various types of endometriosis which are considered the origin of endometriosis-associated ovarian cancer but also the normal endometrium which is considered the origin of endometriosis. We focused on genomic linkage from normal endometrium to ovarian endometriosis and endometriosis-associated ovarian cancer (EAOC) and summarized the current knowledge of the commonality and differentiation of genomic features in the uterine endometrium, endometriosis, and EAOC. In addition, we have proposed molecular mechanism of ovarian carcinogenesis from the normal endometrium via endometriosis based on genomic alterations. This review is expected to contribute to research for the prevention of endometriosis and EAOC.Numerous epidemiological and histopathological studies support the notion that clear cell and endometrioid carcinomas derive from ovarian endometriosis. Accordingly, these histologic types are referred to as “endometriosis-associated ovarian cancer” (EAOC). Although the uterine endometrium is also considered an origin of endometriosis, the molecular mechanism involved in transformation of the uterine endometrium to EAOC via ovarian endometriosis has not yet been clarified. Recent studies based on high-throughput sequencing technology have revealed that cancer-associated gene mutations frequently identified in EAOC may exist in the normal uterine endometrial epithelium and ovarian endometriotic epithelium. The continuum of genomic alterations from the uterine endometrium to endometriosis and EAOC has been described, though the significance of cancer-associated gene mutations in the uterine endometrium or endometriosis remains unclear. In this review, we summarize current knowledge regarding the molecular characteristics of the uterine endometrium, endometriosis, and EAOC and discuss the molecular mechanism of cancer development from the normal endometrium through endometriosis in an effort to prevent EAOC.

High expression of ENPP1 in high-grade serous ovarian carcinoma predicts poor prognosis and as a molecular therapy target.

Recent studies have shown that the expression of ENPP1 is related to differentiation, death, dissemination and chemosensitivity of tumor cells. So far, there is no research in ovarian carcinoma. This study aimed at exploring the role of ENPP1 gene in ovarian carcinoma, the relationship with prognostic indicators and chemotherapy resistance, and investigates the possibility of molecular targeted therapy. The expression of ENPP1 in 41 normal ovarian epithelial tissues, 97 ovarian serous cystadenoma and 103 HGSOC tissues was detected by IHC. In ovarian cancer tissues and ovarian cancer cell lines, mRNA and protein expression of ENPP1 was determined by qRT-PCR and Western blot. The ENPP1 expression was knockdowned by siRNA. Cell proliferation was measured with the BrdU Cell Proliferation ELISA. Cell migration and invasion were detected by Wound-Healing, Transwell migration and Matrigel invasion assay. Caspase 3 activity was determined by the CaspACE. The expression of EMT markers such as E-cadherin, N-cadherin, and Vimentin was measured, and the expression of PCNA and MMP9 was also be detected. The results showed that the expression of ENPP1 was significantly increased in high-grade ovarian serous carcinoma, the number of strong expression was 85.4% (22.3%+63.1%) and only 1.03% (1.03%+0.0%) in serous cystadenoma, but no in normal ovarian epithelium (P< 0.05). And the stronger the expression of ENPP1, the later the FIGO stage and the poorer differentiation of cells (P = 0.001 or <0.001, respectively). However, no correlation was found between the expression of ENPP1 and chemosensitivity. ENPP1 was also highly expressed in ovarian cancer tissues and in epithelial ovarian cancer cell lines (A2780, CaoV3, OVCAR3, SKOV3 and 3ao). After down-regulation of ENPP1 expression by RNA interference, the cell proliferation, migration and invasion of ovarian cancer cell decreased significantly, the expression of apoptosis related gene caspase 3 increased significantly, while the expression of PCNA and MMP9 was significantly down regulated. In addition, EMT biological characteristics of A2780 and SKOV3 cells were also inhibited. In summary, the increased expression of ENPP1 may be related to the occurrence of HGSOC, and indicate that the disease progresses rapidly and the prognosis is poor. ENPP1 may be considered as a potential molecular therapeutic target.

Association among Vitamin D, Retinoic Acid-Related Orphan Receptors, and Vitamin D Hydroxyderivatives in Ovarian Cancer.

Vitamin D and its derivatives, acting via the vitamin D receptor (VDR) and retinoic acid-related orphan receptors γ and α (RORγ and RORα), show anticancer properties. Since pathological conditions are characterized by disturbances in the expression of these receptors, in this study, we investigated their expression in ovarian cancers (OCs), as well as explored the phenotypic effects of vitamin D hydroxyderivatives and RORγ/α agonists on OC cells. The VDR and RORγ showed both a nuclear and a cytoplasmic location, and their expression levels were found to be reduced in the primary and metastatic OCs in comparison to normal ovarian epithelium, as well as correlated to the tumor grade. This reduction in VDR and RORγ expression correlated with a shorter overall disease-free survival. VDR, RORγ, and RORα were also detected in SKOV-3 and OVCAR-3 cell lines with increased expression in the latter line. 20-Hydroxy-lumisterol3 (20(OH)L3) and synthetic RORα/RORγ agonist SR1078 inhibited proliferation only in the OVCAR-3 line, while 20-hydroxyvitamin-D3 (20(OH)D3) only inhibited SKOV-3 cell proliferation. 1,25(OH)2D3, 20(OH)L3, and SR1078, but not 20(OH)D3, inhibited spheroid formation in SKOV-3 cells. In summary, decreases in VDR, RORγ, and RORα expression correlated with an unfavorable outcome for OC, and compounds targeting these receptors had a context-dependent anti-tumor activity in vitro. We conclude that VDR and RORγ expression can be used in the diagnosis and prognosis of OC and suggest their ligands as potential candidates for OC therapy.

Inhibition of AXL enhances chemosensitivity of human ovarian cancer cells to cisplatin via decreasing glycolysis.

Anexelekto (AXL), a member of the TYRO3-AXL-MER (TAM) family of receptor tyrosine kinases(RTK), is overexpressed in varieties of tumor tissues and promotes tumor development by regulating cell proliferation, migration and invasion. In this study, we investigated the role of AXL in regulating glycolysis in human ovarian cancer (OvCa) cells. We showed that the expression of AXL mRNA and protein was significantly higher in OvCa tissue than that in normal ovarian epithelial tissue. In human OvCa cell lines suppression of AXL significantly inhibited cell proliferation, and increased the sensitivity of OvCa cells to cisplatin, which also proved by nude mice tumor formation experiment. KEGG analysis showed that AXL was significantly enriched in the glycolysis pathways of cancer. Changes in AXL expression in OvCa cells affect tumor glycolysis. We demonstrated that the promotion effect of AXL on glycolysis was mediated by phosphorylating the M2 isoform of pyruvate kinase (PKM2) at Y105. AXL expression was significantly higher in cisplatin-resistant OvCa cells A2780/DDP compared with the parental A2780 cells. Inhibition of AXL decreased the level of glycolysis in A2780/DDP cells, and increased the cytotoxicity of cisplatin against A2780/DDP cells, suggesting that AXL-mediated glycolysis was associated with cisplatin resistance in OvCa. In conclusion, this study demonstrates for the first time that AXL is involved in the regulation of the Warburg effect. Our results not only highlight the clinical value of targeting AXL, but also provide theoretical basis for the combination of AXL inhibitor and cisplatin in the treatment of OvCa.

Low Expression of Claudin-7 as Potential Predictor of Distant Metastases in High-Grade Serous Ovarian Carcinoma Patients.

High-grade serous ovarian carcinoma (HGSOC) usually spreads directly into the peritoneal cavity following a transcoelomic dissemination route, although distant hematogenous metastasis exist and have been reported. However, no tumor markers can currently predict the risk of distant metastases in HGSOC. Claudins, belonging to tight-junction proteins, are dysregulated in HGSOC and functionally related to cancer progression. Here we analyzed claudin-3, -4, and -7 expression as potential markers of distant metastases. Using quantitative RT-PCR and immunohistochemistry we assessed the expression of claudins in primary HGSOC tissues, normal ovarian, and normal fallopian tube epithelia and correlated it with clinicopathological features, including the site of metastasis and the route of dissemination. Gene set enrichment analysis was performed on microarray-generated gene expression data to investigate key pathways in patients with distant metastases. We found the overall expression level of claudin-3, -4, and -7 mRNA decreased in HGSOC compared to normal tubal epithelium, currently considered the potential site of origin of many HGSOC. The reduced expression of claudin-7 is significantly associated with the development of distant metastases (p = 0.016), mainly by hematogenous route (p = 0.025). In patients with diminished expression of claudin-7, immunohistochemical staining revealed a heterogeneous pattern of membranous staining with discontinuous expression of claudin-7 along the cell border, indicative of a dischoesive architecture. The estimated reduction in the probability of distant disease is of 39% per unit increase in the level of claudin-7 (p = 0.03). Genes involved in epithelial to mesenchymal transition, hypoxia, and angiogenesis processes resulted strongly associated to hematogenous recurrence. Our data suggest a potential role of claudin-7 in discriminating distant metastatic events in HGSOC patients. The quantification of its expression levels could be a useful tool to identify patient deserving a personalized follow-up in terms of clinical and radiological assessment.

The DNA Cytosine Deaminase APOBEC3B is a Molecular Determinant of Platinum Responsiveness in Clear Cell Ovarian Cancer.

Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention. APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens (n = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates (n = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages. One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival (P = 0.026) and moderately with overall survival (P = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues. These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.

Epithelial-mesenchymal interconversions in ovarian cancer: The levels and functions of E-cadherin in intraabdominal dissemination.

The metastatic process of ovarian cancer (OC) is almost exclusively defined by direct shedding of tumor cells into the abdominal cavity, followed by clustering into multicellular aggregates and posterior peritoneal anchorage. This process relies on dynamic intercellular interactions which are modified by epithelial- mesenchymal interconversions and, therefore, E-cadherin expression variability. Although widely accepted as a tumor suppressor in many types of cancer, E-cadherin is currently known to have a dynamic expression and a much more complex role in OC. First, high E-cadherin expression is considered a sign of metaplasia in the normal ovarian epithelium, due to its association with epithelial growth factor receptor (EGFR) mediated cell proliferation. Subsequently, it is the decreased expression of E-cadherin that allows the acquisition of a more invasive phenotype, leading to the spread of primary tumor cells into the peritoneal fluid. This downregulation seems to depend on complex regulatory mechanisms, from molecular proteolysis to microenvironment interference and epigenetic regulation. E-cadherin cleavage and its resulting fragments appear to be essential to the process of dissemination and even to the formation of multicellular aggregates. Paradoxically, the maintenance of some E-cadherin expression seems to promote intercellular adhesion, resistance, and survival while decreasing cancer response to chemotherapy. Multiple studies have shown that reversing epithelial-mesenchymal transaction (EMT) and increasing E-cadherin expression prevents OC intraperitoneal dissemination, but findings that simultaneously correlate E-cadherin downregulation to higher chemotherapy sensitivity should not be ignored. Nevertheless, EMT and E-cadherin seem to have a potential interest as therapeutic targets in novel approaches to OC treatment.