Transition In Ovarian Cancer Research Articles

Abstract A033: The transcriptional landscape of cisplatin resistance and its relationship with epithelial mesenchymal transition in ovarian cancer

Abstract A033: The transcriptional landscape of cisplatin resistance and its relationship with epithelial mesenchymal transition in ovarian cancer

RIPK4 Promotes Cell Invasion and the Epithelial-Mesenchymal Transition in Ovarian Cancer.

To investigate the clinical role and biological function of receptor-interacting protein kinase 4 (RIPK4) in ovarian cancer (OC). We conducted a comprehensive analysis of the expression and prognostic role of RIPK4 in OC using various public databases including The Cancer Genome Atlas, Oncomine, and Kaplan-Meier plotter. In vitro studies included wound healing, cell migration and invasion, cell proliferation, and cell apoptosis assays as well as vascular mimicry experiments. In vivo studies were conducted using subcutaneous and intraperitoneal xenografts. Our findings revealed that RIPK4 was significantly overexpressed in OC tissue compared to normal ovarian tissue. Moreover, the overexpression of RIPK4 was associated with advanced-stage disease and a poor prognosis in OC patients. RIPK4 silencing resulted in significant inhibition of intraperitoneal tumor growth, invasion, and vascular mimicry in OC cells. Furthermore, downregulation of RIPK4 inhibited the epithelial-mesenchymal transition of OC cells both in vitro and in vivo by promoting the expression of E-cadherin and inhibiting the expression of N-cadherin. The results of this study suggest that RIPK4 may function as an oncogene in the development and prognosis of OC.

The Mechanism and Dynamic Regulation of Epithelial to Mesenchymal Transition in Ovarian Cancer

The Mechanism and Dynamic Regulation of Epithelial to Mesenchymal Transition in Ovarian Cancer

Myricetin suppresses TGF-β-induced epithelial-to-mesenchymal transition in ovarian cancer.

Background: Ovarian cancer (OC) is the second most common gynecological malignancy and has a high mortality rate. The current chemotherapeutic drugs have the disadvantages of drug resistance and side effects. Myricetin, a kind of natural compound, has the advantages of easy extraction, low price, and fewer side effects. Multiple studies have demonstrated the anti-cancer properties of myricetin. However, its impact on OC is still unknown and needs further investigation. Therefore, this study aimed to elucidate the mechanism by which myricetin suppresses transforming growth factor-β (TGF-β) -induced epithelial-to-mesenchymal transition (EMT) in OC through in vivo and in vitro experiments. Methods: In vitro experiments were conducted to evaluate the effects of myricetin on cell proliferation and apoptosis using CCK8 assay, plate clonal formation assay, and flow cytometry. Western blot was employed to evaluate the expression levels of caspase-3, PARP, and the MAPK/ERK and PI3K/AKT signaling pathways. Wound healing, transwell, western blot and immunofluorescence assay were used to detect TGF-β-induced cell migration, invasion, EMT and the levels of Smad3, MAPK/ERK, PI3K/AKT signaling pathways. Additionally, a mouse xenograft model was established to verify the effects of myricetin on OC in vivo. Results: Myricetin inhibited OC proliferation through MAPK/ERK and PI3K/AKT signaling pathways. Flow cytometry and western blot analyses demonstrated that myricetin promoted apoptosis by increasing the expression of cleaved-PARP and cleaved-caspase-3 and the ratio of Bax/Bcl-2 in OC. Furthermore, myricetin suppressed the TGF-β-induced migration and invasion by transwell and wound healing assays. Mechanistically, western blot indicated that myricetin reversed TGF-β-induced metastasis through Smad3, MAPK/ERK and PI3K/AKT signaling pathway. In vivo, myricetin significantly repressed OC progression and liver and lung metastasis. Conclusion: Myricetin exhibited inhibitory effects on OC progression and metastasis both in vivo and in vitro. And it also reversed TGF-β-induced EMT through the classical and non-classical Smad signaling pathways.

Loss of connective tissue growth factor expression promotes remodeling of the extracellular matrix and epithelial-to-mesenchymal transition in ovarian cancer (2294)

Loss of connective tissue growth factor expression promotes remodeling of the extracellular matrix and epithelial-to-mesenchymal transition in ovarian cancer (2294)

Exosome Secretion and Epithelial-Mesenchymal Transition in Ovarian Cancer Are Regulated by Phospholipase D.

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.

Loss of connective tissue growth factor permits mesenchymal transition in ovarian cancer (260)

<b>Objectives:</b> Epithelial-mesenchymal transition (EMT) is a characteristic feature of ovarian cancer (OC) metastasis. Our study aimed to determine the role of Connective Tissue Growth Factor (CTGF) in EMT. <b>Methods:</b> R182 and R2615 are epithelial OC cell lines that are CK18+, b-catenin+, TWIST -, and vimentin+. Time-course experiments +/- Transforming Growth Factor-b (TGF-b) and a TGF-b inhibitor were performed to evaluate the timing of CTGF expression in EMT. R182 and R2615 CTGF knockouts (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector. Phenotypic characterization of wild types (WT) and CTGF KO cell lines were performed with the assays, including scratch wound, proliferation, and anoikis resistance assays. Scratch wound assays were treated with 50 ng/mL and 100 ng/mL concentrations of recombinant CTGF for 48 hours (hr). Cells were plated in triplicate, and confluence was measured. Proliferation was determined by cell count using Biotek Cytation over 72 hr. For anoikis resistance, cells were plated in triplicate in an ultra-low adhesive cell plate with cell growth media. Using Promega CellTiter assay, cell viability was quantified by absorbance at 490 nm at the following time points: 1hr, 24hr, 48hr, and 120hr. Western blots were performed to evaluate the expression of epithelial and mesenchymal markers. RNA sequence analysis was performed, and pathway analysis was evaluated using the iPathway guide. <b>Results:</b> TGF-b induced CTGF expression in both OC cell lines tested. CTGF was induced as early as 30 minutes post-treatment, continued to increase until 6hr, and expression was maintained until 24 hr. No CTGF expression was seen in the mesenchymal mR182 and mR2615 OC cell lines. CTGF KO was confirmed by western blot analysis and indel sequencing. CTGF KO had no effect on the expression of epithelial markers, b-catenin, CK18, and vimentin, but SNAIL, a mesenchymal marker, was induced. Loss of CTGF also had no effect on cell proliferation but promoted anoikis resistance. R182 CTGF KO cells displayed 80% viability when grown in detached conditions for 48hr compared to only 20% viability in WT cells. At 120hr, 30% of KO cells remain viable compared to <10% for WT cells. R2615 WT and KO cells demonstrated similar results. Finally, the scratch assay showed that CTGF KO cells decrease the time to confluence compared to WT. Administration of recombinant CTGF does not rescue the phenotype to WT levels.Fig. 1 <b>Conclusions:</b> CTGF is expressed early in epithelial OC cells during EMT and is regulated by TGF-b. Our data suggest that CTGF permits maintenance of an epithelial phenotype through inhibition of SNAIL expression, and loss of CTGF should be evaluated as a poor prognosis indicator in OC.

Effect of WWOX on epithelial-mesenchymal transition in ovarian cancer cells in vivo, and its association with Elf5/Snail-1 signal pathway

Purpose: To investigate how WW domain-containing oxidoreductase (WWOX) gene affects epithelial-mesenchymal transition (EMT) of ovarian cancer (OC) cells, and the implication of Elf5/Snail-1 pathway in the process.&#x0D; Methods: This study employed female BALB/c nude mice aged 6 weeks. The expression levels of E-cadherin, β-Catenin, N-cadherin, Vimentin, Snail1 and Elf5 were assayed using qRT-PCR. The protein levels of WW1, WW2, E-cad, β-Cat, N-cad, vimentin, Snail-1 and Elf5 were determined using western blotting (WB). Invasion changes in cells in each group were analyzed by Transwell invasion test while WWOX and Elf5 were evaluated by GST pull-down test; furthermore, WWOX and ELF5 were evaluated by co-immunoprecipitation (COIP).&#x0D; Results: Relative to control group, tumor volume and weight of WWOX high-expression mice were significantly decreased, while those of WWOX low-expression group were increased (p &lt; 0.05). The levels of E-cad, Elf5 and β-catenin increased, but those of N-cad, Snail-1 and vimentin decreased in WWOX high expression group. In the WWOX low-expression group, the levels of E-cad, Elf5 and β-cat were down-regulated, while those of N-cad, Snail-1 and vimentin were up-regulated (p &lt; 0.05). The expression of E-cad, Elf5 and β-cat declined; on the other hand, those of N-Cad, Snail-1 and vimentin were elevated after mutation of WW1 domain in WWOX gene (p &lt; 0.05).&#x0D; Conclusion: Based on the in vivo results, the role of WWOX/Elf5/Snail1 signal route in the epithelial-mesenchymal transition of ovarian cancer, and its related mechanisms have been clarified. This provides a new target for gene therapy of ovarian cancer.

Wnt5A and TGFβ1 Converges through YAP1 Activity and Integrin Alpha v Up-Regulation Promoting Epithelial to Mesenchymal Transition in Ovarian Cancer Cells and Mesothelial Cell Activation.

In this paper, we investigate whether Wnt5A is associated with the TGF-β1/Smad2/3 and Hippo-YAP1/TAZ-TEAD pathways, implicated in epithelial to mesenchymal transition (EMT) in epithelial ovarian cancer. We used 3D and 2D cultures of human epithelial ovarian cancer cell lines SKOV-3, OVCAR-3, CAOV-4, and different subtypes of human serous ovarian cancer compared to normal ovary specimens. Wnt5A showed a positive correlation with TAZ and TGFβ1 in high- and low-grade serous ovarian cancer specimens compared to borderline serous and normal ovaries. Silencing Wnt5A by siRNAs significantly decreased Smad2/3 activation and YAP1 expression and nuclear shuttling in ovarian cancer (OvCa) cells. Furthermore, Wnt5A was required for TGFβ1-induced cell migration and invasion. In addition, inhibition of YAP1 transcriptional activity by Verteporfin (VP) altered OvCa cell migration and invasion through decreased Wnt5A expression and inhibition of Smad2/3 activation, which was reverted in the presence of exogenous Wnt5A. We found that the activation of TGFβ1 and YAP1 nuclear shuttling was promoted by Wnt5A-induced integrin alpha v. Lastly, Wnt5A was implicated in activating human primary omental mesothelial cells and subsequent invasion of ovarian cancer cells. Together, we propose that Wnt5A could be a critical mediator of EMT-associated pathways.

Downregulation of RIPK4 Expression Inhibits Epithelial-Mesenchymal Transition in Ovarian Cancer through IL-6.

RIPK4 has been implicated in multiple cancer types, but its role in ovarian cancer (OC) has not been clearly elucidated. Our data from Gene Expression Profiling Interactive Analysis, RT-PCR, and immunohistochemical analysis showed that RIPK4 was expressed at higher levels in OC tissues and cells than in normal ovarian tissues and cells. Increased RIPK4 expression in OC markedly correlated with a worse overall survival than lower RIPK4 expression levels (hazard rate (HR) 1.5 (1.45–1.87); P = 0.001). In functional experiments, RIPK4 downregulation significantly inhibited metastatic behaviours in OC cells. Subsequently, based on data from 593 OC patients in the TCGA database, gene set enrichment analysis revealed that RIPK4 was involved in epithelial-mesenchymal transition (EMT) in OC. At the molecular level, silencing RIPK4 significantly downregulated vimentin, N-cadherin, and Twist expression but induced an increase in the protein level of E-cadherin and inhibited the IL-6 and STAT3 levels. Moreover, IL-6 levels were significantly decreased in RIPK4-silenced OC cells (P < 0.05). The addition of IL-6 to OC cells rescued the suppressive effect of RIPK4 knockdown on EMT. Thus, our data illustrate that downregulation of RIPK4 expression can restrain EMT in OC by inhibiting IL-6. This finding may provide a novel diagnostic and therapeutic target for improving the poor prognoses of OC patients.

KRT7 promotes epithelial‑mesenchymal transition in ovarian cancer via the TGF‑β/Smad2/3 signaling pathway.

Keratin 7 (KRT7) is a member of the keratin gene family. KRT7 is abnormally expressed in various types of cancer and promotes the malignant progression of tumors. However, the role of KRT7 in ovarian cancer remains unclear. The present study aimed to validate the role of KRT7 in ovarian cancer progression. KRT7 expression levels in patients with ovarian cancer were analyzed using data obtained from the Human Protein Atlas and The Cancer Genome Atlas databases. KRT7 mRNA and protein expression levels were upregulated in ovarian cancer tissue compared with normal tissue. KRT7 expression was associated with the grading, staging and poor prognosis of ovarian cancer. The differentially expressed genes affected by KRT7 were primarily enriched in the functions of cell migration, cell adhesion and cell growth. In vitro studies, including a CCK8 assay, were used to detect cell proliferation. In addition, wound healing and transwell assays were performed to analyze cell migration. The results demonstrated that KRT7 overexpression was associated with increased proliferation, migration and epithelial-mesenchymal transition (EMT) of ovarian cancer cells, and the migration and EMT of ovarian cancers cells were decreased following knockdown with KRT7 small interfering RNA. In vivo, knockdown of KRT7 inhibited tumor growth of ovarian cancer. Furthermore, KRT7 regulated EMT in ovarian cancer via the TGF-β/Smad2/3 pathway, and regulated cell-matrix adhesion through integrin-β1-focal adhesion kinase signaling. These results suggest that KRT7 may be a potential molecular marker for prognosis prediction in patients with ovarian cancer.

Effects of activating GABAB1 receptor on proliferation, migration, invasion and epithelial-mesenchymal transition of ovarian cancer cells

ObjectiveThis study aimed to explore the effects of activating GABAB1 receptor by baclofen on proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of ovarian cancer cells.ResultsOne hundred μmol/L, 200 μmol/L and 300 μmol/L were selected as low, medium and high baclofen concentrations respectively. Cells were divided into four groups: Control, 100 μmol/L, 200 μmol/L and 300 μmol/L. Compared with the control group, the viability, colony formation, migration and invasion of SKOV3 cells were inhibited, and the apoptosis of SKOV3 cells were enhanced significantly at 200 μmol/L and 300 μmol/L baclofen. Moreover, they changed significantly with the increase of baclofen concentration. Compared with the control group, the expression of E-cadherin and GABAB1 increased and the N-cadherin expression decreased significantly in 200 μmol/L and 300 μmol/L groups. Higher concentration of baclofen induced higher expression of E-cadherin and lower expression of N-cadherin.ConclusionBaclofen inhibited the proliferation, cloning, migration, invasion and EMT of ovarian cancer cells by activating GABAB1 receptor. These results might contribute a lot to clarify the role and possible mechanism of GABAB1 receptor in ovarian cancer.

Down-regulation of PADI2 prevents proliferation and epithelial-mesenchymal transition in ovarian cancer through inhibiting JAK2/STAT3 pathway in vitro and in vivo, alone or in combination with Olaparib

BackgroundEpithelial ovarian cancer (EOC) is the most lethal disease among female genital malignant tumors. Peptidylarginine deiminase type II(PADI II) has been shown to enhance a variety of cancers carcinogenesis, including ovarian cancer. The purpose of this study was to investigate the biological role of PADI2 in ovarian cancer (OC) and the relative mechanism.MethodsGene Expression Profiling Interactive Analysis (GEPIA) (https://gepia.pku.cn/) and ONCOMINE (https://www.oncomine.org/) were used to analyze PADI2 Gene Expression data. The survival curve for the PADI2 gene was generated by using the online Kaplan–Meier mapping site (https://www.kmplot.com/). We conducted MTT assay, cloning formation assay and EdU cell proliferation assay to detect the cell activity of PADI2 knockdown A2780 and SKOV3 ovarian cancer cells treated with Olaparib. Cell migration and invasion were observed by would healing and transwell assay. The pathway changes after the treatment of PADI2 were detected by transcriptome sequencing and western blot. The role of PADI2 combined with Olaparib treatment in vivo was studied in nude mouse model bearing ovarian cancer tumor.ResultsWe investigated the role of PADI2 on EOC in vitro and in vivo. PADI2 was upregulated in ovarian cancer samples and high PADI2 expression was correlated with poor outcome. Downregulating PADI2 suppressed colony formation, proliferation, migration and invasion of A2780 and SKOV3 cells. Furthermore, downregulating PADI2 and Olaparib combination treatment attenuated the viability, migration and invasion of A2780 and SKOV3 cells. We identified differentially expressed genes in A2780-shPADI2 and SKOV3-shPADI2 cell by transcriptome sequencing analysis and verified that downregulating PADI2 and Olaparib combination treatment suppresses EMT and JAK2/STAT3 signaling pathway in A2780 and SKOV3 cells in vitro and in vivo.ConclusionsDownregulation of PADI2 and Olaparib combination treatment attenuated the proliferation, migration and invasion of A2780 and SKOV3 cells by inhibiting the EMT through JAK2/STAT3 signaling pathway.

MiR-197-3p reduces epithelial-mesenchymal transition by targeting ABCA7 in ovarian cancer cells.

The present study was designed to explore the role of microRNA-197-3p in regulating the epithelial-mesenchymal cellular transition in ovarian cancer. The results showed that miR-197 to be significantly (P < 0.05) downregulated in human ovarian cancer tissues and cell lines. Overexpression of miR-197 significantly (P < 0.05) reduced the proliferation of OVACAR-3 cancer cells. Additionally, the colony formation of the OVACAR-3 cells was inhibited by 59% relative to control. The migration and invasion of the OVACAR-3 cells was inhibited by 64% and 72%, respectively, upon miR-197 overexpression. Western blot analysis showed miR-197 was found to upregulate the expression of E-cadherin, while the expression of N-cadherin, vimentin, and snail proteins was found to decrease significantly (P < 0.05). TargetScan analysis together with dual luciferase assay revealed that miR-197 exerts its effects by targeting ABCA7 in ovarian cancer. ABCA7 was significantly (P < 0.05) overexpressed in ovarian cancer tissues and cell lines. However, silencing of ABCA7 resulted in significant inhibition of cell proliferation, migration, and invasion. Nonetheless, overexpression of ABCA7 could abolish the tumor-suppressive effects of miR-197 on the OVACAR-3 cells. Taken together, miR-197 acts a tumor-suppressive in ovarian cancer and points towards its therapeutic implications in the treatment of ovarian cancer.

Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer.

Gα13, a heterotrimeric G-protein of the Gα12/13 subfamily, is associated with aggressive phenotypes in various human cancers. However, the mechanisms by which Gα13 promotes cancer progression have not been fully elucidated. Here, we demonstrate that the activation of Gα13 induces epithelial-mesenchymal transition in ovarian cancer (OvCa) cells through down-regulation of large tumor suppressor kinase (LATS) 1, a critical component of the Hippo signaling pathway. A synthetic biology approach using a mutant GPCR and chimeric G-protein revealed that Gα13-regulated phosphorylation of LATS1 at serine 909 within its activation loop induced recruitment of the itchy E3 ubiquitin protein ligase to trigger LATS1 degradation. Our findings uncover novel mechanisms through which Gα13 activation induces dysregulation of the Hippo signaling pathway, which leads to aggressive cancer phenotypes, and thereby identify a potential target for preventing the metastatic spread of OvCa.-Yagi, H., Onoyama, I., Asanoma, K., Hori, E., Yasunaga, M., Kodama, K., Kijima, M., Ohgami, T., Kaneki, E., Okugawa, K., Yahata, H., Kato, K. Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer.

TRPM7 promotes the epithelial\u2013mesenchymal transition in ovarian cancer through the calcium-related PI3K / AKT oncogenic signaling

BackgroundThe epithelial-mesenchymal transition (EMT) is crucial for metastasis and positively regulated by calcium-related signaling. The melastatin-related transient receptor potential 7 (TRPM7) regulates a non-selective cation channel and promotes cancer metastasis. However, the mechanisms underlying the action of TRPM7 in ovarian cancer are unclear.MethodsThe expression of TRPM7 and EMT markers (Vimentin, N-cadherin, Twist and E-cadherin) in ovarian cancer samples was detected. TRPM7was knockdown by shRNA in Ovarian cancer cell lines to examine calcium [Ca2+]i, EMT markers and PI3K/AKT markers. Various cellular assays, such as invasion and migration, were performed in vitro, and further confirmed in vivo.ResultsTRPM7 expression is negatively correlated with E-cadherin, but positively with N-cadherin, Vimentin and Twist expression in ovarian cancer samples. TRPM7 depletion inhibited the migration and invasion in SKOV3 and OVCAR3 cells. In addition, TRPM7 silencing decreased the lung metastasis of SKOV3 tumors and prolonged the survival of tumor-bearing mice. Similar to that of TRPM7 silencing, treatment with MK886, a potent 5-lipoxygenase inhibitor to reduce TRPM7 expression, and/or BAPTA-AM, an intracellular calcium chelator, significantly mitigated the Epidermal growth factor (EGF) or Insulin-like growth factors (IGF)-stimulated migration, invasion, and the EMT in ovarian cancer cells by decreasing the levels of intracellular calcium [Ca2+]i. Furthermore, treatment with LY2904002, a PI3K inhibitor, also inhibited the migration, invasion, and treatment with both LY2904002 and BAPTA-AM further enhanced their inhibition in ovarian cancer cells. Moreover, treatment with BAPTA-AM mitigated the IGF-stimulated migration, invasion, particularly in TRPM7-silenced ovarian cancer cells. Finally, TRPM7 silencing attenuated the PI3K/AKT activation, which was enhanced by BAPTA-AM, MK886 or LY2904002 treatment in ovarian cancer cells.ConclusionsTRPM7 silencing inhibited the EMT and metastasis of ovarian cancer by attenuating the calcium-related PI3k/AKT activation. Our findings suggest that TRPM7 may be a therapeutic target for intervention of ovarian cancer.

Abstract A46: Hsp90 regulates Twist1 expression through STAT3 to induce epithelial-mesenchymal transition in ovarian cancer

Abstract Background: Metastatic disease is the leading cause of death from ovarian cancer and its underlying mechanisms are poorly understood. Twist1 is a key driver of epithelial-mesenchymal transition (EMT) and metastasis. Understanding the function and regulation of Twist1 is a vital step in the development of effective treatments for metastatic ovarian cancer. Heat shock protein 90 (Hsp90) is a molecular chaperone that modulates multiple signaling networks, and recent studies have highlighted the roles of extracellular Hsp90 in promoting metastasis in cancer. The roles of Hsp90 in regulating intracellular pathways leading to EMT and metastasis remain largely unknown. Objective: In our study, we tested the hypothesis that Hsp90 promotes EMT in ovarian cancer through the regulation of Twist1 at the transcriptional level. Methods: We treated A2780 and SKOV3 with a Hsp90-specific inhibitor, 17-allylamino-17 demethoxygeldanamycin (17-AAG). The effects of Hsp90 inhibition on Twist1 mRNA expression and promoter activity were measured using quantitative PCR and luciferase reporter assays, respectively. Proximity ligation assays were performed to visualize the effects of 17-AAG on the interaction between Hsp90 and transcription factors, followed by chromatin immunoprecipitation to measure the binding of transcription factors to the Twist1 promoter. Results: Treatment with 17-AAG significantly downregulated Twist1 expression at the mRNA level in A2780, SKOV3, and three ovarian cancer patient-derived cell lines. Hsp90 overexpression substantially induced Twist1 promoter activity while treatment with 17-AAG significantly decreased the activity. Western blotting and immunofluorescent staining revealed the presence of 4 transcription factors known to be clients of Hsp90 and regulators of Twist1, which are β-catenin, signal transducer and activator of transcription 3 (STAT3), hypoxia-inducible factor 1-alpha (HIF-1α), and HIF-1β. We identified that Hsp90 interacts with β-catenin, STAT3, and HIF-1α in our cell lines. We observed that 17-AAG treatment dramatically impaired Hsp90-STAT3 interaction and the binding of STAT3 to the Twist1 promoter. Inhibition of Hsp90 was also shown to block interleukin-6 (IL-6) and transforming growth factor beta (TGF-β)-induced EMT. Conclusion: Taken together, our findings reveal that STAT3 is dependent on Hsp90 to activate Twist1 expression. Hsp90 plays a critical role in enabling STAT3 to bind to the Twist1 promoter and promote Twist1 transcription leading to EMT. We uncovered a previously unrecognized role of Hsp90, which cooperates with STAT3 in the transcriptional regulation of Twist1 in ovarian cancer cells. Inhibiting Hsp90 using small-molecule inhibitors such as 17-AAG may have potential as a therapeutic strategy to prevent EMT and metastasis in ovarian cancer. Citation Format: Kay Yi Chong, Francesca Garofalo, Oluwagbemisola Madarikan, Nicholas Pitruzzello, Cheng-Hsiu Tsai, Jamie Bingham, Yang Yang-Hartwich. Hsp90 regulates Twist1 expression through STAT3 to induce epithelial-mesenchymal transition in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A46.

Abstract 2038: The role of epithelial-mesenchymal transition in ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is one of the leading causes of death due to malignant disease in women. Due to its asymptomatic nature, 80% of patients are diagnosed at late stages, when the disease has metastasized and 5 year survival rates are then around 30%. Typically late stage EOC rapidly develops resistance to chemotherapy. Recently, attention has focused on a possible involvement of EMT: epithelial-mesenchymal-transition- in the development of resistance to chemotherapy drugs. EMT refers to a change in cell morphology with the loss of the apical-basal polarity of a cell, the loss of cell-cell junctions and the gain of a spindle-like shape with migratory and invasive properties. Our work sets out to understand the role of EMT in EOC focusing on its contribution to the development of resistance towards carboplatin and paclitaxel. Firstly, an in-vitro model of EMT was developed with OVCAR-3 EOC cells, using a cocktail of 20ng/ml EGF, 50ng/ml IL-6 and 10ng/ml TGF-β. Western blot results showed significantly increased expression of slug and vimentin with decreased E-cadherin expression in the in-vitro derived EMT model compared to the control untreated cells. In addition, using established drug resistant cell lines of OVCAR-3, PEO1, CaOV3 and SKOV-3 the EMT signature was compared with that of the corresponding parental line. Western blot examination indicated decreased E-cadherin expression (P&amp;gt;0.05) and significantly increased slug and vimentin expression (P&amp;lt;0.05) in the resistant cell lines, similar to the cytokine-treated model of EMT induction. In order to observe the effect of drug resistance on migratory properties, OVCAR-3 (paclitaxel-sensitive and resistant) and PEO1 (carboplatin-sensitive and resistant) were used in scratch/wound healing assays. Drug treatment was carried out and wound closure was measured with CellProfiler after 72 hours. A significantly increased migratory capacity (wound closure) was observed in drug resistant cells in the absence and presence of chemotherapy drugs, when compared with parental cells (P&amp;lt;0.05). In order to clarify any possible role for slug in the induction of drug resistance, we carried out SiRNA silencing of OVCAR-3 cells. Silenced cells (alongside scramble control) were subjected to drug treatment for 48 hours and were then assessed for apoptosis using the annexin assay (FITC and PI). On silencing the slug gene, a significant resensitization to carboplatin could be observed compared to control cells (P&amp;lt;0.05). Further work includes analyses of the panel of drug sensitive and drug resistant (carboplatin and paclitaxel) EOC cell lines to identify other gene changes associated with EMT, using PCR array approaches. This work will help with the identity of candidate biomarkers of clinical drug resistance for EOC patients. Citation Format: Hasanthi Assalaarachchi, Helen M. Coley. The role of epithelial-mesenchymal transition in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2038.

IGHG1 promotes motility likely through epithelial-mesenchymal transition in ovarian cancer.

Ovarian cancer (OC) is one of the leading causes of death for female cancer patients. COC166-9 is an OC-specific monoclonal antibody and we have identified immunoglobulin γ-1 heavy chain constant region (IGHG1) as its antigen. We explore the function of IGHG1 in proliferation, apoptosis and motility of OC cells further in this research. IGHG1 expression in OC specimens was detected through immunohistochemistry. Real-time quantitative polymerase chain reaction (RT-qPCR) or western blotting assay was used to test IGHG1 expression in OC cells. Viability of OC cells was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry or western blotting assay was used to detect cell cycle and apoptosis. Cellular motility was analyzed by using transwell assay and the markers of epithelial-mesenchymal transition (EMT) were tested through immunoblots. Although it exerts negligible effect on the viability and apoptosis of OC cells, IGHG1 could promote migration and invasion of malignant cells in vitro. Mechanistically, IGHG1 increases the expression of N-cadherin and Vimentin while decreases E-cadherin expression. Additionally, IGHG1 expression in OC specimens is higher relative to the paired normal counterparts. Further analysis demonstrates that the increased IGHG1 expression correlates positively with the lymph node metastasis of OC. IGHG1 promotes the motility of OC cells likely through executing the EMT program. Increased IGHG1 expression in OC specimens is associated with the lymph node metastasis.

Linc-ROR induces epithelial-to-mesenchymal transition in ovarian cancer by increasing Wnt/β-catenin signaling.

Long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) is an intergenic long non-coding RNA (lncRNA) previously shown to contribute to tumorigenesis in several malignancies. However, little is known about whether linc-ROR has a role in ovarian cancer progression. In this study, we found that linc-ROR expression was increased in high-grade ovarian serous cancer tissues compared with normal ovarian tissues or normal fallopian tube tissues. Furthermore, the level of linc-ROR expression was associated with ovarian cancer International Federation of Gynecology and Obstetrics stage and lymph node metastasis. Linc-ROR promoted ovarian cancer cell proliferation both in vitro and in vivo, and contributed to cell migration and invasion. Linc-ROR knockdown in ovarian cancer cell lines inhibited the epithelial-to-mesenchymal transition (EMT) program, which led to ovarian cancer cell metastasis through the repression of canonical Wnt/β-catenin signaling. Together, our results indicated that linc-ROR induces EMT in ovarian cancer cells and may be an important molecule in the invasion and metastasis of ovarian cancer.