Ovarian Clear Cell Carcinoma Tissues Research Articles

Tryptophan metabolism enzymes are potential targets in ovarian clear cell carcinoma.

As the second most prevalent subtype of epithelial ovarian cancers, ovarian clear cell carcinoma (OCCC) is known for its chemoresistance to conventional platinum-based therapy. In this work, we examined the tryptophan (Trp) metabolism enzymes' differential expression in patients with OCCC to assess the potential for personalised treatment. A total of 127 OCCC tissues were used to construct tissue microarrays, and immunohistochemistry (IHC) staining of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 was performed. The correlations between Trp enzyme expression and clinical characteristics were analysed. Positive IDO1, IDO2, TDO2 and IL4I1 staining was identified in 26.8%, 94.5%, 75.6% and 82.7% of OCCC respectively. IDO1-positive samples were more common in the chemoresistant group than in the platinum-sensitive group (46.7% vs. 19.8%). Moreover, positive expression of IDO1, TDO2 and IL4I1 was related to advanced stage, metastasis, bilateral tumours, endometriosis and tumour rupture (p < 0.05) respectively. Univariate analysis revealed a significant association between bilateral tumours, lymph node metastasis, advanced stage, distant metastasis and aberrant cytology with a poor prognosis for OCCC, while the absence of residual tumour was correlated with a favourable outcome (p < 0.05). However, only bilateral tumours and lymph node metastases were related to a poor prognosis after multivariate analysis. This is the first study to investigate the expression of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 in OCCC tissues. IDO2, TDO2 and IL4I1 were detected in the majority of OCCC. Clinical traits were correlated with IDO1, IDO2, TDO2 and IL4I1 expression. IDO1 may be used as a therapeutic target given the large percentage of chemoresistant cases with IDO1 expression. These results will aid the development of personalised therapies for OCCC.

Fibroblast Growth Factor 11 (FGF11) Promotes Progression and Cisplatin Resistance Through the HIF-1α/FGF11 Signaling Axis in Ovarian Clear Cell Carcinoma.

A poor prognosis is often associated with ovarian clear cell carcinoma (OCCC) due to its relative resistance to platinum-based chemotherapy. Although several studies have been launched to explore the pathogenesis of OCCC, the mechanism of chemoresistance has yet to be uncovered. Nanostring nCounter PanCancer Pathways Panel was performed to explore the expression profiles of OCCC tissues from patients showing different platinum sensitivity. Bioinformatic analysis was performed to select genes associated with chemoresistance and cell function assays, including colony formation, wound healing, transwell and flow cytometric analysis, were used to explore the role of the target gene in the progression of OCCC and resistance to cisplatin (DDP). Gene expression profiles and bioinformatic analysis verified that the expression of fibroblast growth factor 11 (FGF11) was significantly increased in platinum-resistant OCCC tissues and increased FGF11 expression was related to poorer survival. Downregulation of FGF11 inhibited the proliferation, migration, and invasion, reversing the DDP resistance of OCCC cells. Mechanically, FGF11 was regulated by hypoxia-inducible factor-1α (HIF-1α) to modulate the DDP sensitivity. FGF11 was highly expressed in platinum-resistant OCCC tissues, promoting progression and resistance to DDP through the HIF-1α/FGF11 signaling axis.

Preoperative serum microRNAs as potential prognostic biomarkers in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian carcinoma with poor prognosis. However, no effective biomarkers have been established for predicting unfavorable events, including recurrence and poor prognoses. Serum microRNAs (miRNAs) have been increasingly reported to be useful in predicting a patient's condition and have been recognized as a potentially less-invasive source for liquid biopsy in cancer. Therefore, this study aimed to evaluate serum miRNA profiles from patients with OCCC and to establish biomarker for predicting the prognoses. The GSE106817, which included preoperative serum miRNA profiles of patients with ovarian tumors, was used, and clinical information was investigated. In all, 66 patients with OCCC were included, excluding those with other histological subtypes or insufficient prognostic information. Moreover, miRNA profiles of OCCC tissues were also examined. The median follow-up period was 64.3 (8.0-153.3) months. Based on multivariable Cox regression analyses and the expression of miRNAs in OCCC tissues, miR-150-3p, miR-3195, and miR-7704 were selected as miRNA candidates associated with both progression-free survival (PFS) and overall survival (OS). Then, the prognostic index was calculated based on expression values of 3 serum miRNAs. Kaplan-Meier survival analysis indicated that the prognostic index was significantly predictive of PFS and OS (p=0.004 and p=0.012, respectively). Preoperative serum miRNA profiles of miR-150-3p, miR-3195, and miR-7704 can be used to potentially predict the prognosis of patients with OCCC.

Label-free diagnosis of ovarian cancer using spoof surface plasmon polariton resonant biosensor

Artificial periodic grooves on a metal line can mimic optical surface plasmon polaritons (SPPs) with high cut-off response at microwave frequencies, which is known as spoof SPPs and has recently gained great interest in biosensing due to the strong enhancement of the localized electric field. In this paper, a sensitive spoof SPP biosensor with split ring resonators (SRRs) for ovarian cancer diagnosis is reported for the first time. The SRRs are distributed in series on a metal line to replace the conventional periodic grooves, which maintains the same cut-off frequency and generates a sharp resonance simultaneously. This resonance enhances the localized field by a factor of 250 so that the proposed device is highly sensitive for detecting permittivity difference. The sensitivity in terms of frequency shifting was first investigated by electromagnetic simulation, and then demonstrated by distinguishing sucrose solutions with various concentrations. With the benefit of high sensitivity, the proposed biosensor successfully detects the serous ovarian cancer (SOC) and ovarian clear cell carcinoma (OCCC) tissues. The resonant frequency of the biosensor is 53.990 GHz intrinsically, which shifts to 53.814–53.968, 53.698–53.872, and 53.719–53.845 GHz with the normal, SOC, and OCCC tissues, respectively. The average point of all ovarian tissues is at 53.812 GHz, i.e., well below the normal tissues, which is a reasonable reference for cancer detection. Moreover, the average transmittance of all cancerous tissues is lower than the SOC tissues, which may be used to classify the SOC and OCCC tissues. The proposed planar SPP biosensor reveals a fast, sensitive, and label-free detection approach for ovarian cancers.

LncRNA GAS5 inhibits the proliferation and invasion of ovarian clear cell carcinoma via the miR-31-5p/ARID1A axis.

To investigate the role of the lncRNA growth arrest special 5 (GAS5) in ovarian clear cell carcinoma (OCCC), we measured the expression of GAS5 and miR-31-5p in OCCC tissue samples and OCCC cell lines using RT-qPCR. MTT and colony formation assays were used to measure cell viability and colony formation ability. Cell invasion was determined by Transwell assays. The binding between GAS5 and miR-31-5p as well as miR-31-5p and ARID1A was determined by dual-luciferase reporter assays. The ARID1A protein levels were detected using western blotting. Kaplan-Meier curves were used for the analysis of the 5-year survival rate of patients with OCCC. GAS5 and ARID1A levels were significantly decreased, while miR-31-5p levels were strongly elevated in the OCCC tissues and cell lines. Patients with lower GAS5/ARID1A levels had shorter overall survival times. Overexpression of GAS5 or inhibition of miR-31-5p suppressed cell viability and invasion of OCCC cells and upregulated the protein levels of ARID1A. Moreover, overexpression of miR-31-5p reversed the effects of overexpression of GAS5. Cotransfection with pcDNA3.1-GAS5 and miR-31-5p inhibitor led to the lowest cell viability and cell invasion rates. A dual-luciferase reporter assay was performed to confirm the target relationship between GAS5 and miR-31-5p, as well as between miR-31-5p and ARID1A. LncRNA GAS5 inhibited cell viability and invasion of OCCC through activation of ARID1A by sponging miR-31-5p.

Changes in SLIT2 expression are associated with the migration of human ovarian clear cell carcinoma cells.

Ovarian clear cell carcinoma (OCCC) is characterized by a poor survival of patients, which is mainly due to metastasis and treatment failure. Slit guidance ligand 2 (SLIT2), a secreted protein, has been reported to modulate the migration of neural cells and human cancer cells. However, the effect of changes in SLIT2 expression on the regulation of cell migration in OCCC remains unknown. The present study examined alterations in SLIT2 expression using OCCC cell models, including low- and high-mobility SKOV3 cells, as well as OCCC tissues. DNA methylation analysis suggested that promoter hypermethylation was responsible for the low expression levels of SLIT2 in OCCC cells. The demethylating agent 5-Aza-deoxycytosine was able to restore SLIT2 expression at both the mRNA and protein levels in high-mobility SKOV3 cells that harbored the relevant methylated promoter. Overexpression of SLIT2 inhibited the migration of high-mobility OCCC cells, as well as decreased the protein expression levels of β-catenin, phosphorylated (p)AKT and snail family transcriptional repressor 1 (SNAI1). On the other hand, knockdown of SLIT2 increased the migration of low-mobility OCCC cells, and enhanced the protein expression levels of β-catenin, pAKT and SNAI1. Overall, the results of the present study provided evidence that low expression levels of SLIT2 were associated with increased OCCC cell migration, and that SLIT2 may act as a suppressor gene of cancer cell migration.

Integrative genomic and transcriptomic analysis reveals prognostic markers and immune subtype in Chinese ovarian clear cell carcinoma.

e17529 Background: Ovarian clear cell carcinoma (OCCC) is a subtype of ovarian cancer characterized by chemo-resistance and poor survival. Herein, we performed comprehensive genomic and transcriptomic profiling of OCCC tissues to identify prognostic markers and immune subtype in Chinese patients. Methods: A total of 61 patients were included, comprising 41 early-stage and 19 late-stage tumors. Tissue samples were collected before chemotherapy and subjected to capture-based targeted sequencing using a panel consisting of 520 cancer related genes and whole transcriptomic sequencing. Genetic alterations, tumor mutation burden (TMB), microsatellite instability (MSI), and gene expression were evaluated. Association analysis was performed to evaluate clinical and genetic factors with platinum-sensitivity and treatment effects. A nomogram was constructed to predict survival outcomes. Single sample Gene Set Enrichment analysis (ssGSEA) was performed with gene expression profile and immune gene sets. Results: Genetic alterations were mainly identified in ARID1A (49%), PIK3CA (48%), TP53 (18%), ATM (15%), SMARCA4 (13%) and PRKDC (13%). Co-occurrence of mutations in ARID1A with PIK3CA and PRKDC were observed (p &lt; 0.05). Meanwhile, no alterations were identified in BRCA1/2. Patients harboring ATM mutations tended to be platinum sensitive compared to those wildtype counterparts (p &lt; 0.05). In patients with stage IV OCCC, mutations of ARID1A, PIK3CA, SMARCA4 and the HRR pathway were also related to higher percentage of platinum-sensitivity, though significance was not achieved. Mutations of FGFR2, NOTCH1 and the BER pathway were more frequently identified in patients with stage II to IV than stage I (p &lt; 0.05). Patients harboring alterations at chromosome 8q covering genes such as PRKDC showed better overall survival (OS) (p &lt; 0.05). Both clinical factors and frequently mutated genes were used to construct a nomogram for progression-free survival (PFS) prediction. The clinical factors (stage, platinum response and residual disease) in combination with genetic mutations ( ATM and SMARCA4) showed better performance in predicting PFS than clinical factors alone (concordance index 0.85 vs. 0.74, p &lt; 0.01). Transcriptomic analysis with ssGSEA of immune pathway revealed an immune subtype with enrichment of PD-1 signaling. OCCC patients with this immune subtype showed poor PFS and OS. Integration of genomic and transcriptomic analysis showed that the immune subtype patients had higher mutation rate of PIK3CA. Conclusions: The mutational profiles of OCCC were varied in patients with different platinum response and tumor stages. An immune subtype of OCCC with poor survival and enrichment of PD-1 signaling was identified. Our study identified several potential prognostic markers of OCCC at genetic level and revealed an immune subtype for potential immunotherapy.

Tissue factor pathway inhibitor‑2 is specifically expressed in ovarian clear cell carcinoma tissues in the nucleus, cytoplasm and extracellular matrix.

Tissue factor pathway inhibitor-2 (TFPI-2) is a promising candidate as a serum biomarker of ovarian clear cell carcinoma (OCCC), a lethal histological subtype of epithelial ovarian cancer (EOC). TFPI-2 is a secreted serine protease inhibitor that suppresses cancer progression through the inhibition of matrix protease activities. Previous studies have also identified TFPI-2 in the nucleus, and a possible function of nuclear TFPI-2 as a transcriptional repressor of matrix metalloproteinase-2 (MMP-2) was recently demonstrated. We are currently establishing TFPI-2 as a serum biomarker for OCCC patients; however, TFPI-2 expression in OCCC tissues has not been previously investigated. In the present study, we examined TFPI-2 expression and its localization in 11 OCCC cell lines by western blotting and enzyme-linked immune assay. Four cell lines expressed TFPI-2 in the nucleus, cytoplasm and culture plate–attached extracellular fraction, while four other cell lines expressed TFPI-2 only in the extracellular fraction. In the remaining three cell lines, TFPI-2 was not identified in any fraction. The amount of secreted soluble TFPI-2 showed similar trends to that of the plate-attached fraction. We next investigated the expression levels and distribution of TFPI-2 in surgically resected EOC tissues by immunohistochemistry. In 52 of the 77 (67.5%) OCCC tumors, TFPI-2 expression was detected in at least one of the nuclear, cytoplasmic and extracellular matrix fractions. In contrast, we did not identify TFPI-2 in the other EOC subtypes (n=65). TFPI-2-positive expression distinguished CCC from the other EOC tissues with a sensitivity of 67.5% and specificity of 100%. Although the inherent tumor suppressor function, statistical analyses failed to demonstrate correlations between TFPI-2 expression and clinical parameters, including 5-year overall survival, except for the patient age. In conclusion, we identified TFPI-2 expression in the nucleus, cytoplasm and extracellular matrix in OCCC tissues. The high specificity of TFPI-2 may support its use for diagnosis of OCCC in combination with existing markers.

Tetraspanin 1 promotes endometriosis leading to ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) reportedly develops from endometriosis. However, the molecular mechanism underlying its malignant progression to OCCC remains elusive. This study aimed to identify an essential gene in the malignant transformation of endometriosis to OCCC. We performed RNA sequencing in formalin‐fixed, paraffin‐embedded (FFPE) tissues of endometriosis (n = 9), atypical endometriosis (AtyEm) (n = 18), adjacent endometriosis to OCCC (AdjEm) (n = 7), and OCCC (n = 17). We found that tetraspanin 1 (TSPAN1) mRNA level was significantly increased by 2.4‐ (DESeq2) and 3.4‐fold (edgeR) in AtyEm and by 80.7‐ (DESeq2) and 101‐fold (edgeR) in OCCC relative to endometriosis. We confirmed that TSPAN1 protein level was similarly overexpressed in OCCC tissues and cell lines. In immortalized endometriosis cell lines, TSPAN1 overexpression enhanced cell growth and invasion. Mechanistically, TSPAN1 triggered AMP‐activated protein kinase (AMPK) activity, promoting endometriosis and cell growth. Upregulated levels of TSPAN1 are considered an early event in the development of high‐risk endometriosis that could progress to ovarian cancer. Our study suggests the potential of TSPAN1 as a screening candidate for high‐risk endometriosis.

Over-expression of RALYL suppresses the progression of ovarian clear cell carcinoma through inhibiting MAPK and CDH1 signaling pathways.

Background: The molecular mechanism in the progression of ovarian clear cell carcinoma (OCCC) remains unclear.Objective: This study aimed to investigate the potential function of RAYLY in OCCC.Methods: To validate RAYLY expression, immunohistochemistry, quantitative real-time PCR and western blotting were performed in OCCC tissues and the cell lines of OCCC and epithelial ovarian carcinoma (EOC). Subsequently, the biological effects of RALYL were evaluated through colony formation, and cell proliferation, migration and invasion assays. Finally, RNA-sequencing and gene set enrichment analysis (GSEA) were conducted to explore potential mechanism of RALYL in OCCC.Results: In our study, RALYL was significantly down-regulated in a majority of OCCC tissues compared to adjacent non-tumorous tissues, and OCCC cells had a lower expression level of RALYL than that of EOC cells. OCCC patients with high RALYL expression had a better pathological stage and prognosis. In vitro, over-expression of RALYL inhibited cell proliferation, migration and invasion in OCCC. GSEA analysis and western blot indicated an enrichment of MAPK and CDH1 signaling pathways in OCCC cells without RALYL over-expression.Conclusions: RALYL played an important role in the progression of OCCC, and might serve as a potential prognostic biomarker and novel therapeutic target for OCCC.

P53 and ANXA4/NF‑κB p50 complexes regulate cell proliferation, apoptosis and tumor progression in ovarian clear cell carcinoma.

Annexin IV (ANXA4) is highly expressed in ovarian clear cell carcinoma (OCCC); however, its underlying molecular mechanism in OCCC remains unknown. The present study aimed to identify the molecule that ANXA4 may act on and to determine its underlying molecular mechanism. Immunohistochemistry, co-immunoprecipitation and western blotting were performed to detect the expression and interaction of ANXA4, and its associated proteins. Furthermore, MTT assay, flow cytometry, western blotting and gene expression profile enrichment analysis were performed to identify the potential role and molecular mechanism of ANXA4 in OCCC. The results demonstrated that ANXA4 and nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB) p50 nuclear expression levels were significantly higher in OCCC tissues compared with other subtypes of ovarian cancer, such as serous and mucinous. In addition, a significantly positive correlation was observed between ANXA4 and NF-κB p50 expression in OCCC; however, the expression levels of mutant p53 and ANXA4 were negatively correlated in a linear manner. These results suggest that ANXA4 and NF-κB p50 may be potential independent risk factors for poor prognosis. ANXA4 and NF-κB p50 were demonstrated to interact and their expression was co-localized. The cBioPortal database was used to construct a protein-protein interaction network between ANXA4, NF-κB p50 and p53, and functional pathway analysis indicated that the genes were predominantly enriched in the cell cycle and during apoptosis. Transfection of the ANXA4 gene increased the expression of NF-κB p50, as well as its downstream targets, Cyclin D1 and B-cell lymphoma-2 (Bcl-2). Furthermore, transfection of the ANXA4 gene increased proliferation and decreased apoptosis of OCCC cells. Treatment with the NF-κB inhibitor, BAY 11-7082, decreased Cyclin D1 and Bcl-2 expression levels. Collectively, the results of the present study suggest that wild p53 activates ANXA4 transcription, promotes its expression and enhances NF-κB p50 and ANXA4 interaction. This in turn activates the NF-κB signaling pathway, promotes cell cycle progression and inhibits apoptosis, thus contributing to the malignant progression of OCCC. Thus, ANXA4 and NF-κB p50 may be used as prognostic biomarkers, and may be molecular therapeutic targets in OCCC.

Long non-coding RNA SNHG6 promotes cell proliferation and migration through sponging miR-4465 in ovarian clear cell carcinoma.

Dysregulation of small nucleolar RNA host gene 6 (SNHG6) exerts critical oncogenic effects and facilitates tumourigenesis in human cancers. However, little information about the expression pattern of SNHG6 in ovarian clear cell carcinoma (OCCC) is available, and the contributions of this long non‐coding RNA to the tumourigenesis and progression of OCCC are unclear. In the present study, we showed via quantitative real‐time PCR that SNHG6 expression was abnormally up‐regulated in OCCC tissues relative to that in unpaired normal ovarian tissues. High SNHG6 expression was correlated with vascular invasion, distant metastasis and poor survival. Further functional experiments demonstrated that knockdown of SNHG6 in OCCC cells inhibited cell proliferation, migration and invasion in vitro as well as tumour growth in vivo. Moreover, SNHG6 functioned as a competing endogenous RNA (ceRNA), effectively acting as a sponge for miR‐4465 and thereby modulating the expression of enhancer of zeste homolog 2 (EZH2). Taken together, our data suggest that SNHG6 is a novel molecule involved in OCCC progression and that targeting the ceRNA network involving SNHG6 may be a treatment strategy in OCCC.

The histone methyltransferase WHSC1 is regulated by EZH2 and is important for ovarian clear cell carcinoma cell proliferation

BackgroundWolf-Hirschhorn syndrome candidate gene-1 (WHSC1), a histone methyltransferase, has been found to be upregulated and its expression to be correlated with expression of enhancer of zeste homolog 2 (EZH2) in several cancers. In this study, we evaluated the role of WHSC1 and its therapeutic significance in ovarian clear cell carcinoma (OCCC).MethodsFirst, we analyzed WHSC1 expression by quantitative PCR and immunohistochemistry using 23 clinical OCCC specimens. Second, the involvement of WHSC1 in OCCC cell proliferation was evaluated by MTT assays after siRNA-mediated WHSC1 knockdown. We also performed flow cytometry (FACS) to address the effect of WHSC1 on cell cycle. To examine the functional relationship between EZH2 and WHSC1, we knocked down EZH2 using siRNAs and checked the expression levels of WHSC1 and its histone mark H3K36m2 in OCCC cell lines. Finally, we checked WHSC1 expression after treatment with the selective inhibitor, GSK126.ResultsBoth quantitative PCR and immunohistochemical analysis revealed that WHSC1 was significantly overexpressed in OCCC tissues compared with that in normal ovarian tissues. MTT assay revealed that knockdown of WHSC1 suppressed cell proliferation, and H3K36me2 levels were found to be decreased in immunoblotting. FACS revealed that WHSC1 knockdown affected the cell cycle. We also confirmed that WHSC1 expression was suppressed by EZH2 knockdown or inhibition, indicating that EZH2 is upstream of WHSC1 in OCCC cells.ConclusionsWHSC1 overexpression induced cell growth and its expression is, at least in part, regulated by EZH2. Further functional analysis will reveal whether WHSC1 is a promising therapeutic target for OCCC.

Abstract LB-100: Targeting TMEM205 mechanotransduction pathway using oncolytic virus therapy in ovarian clear cell carcinoma (OCCC)

Abstract Objectives: Ovarian clear cell carcinoma (OCCC) is an aggressive form of ovarian cancer and unfavorable treatment outcomes have been attributed to its relative chemoresistance &amp; recurrence, limiting the available treatment options. Therefore, identifying novel unique protein(s) involved in OCCC chemoresistance and designing a therapeutic modality selectively targeting those proteins would make a significant impact on therapy. TMEM205 is one such protein which has been recently linked to cisplatin resistance in human epidermoid carcinoma, but the precise molecular mechanism of this resistance still needs to be elucidated. This proposed study was planned to a) identify the role of TMEM205 expression in OCCC b) mechanotransduction pathway of TMEM205 c) evaluate the in vitro and in vivo efficacy of oncolytic virus (oHSV) as a single agent or in combination with cisplatin. Methods: Human patient tissue samples from OCCC patients, OCCC cell lines like JHOC, OVTOKO, OVISE and ES2 and exosomes were analyzed for the expression of TMEM205 using Western blot, IHC/ICC and real time qPCR. Exosomes were isolated from conditioned medium and characterized using NTA. Antibody feeding assays and plasma membrane internalization assays were conducted to address the endocytosis and recycling behavior of TMEM205. TMEM205 overexpression and knockdown experiments were performed in vitro in OVTOKO cells and in vivo in nude mice, to confirm the role of TMEM205 in OCCC. Cisplatin accumulation in cells as well as DNA and exosomes was quantified using ICP-MS and dot blot. oHSV virus therapy was further tested in vitro or in vivo-alone or combined with cisplatin treatment. Results: The OCCC tissues, cell lines and exosomes exhibit medium to high expression of TMEM205. Mechanistically, TMEM205 undergoes ligand independent constitutive endocytosis, co-localizes with Rab 11 to be a part of the late recycling endosomes with only 6% of TMEM205 being recycled back to the plasma membrane. Knockdown of TMEM205 triggers more intracellular cisplatin accumulation in comparison to the exosomes derived from them. Further, TMEM205 interacts with glycoprotein C of oHSV and both of these proteins undergo ubiquitinylation to ultimately get shuttled outside the cell via exosomes. We also report that oHSV pretreatment followed by treatment with cisplatin (CP) increases apoptosis and decreases cell survival, in vitro as well as in vivo tumor growth. Conclusions: TMEM205 imparts drug resistance via exosomes and knockdown of TMEM205 in OCCC contributes to enhanced chemotherapy sensitivity. The combination therapy of oHSV with cisplatin is more effective than oHSV or cisplatin treatment alone and reduces the overall tumor burden of OCCC mice. This will pave the way for exploring the clinical implications of TMEM205 in OCCC, and evaluating whether TMEM205 expression is key to higher risk of resistance and recurrence seen in OCCC. Citation Format: Uksha Saini. Targeting TMEM205 mechanotransduction pathway using oncolytic virus therapy in ovarian clear cell carcinoma (OCCC) [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 LB-100.

KIF20A expression as a prognostic indicator and its possible involvement in the proliferation of ovarian clear‑cell carcinoma cells.

Kinesin family member 20A (KIF20A), which is involved in cytokinesis and intracellular transportation, has been recently reported to be upregulated in several malignancies and may contribute to chemotherapeutic resistance. We examined the distribution and expression of KIF20A in clear-cell carcinoma (CCC) of the ovary to elucidate its clinical significance and molecular mechanism. Paraffin sections from ovarian CCC tissues (N=43) were immunostained with KIF20A antibody, and the staining intensities were semi-quantitatively evaluated. Furthermore, we investigated whether silencing of KIF20A contributes to the proliferation-inhibitory potential using CCC cells. During the observational period, 18 patients (41.9%) developed recurrence. The median time to recurrence was 11.5 months. Patients in the high KIF20A expression group showed poorer progression-free survival (PFS) and overall survival (OS) than those in the low expression group (P=0.0443 and P=0.0478, respectively). In multivariable analyses, KIF20A expression was also a significantly independent indicator of PFS and a marginally significant indicator of OS [PFS: HR (high vs. low), 5.488; 95% CI, 1.410–24.772 (P=0.0136); OS: HR, 2.835; 95% CI, 0.854–11.035, (P=0.0897)]. In in vitro studies, the ovarian CCC cell proliferation was significantly decreased by KIF20A silencing or in the presence of KIF20A inhibitor in CCC cells. Cell cycle G2/M arrest and a higher apoptosis-induced fraction were more frequently observed in si-KIF20A-transfected CCC cells than in the control cells. Although the present study was preliminary, these data indicate the possible involvement of KIF20A in the proliferation of CCC, suggesting that targeting this molecule may contribute to reversing the malignant potential consequently affecting the oncologic outcome of CCC patients.

Identification of somatic genetic alterations in ovarian clear cell carcinoma with next generation sequencing.

Ovarian clear cell carcinoma (OCCC) is the most refractory subtype of ovarian cancer and more prevalent in Japanese than Caucasians (25% and 5% of all ovarian cancer, respectively). The aim of this study is to discover the genomic alterations that may cause OCCC and effective molecular targets for chemotherapy. Paired genomic DNAs of 48 OCCC tissues and corresponding noncancerous tissues were extracted from formalin-fixed, paraffin embedded specimens collected between 2007 and 2015 at Tohoku University Hospital. All specimens underwent exome sequencing and the somatic genetic alterations were identified. We divided the cases into three clusters based on the mutation spectra. Clinical characteristics such as age of onset and endometriosis are similar among the clusters but one cluster shows mutations related to APOBEC activation, indicating its contribution to subset of OCCC cases. There are three hypermutated cases (showing 12-fold or higher somatic mutations than the other 45 cases) and they have germline and somatic mismatch repair gene alterations. The frequently mutated genes are ARID1A (66.7%), PIK3CA (50%), PPP2R1A (18.8%), and KRAS (16.7%). Somatic mutations important for selection of chemotherapeutic agents, such as BRAF, ERBB2, PDGFRB, PGR, and KRAS are found in 27.1% of OCCC cases, indicating clinical importance of exome analysis for OCCC. Our study suggests that the genetic instability caused by either mismatch repair defect or activation of APOBEC play critical roles in OCCC carcinogenesis.

Abstract 5888: Tmem 205-mediated cisplatin resistance in ovarian clear cell carcinoma (OCCC) is overcome by herpes simplex virus (oHSV)/cisplatin combination therapy

Abstract Objectives: Ovarian clear cell carcinoma (OCCC) is an aggressive form of ovarian cancer and unfavorable treatment outcomes have been attributed to its relative chemoresistance and recurrence, limiting the available treatment options. Therefore, identifying novel unique protein(s) involved in OCCC chemoresistance and designing a therapeutic modality selectively targeting those proteins would make a significant impact on therapy. TMEM 205 is one such protein which has been recently linked to cisplatin resistance in human epidermoid carcinoma, but the precise molecular mechanism of this resistance still needs to be elucidated. This proposed study was planned to a) decipher the role of TMEM205 expression in OCCC (relative to HGSOC) b) mechanism by which TMEM205 imparts chemoresistance c) evaluate the in vitro and in vivo efficacy of oHSV as a single agent or in combination with standard chemotherapy Methods: Human patient tissue samples from OCCC patients as well as OCCC cell lines like JHOC, OVTOKO, OVISE and ES2 were analyzed for the expression of TMEM205 using Western blot, IHC/ICC and real time qPCR. In situ pO2 was measured in the cells as well as mice using EPR oximetry. Exosomes were isolated and characterized using NTA. TMEM205 overexpression and knockdown experiments were performed in vitro in OVTOKO cells to confirm the role of TMEM205 in OCCC. Cispaltin accumulation in cells and exosomes was quantified using ICP-MS. oHSV virus therapy was further tested in vitro or in vivo-alone or combined with cisplatin treatment. Results demonstrate that the OCCC tissues as well as cell lines have high expression of TMEM205. OCCC cells and mice injected with these cells are severely hypoxic and cisplatin accumulation is more in the exosomes derived from them, which goes down with the knockdown of TMEM205. oHSV treatment suppresses TMEM205 expression in OVTOKO and JHOC cells. Further, ICC showed that TMEM205 and oHSV co-localize in the cells, indicating that oHSV may interact with TMEM205 and suppress its expression and activity. oHSV pretreatment followed by treatment with cisplatin (CP) increases apoptosis and decreases cell survival, in vitro within 48 hours as well as in vivo. Conclusions: TMEM205 mediates drug resistance through exosomes and knockdown of TMEM205 in OCCC contributes to enhanced chemotherapy sensitivity. This will pave the way for exploring the clinical implications of TMEM205 in OCCC, and evaluating whether TMEM205 expression is key to higher risk of resistance and recurrence seen in OCCC. Citation Format: Uksha Saini, Maria Riley, Kalpana Deepa Dorayappan, Chelsea Bolyard, Balveen Kaur, Roman Zingarelli, Ikuo Konishi, Periannan Kuppusamy, George Larry Maxwell, David E. Cohn, Karuppaiyah Selvendiran. Tmem 205-mediated cisplatin resistance in ovarian clear cell carcinoma (OCCC) is overcome by herpes simplex virus (oHSV)/cisplatin combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5888. doi:10.1158/1538-7445.AM2017-5888

Abstract 1397: Antisense oligo nucleotide of Annexin A4 improved platinum resistance in ovarian clear cell cancer

Abstract Introduction: Ovarian cancer in Japan are classified as clear cell carcinoma (CCC) more than 20 %, this percentage is higher than in Europe and United States. Besides, it is well known that CCC of ovary is highly resistant to cancer chemotherapy including carboplatin and paclitaxel treatment. We reported that Annexin A4 protein was overexpressed in ovarian CCC tissues by immunohistochemical analysis. Elevated Annexin A4 level has been detected in various epithelial cancer cell lines and have reported associating with chemoresistance to platinum-based cancer drugs. To overcome the platinum chemoresistance, we thought antisense oligonucleotides (ASOs) to be a good therapeutic option in a way of highly specific therapy for improving chemoresistance by suppressing the expression of Annexin A4 in cancer cells. Methods: We generated ASO targeting Annexin A4 with 2’, 4’-bridged nucleic acid. And we analyzed suppression of Annexin A4 in ASO-transfected RMG-I cell line (CCC) in vitro using real time PCR and western blotting. In 16 types of ASOs targeting Annexin A4, 2 ASOs were eligible. Cells were seeded in 96-well plates (2,000 cells per well). Next day, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50 values) were calculated. Intracellular platinum accumulation in Annexin A4 overexpressing cells was analyzed. To assess the improvement of platinum resistance in vivo, we used ICR nu/nu mice xenografted subcutaneously with RMG-I cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice. Results: By realtime PCR analysis, among strong 16 types of ASOs targeting Annexin A4, 2 ASOs showed strong knockdown efficiency (about 80% knockdown) compared to negative control ASOs. Western blotting analysis showing knockdown of Annexin A4 expression was observed in Annexin A4 ASO transfected cells compared to no treatment or control ASOs in vitro. ASO-transfected RMG-I cells was less resistant to cisplatin (IC50 = 3.3μM) compared with control cells (IC50 = 5.2μM) Same result were obtained with carboplatin. Platinum resistance was significantly improved in treated with Annexin A4 ASO and cisplatin compared to control ASO and cisplatin treated group in vivo. Conclusion: By transfection of ASOs targeting Annexin A4, platinum resistance have improved in vivo and in vitro, Annexin A4 have associated with efflux of platinum anti-tumor drug. In conclusion, antisense oligonucleotides for Annexin A4 will be a therapeutic option for ovarian clear cell carcinoma with chemoresistance to platinum antitumor drug. Citation Format: Reisa Kakubari, Satoshi Nakagawa, Tadashi Iwamiya, Eiji Kobayashi, Shinnya Matsuzaki, Yutaka Ueda, Kiyoshi Yoshino, Yuya Kasahara, Satoshi Obika, Tadashi Kimura, Satoshi Serada, Tetsuji Naka, Minoru Fujimoto. Antisense oligo nucleotide of Annexin A4 improved platinum resistance in ovarian clear cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2017-1397

Down-regulation of ARID1A is sufficient to initiate neoplastic transformation along with epigenetic reprogramming in non-tumorigenic endometriotic cells

The chromatin remodeler AT-Rich Interactive Domain 1A (ARID1A) is frequently mutated in ovarian clear cell carcinoma (OCCC) and endometriosis precursor lesions. Here, we show that knocking down ARID1A in an immortalized endometriosis cell line is sufficient to induce phenotypic changes indicative of neoplastic transformation as evidenced by higher efficiency of anchorage-independent growth, increased propensity to adhere to collagen, and greater capacity to invade basement membrane extract in vitro. ARID1A knockdown is associated with expression dysregulation of 99 target genes, and many of these expression changes are also observed in primary OCCC tissues. Further, pathway analysis indicates these genes fall within networks highly relevant to tumorigenesis including integrin and paxillin pathways. We demonstrate that the down-regulation of ARID1A does not markedly alter global chromatin accessibility or DNA methylation but unexpectedly, we find strong increases in the active H3K27ac mark in promoter regions and decreases of H3K27ac at potential enhancers. Taken together, these data provide evidence that ARID1A mutation can be an early stage event in the oncogenic transformation of endometriosis cells giving rise to OCCC.

Targeting FXYD2 by cardiac glycosides potently blocks tumor growth in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is an aggressive neoplasm with a high recurrence rate that frequently develops resistance to platinum-based chemotherapy. There are few prognostic biomarkers or targeted therapies exist for patients with OCCC. Here, we identified that FXYD2, the modulating subunit of Na+/K+-ATPases, was highly and specifically expressed in clinical OCCC tissues. The expression levels of FXYD2 were significantly higher in advanced-stage of OCCC and positively correlated with patients' prognoses. Silencing of FXYD2 expression in OCCC cells inhibited Na+/K+-ATPase enzyme activity and suppressed tumor growth via induction of autophagy-mediated cell death. We found that high FXYD2 expression in OCCC was transcriptionally regulated by the transcriptional factor HNF1B. Furthermore, up-regulation of FXYD2 expression significantly increased the sensitivity of OCCC cells to cardiac glycosides, the Na+/K+-ATPase inhibitors. Two cardiac glycosides, digoxin and digitoxin, had a great therapeutic efficacy in OCCC cells in vitro and in vivo. Taken together, our results demonstrate that FXYD2 is functionally upregulated in OCCC and may serve as a promising prognostic biomarker and therapeutic target of cardiac glycosides in OCCC.