Human Ovarian Clear Cell Adenocarcinoma Research Articles

Noncanonical IL6 Signaling-Mediated Activation of YAP Regulates Cell Migration and Invasion in Ovarian Clear Cell Cancer.

Ovarian clear cell adenocarcinoma (OCCA) is characterized by a particularly poor response to conventional chemotherapy and a short overall survival time in women with established disease. The development of targeted treatments for OCCA relies on a better understanding of its molecular characteristics. IL6 is strongly expressed in OCCA and may therefore provide a novel therapeutic target. Here we use CRISPR/Cas9 and conditional short hairpin interfering RNA to perform loss-of-function studies in human OCCA cell lines to explore the requirement for IL6 in vitro and in vivo. While reduction of IL6 expression exerted limited effects in vitro, its attenuation significantly impaired tumor growth and neovascularization in vivo. In contrast to typical signaling via STAT3, IL6 in OCCA signaled via a noncanonical pathway involving gp130, Src, and the Hippo pathway protein YAP. A high-throughput combination drug screen identified agents that enhanced cell killing following reduction of IL6 signaling. Intersection of screen hits obtained from two cell lines and orthogonal approaches to attenuation of IL6 yielded AKT and EGFR inhibitors as enhancers of the inhibitory monoclonal IL6 receptor antibody tocilizumab. This study defines for the first time the requirements for, and mechanisms of, signaling by IL6 in human OCCA cell lines and identifies potential combinatory therapeutic approaches. Given the molecular diversity of OCCA, further in vitro and in vivo studies are warranted to determine whether such approaches will overcome the limited efficacy of tocilizumab observed in ovarian cancer to date. SIGNIFICANCE: This study defines the requirements for and mechanisms of noncanonical signaling by IL6 in human ovarian clear cell adenocarcinoma cell lines and identifies combinatory therapeutic approaches to be explored clinically.

Establishment and characterization of a novel ovarian clear cell adenocarcinoma cell line, TU-OC-1, with a mutation in the PIK3CA gene

A new cell line of human ovarian clear cell adenocarcinoma (CCC), TU-OC-1, was established and characterized. The cells showed a polygonal-shaped morphology and grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 64 to 90. A low rate of proliferation was observed, similar to other CCC cell lines tested (OVTOKO, RMG-I, and OVAS), and the doubling time was 38.4h. The respective IC50 values of cisplatin and paclitaxel were 12.2μM and 58.3nM. Mutational analysis revealed that TU-OC-1 cells harbored a PIK3CA mutation at codon 542 (E542K) in exon 9, which is a mutation hot spot on this gene. We observed that phosphorylated Akt protein was overexpressed in TU-OC-1 cells by western blot analysis. Heterotransplantation to nude mice produced tumors that reflected the original. This cell line may be useful to study the chemoresistant mechanisms of CCC and contribute to novel treatment strategies.

Analysis of mTOR Inhibition-Involved Pathway in Ovarian Clear Cell Adenocarcinoma

This study was designed to clarify the mechanism of the mammalian target of rapamycin (mTOR)-hypoxia inducible factor-1 (HIF-1) pathway using the cultured cell strain derived from human ovarian clear cell adenocarcinoma (CCA). Everolimus (a derivative of rapamycin)-treated cells and non-treated cells did not show any difference in mTOR expression. But, phosphorylated-mTOR (p-mTOR) expression significantly decreased in the treated cells, and mTOR-related factors such as phosphorylated-4E-BP1 (p-4E-BP1), HIF-1α, and vascular endothelial growth factor (VEGF) in the downstream region of mTOR revealed a marked decrease in expression. The analysis of influences of the drug on the HIF-1α degradation system showed an increase in von-Hippel Lindau (VHL) expression in the treated cells. Increase of cleaved caspase-3, one of key factors involved in apoptosis, was also shown in the treated cells. In the next step, using nude mice implanted with RMG-1 cells, a decrease in tumor size was demonstrated in 4 of the 7 mice which were orally administered with everolimus. As a result, it was suggested that everolimus administration would be helpful as an anti-tumor therapy for CCA not only via down-regulation of p-mTOR but also degradation of HIF-1α by VHL and induction of apoptosis by cleaved caspase-3.

HOXA10 is Overexpressed in Human Ovarian Clear Cell Adenocarcinoma and Correlates With Poor Survival

Human homeobox gene (HOX) A10 is a homeobox allotype gene of the HOXA family in the HOX family. Human homeobox gene A10 may play an important role in cancer development. However, the role of HOXA10 in the carcinogenesis of ovarian clear cell adenocarcinoma (OCCA) has not been established. We have evaluated the prognostic significance of HOXA10 expression for human OCCA and the effects of HOXA10 on proliferation, motility, and invasion of OCCA cells. We found that HOXA10 was not expressed in normal ovarian epithelium, ovarian endometrial cysts, and ovarian serous carcinomas, but 20 (68.9%) of the 29 OCCAs were positive for the expression of HOXA10. Human homeobox gene A10 expression was negatively correlated to the 5-year survival of OCCA patients (R = -0.442, P = 0.043). When a HOXA10 expression vector was stably transfected into a human OCCA cell line, ES-2, the proliferation rate of ES-2-HOXA10 was much higher than the vector control, the motility of ES-2-HOXA10 cells was significantly increased compared with the control (P < 0.05), and the invasion of ES-2-HOXA10 cells was also much higher than the vector control (P < 0.01 at 5 hours and 12 hours after scratching). In conclusion, HOXA10 was overexpressed in OCCA and was correlated with poor survival. HOXA10 promotes proliferation, migration, and invasion of OCCA cells. Human homeobox gene A10 could be a promising prognostic marker for OCCA.

1042 Human homeobox gene (HOX) A10 is overexpressed in human ovarian clear cell adenocarcinoma and correlates with poor survival

Heat shock protein 60 (HSP60) participates in protein assembly, folding and transport. Increased HSP60 mRNA expression is associated with cisplatin resistance in some in vitro models and with shorter survival among ovarian cancer patients. HSP60 mRNA expression was quantitated in three independent model systems by reverse-transcription PCR (A2780 human ovarian carcinoma cell line and sublines selected for cisplatin or oxaliplatin resistance in vitro and also in the UCRU-BL13 human bladder carcinoma cell line and a cisplatin-resistant subline). Increased HSP60 mRNA expression was observed in all resistant sublines (range 2.5–15 fold), correlated with relative resistance to cisplatin and oxaliplatin. No differences in HSP60 gene copy number were apparent in resistant sublines. These data provide further evidence of a strong association between in vitro resistance to platinum compounds and increased HSP60 mRNA expression.

Establishment and Characterization of the RMG-V Cell Line from Human Ovarian Clear Cell Adenocarcinoma

A cell line, designated as RMG-V, was established from a patient with clear cell adenocarcinoma of the ovary. The cell line has grown without interruption and has been propagated continuously by serial passaging (more than 36 times) over 5 years. The cells are spindle-shaped, display neoplastic and pleomorphic features, and grow in a jigsaw puzzle-like arrangement while forming monolayers without contact inhibition. These cells proliferate rapidly, and the population doubling time is about 15.5 hours. The number of chromosomes ranges between 77 and 85, with a modal number of 83.

Human monoclonal antibody for ovarian clear cell carcinoma-2, a human monoclonal antibody with antitumor activity against ovarian cancer cells that recognizes CA125-like antigen

In recent years, antibody therapy employing monoclonal antibodies has become a new approach for treating cancer. This study was performed to establish a human monoclonal antibody recognizing an epitope related to CA125 using KM mice and to assess its reactivity with ovarian cancer cells. A human ovarian clear cell adenocarcinoma cell line (RMG-I) was used to immunize KM mice, and hybridoma supernatant was obtained by a standard method employing enzyme-linked immunosorbent assay screening. Next, selection of hybridomas was performed with two antibodies (MA602-1 and MA602-6) and a sandwich immunoassay for CA125-like antigen, and then the limiting dilution was used to obtain a human monoclonal antibody. Immunohistochemical reactivity of this antibody (human monoclonal antibody for ovarian clear cell carcinoma-2 [HMOCC-2]) with ovarian cancer was assessed, while its specificity was analyzed by Western blotting. Various antibodies were used to identify the epitope targeted by HMOCC-2. Finally, the antitumor effect of HMOCC-2 was assessed by intraperitoneal administration to SCID (severe combined immunodeficiency) mice with heterografts of RMG-I tumors. HMOCC-2 showed a positive reaction with 60% (63/105) of ovarian cancer specimens. Western blotting of the membrane fraction of RMG-I revealed several bands at 120-250 kd. HMOCC-2 recognized the CA125-like antigens identified by several antibodies. HMOCC-2 also exhibited significant antitumor activity (P < 0.01) against ovarian cancer heterografts. HMOCC-2 reacts specifically with ovarian cancer cells via a target epitope analogous to that of CA125 and also exhibits activity against ovarian tumors. These findings suggest that it may have the potential to be employed clinically for molecular-targeting therapy.

The autocrine effect of activin A on human ovarian clear cell adenocarcinoma cells

The functions of activin, a member of TGF-beta superfamily, in ovarian clear cell adenocarcinoma remain unsolved, although we recently found that inhibin betaA-subunit, activin A, activin receptor type IA, type IB, type IIA, type IIB, Smad2, Smad3 and Smad4 were localized in tumor cells of the ovarian clear cell adenocarcinoma tissue by immunohistochemistry. In the present study, in order to investigate the role of activin concerning cell growth in ovarian clear cell adenocarcinoma cells, we determined the production of activin A and inhibin A, and the expression of activin receptors and Smads using the human ovarian clear cell adenocarcinoma cell line JHOC-5. Moreover, we examined the effects of activin A on the activation of activin signaling pathway and on the proliferation in JHOC-5 cells. We detected a measurable amount of activin A in the culture medium of JHOC-5 cells, although inhibin A was not detected. The expression of activin receptor type IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 was observed in JHOC-5 cells. Activin A induced a significant increase in proliferation of JHOC-5 cells compared with the untreated control. On the other hand, activin A did not affect the growth of JHOC-5 cells and no statistically significant difference was observed in the presence of follistatin which is a specific binding protein of activin. Phosphorylated Smad2, an activated form of Smad2, was detected both in treated JHOC-5 cells and in untreated cells by activin A. Activin A significantly increased the expression of phosphorylated Smad2 in JHOC-5 cells. Therefore, it is possible that activin has autocrine roles in tumor growth of ovarian clear cell adenocarcinoma cells.

Proliferative activity of early ovarian clear cell adenocarcinoma depends on association with endometriosis

Objective To investigate differences in the biological characteristics of ovarian clear cell adenocarcinoma based on the presence/absence of endometriosis and tumor proliferative activity. Methods Stage I ovarian clear cell adenocarcinoma patients were divided into groups with and without endometriosis, and immunohistochemical expression of proliferating cell nuclear antigen was determined in surgical specimens. Then xenograft models of human ovarian clear cell adenocarcinoma with or without human ectopic endometrium were created in severe combined immunodeficiency mice, and tumor growth was assessed from the wet weight and the bromodeoxyuridine uptake. Furthermore, a xenograft model of human endometriosis was made with or without ovarian clear cell adenocarcinoma and cytokine production was investigated. Results The proliferating cell nuclear antigen labeling index was significantly lower in the tumors of patients with endometriosis compared to the tumors of patients without endometriosis. In tumor-bearing mice, the tumor weight and bromodeoxyuridine uptake were both significantly lower when ovarian clear cell adenocarcinoma was associated with endometriosis than in its absence. Release of transforming growth factor-β1 and interleukin-6 from the ectopic human endometrium was greater in the presence of clear cell adenocarcinoma than without it, and transforming growth factor-β1 levels showed a significant difference. Conclusion The proliferative activity of early ovarian clear cell adenocarcinoma seems to depend on the association of this cancer with endometriosis. When endometriosis is associated with ovarian clear cell adenocarcinoma, there is a change of its cytokine production that may inhibit tumor growth.

Establishment and Characterization of Two Human Ovarian Clear Cell Adenocarcinoma Lines from Metastatic Lesions with Different Properties

Two permanent human ovarian clear cell adenocarcinoma lines (OVISE and OVTOKO) were established from metastatic tumors of two patients who were treated with five to six courses of CAP chemotherapy. The two cell lines grow on monolayers and showed a variety in both size and shape: small or moderately sized cuboidal cells, columnar cells, spindle-shaped cells, and malignant tumor giant cells. The cell lines have been in culture for 4 to 6 years, the passage number varying from 160 to 220. The mean population-doubling time of the two cells was 60 to 70 hr. The OVISE cells shed tumor-associated antigens CA19-9, CA125, and TPA in the culture medium, whereas the OVTOKO cells did not secrete them at detectable levels. Immunohistochemical analysis showed that coexpression of cytokeratins and vimentin was preserved in the two cell lines, which is a feature of cultured epithelial origin. Cytokeratin polypeptides 7, 8, 18, and 19 were expressed in both cell lines. The EGF receptor was more intensely expressed in the OVTOKO cells than in the OVISE cells. The estrogen and progesterone receptors were negative in both cell lines. The two cell lines showed no chemosensitivity to anticancer drugs including cisplatin, doxorubicin, cyclophosphamide, and etoposide. Hetero-transplantation of the two cell lines reflected the origin of cells. Intraperitoneal transplantation of the OVTOKO cells yielded peritoneal implantation and distant metastasis, whereas that of the OVISE cells showed no dissemination and metastasis. These new ovarian clear cell adenocarcinoma lines will provide a relevant experimental system for further investigations into the intrinsic alterations responsible for malignant progression and chemoresistance.

Establishment of HUOCA-II, a Human Ovarian Clear Cell Adenocarcinoma Cell Line, and Its Angiogenic Activity

A cell line designated "HUOCA-II" was established from a human ovarian clear cell adenocarcinoma. The HUOCA-II cells, which were oval, spindle, or polygonal and had neoplastic and pleomorphic features, grew in multiple layers without contact inhibition. The cell line grew fast (population doubling time, 24 hr), and 55 serial passages were carried out within 11 months. The chromosomal number ranged around 46, and no karyological abnormality was found in G-band karyotyping. When heterotransplanted into the subcutis of BALB/c nude mice, HUOCA-II cells produced a poorly differentiated clear cell adenocarcinoma. The tumor angiogenesis factor (TAF) of a molecular weight of about 14,000 was purified from the conditioned medium of HUOCA-II cells, and neovascularization was detected by bioassay with the use of the chorioallantoic membrane of a chick embryo. This TAF also stimulated the growth of endothelial cells in an in vitro culture system.