Clear Cell Carcinoma Cell Lines Research Articles

Preclinical Activity of Plitidepsin Against Clear Cell Carcinoma of the Ovary

To evaluate the antitumor effects of Plitidepsin against clear cell carcinoma (CCC) of the ovary. The expression of eEF1A2 in ovarian cancer was assessed by immunohistochemistry. Using ovarian CCC cell lines, the antitumor effect of Plitidepsin was assessed both in vitro and in vivo. By over-expressing or knocking down the eEF1A2 expression, we investigated the role of eEF1A2 in the sensitivity of CCC cells to Plitidepsin. Immunoreactivity to eEF1A2 was observed in 76.2% of CCC, which was significantly higher than other histological subtypes of ovarian cancer. Plitidepsin exhibited significant antitumor activity toward chemonaive and chemoresistant CCC cells both in vitro and in vivo. Ectopic expression of eEF1A2 in CCC cells resulted in increased sensitivity to Plitidepsin. In contrast, eEF1A2 knockdown decreased sensitivity of CCC cells to plitidepsin. Plitidepsin, a novel anti-cancer agent that targets eEF1A2, may be a promising agent for treating ovarian CCC.

Abstract 2005: ZDHHC7 contributes to ferroptosis resistance through YAP localization in ovarian clear cell carcinoma

Abstract [Objective] Ovarian clear cell carcinoma (CCC) shows unique clinical features including an association with benign endometriosis which contains iron-rich fluid, and poor prognosis because of resistance to chemotherapy. Therefore, elucidating the basic mechanism as well as new treatment strategy of ovarian CCC is urgently needed. Recently, ferroptosis, which is characterized by the iron-dependent accumulation of lipid ROS, was reported to affect the malignant behaviors. Also, some reports showed ferroptosis regulation by Hippo signaling. ZDHHC7 is reported as the molecule regulating yes-associated protein (YAP), a key molecule of Hippo signature and is one of the ovarian CCC gene signature which we previously identified. We explored the impact of ferroptosis in ovarian CCC by investigating YAP localization.[Methods] We performed immunohistochemistry (IHC) of malondialdehyde (MDA; a marker of lipid reactive oxygen species, ROS) and 8-Oxo-2'-deoxyguanosine (8-OHdG, a marker of oxidative stress DNA damage) to analyze the status of lipid ROS with ovarian cancer patients. We also performed IHC of YAP and ZDHHC7 to evaluate them in clinical status. We calculated IC50 values of erastin (ferroptosis inducer) with seventeen ovarian cancer cell lines. Functional annotation clustering of microarray expression data was performed using Metascape. ZDHHC7 knockdown and YAP-5SA mutant, or YAP-activated form of ovarian CCC cell lines were generated and applied for in vitro assays.[Results] IHC analysis indicated higher MDA expression (p=0.0354) and 8-OHdG (p<0.0001) in CCC compared to high-grade serous carcinoma (HGSC), suggesting higher lipid ROS as well as oxidative stress status in ovarian CCC. IC50 values of erastin were significantly higher in CCC than in other types of ovarian cancer (p<0.01). Categorical analysis revealed that genes related to Hippo signaling were enriched in erastin-resistant cell lines. IHC analyses indicated low nuclear expression of YAP and high expression of ZDHHC7 in CCC samples (p=0.0010). The cases with low expression of nuclear YAP showed significantly poorer prognosis compared to high expression cases (p=0.0075) in ovarian CCC. YAP-5SA mutant (YAP-active-mutant) CCC cells showed higher sensitivity to erastin and ML210 (ferroptosis inducer) than control in ovarian CCC cell lines. ZDHHC7 knockdown of CCC cells induced nuclear localization of YAP and showed decreased IC50 values of erastin and ML210 compared to control cells.[Conclusion] Although lipid ROS is accumulated in ovarian CCC, ovarian CCC showed ferroptosis resistance through YAP localization regulated by ZDHHC7. Citation Format: Yoko Furutake, Ken Yamaguchi, Masayo Ukita, Mana Taki, Koji Yamanoi, Junzo Hamanishi, Takashi Kobayashi, Masaki Mandai. ZDHHC7 contributes to ferroptosis resistance through YAP localization in ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2005.

Hypoxia-inducible Factor-1α Suppression in Ovarian Clear-cell Carcinoma Cells by Silibinin Administration.

Advanced ovarian clear-cell carcinoma (CCC) fails to respond to standard chemotherapy, and has a poor prognosis. Since hypoxia-inducible factor-1 (HIF-1) stimulates various genes involved in cancer, we aimed to examine the efficacy of silibinin, an active component of milk thistle belonging to Asteraceae, in suppressing HIF-1 activity, and elucidate the underlying mechanism in human CCC cell lines. Human ovarian CCC cell lines HAC-2, OVISE, and RMG-1 were treated with 500 μM silibinin for 4 h under normoxic and hypoxic conditions. Using DNA microarray, we analysed genes whose expression modulated more than 2-fold in response to hypoxia, whereas HIF-1α expression was measured using ELISA. Silibinin treatment decreased HIF-1α protein in all cell lines, and eIF4E2 and RPS6 mRNA in HAC-2 and RMG-1 cells. Silibinin suppressed HIF-1α protein under hypoxic conditions in CCC cell lines and could be a potential anti-cancer drug.

High Expression of p21 as a Potential Therapeutic Target in Ovarian Clear-cell Carcinoma.

DNA damage response (DDR), wherein p21 is a cell fate determinant, is a potential cancer therapeutic target. Molecular expression during DDR was explored in ovarian clear-cell carcinoma (CCC). CHK1, CHK2, TP53 and p21 expression in DDR was examined using immunostaining in surgical sections of CCC (n=22). Molecular alterations in two types of CCC cell lines, JHOC-5 and JHOC-9, were investigated using western blot analysis. Expression of DDR-associated molecules was noted in most patients. While high p21 expression was found in half of the patients, the remaining patients exhibited low p21 expression. Treatment with UC2288, a p21 inhibitor, attenuated proliferation of both cell lines, more prominently in JHOC-9, resulting in reduced viability and subsequent apoptosis. p21 Inhibitor induced cell death in cells with high p21 expression, suggesting that p21 suppression can be a therapeutic strategy to treat patients with CCC.

Abstract A73: BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines

Abstract Clear-cell ovarian carcinoma is a rare histologic subtype of epithelial ovarian cancers. Clear-cell ovarian cancers tend to be less responsive to platinum-based chemotherapy compared to high-grade serous ovarian cancers. In a dataset of large-scale CRISPR knockout and RNAi screens of cancer cell lines (depmap.org), a subset of ovarian clear-cell carcinoma cell lines were sensitive to knockout or knockdown of BCL2L1, encoding antiapoptotic protein BCL-XL. Most clear-cell ovarian cancer cell lines also express BCL2L1 mRNA and protein. We therefore tested a panel of ovarian cancer cell lines for response to BH3 mimetic drugs, which inhibit different members of the BCL-2 family of antiapoptotic proteins. Initial data showed that a subset of ovarian clear-cell carcinoma cell lines was sensitive to single-agent BCL-XL inhibition with A1331852 and dual BCL-2/BCL-XL inhibition with ABT-263. In contrast, these cell lines were relatively insensitive to single-agent BCL-2 inhibition or MCL1 inhibition. We are currently applying BH3 profiling, a functional assay for apoptotic priming, to test the response of ovarian clear-cell carcinoma cell lines to BCL-XL inhibition using a BCL-XL specific peptide, HRK. Collectively, these studies suggest that ovarian clear-cell carcinomas may be sensitive to BCL-XL inhibition, either alone or in combination with chemotherapy. Citation Format: Elizabeth H. Stover, Maya K. Gelbard, Ursula A. Matulonis, Matthew Meyerson. BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A73.

Pathology of clear cell carcinoma of the ovary: A basic view based on cultured cells and modern view from comprehensive approaches.

Clear cell carcinoma (CCC) is a distinct histologic type of ovarian carcinoma. CCC is more frequent in Japan than in the Western world. CCC is chemo-resistant and often associated with paraneoplastic thromboembolism. Histologically, CCC is characterized by both cancer cells and stromas, being concordant with the cytological features. Clear cells contain abundant glycogen. Hepatocyte nuclear factor-1β is a specific marker of CCC, and is likely to be involved in glucose metabolism. Extracellular matrix (ECM)-deposited stroma and plasma cell-rich inflammatory stroma are characteristic stromas of CCC. Studies using CCC cell lines showed that CCC cells produce ECMs and stimulate plasma cell differentiation in a paracrine manner. Most CCCs, as well as endometrioid carcinomas, originate from ovarian endometriosis. This is supported by molecular genetic data, although it remains unclear why different histologic types originate from the same precursor. CCC and endometrioid carcinoma are Lynch syndrome-associated ovarian carcinomas. Recent comprehensive studies indicate that CCC is distinct not only in terms of histology but also in genomics, epigenomics and transcriptomics. This review summarizes the pathology of ovarian CCC along with a basic view based on cultured cells, and refers to recent genetic and omic data.

CDNA expression library screening revealed novel functional genes involved in clear cell carcinogenesis of the ovary in vitro

In order to identify genes involved in the pathogenesis of clear cell carcinoma of the ovary (CCC), functional screening using a cDNA expression library was performed. We extracted mRNA from a CCC cell line (RMG-1), established a cDNA library using a retroviral vector, transfected that library into mouse NIH3T3 cells and sequenced the resultant foci. The tissue-type specific expression of isolated genes and their transforming activities were evaluated. Seven genes were isolated. Of these genes, the mRNA expression of SEC61B and DVL1 is significantly stronger in CCC than in other histological types (p < .05). Immunohistochemical staining reveals the stronger expression of SEC61B and C1ORF38 than normal ovarian tissues (p < .05). Focus formation is confirmed by the transfection of SEC61B, C1ORF38, and DVL1 into NIH3T3 cells. The present study identified novel genes including SEC61B, C1ORF38, and DVL1, involved in the pathogenesis of CCC. These genes may be additional therapeutic targets for CCC. Impact statement What is already known on this subject? Several important genetic abnormalities, including ARID1A and PIK3CA mutations, have been reported in ovarian clear cell carcinoma (CCC). What the results of this study add? SEC61B, C1ORF38, and DVL1 were newly detected as candidate genes involved in ovarian clear cell carcinogenesis. What the implications are of these findings for clinical practice and/or further research? Functional screening using a cDNA expression library may be a useful technique to identify functional genes for pathogenesis. The information obtained using this technique may provide new therapeutic targets of CCC.

Abstract GMM-036: COMPREHENSIVE GENOMIC, PROTEOMIC, AND EXPERIMENTAL CHARACTERIZATION OF OVARIAN CLEAR CELL CARCINOMA CELL LINES FOR IMPROVED DRUG DEVELOPMENT

Abstract PURPOSE: Ovarian cancer is a heterogeneous disease with multiple histological subtypes and a wide range of genetic aberrations. While high-grade serous carcinoma (HGSC) is the most common type, clear cell carcinoma (CCC) of the ovary is notoriously the most challenging to treat and exhibits very low response rates to standard therapies. However, efforts to understand CCC and develop new therapeutic approaches to this subtype have been limited because it represents a minority of ovarian cancer cases in the United States and Europe. In contrast, CCC accounts for more than 30% of all ovarian cancer in Japan. To improve the survival of patients with ovarian CCC, a deeper understanding of the molecular features of available model systems is needed. Our goal is to characterize a panel of CCC cell lines genomically and functionally and identify those that can serve as tractable model systems for future in vivo drug discovery studies. METHOD: First, we characterize 9 CCC cell lines (ES-2, TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-7, JHOC-9, and OVISE) with whole exome sequence and proteomics approaches (reverse phase protein array; RPPA). Secondly, we performed in vitro assays, including soft-agar colony formation assays and MTT assays using standard chemotherapies. Thirdly, to test in vivo tumorigenic potential, by injecting 5 million cells of luciferized CCC lines in NSG female mice using both the subcutaneous route and the intraperitoneal route. Imaging was performed weekly using the In Vivo Imaging System. RESULTS: Among the 9 CCC lines, seven (TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-9, and OVISE) harbor ARID1A mutation, which is the most prevalent mutation in CCC (50%). The second common mutation in CCC (40%), PIK3CA mutation, and was detected in 4 cells (TOV21G, OVMANA, OCI-C5x, and OVISE). The ES-2 line has TP53 and BRAF mutation and its genomic profile is inconsistent with CCC. A BRCA2 mutation was found in OVTOKO and JHOC-5, finding not previously reported. Principal component analysis of RPPA data showed distinct clusters between the 9 CCC lines and the 6 HGSC lines. Interestingly, we also observed two distinct clusters within the CCC lines. Consistent with our genomic analysis, ES-2 correlated with HGSC lines based on RPPA data. In our in vitro drug studies, OVTOKO, OCI-C5x, and OVISE exhibited a Carboplatin/Paclitaxel resistance phenotype. In xenograft study, 4 cell lines (ES-2, TOV21G, OVTOKO, and OCI-C5x) formed tumor within a month, suggesting they are useful tools for in vivo studies. In contrast, OVMANA, JHOC-7, JHOC-9, and OVISE take more than 100 days to form the tumors. Soft-agar colony formation results correlated with how long it took to develop xenograft tumor in each line. CONCLUSION: Our genomic studies identified aberrations in CCC lines not previously described. The proteomics data revealed two clusters within the CCC lines that might represent functionally distinct groups. Finally, we identified four cell lines (ES-2, TOV21G, OVTOKO, and OCI-C5x) that readily form tumors in mice and could be used for future in vivo drug studies. However, ES-2 appears to cluster more closely with HGSC and may not represent the CCC histotype. Citation Format: Yasuto Kinose, Dorothy Hallberg, Gordon Mills, Tan Ince, Victor Velculescu, Fiona Simpkins, Ronny Drapkin. COMPREHENSIVE GENOMIC, PROTEOMIC, AND EXPERIMENTAL CHARACTERIZATION OF OVARIAN CLEAR CELL CARCINOMA CELL LINES FOR IMPROVED DRUG DEVELOPMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-036.

The HNF-1β-USP28-Claspin pathway upregulates DNA damage-induced Chk1 activation in ovarian clear cell carcinoma.

Transcription factor hepatocyte nuclear factor 1-beta (HNF-1β) enhances checkpoint kinase 1 (Chk1) activation and promotes G2/M cell cycle progression in ovarian clear cell carcinoma (CCC) following exposure to diverse genotoxic agents including bleomycin. However, the underlying mechanism leading to checkpoint activation of HNF-1β still remains largely unknown. To clarify the effects of HNF-1β on cell cycle checkpoints, human CCC cell lines were transfected with siRNAs targeting HNF-1β, Claspin, USP28, or a control vector. Ubiquitination and stabilization of Claspin protein by HNF-1β was assessed by immunoprecipitation. Loss-of-function studies using RNAi-mediated gene silencing indicated that HNF-1β facilitated the Claspin expression after treatment with a genotoxic agent bleomycin, resulting in accumulation of phosphorylated Chk1 (p-Chk1) and promotion of survival in CCC cell lines. This study showed for the first time that USP28, a de-ubiquitinase crucial for Claspin expression, is one target gene of HNF-1β. Knockdown of endogenous USP28 suppressed the Claspin expression and p-Chk1 activation and cell viability. Our findings identify a novel pathway of the HNF-1β―USP28―Claspin―Chk1 axis in checkpoint signal amplification in response to DNA damage. Targeting this pathway may represent a putative, novel, anticancer strategy in ovarian CCC.

Novel potential photodynamic therapy strategy using 5-Aminolevulinic acid for ovarian clear-cell carcinoma.

Photodynamic therapy (PDT) is known as a minimally invasive treatment for cancer. 5-Aminolevulinic acid (ALA) is a precursor of the photosensitizing agent protoporphyrin IX (PpIX). Patients with ovarian clear-cell carcinoma (CCC) have poorer prognoses than those of patients with other histological CCC types. We evaluated the efficacy of ALA-PDT on CCC cells in vitro. We used seven human CCC cell lines to measure the cytotoxicity of ALA-PDT. PpIX production in cancer cells was measured using a micro-plate reader. Quantitative real-time PCR was performed to assess the mRNA levels of genes involved in the accumulation of PpIX in cancer cells. Additionally, we measured the enhancement in cytotoxicity with the use of an ABCG2 inhibitor. We found that three cell lines were highly sensitive to ALA-PDT. In contrast, one cell line was resistant to ALA-PDT. The cytotoxicity of ALA-PDT varied among CCC cell lines. The IC50 values of ALA-PDT for the CCC cell lines had a wide range (30-882μM). The cytotoxicity of ALA-PDT was correlated with the intracellular PpIX accumulation. The cell lines sensitive to ALA-PDT expressed PEPT1 (an ALA uptake transporter). The cell line resistant to ALA-PDT expressed ABCG2 (a PpIX export transporter). In the resistant cell line, a combination treatment with both ALA and an ABCG2 inhibitor resulted in the promotion of cytotoxic sensitivity. The present study revealed the efficacy of ALA-PDT against CCC with chemoresistance in vitro.

Low uptake of fluorodeoxyglucose in positron emission tomography/computed tomography in ovarian clear cell carcinoma may reflect glutaminolysis of its cancer stem cell-like properties.

The characteristics of ovarian cancers that showed low activation of glycolysis were investigated. Using medical records of patients with ovarian cancers who had undergone fluorodeoxyglucose positron emission tomography/computed tomography(FDG-PET/CT) prior to their primary surgery at the University of Tokyo Hospital between 2010 and2015, we identified cases with a low uptake of FDG in PET/CT. We considered the maximum standardized uptake value(SUVmax) as the degree of glucose uptake. We investigated the properties which may account for the low activation of glycolysis invitro. The expression level of alanine, serine, cysteine-preferring transporter2(ASCT2, a glutamine influx transporter), system L-type amino acid transporter1(LAT1, a glutamine efflux transporter) and glucose transporter1(GLUT1, a glucose influx transporter) were investigated by western blotting. The phosphorylation level of AMP-activated protein kinase(AMPK), which is one of the metabolic sensors, was also investigated. Most of the cases with a low uptake SUVmax were limited to patients with ovarian clear cell carcinoma(CCC). We obtained cancer stem cell(CSC)-like properties from CCC cell lines, and compared the expression levels of transporters between non-CSCs and CSCs. Whereas the expression level of ASCT2 was nearly unchanged between non-CSCs and CSCs, the expression levels of LAT1 and GLUT1 were decreased in CSCs compared to non-CSCs. The phosphorylation level of AMPK was reduced in CSCs compared to non-CSCs. Inconclusion, we suggested that ovarian CCC showed low activation of glycolysis, and this may reflect glutaminolysis of its CSC-like properties.

Preclinical Investigations of PM01183 (Lurbinectedin) as a Single Agent or in Combination with Other Anticancer Agents for Clear Cell Carcinoma of the Ovary.

ObjectiveThe objective of this study was to evaluate the antitumor effects of lurbinectedin as a single agent or in combination with existing anticancer agents for clear cell carcinoma (CCC) of the ovary, which is regarded as an aggressive, chemoresistant, histological subtype.MethodsUsing human ovarian CCC cell lines, the antitumor effects of lurbinectedin, SN-38, doxorubicin, cisplatin, and paclitaxel as single agents were assessed using the MTS assay. Then, the antitumor effects of combination therapies involving lurbinectedin and 1 of the other 4 agents were evaluated using isobologram analysis to examine whether these combinations displayed synergistic effects. The antitumor activity of each treatment was also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines. Finally, we determined the effects of mTORC1 inhibition on the antitumor activity of lurbinectedin-based chemotherapy.ResultsLurbinectedin exhibited significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. An examination of mouse CCC cell xenografts revealed that lurbinectedin significantly inhibits tumor growth. Among the tested combinations, lurbinectedin plus SN-38 resulted in a significant synergistic effect. This combination also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Everolimus significantly enhanced the antitumor activity of lurbinectedin-based chemotherapies.ConclusionsLurbinectedin, a new agent that targets active transcription, exhibits antitumor activity in CCC when used as a single agent and has synergistic antitumor effects when combined with irinotecan. Our results indicate that lurbinectedin is a promising agent for treating ovarian CCC, both as a first-line treatment and as a salvage treatment for recurrent lesions that develop after platinum-based or paclitaxel treatment.

Lipocalin 2 attenuates iron-related oxidative stress and prolongs the survival of ovarian clear cell carcinoma cells by up-regulating the CD44 variant

ABSTRACTOvarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.

Fibroblast growth factor receptor 2 is associated with poor overall survival in clear cell carcinoma of the ovary and may be a novel therapeutic approach.

We previously found that gene and protein expression of fibroblast growth factor receptor (FGFR) 2 were increased in ovarian clear cell carcinoma (CCC); here, we examined FGFR2 expression in CCC tumor tissues and its correlation with clinical parameters. We also analyzed the effect of an FGFR inhibitor on the growth of CCC cells to investigate whether FGFR2 could be a therapeutic target for this disease. We analyze the protein expression of FGFR2 by immunohistochemical staining in CCC from 112 patients and evaluated the association of these molecular parameters with clinical outcome. We treated the 11 CCC cell lines with an FGFR inhibitor, and then assessed cell viability, the expression of protein in FGFR2 signaling pathway, and cell cycle distribution. The expressions of FGFR2 were found in 96% of CCC. The 5-year survival rate for patients with a moderate or strong expression of FGFR2 was significantly lower than that for those with an absent or poor expression of FGFR2 (54% vs 79%). Multivariable analysis revealed that FGFR2 expression and disease stage were independent prognostic factors. The FGFR inhibitor effectively suppressed the growth of CCC cells with induction of G1 cell cycle arrest and down-regulated the expression of phosphorylated Akt and phosphorylated ERK. FGFR2 is an important biomarker predictive of patient outcome and is a potential target for CCC. Further study is warranted for FGFR inhibitor to treat CCC.

Preclinical Efficacy for AKT Targeting in Clear Cell Carcinoma of the Ovary

The aim of this study was to determine the role of AKT as a therapeutic target in ovarian clear cell carcinoma (CCC), an aggressive, chemoresistant histologic subtype of ovarian cancer. AKT activation was assessed by immunohistochemistry (IHC) using human tissue microarrays of primary ovarian cancers, composed of both CCC and serous adenocarcinoma (SAC). The growth-inhibitory effect of AKT-specific targeting by the small-molecule inhibitor, perifosine, was examined using ovarian CCC cell lines in vitro and in vivo. Finally, the activity of perifosine was examined using in CCC-derived tumors that had acquired resistance to anti-VEGF or chemotherapeutics such as bevacizumab or cisplatin, respectively. Interestingly, AKT was frequently activated both in early-stage and advanced-stage CCCs. Treatment of CCC cells with perifosine attenuated the activity of AKT-mTORC1 signaling, inhibited proliferation, and induced apoptosis. The effect of perifosine was more profound under conditions of high AKT activity compared with low AKT activity. Increased AKT activation and enhanced sensitivity to perifosine were observed in the context of cisplatin-resistant CCC. Treatment with perifosine concurrently with cisplatin significantly enhanced the antitumor effect of cisplatin. Moreover, perifosine showed significant antitumor activity in CCC-derived tumors that had acquired resistance to bevacizumab or cisplatin. Collectively, these data reveal that AKT is frequently activated in ovarian CCCs and is a promising therapeutic target in aggressive forms of ovarian cancer. AKT-targeted therapy has value in a first-line setting as well as a second-line treatment for recurrent disease developing after platinum-based chemotherapy or bevacizumab treatment.

Cystatin B as a potential diagnostic biomarker in ovarian clear cell carcinoma.

Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in EOCs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.

Combination treatment with trabectedin and irinotecan or topotecan has synergistic effects against ovarian clear cell carcinoma cells.

The objective of this study was to investigate the chemotherapeutic agents that produce the strongest synergistic effects when combined with trabectedin against ovarian clear cell carcinoma (CCC), which is regarded as an aggressive chemoresistant histological subtype. Using 4 human CCC cell lines (RMG1, RMG2, KOC7C, and HAC2), the cytotoxicities of trabectedin, SN-38, topotecan, doxorubicin, cisplatin, and paclitaxel as single agents were first assessed using the MTS assay. Then, the cytotoxicities of combination treatments involving trabectedin and 1 of the other 4 agents were evaluated by isobologram analysis to examine whether these combinations displayed synergistic, additive, or antagonistic effects. The antitumor activities of the combination treatments were also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines, which were derived from the parental CCC cells by continuously exposing them to cisplatin or paclitaxel. Finally, we determined the effect of everolimus on the antitumor efficacy of trabectedin-based combination chemotherapy. Concurrent exposure to trabectedin and SN-38 or topotecan resulted in synergistic interactions in all 4 CCC cell lines. Among the tested combinations, trabectedin plus SN-38 was the most effective cytotoxic regimen. The combination of trabectedin plus SN-38 also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Treatment with everolimus significantly enhanced the antitumor activity of trabectedin plus SN-38 or topotecan. Combination treatment with trabectedin and SN-38 displays the greatest cytotoxic effect against ovarian CCC. Our in vitro study provides the rationale for future clinical trials of trabectedin plus irinotecan with or without everolimus in patients with ovarian CCC in both the front-line chemotherapy setting and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel.

X-chromosome-linked inhibitor of apoptosis as a key factor for chemoresistance in clear cell carcinoma of the ovary.

Background:X-chromosome-linked inhibitor of apoptosis (XIAP) is one of the anti-apoptotic proteins leading to chemoresistance in several cancers. The aim of this study is to evaluate the impact of XIAP expression upon ovarian clear cell carcinoma (CCC) that has a platinum-resistant phenotype.Methods:Tissue microarrays made from 90 CCC patients were analysed for immunohistochemical expression levels of XIAP, c-Met, p-Akt and Bcl-XL. In addition, CCC cell lines were evaluated whether XIAP silencing could modulate sensitivity to platinum agent in vitro.Results:High XIAP expression was observed in 30 (33%) of 90 CCC cases, and was associated with c-Met (<0.01) and Bcl-XL (<0.01) expression. Cases with high XIAP expression had lower response rate to primary platinum-based chemotherapy (10% vs 65%, P=0.02). In stages II–IV tumours, high XIAP expression was related with worse progression-free survival (PFS, P=0.02). Furthermore, high XIAP expression was identified as an independent worse prognostic factor for PFS and overall survival. Finally, downregulation of XIAP using XIAP-specific small interfering RNA increased sensitivity to cisplatin in human cancer cells derived from CCC.Conclusions:X-chromosome-linked inhibitor of apoptosis expression was correlated with chemoresistance of primary chemotherapy, and identified as a prognostic marker for CCC. X-chromosome-linked inhibitor of apoptosis could be a candidate for new therapeutic target in CCC.

Epigenetic determinants of ovarian clear cell carcinoma biology.

Targeted approaches have revealed frequent epigenetic alterations in ovarian cancer, but the scope and relation of these changes to histologic subtype of disease is unclear. Genome-wide methylation and expression data for 14 clear cell carcinoma (CCC), 32 non-CCC and four corresponding normal cell lines were generated to determine how methylation profiles differ between cells of different histological derivations of ovarian cancer. Consensus clustering showed that CCC is epigenetically distinct. Inverse relationships between expression and methylation in CCC were identified, suggesting functional regulation by methylation, and included 22 hypomethylated (UM) genes and 276 hypermethylated (HM) genes. Categorical and pathway analyses indicated that the CCC-specific UM genes were involved in response to stress and many contain hepatocyte nuclear factor (HNF) 1-binding sites, while the CCC-specific HM genes included members of the estrogen receptor alpha (ERalpha) network and genes involved in tumor development. We independently validated the methylation status of 17 of these pathway-specific genes, and confirmed increased expression of HNF1 network genes and repression of ERalpha pathway genes in CCC cell lines and primary cancer tissues relative to non-CCC specimens. Treatment of three CCC cell lines with the demethylating agent Decitabine significantly induced expression for all five genes analyzed. Coordinate changes in pathway expression were confirmed using two primary ovarian cancer datasets (p < 0.0001 for both). Our results suggest that methylation regulates specific pathways and biological functions in CCC, with hypomethylation influencing the characteristic biology of the disease while hypermethylation contributes to the carcinogenic process.

Effects of SIPL1 screened by suppression subtractive hybridization (SSH) on biological function and drug resistance of renal cell carcinoma cells

To screen the differentially expressed genes in human renal clear-cell carcinoma (RCC) cells using suppression subtractive hybridization (SSH), and to explore their biological function and underlying mechanism in RCC cells. Total RNAs were extracted from human renal clear-cell carcinoma cell line RLC-310 and human normal renal cell line HK-2 cells, and SSH technology was used to construct a RCC cell library of differential expression genes and to screen the most differentially expressed genes. RNA interference vector was constructed to silence the expression of the differentially expressed gene SIPL1 in human renal cell lines RLC-310 and GRC-1. Proliferation index was estimated by cell counting, MTT and tumor xenograft assay. Cell cycle analysis was performed using fluorescence activated cell sorting. Drug resistance potential to adriamycin was assessed by MTT. A subtractive cDNA library of highly expressed genes in the RCC cells was constructed and 12 differentially expressed genes were screened from the subtractive library, in which SIPL1 was the most differently expressed gene in the RCC cell line. SIPL1 overexpression in the RCC cells and clinical samples was confirmed by RT-PCR and Western blot analyses. The shRNA expression plasmid targeting to SIPL1 gene was constructed and transfected into RLC-310 and GRC-1 cells, resulting in downregulation of SIPL1. SIPL1 knockdown inhibited the cell proliferation (P < 0.05) and tumorgenesis. The tumor weights formed by RLC-310 cells transfected with SIPL1 shRNA was (0.22 ± 0.07)g and that of negative control vector was (0.85 ± 0.06)g. The tumor weight formed by GRC-1 cells was (0.32 ± 0.07)g and that of control vectors was (1.21 ± 0.11)g (P < 0.05). SIPL1 shRNA-transfected RLC-310 cells showed that more cells were arrested at G0/G1 phase [(71.13 ± 4.58)%] than that in the negative control RLC-310 cells [(53.27 ± 3.34)%, P < 0.05]. The proportion of G0/G1 phase in the SIPL1 shRNA transfected GRC-1 cells was (73.83 ± 3.97)%, significantly higher than that of (59.33 ± 3.03)% in the negative control GRC-1 cells (P < 0.05), and enhanced their sensitivity to adriamycin (P < 0.05). Silence of SIPL1 caused inactivation of AKT signaling and up-regulated expression of P27(Kip1) and P21(Cip1) proteins. A differentially expressed gene SIPL1 in the renal clear-cell carcinoma is successfully screened using SSH technology. SIPL1 functions as an oncogene in RCC, and may become a novel molecular target for RCC diagnosis and therapy.