Clear Cell Carcinoma Cell Research Articles

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.

Potential Role of mTORC2 as a Therapeutic Target in Clear Cell Carcinoma of the Ovary

The goal of this study was to examine the role of mTOR complex 2 (mTORC2) as a therapeutic target in ovarian clear cell carcinoma (CCC), which is regarded as an aggressive, chemoresistant histologic subtype. Using tissue microarrays of 98 primary ovarian cancers [52 CCCs and 46 serous adenocarcinomas (SAC)], activation of mTORC2 was assessed by immunohistochemistry. Then, the growth-inhibitory effect of mTORC2-targeting therapy, as well as the role of mTORC2 signaling as a mechanism for acquired resistance to the mTOR complex 1 (mTORC1) inhibitor RAD001 in ovarian CCC, were examined using two pairs of RAD001-sensitive parental (RMG2 and HAC2) and RAD001-resistant CCC cell lines (RMG2-RR and HAC2-RR). mTORC2 was more frequently activated in CCCs than in SACs (71.2% vs. 45.7%). Simultaneous inhibition of mTORC1 and mTORC2 by AZD8055 markedly inhibited the proliferation of both RAD001-sensitive and -resistant cells in vitro. Treatment with RAD001 induced mTORC2-mediated AKT activation in RAD001-sensitive CCC cells. Moreover, increased activation of mTORC2-AKT signaling was observed in RAD001-resistant CCC cells compared with the respective parental cells. Inhibition of mTORC2 during RAD001 treatment enhanced the antitumor effect of RAD001 and prevented CCC cells from acquiring resistance to RAD001. In conclusion, mTORC2 is frequently activated, and can be a promising therapeutic target, in ovarian CCCs. Moreover, mTORC2-targeted therapy may be efficacious in a first-line setting as well as for second-line treatment of recurrent disease developing after RAD001-treatment.

Analysis of the antitumor activity of gemcitabine and carboplatin against ovarian clear-cell carcinoma using the DNA damage marker &gamma;H2AX

BackgroundDifferences in the incidence and type of DNA damage induced by antitumor agents for ovarian clear-cell carcinoma (CCC) were determined in two CCC cell lines, using γH2AX.Materials and methodsThe antitumor activity of gemcitabine (GEM) and carboplatin (CBDCA) were examined using cultured cell lines of CCC (OVISE and RMG-I). Each cell line was treated with GEM and CBDCA, the cells were collected, fixed, and then reacted with anti-γH2AX antibody. γH2AX and nuclear DNA were then simultaneously detected by flow cytometry using fluorescein isothiocyanate and propidium iodide, respectively, to determine the amounts of γH2AX formed in each cell-cycle phase.ResultsAfter administration of GEM, both cell lines showed DNA damage and cell-cycle arrest in the S and G2/M phases, and increased apoptosis. Similarly, with CBDCA, OVISE showed S- and G2/M-phase arrest, while RMG-I showed G2/M-phase arrest.ConclusionThe mechanism of action of GEM and CBDCA in CCC cell lines was elucidated using γH2AX as a DNA damage marker. Our findings suggested that concomitant use of GEM plus CBDCA may be effective in the treatment of CCC.

Cytokine gene expression signature in ovarian clear cell carcinoma

Cytokine expression in a tumor microenvironment can impact both host defense against the tumor and tumor cell survival. In this study, we sought to clarify whether the cytokine gene expression profile could have clinical associations with ovarian cancer. We analyzed the expression of 16 cytokine genes (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-γ, TNF-α, IL-6, HLA-DRA, HLA-DPA1 and CSF1) in 50 ovarian carcinomas. Hierarchical clustering analysis of these tumors was carried out using Cluster software and differentially expressed genes were examined between clear cell carcinoma (CCC) and other subtypes. Following this examination we evaluated the biological significance of IL-6 knockdown in CCC. Unsupervised hierarchical clustering analysis of cytokine gene expression revealed two distinct clusters. The relationship between the two clusters and clinical parameters showed statistically significant differences in CCC compared to other histologies. CCC showed a dominant Th-2 cytokine expression pattern driven largely by IL-6 expression. Inhibition of IL-6 in CCC cells suppressed Stat3 signaling and rendered cells sensitive to cytotoxic agents. The unique cytokine expression pattern found in CCC may be involved in the pathogenesis of this subtype. In particular, high IL-6 expression appears likely to be driven by the tumor cells, fueling an autocrine pathway involving IL-6 expression and Stat3 activation and may influence survival when exposed to cytotoxic chemotherapy. Modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for CCC.

Are platinum agents, paclitaxel and irinotecan effective for clear cell carcinoma of the ovary? DNA damage detected with γH2AX induced by anticancer agents

ObjectivesDifferences in the incidences and types of DNA damage induced by antitumor agents for clear cell carcinoma (CCC) were determined in 2 ovarian CCC cell lines using γH2AX.Material and methodsThe antitumor activity of anticancer agents, CDDP, CBDCA, PTX and SN-38, was examined using ovarian clear cell carcinoma cultured cell lines (OVISE and RMG-I). After culture, each cell line was treated with each anticancer agent, the cells were collected, fixed, and then reacted with the anti-γH2AX antibody. γH2AX and nuclear DNA were then simultaneously detected by flow cytometry using FITC and propidium iodide, respectively, to determine γH2AX in each cell cycle phase.ResultsAfter administration of CDDP, DNA damage was frequent in S-phase cells, while cell-cycle arrest occurred in the G1 and G2/M phases and γH2AX did not increase in CDDP-resistant cells. Sensitivities to CDDP and CBDCA differed between the two cell lines. The antitumor effect of PTX is induced by G2/M arrest, and combination treatment with CBDCA, inducing DNA damage in G2/M-phase cells, might be effective.ConclusionsThis is the first study in Japan to evaluate the antitumor activity of anticancer agents by focusing on the relationship between the cell cycle and DNA damage using γH2AX as an indicator. The immunocytochemical method used in this study detects γH2AX, which indicates DNA damage even at very low concentrations and with high sensitivity. Therefore, a promising method of easily and rapidly identifying agents potentially effective against CCC.

Abstract 3740: NVP-BEZ235, a dual PI3K/ mTOR inhibitor, suppress tumorigenicity of ovarian clear cell carcinoma cells.

Abstract 1 [Background] Ovarian clear cell carcinoma (CCC) has shown the resistance to the standard chemotherapy for epithelial ovarian cancer (EOC). Therefore, patients with CCC have a worse prognosis than those with ovarian serous adenocarcinoma (SAC), which is a dominant subtype in EOC. Recently, it has shown that CCC has a high frequency of activating mutations of PIK3CA. Thus, PI3K-Akt pathways may be an attractive target of therapy. NVP-BEZ235 (BEZ235) is a novel, orally bioavailable imidazoquinoline that potently and reversibly inhibits class 1 PI3K activity. BEZ235 also binds directly to the mTOR ATP-binding domain and directly inhibits its catalytic activity, thereby inhibiting both mTOR complex 1 (mTORC1) and mTORC2. There are few reports on the activity of BEZ235 against ovarian carcinoma cells. The purpose of the present study was to determine whether the drug targeting the PI3K-Akt-mTOR pathway suppresses cell proliferation and tumorigenicity of CCC. [Material&amp; Methods] We used eight CCC cell lines (OVISE, SMOV-2, KK, TU-OC-1, OVTOKO, KOC-7c, OVMANA, and RMG-I) and five SAC cell lines (KOC-2s, KF, TU-OS-3, TU-OS-4, SHIN-3). IC50 to BEZ235 was determined on the basis of the dose-effect curves, using WST-8 assay. Protein expressions of Akt, phospho(p)-Akt, mTOR, pmTOR, p70S6K, pp70S6K, 4E-BP1, and p4E-BP1 were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Apoptotic cells were assessed by Annexin V-FITC/PI double staining. We also investigated the effects of BEZ235 on tumor growth in a nude mice xenograft model. Mice bearing subcutaneous tumor of OVISE were received 3-weeks treatment with vehicle or BEZ235 (25mg/kg/day or 50mg/kg/day). The two perpendicular axes of the tumor diameter was measured in control and treated groups using a caliper twice weekly, and tumor volume was calculated, using the following formula: length x (width)*2 x (≥/6). [Results] IC50 to BEZ235 for CCC cell lines ranged from 44 to 777 nM (median:332.5nM) and those for SAC cell lines ranged from 779 to 25400 nM (median: 1004nM). We confirmed that all CCC cell lines exhibited protein expressions of pAkt and pmTOR. pAkt, p-p70S6K and p4E-BP1 expressions in CCC cells (OVISE and KK) were suppressed after exposure to BEZ235 in a dose-dependent manner. The both cell lines exhibited G1 cell cycle arrest after exposure to 10 or 100nM BEZ235, and apoptotic cells were induced by higher concentrations of the agent. Finally, treatment of the mice with BEZ235 at doses 25mg/kg/day or 50mg/kg/day significantly suppressed tumor growth in a xenograft model. [Conclusion] These results suggest that the PI3K-Akt-mTOR pathway is a potential therapeutic target for CCC and that NVP-BEZ235 is worth exploring as a therapeutic agent for CCC. 1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3740. doi:1538-7445.AM2012-3740

Abstract 4225: Annexin A4 induces chemoresistance for multiple drugs in ovarian clear cell carcinoma

Abstract BACKGROUND: Platinum drugs play the key role for postoperative adjuvant therapy and treatment in progress and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtypes of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin. However, it remains unclear whether targeting Anx A4 is effective for improving the sensitization against chemotherapy, like platinum drugs. The aim of this study was to evaluate the role of Anx A4 in chemoresistance in ovarian CCC. MATERIAL AND METHODS: RMG-I cell lines were used to establish the stable knockdown of Anx A4. Plasmids encoding short hairpin RNAs (shRNAs) targeting Anx A4 were transfected. Chemoresistance was assessed by IC50 value in various drugs. RESULTS: Compared with control cells, knockdown of Anx A4 gene expression in RMG-I cells induced chemosentisitzation around 2-fold to cisplatin (IC50: 11.2 μM to 5.8 μM, P&amp;lt;0.01) and Carboplatin (IC50: 130.4 μM to 63.3 μM, P&amp;lt;0.01). Unexpectedly, knockdown of Anx A4 also improved the chemosentisitzation of epirubicin (IC50: 256.7 nM to 180.3 nM, P&amp;lt;0.01) and Paclitaxel (IC50: 5.5 nM to 2.9 nM, P&amp;lt;0.01). In consistent with the improved chemosensitivity, drugs induced p53 was markedly increased by knockdown of Anx A4 in RMG-I cells. In xenograft models, significant chemosensitization toward cisplatin was demonstrated by knockdown of Anx A4 in RMG-I cells. CONCLUSIONS: Inhibition of Anx A4 induced chemosensitization in ovarian CCC cells. Anx A4 blockade might be a novel therapeutic target of chemoresistance in patients with ovarian CCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4225. doi:1538-7445.AM2012-4225

Glypican‐3 could be an effective target for immunotherapy combined with chemotherapy against ovarian clear cell carcinoma

Glypican-3 (GPC3) is useful not only as a novel tumor marker, but also as an oncofetal antigen for immunotherapy. We recently established HLA-A2-restricted GPC3(144-152) peptide-specific CTL clones from hepatocellular carcinoma patients after GPC3(144-152) peptide vaccination. The present study was designed to evaluate the tumor reactivity of a HLA-A2-restricted GPC3(144-152) peptide-specific CTL clone against ovarian clear cell carcinoma (CCC) cell lines. The GPC3(144-152) peptide-specific CTL clone could recognize HLA-A2-positive and GPC3-positive ovarian CCC cell lines on interferon (IFN)-γ enzyme-linked immunospot assay and showed cytotoxicity against KOC-7c cells. The CTL clone recognized naturally processed GPC3-derived peptide on ovarian CCC cells in a HLA class I-restricted manner. Moreover, we confirmed that the level of GPC3 expression was responsible for CTL recognition and that subtoxic-dose chemotherapy made tumor cells more susceptible to the cytotoxic effect of CTL. Thus, it might be possible to treat ovarian CCC patients by combining chemotherapy with immunotherapy. Our data suggest that GPC3 could be an effective target for immunotherapy against ovarian CCC.

The Activity of Trabectedin As a Single Agent or in Combination with Everolimus for Clear Cell Carcinoma of the Ovary

The objective of this study was to evaluate the antitumor efficacy of trabectedin in clear cell carcinoma (CCC) of the ovary, which is regarded as an aggressive, chemoresistant, histologic subtype. Using 6 human ovarian cancer cell lines (3 CCC and 3 serous adenocarcinomas), the antitumor effects of trabectedin were examined in vitro, and we compared its activity according to histology. We next examined the antitumor activity of trabectedin in both cisplatin-resistant and paclitaxel-resistant CCC cells in vitro. Then, the in vivo effects of trabectedin were evaluated using mice inoculated with CCC cell lines. Using 2 pairs of trabectedin-sensitive parental and trabectedin-resistant CCC sublines, we investigated the role of mTOR in the mechanism of acquired resistance to trabectedin. Finally, we determined the effect of mTOR inhibition by everolimus on the antitumor efficacy of trabectedin in vitro and in vivo. Trabectedin showed significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. Mouse xenografts of CCC cells revealed that trabectedin significantly inhibits tumor growth. Greater activation of mTOR was observed in trabectedin-resistant CCC cells than in their respective parental cells. The continuous inhibition of mTOR significantly enhanced the therapeutic efficacy of trabectedin and prevented CCC cells from acquiring resistance to trabectedin. Trabectedin is a promising agent for CCC as a first-line chemotherapy and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel. Moreover, trabectedin combined with everolimus may be more efficacious for the management of CCC.

Novel mechanism of reduced proliferation in ovarian clear cell carcinoma cells: Cytoplasmic sequestration of CDK2 by p27

Objective Ovarian clear cell carcinoma (CCC) carries a poor prognosis because of its insensitivity to chemotherapy. We previously found an association between reduced proliferation of CCC and chemoresistance; here we investigated the mechanism of the reduced proliferation. Methods We assessed cell cycle function by measuring the activity of cyclin-dependent kinases (CDKs) and the protein expression of cyclins, the CDK inhibitors, and p53 in 22 ovarian cancer cell lines and 60 human ovarian cancer specimens. We examined the cellular location of p27, p27 phosphorylated at threonine 157 (p27 Thr157), and CDK2 protein by confocal microscopy and western blotting. We tested the effect of the inhibitor of phosphatidylinositol-3-kinase (PI3K) and small interfering RNA against p27 (si-p27) in two CCC cell lines (RMG-I, SMOV-2). Results CCC cells had lower CDK2 activity and higher p27 expression than serous adenocarcinoma (SA) cells. Low CDK2 activity correlated with high p27 protein expression. p27 Thr157 sequestered CDK2 in the cytoplasm, but PI3K inhibitor or si-p27 maintained CDK2 in the nucleus and restored its activity. In human specimens, CDK2 was mostly in the cytoplasm and was spatially associated with p27; CDK2 activity was lower in the CCC than in the SA specimens. si-p27 enhanced the cytotoxic effect of cisplatin, doxorubicin, and gemcitabine in both RMG-I cells and SMOV-2 cells. Conclusions Reduced CDK2 activity via the cytoplasmic sequestration of CDK2 by p27 Thr157 may contribute to suppression of CCC proliferation. A prospective study is needed to determine whether the cytoplasmic sequestration of CDK2 results in the chemoresistance of CCC.

Abstract 4497: NVP-BEZ235, a dual PI3K/ mTOR inhibitor, may be effective for ovarian clear cell carcinoma cells

Abstract [Background] NVP-BEZ235 (BEZ235) is a novel, orally bioavailable imidazoquinoline that potently and reversibly inhibits class 1 PI3K activity by binding to its ATP-binding domain. BEZ235 also binds directly to the mTOR ATP-binding domain and directly inhibits its catalytic activity, thereby inhibiting both TORC1 and TORC2. BEZ235 is currently in phase I testing in patients with solid tumors. There are few reports on the activity of BEZ235 against ovarian carcinoma cells. Ovarian clear cell carcinoma (CCC) has shown the resistance to the standard chemotherapy for epithelial ovarian cancer (EOC). Therefore, patients with CCC have a worse prognosis than those with ovarian serous adenocarcinoma (SAC), which is a dominant subtype in EOC. However, little has been known about genetic alterations in each histologic subtype of EOC. Recently, it has shown that CCC has a high frequency of activating PIK3CA mutations. The purpose of the current study was to determine whether the drug targeting the PI3K-Akt-mTOR pathway suppresses cell proliferation of CCC. [Material&amp; Methods] We used eight CCC cell lines (KK, OVISE, SMOV-2, KOC-7c, TUOC-1, OVTOKO, OVMANA, and RMG-I) and four SAC cell lines (SHIN-3, TUOS-4, KOC-2s, SK-OV-3) in this study. IC50 to BEZ235 was determined on the basis of the dose-effect curves, using WST-8 assay. Protein expressions of Akt, phospho(p)-Akt(Ser473), mTOR, pmTOR(Ser2448), p70S6K, pp70S6K(Thr389), 4E-BP1, and p4E-BP1(Ser37/46) were examined by western blotting. [Results] IC50 to BEZ235 for CCC cell lines ranged from 44 to 777 nM and those for SAC cell lines ranged from 989 to 25400 nM. All 8 CCC cell lines showed Akt, mTOR, pmTOR, and p70S6K expressions. Six cell lines showed pAkt expression, and 2 of the 6 cell lines showed both pAkt and p4E-BP1 expressions. Remaining 2 cell lines showed pp70S6K expression. pAkt, p-p70S6K and p4E-BP1 expression in CCC cells were suppressed after exposure to BEZ235 in a dose-dependent manner. [Conclusion] These results suggest that the PI3K/Akt/mTOR pathway is a potential therapeutic target for CCC and that NVP-BEZ235 is worth exploring as therapeutic agents for CCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4497. doi:10.1158/1538-7445.AM2011-4497

Glypican-3 expression predicts poor clinical outcome of patients with early-stage clear cell carcinoma of the ovary

BackgroundGlypican-3 (GPC3), a membrane-bound heparan sulphate proteoglycan, may play a role in promoting cancer cell growth and differentiation. Recent studies reported that GPC3 is overexpressed in clear cell carcinoma (CCC)...

Growth-suppressing function of glypican-3 (GPC3) via insulin like growth factor II (IGF-II) signaling pathway in ovarian clear cell carcinoma cells

Objective Ovarian clear cell carcinoma (CCC) is well known to be highly resistant to platinum-based chemotherapy. Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, is overexpressed in only CCC of epithelial ovarian carcinoma subtypes. The purpose of this study was to identify the role of GPC3 in ovarian CCC. Methods To evaluate the function of GPC3 in ovarian CCC cells, we generated an ovarian cancer cell line, KOC7C cells stably transfected with plasmids encompassing shRNA targeting GPC3 (shGPC cells), and compared cell growth and the colony-forming ability to control shRNA-transfected cells (shCon cells). Results We showed that shGPC3 cells significantly increased cell growth and the colony-forming potential compared with shCon cells in 1% serum containing medium with 100 ng/ml IGF-II. Furthermore, these effects were significantly attenuated by pretreatment with 1 μM wortmannin (an inhibitor of PI3K/Akt). Conclusions We have demonstrated for the first time the presence of elevated levels of GPC3 protein associated with cell growth inhibition in CCC cells. Our data suggest that GPC3 has the potential to become a novel therapeutic target for ovarian CCC patients.

Vascular Endothelial Growth Factor Is a Promising Therapeutic Target for the Treatment of Clear Cell Carcinoma of the Ovary

This study examines the role of vascular endothelial growth factor (VEGF) as a therapeutic target in clear cell carcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histologic subtype. Immunohistochemical analysis using tissue microarrays of 98 primary ovarian cancers revealed that VEGF was strongly expressed both in early-stage and advanced-stage CCC of the ovary. In early-stage CCCs, patients who had tumors with high levels of VEGF had significantly shorter survival than those with low levels of VEGF. In vitro experiments revealed that VEGF expression was significantly higher in cisplatin-refractory human CCC cells (RMG1-CR and KOC7C-CR), compared with the respective parental cells (RMG1 and KOC7C) in the presence of cisplatin. In vivo treatment with bevacizumab markedly inhibited the growth of both parental CCC cell-derived (RMG1 and KOC7C) and cisplatin-refractory CCC cell-derived (RMG1-CR and KOC7C-CR) tumors as a result of inhibition of tumor angiogenesis. The results of the current study indicate that VEGF is frequently expressed and can be a promising therapeutic target in the management of CCC. Bevacizumab may be efficacious not only as a first-line treatment but also as a second-line treatment of recurrent disease in patients previously treated with cisplatin.

Abstract 1619: ERK-targeted therapy suppresses tumorigenicity in ovarian clear cell carcinoma and is modulated by ERK-binding protein PEA-15

Abstract Ovarian clear cell carcinoma (CCC) has shown resistance to the current standard chemotherapy used for epithelial ovarian cancer (EOC). Therefore, developing a novel therapeutic strategy is imperative to improve the prognosis for patients with CCC. Epidermal growth factor receptor (EGFR) is frequently expressed in EOC. PEA-15 is a 15-kDa phosphoprotein that slows cell proliferation by binding to and sequestering ERK in the cytoplasm. The purpose of the current study was to determine whether the EGFR-ERK pathway is a therapeutic target in CCC and whether PEA-15 modulates ERK-targeted therapy in CCC. We screened a panel of four CCC cell lines (RMG-I, SMOV-2, OVTOKO and KOC-7c) and performed Western blotting to detect the expression levels of EGFR, pEGFR, ERK and pERK. RMG-I and SMOV-2 showed high EGFR expression, while OVTOKO and KOC-7c showed lower expression. We next performed WST-1 assay and showed that erlotinib inhibited cell proliferation in high EGFR-expressing cell lines (IC50 = 0.1 μM for RMG-I and 1 μM for SMOV-2) by suppressing pEGFR and pERK expression and inducing G1 arrest. Further, transfection with constitutive active MEK1 significantly reduced the sensitivity to erlotinib in RMG-I cells (p&amp;lt;0.005), suggesting that erlotinib sensitivity is partially dependent on ERK. Therefore, we hypothesized that ERK inhibition suppresses tumorigenicity in CCC. We examined the sensitivity of CCC to MEK inhibitor AZD6244 (ARRY-142886) and observed that the erlotinib-sensitive cell lines were also sensitive to AZD6244 (IC50 = 0.79 μM for RMG-I and 0.85 μM for SMOV-2) by suppressing pERK expression and inducing G1 arrest. We next investigated the possible mechanistic role of PEA-15 in the therapeutic effect of the ERK-targeted therapy with AZD6244. Phosphorylated PEA-15 at ser116 [pPEA-15(S116)] levels increased after AZD6244 treatment in the sensitive cell lines. Transient transfection with phosphomimetic mutant PEA-15 (S116D) reduced cell viability, sensitizing the insensitive cell lines to AZD6244, implying that pPEA-15 (S116) confers sensitivity to AZD6244 in CCC cells. Further, both AZD6244 (50mg/kg/d) and erlotinib (100mg/kg/d) significantly suppressed tumor growth (p &amp;lt; 0.05) in a CCC xenograft model. As expected, the levels of pERK1/2 and Ki-67 expression were markedly reduced in the AZD6244 treated mice groups. The expression of both PEA-15 and pPEA-15 (S116) expressions was also increased in the AZD6244 treatment groups in vivo. However, at the doses used, AZD6244 was better tolerated than erlotinib in terms of weight loss and skin effects (severe skin peeling). We concluded that the MEK/ERK pathway is a potential therapeutic target for CCC and that AZD6244 is worth exploring as a therapeutic agent for patients with CCC. Further studies are warranted to determine if PEA-15 may be useful to predict sensitivity to ERK-targeted therapy in CCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1619.

DNA Copy Numbers Profiles in Affinity-Purified Ovarian Clear Cell Carcinoma

Advanced ovarian clear cell carcinoma (CCC) is one of the most aggressive ovarian malignancies, in part because it tends to be resistant to platinum-based chemotherapy. At present, little is known about the molecular genetic alterations in CCCs except that there are frequent activating mutations in PIK3CA. The purpose of this study is to comprehensively define the genomic changes in CCC based on DNA copy number alterations. We performed 250K high-density single nucleotide polymorphism array analysis in 12 affinity-purified CCCs and 10 CCC cell lines. Discrete regions of amplification and deletion were also analyzed in additional 21 affinity-purified CCCs using quantitative real-time PCR. The level of chromosomal instability in CCC as defined by the extent of DNA copy number changes is similar to those previously reported in low-grade ovarian serous carcinoma but much less than those in high-grade serous carcinoma. The most remarkable region with DNA copy number gain is at chr20, which harbors a potential oncogene, ZNF217. This discrete amplicon is observed in 36% of CCCs but rarely detected in serous carcinomas regardless of grade. In addition, homozygous deletions are detected at the CDKN2A/2B and LZTS1 loci. Interestingly, the DNA copy number changes observed in fresh CCC tissues are rarely detected in the established CCC cell lines. This study provides the first high resolution, genome-wide view of DNA copy number alterations in ovarian CCC. The findings provide a genomic landscape for future studies aimed at elucidating the pathogenesis and developing new target-based therapies for CCCs.

Enhanced expression of Annexin A4 in clear cell carcinoma of the ovary and its association with chemoresistance to carboplatin

Clear cell carcinoma (CCC) of the ovary is known to be highly resistant to platinum-based chemotherapy. The purpose of our study was to identify a candidate protein that is associated with chemoresistance of CCC and to investigate the specific mechanism of chemoresistance conferred by the identified protein. Enhanced expression of Annexin A4 (Anx A4) was identified in ovarian CCC cells using 2-D differential gel electrophoresis (2D-DIGE) and mass spectrometry. Anx A4 levels were elevated in CCC cells compared with non-CCC cells as determined by real-time RT-PCR and Western blot analysis. Immunohistochemical analysis of Anx A4 was performed in 126 epithelial ovarian cancer tissue samples and demonstrated significantly elevated levels of Anx A4 protein levels in ovarian CCC tumors compared with ovarian serous and endometrioid tumors (p < 0.01). Anx A4-transfected ovarian non-CCC cells were more resistant to carboplatin (IC50 = 42 microM) compared with control cells (IC50 = 23 microM) as determined by modified MTT assay. Intracellular platinum levels were significantly lower in Anx A4-transfected cells compared with control cells after carboplatin treatment (p = 0.0020) and after an additional 360 min of carboplatin-free incubation (p = 0.0004), as measured by atomic absorption spectrophotometry. Expression of Anx A4 is elevated in ovarian CCC tumors and is associated with chemoresistance in cultured ovarian cancer cells. These results demonstrate that Anx A4 confers chemoresistance in ovarian cancer cells in part by enhancing drug efflux. Thus, Anx A4 may represent a novel therapeutic target of chemoresistance in patients with ovarian CCC.

Frequent Activating Mutations of PIK3CA in Ovarian Clear Cell Carcinoma

Ovarian clear cell carcinoma (CCC) is one of the most malignant types of ovarian carcinomas, particularly at advanced stages. Unlike the more common type of ovarian cancer, high-grade serous carcinoma, ovarian CCC is often resistant to platinum-based chemotherapy, and therefore an effective treatment for this tumor type at advanced stages is urgently needed. In this study, we analyzed 97 ovarian CCCs for sequence mutations in KRAS, BRAF, PIK3CA, TP53, PTEN, and CTNNB1 as these mutations frequently occur in other major types of ovarian carcinomas. The samples included 18 CCCs for which affinity-purified tumor cells from fresh specimens were available, 69 microdissected tumors from paraffin tissues, and 10 tumor cell lines. Sequence mutations of PIK3CA, TP53, KRAS, PTEN, CTNNB1, and BRAF occurred in 33%, 15%, 7%, 5%, 3%, and 1% of CCC cases, respectively. Sequence analysis of PIK3CA in 28 affinity-purified CCCs and CCC cell lines showed a mutation frequency of 46%. Samples with PIK3CA mutations showed intense phosphorylated AKT immunoreactivity. These findings demonstrate that ovarian CCCs have a high frequency of activating PIK3CA mutations. We therefore suggest that the use of PIK3CA-targeting drugs may offer a more effective therapeutic approach compared with current chemotherapeutic agents for patients with advanced-stage and recurrent CCC.

Relation Between Laminin-5 γ2 Chain and Cell Surface Metalloproteinase MT1-MMP in Clear Cell Carcinoma of the Ovary

Ovarian clear cell carcinoma (CCC) characteristically shows stromal accumulation of the basement membrane material laminin-5 (LN-5). LN-5 works not only as a substrate for cell anchorage but also as a stimulator for cell migration. Our previous study showed that CCC cell increased migration over excessive LN-5 in vitro. However, it remains unclear why LN-5 rather promotes cell migration than cell anchorage. A recent study has shown that the processing of the LN-5 gamma 2 chain by membrane type 1-matrix metalloproteinase (MT1-MMP) was responsible for increasing cell migration activity but reducing cell anchoring activity of LN-5. In the present study, 3 CCC cell lines were examined for LN-5 gamma 2 chain levels by Western blotting. Processed gamma 2 chain (105 kDa) was detected in the extracellular matrix of all 3 CCC cell lines in vitro. Immunocytochemically they expressed MT1-MMP on the cell surface. In surgical specimens, 22 of 30 (73%) CCCs showed membranous expression of MT1-MMP, and stromal accumulation of the LN-5 gamma 2 chain, especially in the area of papillary architecture. These results indicate that proteolytic processing of the LN-5 gamma 2 chain by MT1-MMP is involved in cell migration and subsequent architectural organization in ovarian CCC.

Overexpression of osteopontin in clear cell carcinoma of the ovary: close association with HNF‐1β expression

Transcription factor hepatocyte nuclear factor (HNF)-1beta is selectively expressed in clear cell carcinoma (CCC) of the ovary. One of the potential HNF-1beta target genes is osteopontin (OPN). Although elevation of OPN mRNA has been reported in CCC, it remains unclear whether CCC cells overexpress OPN protein. The aim was to investigate the expression of OPN protein and its correlation with HNF-1beta status in CCC. Three CCC and two serous adenocarcinoma (SA) cell lines were evaluated for expression of OPN by reverse transcriptase-polymerase chain reaction and immunocytochemistry. OPN expression, at both the mRNA and protein levels, was higher in the three CCCs than in the two SAs. HNF-1beta expression was detected in the CCCs but not in the SAs. Subsequently, 60 surgical specimens (30 CCCs and 30 SAs) were examined immunohistochemically for expression of OPN and HNF-1beta. All 30 CCCs showed immunopositivity for both OPN and HNF-1beta. The 12 (40%) CCCs with a high OPN score all had a high HNF-1beta score. In contrast, SAs rarely showed immunoreactivity for OPN or HNF-1beta. OPN expression is elevated in ovarian CCC and is closely associated with HNF-1beta overexpression. HNF-1beta is likely to participate in OPN up-regulation in CCC.