Abstract 1619: ERK-targeted therapy suppresses tumorigenicity in ovarian clear cell carcinoma and is modulated by ERK-binding protein PEA-15
Authors: Oishi Tetsuro , Bartholomeusz Chandra , Kazansky Anna , Akar Ugur , Krishunamurthy Savitori , Esteva Francisco J. , Itamochi Hiroaki , Ueno Naoto T. 
Published: February 2011

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&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 &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.