Clear cell carcinoma (CCC) of the ovary, a distinct histologic entity in the World Health Organization classification of ovarian tumors, behaves differently than other epithelial ovarian cancers. CCC contributes only 4% to 12% of epithelial ovarian cancers but its prognosis is very poor (median survival, 12.7 months in advanced disease), in part because of its resistance to conventional platinum- or taxane-based chemotherapy. We previously found that the low proliferation (by Ki-67 staining) characteristic of CCC may contribute to its resistance to chemotherapy (Itamochi et al., Obstet Gynecol., 2002, 100:281); here we investigated the molecular mechanism of this reduction in proliferation. We profiled cell cycle of 13 CCC and 9 ovarian serous adenocarcinoma cell lines. These profiles consist of measures of CDK activity (CDK1, -2, -4, and -6) and the protein expression of cyclins B1, D1, and E; CDK inhibitors p16, p21, and p27; and p53 used the novel device (Sysmex, Kobe, Japan). In this cell cycle profile, lower CDK2 activity and higher p27 expression were observed in CCC cells compared with serous adenocarcinoma cells (p < 0.05), and low CDK2 activity correlated with high p27 protein expression (P < 0.05). Because unphosphorylated p27 in the nucleus has been shown to suppress CDK2 activity in breast cancer cells (Liang et al., Nature Med., 2002, 8:1153), we examined the intracellular locations of p27, phosphorylated p27 (p-p27), and CDK2 protein in CCC cells by confocal microscopy and western blotting. CDK2 colocalized with p-p27 (phosphorylated at Thr157) in the cytoplasm, suggesting that low CDK2 activity in CCC cells is due to phosphorylation of p27 and sequestration of CDK2 in the cytoplasm. Because phosphorylation of p27 at Thr157 by Akt allows p27 to be moved into the cytoplasm, we treated 2 CCC cell lines (RMG-I, SMOV2) with LY294002, an inhibitor of Akt. We found that CDK2 and p27 were colocalized in the nucleus at 24 h and that CDK2 activity was restored by 1-6 hours after LY294002 treatment. However, CDK2 activity decreased rapidly thereafter, presumably because of the coexistence of unphosphorylated p27 in the nucleus. We conclude that phosphorylation of p27 by Akt inactivates CDK2 by sequestering both p27 and CDK2 in the cytoplasm in CCC cells. The cytoplasmic colocalization of CDK2 by phosphorylated p27 represents a novel mechanism by which CDK2 activity is inhibited and may contribute to reduced cell proliferation of CCC. Transiently increasing CDK2 activity to increase cell proliferation may be a novel targeted therapy to reverse chemoresistance of CCC to cell-cycle-dependent chemotherapeutic agents (e.g., paclitaxel, docetaxel, capecitabine).
[Proc Amer Assoc Cancer Res, Volume 46, 2005]