Abstract B13: Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2

Abstract

Abstract Calcium/Calmodulin-dependent protein kinase kinase 2 (CaMKK2) has been implicated in the regulation of G1/S phase cell cycle progression in prostate and breast cancer but has not been studied in ovarian cancer (OvCa). We report here the following. CaMKK2 is abundantly expressed in high grade serous papillary cystadenocarcinoma. Knockdown (KD) of CaMKK2 expression using RNAi in OVCAR-3 OvCa cells resulted in slowing of cell growth, decreased cyclin D1 protein and mRNA, decreased phosphorylation of the tumor suppressor protein Rb at Ser807/811 and decreased DNA synthesis, indicating an arrest at the G1/S interface. CaMKK2 KD decreased cell viability, increased PARP cleavage and increased Caspase-3/7 activity suggesting apoptosis induction by CaMKK2 loss. CaMKK2 KD decreased p-Akt at Thr308 to a similar extent (59.3%) as that of PDK1 KD (50.4%) in OVCAR-3 cells. Combined CaMKK2 and PDK1 KDs led to an additive (80.7%) decrease in p-Akt suggesting that CaMKK2 regulates Akt activating phosphorylation independently of the canonical-PDK1 pathway. Additive effects of combined PDK1/CaMKK2 KD on p-Akt Thr308 in SKOV-3 OvCa cells were also obtained. The ability of baculovirus expressed and purified Akt to phosphorylate a specific peptide substrate was enhanced at least 9-fold by purified CaMKK2, whereas kinase activity of Akt T308A/S473A was not increased by CaMKK2. Direct phosphorylation of baculovirus expressed Akt at Thr308 by CaMKK2 was confirmed by Western blotting using Akt phospho-specific antibodies. Altogether these data show that CaMKK2 regulates cell growth and viability in OvCa cells and directly phosphorylates Akt to promote its activity via a non-PDK1-dependent pathway. These data indicate that CaMKK2 may be a novel therapeutic target for OvCa in combination with a PI3K inhibitor. Citation Format: Angela M. Gocher, Thomas F. Franke, Gissou Azabdaftari, Loukia G. Karacosta, Arthur M. Edelman. Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B13.