Abstract
Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G1/S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca2+i chelation or CaM inhibition. Ionomycin-induced Ca2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.
Highlights
Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells
We observed that Ca2؉/ calmodulin (CaM)-dependent protein kinase kinase 2 () (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov3)
Protein translation is mediated by Akt phosphorylation of PRAS40 leading to the release of mTORC1 from an inhibited state allowing for its phosphorylation of the p70 ribosomal protein S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) [10]
Summary
CaMKK2 is normally expressed at low levels in non-neural tissues [18] but is markedly increased in prostate cancer and has been implicated in prostate and breast oncogenesis [25,26,27,28]. The additivity of PI3K and CaMKK inhibition was shown in the third cell line examined, Caov-3 (supplemental Fig. S2E) Together, these results support a mechanism by which CaMKK2 directly phosphorylates Akt at Thr-308 independently of the PI3K/PDK1 pathway, leading to subsequent phosphorylation at Ser-473 presumably as a consequence of the activation of mTORC2. Regulation of the G1/S phase cell cycle regulators shown in Fig. 10 and supplemental Fig. S3, B and C may represent both Akt-dependent and -independent roles for CaMKK2, which is suggested by the greater than additive effect of combined PDK1 and CaMKK2 knockdowns on cell growth (Fig. 5E) Together, these data suggest that CaMKK2 is important for G1/S phase cell cycle progression and for maintaining the viability of OVCa cells. This model may provide both a framework for further investigation at the molecular level and study of whether CaMKK2 represents a novel and promising target for OVCa therapy in combination with carboplatin and/or PI3K inhibitors
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