Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 Phosphorylates and Activates Oncogenic Akt in Ovarian Cancer Cells
Gocher, Angela M.
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Hyperactivation of Akt is associated with oncogenic changes in the growth, survival and chemoresistance of cancer cells. The growth factor-dependent Phosphoinositide 3-kinase (PI3K)/Phosphoinositide-dependent kinase 1 (PDK1) pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr308. I observed that Ca 2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (beta) (CaMKK2) is highly expressed in high-grade serous ovarian cancer and investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and viability and in the regulation of Akt downstream targets involved in the G1/S phase transition and apoptosis. CaMKK2 knockdown or inhibition, decreased Akt phosphorylation at Thr308 and Ser473 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 Akt phosphorylation, consistent with direct Akt phosphorylation by CaMKK2. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr308 in a Ca 2+ /CaM-dependent manner. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via promotion of Akt stability (p-Akt Thr450), activation of PDK1 (p-PDK1 Ser241) or inhibition of the mTOR1/S6K1/IRS-1 negative feedback loop (p-IRS1 Ser636/639). In OVCa cells, Akt phosphorylation at Thr308 was significantly inhibited by intracellular Ca2+-chelation and CaM inhibition. Ionomycin-induced Ca2+-influx promoted p-Akt, an effect blocked by CaM inhibition, PDK1, and/or CaMKK2, siRNAs and by PI3K and/or CaMKK inhibitors. Additionally, p-Akt Thr308 in response to epidermal growth factor was blocked by PDK1, but not CaMKK2, knockdown, suggesting that CaMKK2 phosphorylation of Akt is independent of growth factors. CaMKK2 knockdown inhibited activation of the stress kinase AMPK caused by glucose deprivation, suggesting that CaMKK2 modulates a balance between cellular growth and energy conservation by Akt and AMPK and the differential regulation by CaMKK2 may be controlled in response to metabolic requirements. CaMKK2 knockdown in OVCa cells potentiated the effects of the current OVCa therapies, the Pt-chemotherapeutic carboplatin and the PI3K inhibitor PX-866, on the reduction of cell proliferation and survival. Together these results suggest that CaMKK2 represents a growth factor/PI3K/PDK1-independent, and Ca 2+ /CaM-dependent, mechanism for Akt activation. These published findings imply that CaMKK2 may be a mechanism for cancer cells to sustain Akt activation to promote cell growth and survival during therapy with PI3K/PDK1 inhibitors, suggesting that CaMKK2 represents a novel and therapeutic target for OVCa (1).