Abstract P2-07-06: Role of CaMKK2-AMPK Signaling in Breast Cancer Metabolic and Hypoxic Stress Responses

Background: Adenosine monophosphate-activated protein kinase (AMPK) is a major regulator of energy homeostasis at both the cellular and whole-body levels. AMPK is activated by elevated intracellular AMP level and by phosphorylation on threonine 172 (T172) in the activation loop of the α catalytic subunit by upstream kinases, LKB1 and Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Once activated, AMPK switches on ATP-producing pathways while switching off ATP-consuming processes. Recent studies suggest that AMPK also controls processes relevant to tumor development, including protein synthesis, cell cycle progression, cell growth, survival and stress-induced autophagy. The purpose of our study is to determine the role of CaMKK2 as an AMPK upstream kinase in breast cancer cells, as well as its function in regulating cell proliferation, migration and survival. We chose the prototypical ERα + MCF-7 breast cancer cell line as our research model since this cell line does not express CaMKK1, which has similar functions to CaMKK2 but cannot activate AMPK. Materials and Methods: Illumina cDNA expression array was preformed on MCF-7, T-47D and MDA-MB-231 breast cancer cell lines, and normalized by rank-invariant method. MCF-7 cells with suppressed CaMKK2 or LKB1 expression were established using lentiviral gene specific shRNA interference. Stable knockdown cells (shNon-Target, shCaMKK2, shLKB1) were subject to acute and chronic stress conditions (i.e. ionomycin, 2-deoxy-D-glucose, hypoxia) and signaling events were evaluated using immunoblot analysis. Proliferation, anchorage-independent growth and motility of the knockdown cells were determined by MTT, soft agar formation and in vitro migration assay. Xenograft tumor studies were performed by inoculating estradiol-supplemented athymic nude mice with shNon-Target, shCaMKK2 and shLKB1 MCF-7 cells into the mammary fat pad. Tumor growth was determined by external caliper measurements twice a week. Forty-five days post-injection, mice were euthanized, tumors removed, weighed and prepared for histological, protein and RNA analyses. Results: CaMKK2 and LKB1 mRNAs are expressed at a similar level in the ERα + and ERα — breast cancer cell lines tested in the Illumina cDNA expression array. CaMKK2 activated AMPK phosphorylation (T172) under increased intracellular Ca2+ level induced by ionomycin. Under acute stress conditions, CaMKK2 functioned as the predominant AMPK upstream kinase. However, when cells are subject to long-term metabolic and hypoxic stress, CaMKK2 and LKB1 were both required for AMPK activation. Loss of CaMKK2 in MCF-7 cells promoted a moderate growth advantage under metabolic stress, increased soft agar colony formation and elevated cell migration capacity. CaMKK2 knockdown xenografts demonstrated less ductal structure formation, decreased E-Cadherin expression and reduced angiogenesis, as well as reduced VEGF and Glut1 mRNA levels in vivo. Conclusions: We have demonstrated that Ca2+-CaMKK2 signaling plays an important role in regulating breast cancer cell proliferation, migration and survival pathways. These findings suggest that Ca2+ activation through CaMKK2 promotes AMPK signaling, survival and angiogenesis in response to stress conditions in breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-07-06.