One of the hallmarks of cancer is reprogramming of energy metabolism, characterized by a shift to aerobic glycolysis (Warburg effect). A major regulator of this phenomenon is the IGF-1/Pi3K/AKT pathway. Klotho is a transmembrane protein, which can be cleaved, shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. Klotho is a potent tumor suppressor in BC. As klotho is a potent inhibitor of the IGF-1/Pi3K/AKT pathway in BC, we hypothesized that it may revert the metabolic switch in BC cells. We first analyzed the effect of klotho on the energy sensor enzyme AMP-activated kinase (AMPK) and its down-stream effector acetyl CoA carboxylase (ACC). Overexpression of klotho, or treatment with the soluble protein, elevated AMPK and ACC phosphorylation in three BC cell lines. AMPK is activated by the tumor suppressor liver kinase B1 (LKB1) and elevated AMP/ATP ratio. Expression of a dominant negative LKB1 prevented activation of AMPK by klotho and decreased the ability of klotho to inhibit cell growth and migration, implying that klotho tumor suppressor activities are LKB1 dependent. We next analyzed the effects of klotho on critical components of glucose metabolism. Treatment with soluble klotho reduced expression of the glucose transporter GLUT1 and the key glycolytic enzymes hexokinase2 (HK2), phosphofructokinase1 (PFK1), pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase kinase 1(PDK1). Using the Seahorse analyzer we noted reduced oxidative mitochondrial metabolism and glycolysis following klotho treatment. Energy is obtained essentially in the mitochondria with the transfer of protons across the inner membrane that produces ATP. We, therefore, analyzed klotho effect on the mitochondria membrane potential and noted reduction in the mitochondria potential following treatment with klotho. Finally, we examined the effects of klotho on specific metabolites and also conducted an NMR-based metabolic profiling. In accordance with the alterations in signaling pathways and levels of glycolytic enzymes, klotho reduced glucose uptake and inhibited lactate, pyruvate and 3-hydroxy-butyrate production. Furthermore, klotho also inhibited ATP production. Taken together, our data indicate klotho as a regulator of metabolic activity in BC and suggest that reversal of the metabolic switch is a key mechanism of klotho-mediated tumor suppressor activities.

Citation Format: Tami Rubinek, Ido Wolf. The tumor suppressor klotho: A master regulator of metabolism in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5417. doi:10.1158/1538-7445.AM2017-5417