Abstract 1184: The tumor suppressor klotho: a master regulator of metabolism in breast cancer
Authors: Shmulevich Riva , Rubinstein Tammi , Scherf Taly , Wolf Ido , Rubinek Tami 
Published: August 2015

Abstract
One of the hallmarks of cancer is reprogramming of energy metabolism, characterized by a shift to aerobic glycolysis (Warburg effect). This metabolic switch provides cancer cells appropriate conditions required for enhanced proliferation and survival. 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. We identified klotho as a tumor suppressor in breast and pancreatic cancers and recent data indicate it as a potent tumor suppressor in a wide array of malignancies. As klotho is a potent inhibitor of the IGF-1/Pi3K/AKT pathway in breast cancer, we hypothesized it may revert the metabolic switch in cancer cells.
A master regulator of energy metabolism is the energy sensor enzyme, AMP-activated kinase (AMPK). We first analyzed the effect of klotho on AMPK and its down-stream effector acetyl CoA carboxylase (ACC) in MCF-7, T47D and MDA-MB-231 breast cancer cell lines. These cell lines represent different disease phenotypes. Overexpression of klotho, or treatment with the soluble protein, AMPK and ACC phosphorylation in all three cell lines. AMPK is activated by the tumor suppressor liver kinase B1 (LKB1) and elevated AMP/ATP ratio or by calcium/calmodulin-dependent protein kinase kinase (CAMKK) 1β. LKB1 was expressed in all cell lines, although lower expression was noted in MD-MB-231 cells. Expression of a dominant negative LKB1 prevented activation of AMPK by klotho. Furthermore, it decreased the ability of klotho to inhibit cell growth and migration, implying that klotho tumor suppressor activity may be 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).
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, for the first time, as a regulator of metabolic activity in breast cancer and suggest that reversal of the metabolic switch is a key mechanism of klotho-mediated tumor suppressor activities.
Citation Format: Riva Shmulevich, Tammi Rubinstein, Taly Scherf, Ido Wolf, Tami Rubinek. The tumor suppressor klotho: a master regulator of metabolism in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2015-1184