Activation of the IGF pathway plays an important role in breast cancer pathogenesis. Recent data indicate the aging suppressor klotho as a potent inhibitor of IGF-1 receptor (IGFR) activation. Klotho is a transmembrane protein, which is expressed in the brain and kidney, and also in various endocrine-related tissues. We have shown previously high klotho expression in normal breast, but low expression in breast cancer and noted an association between low klotho expression and adverse tumor characteristics. Moreover, klotho overexpression inhibited growth of breast cancer cells. We aimed to analyze mechanisms associated with klotho-induced growth inhibition and modulation of the IGF pathway. Also, as klotho extracellular domain can be shed and serve as a circulating hormone, we aimed to elucidate its activities in breast cancer.
 Klotho effects on proliferation were assessed using MTT and colony assays in MCF-7 and MDA-MB-231 breast cancer cells and in the non-tumorous HEK293 cells. While klotho overexpression reduced breast cancer cells proliferation, it had no effect on HEK293 cells. Moreover, klotho silencing in MCF-7 cells, which express detectable klotho levels, enhanced their proliferation. Klotho effects on the IGF pathway were analyzed in MCF-7 cells. We conducted immunoprecipitation assays and identified, for the first time, direct interaction between klotho and the IGFR. Moreover, localization studies, using GFP-tagged klotho, revealed translocation of klotho from the cytoplasm to the cell membrane following IGF stimulation. Klotho overexpression in breast cancer cells inhibited IGF-induced phosphorylation of the IGFR and its downstream targets, but activated GSK3beta, a negative regulator of the IGF pathway, and increased expression of p53 and of the tumor suppressor gene C/EBPbeta. Klotho silencing using siRNA in MCF-7 cells induced opposite effects. Importantly, klotho overexpression did not inhibit IGF pathway activation in HEK293 cells, thus suggesting cell-type dependent activity.
 In order to assess the activity of klotho extracellular soluble form, breast cancer cells were treated with either recombinant soluble klotho or with a conditioned media taken from klotho overexpressing cells. Complete inhibition of IGFR activation was observed following either treatment, and soluble klotho at a concentration of 1 nM was sufficient to induce maximal effect.
 These data indicate, for the first time, cell-type specific growth suppression and IGF pathway inhibition by klotho. Two novel aspects of klotho activity were identified: interaction with the IGFR and changes in subcellular localization. Importantly, our data may indicate soluble klotho as a potent inhibitor of the IGF pathway in breast cancer. As soluble klotho is an endogenous hormone, its use as a novel therapeutic model may be feasible and should be further explored.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA