Klotho is a transmembranal protein implicated in aging and in calcium and phosphate metabolism. Klotho-deficient mice manifest a syndrome resembling accelerated aging, whereas, klotho over expression in mice extends life span. Klotho can be cleaved, shed and act as a circulating hormone. We have recently identified klotho as a potent tumor suppressor in breast and pancreatic cancers. We found that klotho is epigenetically silenced in these cancers and treatment with klotho inhibits proliferation of cancer cells in vitro and in vivo. We also showed that klotho inhibits bFGF-induced ERK phosphorylation. The ERK cascade plays a major role in tumorigenesis. Upon activation of the ERK cascade, ERK1/2 are phosphorylated and translocate to the nucleus, where they phosphorylate numerous substrates.

Using visualization of endogenous and GFP-tagged ERK2 and cell fractionation studies, we discovered that klotho inhibits nuclear translocation of phosphorylated ERK1/2. Furthermore, upon either klotho transfection or treatment with soluble klotho we noted activation of cytoplasmatic targets, but not of nuclear targets of ERK2. Thus RSK, an ERK2 cytoplasmic target, is activated following klotho co-treatment with bFGF. On the other hand, the ERK2 nuclear target Elk exhibited reduced activity under these conditions. That was evidenced by decreased Elk phosphorylation, and a decline in the mRNA expression of Elk-regulated gene cFOS.

One of klotho activities is regulation of calcium homeostasis through activation of the calcium channels TRPV5/6, resulting in increased intra-cellular levels of calcium. Interestingly, increased calcium levels have been shown recently to prevent nuclear translocation of activated ERK2. We hypothesized that TRPV6 may mediate klotho effects toward ERK1/2. Indeed, using visualization of GFP-tagged TRPV6 we noted that klotho recruited TRPV6 to the cell membrane and using co-immunoprecipitation noted an interaction between klotho and TRPV6 in breast cancer cells. Furthermore, overexpression of TRPV6 inhibited colony formation of these cells and the effect was augmented by co-transfection with klotho. Elevation in calcium levels by TRPV6 is expected to affect genes involved in regulating cellular calcium levels. Therefore, we analyzed the effects of klotho on the calcium binding protein S100A6, and on stromal interaction molecule-1 (STIM-1) which activates the "store-operated" ORAI1 calcium ion channels in plasma membrane. We noted, for the first time, that klotho down-regulated these genes.Taken together, our data indicate novel activities of klotho in cancer cells: regulation of intracellular calcium trafficking and dissociation between activation and nuclear translocation of ERK1/2.

Citation Format: Shiri Shahmoon, Tami Rubinek, Ido Wolf. Klotho-induced dissociation between activation and nuclear localization of extracellular signal-regulated kinase (ERK) 1/2: A novel growth inhibitory mechanism in breast cancer cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2013-541