Abstract 4507: Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer
Authors: Etan Tal , Rubinstein Tammi , Rubinek Tami , Shahmoon Shiri , Varol Chen , Zigmond Ehud , Rosin-Arbesfeld Rina , Barshack Iris , Wolf Ido 
Published: July 2017

Abstract
Klotho is a transmembrane protein, which can be shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor and as an inhibitor of the IGF-1 pathway. Recent data indicate klotho as a tumor suppressor in an array of malignancies, including colorectal cancer (CRC). We aimed to decipher the role of klotho as a tumor suppressor in CRC. Analysis of a public database (Oncomine) indicated reduced expression of klotho in CRC. Two models were employed to study the effect of klotho in vivo. Using the azoxymethane inflammation model, we discovered that klotho inhibited colon polyp formation. Using an orthotopic model consisting of direct injection of MC38 cells to the mice colons, we found that klotho inhibited colonic obstruction and tumor formation. Similarly, klotho inhibited colony formation and proliferation of CRC cell lines. Aberrant activation of the canonical Wnt pathway is implicated in CRC development and progression. β-catenin, the key effector of this pathway, functions with T-cell factor/lymphoid-enhancer-factor (TCF/LEF) to activate expression of Wnt target genes. While we did not see an effect of klotho on the IGF-1 pathway, it reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. Yet, the inhibitory effect of klotho on CRC cell colony formation was only partially rescued by a constitutively active β-catenin. These data indicate on additional mechanisms involved in the TS activity of klotho. Thus, we conducted an RNA expression array and observed involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Further studies showed that klotho induced elevation in XBP1 RNA levels, GRP78 protein levels, and eIF2α phosphorylation, all indicative of UPR regulation. Importantly, pharmacologic inhibition of ER stress and UPR overcame the growth suppression effect of klotho in CRC cells. Our data indicate klotho as a potent tumor suppressor in CRC, and suggest its role at very early stages of tumor formation, already at polyp formation. Our data also indicate, for the first time, klotho as a regulator of ER stress and UPR in cancer. These findings may pave the way for the development of novel therapeutic strategies based on enhancement of UPR.
Citation Format: Tal Etan, Tammi Rubinstein, Tami Rubinek, Shiri Shahmoon, Chen Varol, Ehud Zigmond, Rina Rosin-Arbesfeld, Iris Barshack, Ido Wolf. Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal 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 4507. doi:10.1158/1538-7445.AM2017-4507