Endocrine Abstracts

Background: Klotho is a 1012 amino acids transmembrane protein, composed of two internal repeats, KL1 and KL2, that can be cleaved, shed and act as a circulating hormone. Klotho regulates several pathways, including fibroblast growth factor 23 (FGF23), Wnt-β/catenin and the cation channels TRPV and TRPC. Our group and other labs identified klotho as a potent tumor suppressor in breast and pancreatic cancers and that KL1 domain is responsible for this activity. Yet, the mechanism mediating this activity remains unresolved.

Aim: Reveal the role of C-terminal and N-terminal parts within KL1 and understand their association to the tumor suppressor activity.

Methods: Expression vectors of truncated KL1 included C-terminal truncations: KL340, KL320, and N-terminal truncation: KL88-340. We assessed their anti-cancer activity or regulation of the Wnt-β/catenin pathway by colony formation assay and co-expression in cancer cells. We used MCF-7, MDA-MB-231, PANC1, MIAPaca-2, HCT116. We then performed RNA seq analysis on MCF7 cells over-expressing either KL1 or one of the C-terminal truncated KL340, KL320 to decipher global transcriptome differences.

Results: Over expression of C-terminal truncated protein KL340 inhibited colony formation, unlike KL320 in pancreatic and colon cancer. In breast cancer KL340 partially inhibited colony formation. N-terminal truncated proteins lost the ability to inhibit colony formation. Investigation of the Wnt-β/catenin signaling showed that only KL1 and KL340 decreased the levels of wnt3a in all cancer cell lines. Gene expression profile in MCF7 cells overexpressing KL1 and KL340 reflected their tumor suppressor activity.

Conclusions: This study reveals that klotho’s anti-cancer effect may be mediated by an N-terminal sequence. KL340 is the shortest truncation that retains the significant tumor suppressor activity of KL1. KL340 and KL1 induce a wide systemic transcriptome change in breast cancer, affecting cell survival. The structural features of the protein gained by the N-terminus remains questionable. Moreover, the 20 amino acids missing in KL320 compared to KL340 are critical for the tumor suppressor function and require further investigation.