Revealing the anti-cancer region within KL1 domain of the hormone Klotho

Klotho, a 1012 amino acid transmembrane protein, has emerged as a potent tumorsuppressor in different cancer types. Klotho is composed of two internal repeats KL1 andKL2, and the anti-cancer effect is primarily attributed to the 540 amino-acid KL1 domain.Despite its significant role in regulating various cancer-related pathways, the precisemechanism underlying its anti-cancer activity remains unresolved. In this study, weaimed to identify the sequence responsible for the tumor suppressor function of klothoand gain deeper insights into its mechanism of action. To accomplish this, we generatedexpression vectors of truncated KL1 at the C-terminal and N-terminal regions andevaluated their anti-cancer activity using breast, colon and pancreatic cancer cell lines.Our findings demonstrate that KL340 effectively inhibited colony formation of cancercells similar to KL1, while KL320 lost this activity. Furthermore, investigations into theinvolvement of the Wnt pathway in mediating klotho39;s anti-cancer effects revealed thatKL1 and KL340 significantly decreased Wnt3a protein levels, whereas KL320 did notaffect Wnt3a expression. Transcriptomic analysis of KL1 and KL340 further supportedtheir tumor suppressor activity and unveiled numerous cancer-associated pathwayspotentially implicated in mediating their anti-tumorigenic effects and suppressing cellsurvival. Interestingly, the -fold predictor tool highlighted distinct differences in theand sheets of the TIM barrel fold of the truncated klotho constructs, adding to ourunderstanding of their structural variations. This study elucidates that the 340 N-terminalamino acids of the klotho protein possess the tumor suppressor activity and reveals acritical role of the 20 amino acids residing in the 320-340 sequence for this function. Thecomprehensive insights gained from this study enhance our understanding of klotho39;stumor suppressor activity and provide a foundation for the development of klotho-basedtherapeutic approaches for cancer treatment.