Tumor Suppressor Activity of Klotho in Breast Cancer Is Revealed by Structure–Function Analysis

Abstract

Klotho is a transmembrane protein containing two internal repeats, KL1 and KL2, both displaying significant homology to members of the b-glycosidase family. Klotho is expressed in the kidney, brain, and various endocrine tissues, but can also be cleaved and act as a circulating hormone. Klotho is an essential cofactor for binding of fibroblast growth factor 23 (FGF23) to the FGF receptor and can also inhibit the insulin-like growth factor-1 (IGF-1) pathway. Data from a wide array of malignancies indicate klotho as a tumor suppressor; however, the structure-function relationships governing its tumor suppressor activities have not been deciphered. Here, the tumor suppressor activities of the KL1 and KL2 domains were examined. Overexpression of either klotho or KL1, but not of KL2, inhibited colony formation by MCF-7 and MDA-MB-231 cells. Moreover, in vivo administration of KL1 was not only well tolerated but significantly slowed tumor formation in nude mice. Further studies indicated that KL1, but not KL2, interacted with the IGF-1R and inhibited the IGF-1 pathway. Based on computerized structural modeling, klotho constructs were generated in which critical amino acids have been mutated. Interestingly, the mutated proteins retained their tumor suppressor activity but showed reduced ability to modulate FGF23 signaling. These data indicate differential activity of the klotho domains, KL1 and KL2, in breast cancer and reveal that the tumor suppressor activities of klotho can be dissected from its physiologic activities.Implications: These findings pave the way for a rational design of safe klotho-based molecules for the treatment of breast cancer.

Klotho, an anti-aging gene, acts as a tumor suppressor and inhibitor of IGF-1R signaling in diffuse large B cell lymphoma

“…Recently, Stromberg et al reported the involvement of IGF-1R and the antitumor effects of specific IGF-1R inhibitors in DLBCL tumorigenesis [ 25 ]. Structure-function analysis of Klotho indicated that Klotho could interact with the IGF-1R [ 43 ]. Klotho-induced inhibition of IGF-1R signaling may act as a novel mechanism involved in the development of DLBCL.…”

New Insights into the Mechanism of Action of Soluble Klotho

“…α-Klotho acts as a tumor suppressor by inhibiting insulin/IGF-1 signaling in breast cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, and ovarian cancer ( 57 , 59 , 65 , 73 – 76 ). Overexpression of α-Klotho or treatment with sKl inhibits insulin/IGF-1-mediated downstream effectors IRS-1, Akt1, and ERK1/2 in cancer cells ( 57 , 59 , 65 , 67 , 73 – 76 ). The tumor suppressive activity has been attributed to its KL1 domain ( 59 , 76 ).…”

“…Overexpression of α-Klotho or treatment with sKl inhibits insulin/IGF-1-mediated downstream effectors IRS-1, Akt1, and ERK1/2 in cancer cells ( 57 , 59 , 65 , 67 , 73 – 76 ). The tumor suppressive activity has been attributed to its KL1 domain ( 59 , 76 ).…”

Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease

“…While not impossible to perform our experiments with transfected Klotho plasmids with and without PTCs, it is challenging having to use the large, native 50 kb gene, since splicing is eliminated in cDNA translation. As expected, "secreted Klotho" cDNA leads to protein production for this reason (10,42,43), which is likely nonphysiological, in spite of the biological effects of overexpressed secreted Klotho, essentially amounting to KL1 domain effects (3,(44)(45)(46)(47)(48)(49)(50). Notably, the antibody we used to detect Klotho (KM2076) would detect the KL1 domain in any KL1-containing form of Klotho (13,51).…”

“…For example, Lu et al found that ovarian carcinoma patients positive for alternative Klotho mRNA have a higher tumor grade (17), which is interesting in light of the generally accepted view of Klotho as a tumor suppressor (44,46,60,61), including in ovarian carcinoma (62,63). However, higher alternative Klotho expression levels may not implicate effects of Klotho protein in whichever form and may even indicate dysregulated splicing or NMD dysfunction, which would lead to ER stress and may therefore reflect general cellular health.…”