Epigenetic silencing of the tumor suppressor klotho in human breast cancer

Abstract

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.

Klotho/FGF23 Axis in Chronic Kidney Disease and Cardiovascular Disease

“…In CKD, accumulated indoxyl sulfate or p-cresyl sulfate in the blood induces hypermethylation of the Klotho gene and decreased Klotho expression in CKD [81,82] . The hyperacetylation of histone in the Klotho promoter also inhibits Klotho expression [83] . TNF and TNF-like weak inducer of apoptosis-downregulated renal Klotho expression can be blunted by inhibition of histone deacetylase [84] , indicating that cytokine-induced elevation of histone acetylation is associated with downregulation of Klotho expression.…”

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

“…We have previously shown that klotho is abundantly expressed in normal breast and pancreatic tissues and that upon malignant transformation its expression is reduced (9,10,17). Restoring klotho expression, or treatment with the soluble protein, inhibited proliferation of breast and pancreatic cancer cells (9,10), and further studies indicated it as an inhibitor of the IGF-1 pathway (4,9).…”

The relevance of α-KLOTHO to the central nervous system: Some key questions

“…This phenomenon has been suggested to underlie the tightly controlled tissue-specific pattern of α-Klotho expression (Azuma et al, 2012) and is reported to increase with age (King et al, 2012). Interestingly, increased KL promoter methylation has also been described in a number of cancers, including cervical carcinoma, colorectal, breast and gastric cancers Pan et al, 2011;Rubinek et al, 2012;Wang et al, 2011). Fascinatingly, promoter methylation may be important to our observations.…”