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.