Methylation of cystatin M promoter is associated with unfavorable prognosis in operable breast cancer

Abstract

The methylation status of cystatin M (CST6) gene in breast tumors was investigated and its prognostic significance as a novel breast cancer biomarker was evaluated. Using methylation-specific PCR (MSP), CST6 promoter methylation was examined in 134 formalin fixed paraffin-embedded tissues (FFPEs): 10 pairs of breast tumors and their surrounding normal tissues, 10 breast fibroadenomas, 11 normal breast tissues and 93 breast tumors. Methylation of CST6 promoter was observed in 2/21 (9.5%) noncancerous breast tissues, 1/10 (10%) benign breast tumors (fibroadenomas) and 52 (55.9%) operable breast cancer tumor samples. CST6 was rarely methylated in the normal tissue surrounding the tumor (10%). During the follow-up period, 24 (25.8%) patients relapsed and 19 (20.4%) died. CST6 methylation was detected in 19 (79.2%) of patients who relapsed and in 15 (78.9%) of patients who died. Disease-free-interval (DFI) and overall survival (OS) were significantly associated with CST6 promoter methylation (p 5 0.004 and p 5 0.001 respectively). Multivariate analysis revealed that CST6 methylation is an independent prognostic factor for DFI (HR 5 3.484; 95% CI: 1.155-10.511; p 5 0.027). and OS (HR 5 9.190; 95% CI: 1.989-42.454; p 5 0.004). CST6 promoter methylation status in tumor cells seems to provide important prognostic information in operable breast cancer and merits to be further evaluated and validated in a larger cohort of patients. ' 2009 UICC

DNA Methylation of Tumor Suppressor and Metastasis Suppressor Genes in Circulating Tumor Cells

“…A recent functional epigenetic study identified CST6 as 1 of 8 genes that showed frequent (Ͼ30%) tumor-specific promoter region hypermethylation associated with transcriptional silencing and that its re- expression suppressed the growth of renal cell carcinoma cell lines (36 ). Moreover, according to our recent findings, CST6 methylation is a promising biomarker for prediction of relapses and deaths in operable breast cancer (24 ). Here we provide evidence that the CST6 promoter is methylated in CTCs.…”

“…MSP products were fractionated on 2% agarose gels containing 40 mmol/L Tris-acetate/1.0 mmol/L EDTA (pH 8.0). To avoid false-negative results, we amplified SBtreated DNA in 2 separate MSP reactions, 1 with a set of primers specific for methylated and 1 for unmethylated CST6 promoter sequences (24 ). We used human placental genomic DNA (gDNA; Sigma-Aldrich) methylated in vitro with SssI methylase (NEB), after SB conversion, as a fully methylated (100%) MSP positive control; we used the same unmethylated placental gDNA, after SB conversion, as a negative MSP control.…”

“…We extensively evaluated the analytical sensitivity and specificity of the MSP assay for CST6 promoter methylation in our previous study (24 ). The analytical sensitivity and specificity of the MSP assays for BRMS1 and SOX17 promoter methylation have been evaluated (26,29,33 ).…”

“…Indeed, exogenous cystatin M expression in a breast cancer cell line resulted in significant reduction of cell proliferation, migration, Matrigel invasion, and adhesion to endothelial cells (20 ). CST6 promoter methylation provides important prognostic information in patients with operable breast cancer (24 ).…”

SOX17 Promoter Methylation in Circulating Tumor Cells and Matched Cell-Free DNA Isolated from Plasma of Patients with Breast Cancer

“…The breast cancer cell line MCF-7 was used as a positive control in MSP reactions for the detection of SOX17 promoter methylation. Genomic DNA (gDNA) from both FFPEs and MCF-7 cells was isolated with the High Pure PCR template preparation kit (Roche) as previously described (19 ). DNA concentration was determined in the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies).…”