Breast cancer metastasis suppressor-1 promoter methylation in primary breast tumors and corresponding circulating tumor cells

Abstract

Breast cancer metastasis suppressor-1 (BRMS1) differentially regulates the expression of multiple genes, leading to metastasis suppression without affecting orthotopic tumor growth. For the first time, BRMS1 promoter methylation was evaluated as a prognostic biomarker in primary breast tumors and a subset of corresponding circulating tumor cells (CTC). Formalin-fixed paraffin embedded samples were analyzed for BRMS1 methylation status using methylation-specific PCR in a human specimen cohort consisting of noncancerous tissues, benign fibroadenomas, and primary breast tumors, including some with adjacent noncancerous tissues. Peripheral blood mononuclear cells from a large subset of these patients were fixed in cytospins and analyzed. In addition, BRMS1 expression in cytospins was examined by double-immunofluorescence using anti-BRMS1 and pan-cytokeratin antibodies. BRMS1 promoter methylation was not detected in noncancerous breast tissues or benign fibroadenomas; however, methylation was observed in more than a third of primary breast tumors. Critically, BRMS1 promoter methylation in primary tumors was significantly associated with reduced disease-free survival with a trend toward reduced overall survival. Similarly, a third of cytospin samples were positive for the presence of CTCs, and the total number of detected CTCs was 41. Although a large fraction of CTCs were negative or maintained low expression of BRSM1, promoter methylation was observed in a small fraction of samples, implying that BRSM1 expression in CTCs was either downregulated or heterogeneous. In summary, these data define BRMS1 promoter methylation in primary breast tumors and associated CTCs.

Implications: This study indicates that BRSM1 promoter methylation status has biomarker potential in breast cancer.

Figure 1

Schematic diagram of the workflow of the study

Figure 2

A) Analytical sensitivity of nested MSP for BRMS1 using methylated primer set and synthetic mixtures, containing : 1) DNA marker 50bp, 2) Negative control: dH₂O, 3) 0%, 4) 0.1%, 5) 1%, 6) 10%, 7) 50% SB converted positive control (100% methylated DNA), B) Analytical specificity of nested MSP for BRMS1 using methylated primer set and control samples: 1) DNA marker 50bp, 2) Negative control: dH₂O, 3) unconverted DNA, 4) SB converted placental DNA (0% methylated), 5) SB converted DNA from the MCF-7 cell line and 6) 100% methylated DNA, C) Nested MSP for BRMS1 promoter for methylated sequences: 1) DNA marker 50bp, 2) Negative control: dH₂O, 3–6) non-cancerous breast tissues, 7-15) breast tumors, 16) MSP positive control (100% methylated DNA).

Figure 3

BRMS1 expression in cytospin stained CTC of early breast cancer patients. Cells were stained with A45-B/B3 antibody (green), BRMS1 antibody (red) and Dapi (blue). Quantification of the samples was performed with the ARIOL system and scored as A):BRMS1 high expression, (B): BRMS1 low expression, C) negative for BRMS1. D): Heat map for BRMS1 expression and matched DNA samples for BRMS1 promoter methylation in cytospin stained CTC (n=39): black: no CTC cells, red: no BRMS1 expression at upper row, BRMS1 methylation at lower row, green: normal BRMS1 expression at upper row, BRMS1 non-methylated at lower row, brown: low BRMS1 expression

Figure 3

BRMS1 expression in cytospin stained CTC of early breast cancer patients. Cells were stained with A45-B/B3 antibody (green), BRMS1 antibody (red) and Dapi (blue). Quantification of the samples was performed with the ARIOL system and scored as A):BRMS1 high expression, (B): BRMS1 low expression, C) negative for BRMS1. D): Heat map for BRMS1 expression and matched DNA samples for BRMS1 promoter methylation in cytospin stained CTC (n=39): black: no CTC cells, red: no BRMS1 expression at upper row, BRMS1 methylation at lower row, green: normal BRMS1 expression at upper row, BRMS1 non-methylated at lower row, brown: low BRMS1 expression

Figure 4

A) Kaplan-Meier estimates of disease-free interval (DFI) for early breast cancer patients with (green) or without (blue) BRMS1 promoter methylation (p=0.009), B) Kaplan-Meier estimates of overall survival (OS) for early breast cancer patients with (green) or without (blue) BRMS1 promoter methylation (p= 0.071)