PURPOSE: Down-regulation of Laminin-5 (LN5)-encoding genes (LAMA3, LAMB3, and LAMC2) has been reported in various human cancers. However, the mechanism of inactivation was not clearly understood until recently. In this study, we investigated the loss of expression of three LN5-encoding genes in breast cancer cell lines and elucidated the mechanism of silencing of the genes in breast cancer cell lines and tumors. EXPERIMENTAL DESIGN: We examined the expression of the three LN5-encoding genes by reverse transcription-PCR in breast cancer cell lines (n = 20). To elucidate the mechanism of silencing, we treated expression negative cell lines (n = 5) with a demethylating agent and examined restoration of expression by reverse transcription-PCR. By using methylation-specific primers designed by us, we validated the methylation status of the promoter regions in breast cancer cell lines using methylation-specific PCR. We additionally studied the methylation patterns in primary breast tumors (n = 74) and correlated the data with clinical parameters. RESULTS: We observed varied losses of expression (10-55%) of LN5-encoding genes in breast cancer cell lines. Expression of one or more genes was lost in 65% of breast cancer cell lines. Treatment of expression negative cell lines with demethylating agent restored expression in all cases. Methylation frequencies of LAMA3, LAMB3, and LAMC2 genes in 20 breast cancer cell lines were 40, 5, and 15%, respectively. The concordances between loss of expression and methylation in 20 breast cancer cell lines for the three genes (85-95%) were statistically significant. Nonmalignant breast tissues (n = 30) had very low frequencies of methylation (0-7%). In 74 breast tumors, methylation frequencies LAMA3, LAMB3, and LAMC2 were 44, 4, and 20%, respectively. The differences in methylation frequencies between cell lines and tumors were not statistically significant for all of the three genes. The methylation frequencies of LAMA3 and mean chain methylation index in cell lines and tumors were significantly different from methylation frequencies in nonmalignant tissues, and they were significantly higher in high stage and large size tumors as compared with low-stage and small size tumors. LAMA3 promoter methylation frequency in breast tumors was associated with increased tumor stage (P < 0.001) and tumor size (P < 0.001). CONCLUSIONS: Our results demonstrate epigenetic inactivation of LN5-encoding genes in breast cancers and association of LAMA3 promoter methylation with increased tumor stage and tumor size. Our findings are of biological interest and potentially of clinical importance.