Aberrant promoter methylation and silencing of laminin-5-encoding genes in breast carcinoma.

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.

Differential inactivation of caspase-8 in lung cancers.

Caspase-8 (CASP8) is an apoptosis inducing cysteine protease which is activated through the formation of a death-inducing signaling complex when death receptors are complexed to their specific ligands. Recent reports indicate that CASP8 expression is lost via a combination of promoter methylation and allelic loss in subset of neuroblastomas. We investigated the state of the gene in lung tumors and cell lines. RT-PCR studies indicated that gene expression was lost in most (27 of 34, 79%) of small cell lung carcinoma (SCLC) cell lines, but expression was retained in all 22 non-SCLC (NSCLC) lines tested. Loss of gene expression at the RNA level was associated with absent protein expression by Western blotting and lack of CASP8 enzymatic activity. Methylation of the promoter region of the CASP8 gene was present in 16 of 27 (59%) of the SCLC lines lacking gene expression. All methylated cell lines lacked the presence of an unmethylated allele indicating biallelic methylation or loss of non-methylated allele. Promoter methylation was absent in all SCLC and NSCLC cell lines retaining gene expression, and all of these lines had the unmethylated form of the gene. One non-expressing SCLC cell line, NCI-H82, had a homozygous deletion at 2q33 encompassing the chromosomal location of the CASP8 gene. The mechanism of gene inactivation in the remaining 10 of 27 (37%) non-expressing SCLC cell lines is unknown. Using five polymorphic markers for 2q33 a high frequency of allelic loss was present in SCLC lines. Analyses of fresh tumors showed that 15 of 43 (35%) of the SCLC, seven of 40 (18%) of bronchial carcinoids and none of 44 NSCLC tumors had CASP8 promoter methylation. Because only approximately 60% of SCLC cell lines lacking CASP8 expression were methylated, extrapolating from the cell line data, we estimate that approximately 58% of SCLC and 30% of bronchial carcinoids lack CASP8 expression. Thus, CASP8 expression is absent in a subset of both high grade (SCLC) and low grade (carcinoid) neuroendocrine lung tumors but not in NSCLC, which usually lack neuroendocrine features. CASP8 may function as a tumor suppressor gene in neuroendocrine lung tumors.