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Epigenetic Inactivation of Laminin-5-encoding Genes in Lung Cancers¹

Hamon Center for Therapeutic Oncology Research [U. G. S., S. T., A. P., J. D. M., A. F. G.] and Departments of Pathology [U. G. S., A. F. G.] and Internal Medicine [J. D. M.], University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593; Division of Molecular Oncology, Aichi Cancer Center, Nagoya 464-8681, Japan [T. T.]; and Laboratoire de Pathologie Cellulaire, Centre Hospitalier Regional Universitaire, Grenoble 38043, France [E. B.]

Purpose: We investigated the loss of expression of three laminin-5 (LN5)-encoding genes in lung cancer cell lines and elucidated the mechanism of inactivation of the genes in lung cancer cell lines and tumors.

Experimental Design: We examined the expression of LN5-encoding genes by reverse transcription-PCR in 49 lung cancer cell lines. To elucidate the mechanism of gene silencing, we treated expression-negative cell lines (two for each gene) with a demethylating agent and examined the restoration of expression by reverse transcription-PCR. We dissected out the methylation patterns of CpG sites unique to the promoter regions of LN5-encoding genes by bisulfite genomic sequencing of expression-negative cell lines. We designed methylation-specific primers and validated the methylation status of the promoter regions in lung cancer cell lines using methylation-specific PCR. We further studied the methylation patterns of primary non-small cell lung cancer [NSCLC (n = 36)], small cell lung cancer [SCLC (n = 26)], and carcinoids (n = 24) tumors.

Results: We observed frequent losses of expression in NSCLC (20–60%) and SCLC (65–86%) cell lines. Expression of one or more genes was lost in 90% of SCLC cell lines and 65% of NSCLC cell lines. Treatment of expression-negative cell lines with demethylating agent restored expression in all of the cases. Methylation of LN5-encoding genes was present more frequently in SCLC cell lines (60–80%) than in NSCLC cell lines (15–60%), and at least one gene was methylated in 95% of SCLC and 60% of NSCLC cell lines. The concordances between loss of expression and methylation in 40 lung cancer cell lines for the three genes (90–95%) were statistically significant. Methylation was more frequent in SCLC tumors (58–77%) than in NSCLC tumors (22–42%) and carcinoids (13–33%), and at least one gene was methylated in 92% of SCLC tumors, 47% of NSCLC tumors, and 33% of carcinoids.

Conclusions: Our results demonstrate frequent epigenetic inactivation of LN5-encoding genes in lung cancers, and these findings are of biological interest and are potentially of clinical importance.

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