Related Experiment Videos
Epigenetic inactivation of laminin-5-encoding genes in lung cancers
Ubaradka G Sathyanarayana1, Shinichi Toyooka, Asha Padar
1Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593, USA.
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.
Insights
Epigenetic silencing of laminin-5 (LN5) genes occurs frequently in lung cancers. Demethylation restores LN5 gene expression, indicating a potential therapeutic target for lung cancer.
Area of Science:
- Oncology
- Molecular Biology
- Epigenetics
Background:
- Lung cancer exhibits frequent gene expression alterations.
- Laminin-5 (LN5) is a key extracellular matrix protein with a role in cell adhesion and migration.
- The epigenetic regulation of LN5-encoding genes in lung cancer is not fully understood.
Purpose of the Study:
- To investigate the loss of expression of three laminin-5 (LN5)-encoding genes in lung cancer cell lines.
- To elucidate the mechanism of LN5 gene inactivation in lung cancer cell lines and tumors.
- To assess the potential clinical relevance of LN5 gene epigenetic alterations.
Main Methods:
- Examined LN5 gene expression using reverse transcription-PCR in 49 lung cancer cell lines.
- Investigated gene silencing mechanisms by treating expression-negative cell lines with a demethylating agent.
- Analyzed promoter methylation patterns using bisulfite genomic sequencing and methylation-specific PCR.
- Assessed methylation status in primary lung cancer tumors (NSCLC, SCLC, carcinoids).
Main Results:
- Frequent loss of LN5 gene expression observed in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines (up to 86%).
- Demethylating agent treatment restored expression in all tested cell lines, confirming epigenetic silencing.
- Methylation of LN5 genes was significantly more frequent in SCLC (60-80%) than NSCLC (15-60%) cell lines and tumors.
- High concordance (90-95%) between gene expression loss and promoter methylation in lung cancer cell lines.
Conclusions:
- Frequent epigenetic inactivation of LN5-encoding genes through promoter methylation is demonstrated in lung cancers.
- These findings highlight the biological significance of epigenetic alterations in lung tumorigenesis.
- Epigenetic silencing of LN5 genes represents a potentially important mechanism in lung cancer development and may offer clinical implications.