Epigenetic inactivation of laminin-5-encoding genes in lung cancers.
OBJECTIVE
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.


METHODS
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.
Sathyanarayana
Ubaradka G
UG
Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593, USA.
Toyooka
Shinichi
S
Padar
Asha
A
Takahashi
Takashi
T
Brambilla
Elizabeth
E
Minna
John D
JD
Gazdar
Adi F
AF
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002276:Carcinoid Tumor / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D018288:Carcinoma, Small Cell / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000378:metabolism*; D045744:Cell Line, Tumor; D018899:CpG Islands; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D000077209:Decitabine; D006801:Humans; D007797:Laminin / Q000096:biosynthesis; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008745:Methylation; D008957:Models, Genetic; D009061:Mouth Mucosa / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D000097794:Kalinin
D001374:Azacitidine; D015815:Cell Adhesion Molecules; D004247:DNA; D000077209:Decitabine; D007797:Laminin; D013447:Sulfites; D000097794:Kalinin; C400567:LAMC2 protein, human; C095567:laminin alpha 3