Transcript-Specific DNA Methylation Alterations of the RASSF1 Locus in Cancer Cells

The locus encoding Ras association domain family member 1 (RASSF1) encodes multiple transcripts with opposing roles in cancer, such as RASSF1A (tumor suppressor), RASSF1C (oncogene), and the lncRNA RASSF1-AS1 (function undefined). Although DNA methylation-mediated repression of RASSF1A expression has been extensively studied in different cancer types, the epigenetic regulation of RASSF1C and RASSF1-AS1 is unclear. We profiled gene expression and promoter DNA methylation of RASSF1A, RASSF1C, and RASSF1-AS1 across 11 tumor cell lines, quantified RASSF1A methylation in lung cancer tissues and plasma by quantitative methylation-specific PCR (qMSP), and integrated single-CpG methylation (pyrosequencing) with in silico transcription factor binding site (TFBS) prediction.

We found that RASSF1A promoter hypermethylation was strongly and inversely associated with its mRNA levels. In contrast, RASSF1C promoter methylation did not correlate with expression. RASSF1-AS1 showed low promoter methylation accompanied by high expression. In clinical samples, RASSF1A methylation was detected in tissue biopsy (54% of cases) and plasma (42% of cases) from lung cancer patients, whereas no methylation was detected in 85% of control individuals, regardless of their smoking history. Finally, the analysis of DNA methylation at specific CpG sites combined with the prediction of TFBS in the evaluated promoter regions allowed the identification of binding domains overlapping differentially methylated regions. Notably, the RASSF1C promoter region exhibited a higher TFBS frequency containing CpG sites with low methylation levels, as determined by pyrosequencing.

These findings highlight isoform-specific epigenetic regulation at the RASSF1 locus and suggest that RASSF1A methylation may represent a promising minimally invasive marker in lung cancer.