Helicase-like Transcription Factor (HLTF) expression and gene methylation in FFPE colon tumors

Background : HLTF, beside its roles in DNA binding, transcriptional and chromatin remodeling, is involved in post-replication DNA repair through its translocase and E3 ubiquitin ligase activities. HLTF is a tumor suppressor, and hypermethylation of its promoter was observed in 30-50% of gastrointestinal cancers. Moreover, HLTF inactivation in Apc-/+ mice induced the transition from colon adenocarcinoma to a carcinoma with high chromosomal instability. The epigenetic heterogeneity of the HLTF promoter could be related to the CpG island methylator phenotype of tumor suppressor genes in colorectal cancers. In the present study we started to investigate HLTF expression in colon cancer progression in relation to the epigenetic status of its gene promoter by using pyrosequencing.Methods : The HLTF promoter contains a 653 bp CpG island with 59 CpG sites (UCSC) among which 11 CpG sites were selected for this study. Genomic DNA was extracted from (a) HeLa (cervix adenocarcinoma) and RKO (colon carcinoma) cells and (b) from formalin-fixed paraffin-embedded (FFPE) colon tumors. DNA was treated with bisulfite and amplified by PCR the HLTF promoter fragment containing the 11 CpG sites. Pyrosequencing was performed to determine the relative methylation frequency at each site (HLTF PyroMark CpG Assay, Qiagen). HLTF expression was evaluated by immunohistochemistry (IHC) on tissue sections, and by RT-PCR and immunodetection on Western blot for the cell lines.Results : There was a negative correlation between promoter methylation and HLTF expression at the RNA and protein levels in RKO and HeLa cells. The average methylation rates were 92% for RKO and 25% for HeLa cells which presented HLTF repression and expression, respectively. IHC staining for HLTF was performed in 14 FFPE colon tumors and good pyrosequencing signals was obtained for DNA extracted from 6/14 samples. Three tumor groups were defined, based on the average methylation rate in the HLTF promoter : (a) < 30% ; (b) = 50% ; and (c) > 60%. In groups (a) and (b), the tissue was still well organized and HTLF protein expression was detected by IHC in the glandular epitelium. However, in group (c), the tumors were disorganized and HLTF protein expression was low or undetectable. These preliminary results suggest a correlation between 3 criteria : HLTF immunodetection, tissue organization and low methylation of the HLTF promoter. Two colon tumors did not express HLTF and presented a hypermethylated promoter.
Conclusion : We could optimize pyrosequencing methylation analysis of genomic DNA extracted from 6 FFPE colon tumors, and confirmed that higher HLTF promoter methylation was associated with its repression in correlation with worse tumor phenotypes. This sensitive technique will now be applied to larger number of samples and additional CpG sites will be assessed.