Frequent Silencing of Low Density Lipoprotein Receptor-Related Protein 1B (LRP1B ) Expression by Genetic and Epigenetic Mechanisms in Esophageal Squamous Cell Carcinoma

Abstract

Low-density lipoprotein receptor-related protein 1B (LRP1B) is frequently deleted in tumors of various types, but its status and expression in esophageal squamous cell carcinomas (ESCs) have never been reported. In the course of a program to screen ESC cell lines for copy-number aberrations using array-based comparative genomic hybridization, we identified a homozygous deletion of LRP1B. Genomic PCR experiments revealed homozygous deletions of LRP1B in additional ESC cell lines (total, 6 of 43; 14.0%) and in primary esophageal tumors (30 of 70; 42.9%). Moreover, expression of LRP1B mRNA was frequently silenced in ESC lines without homozygous deletions (14 of 37; 37.8%). Using bisulfite-PCR analysis and sequencing, we found that LRP1B-nonexpressing cells without homozygous deletions were highly methylated at a CpG island of LRP1B, a sequence possessing promoter activity. Treatment with 5-aza-2-deoxycytidine restored expression of LRP1B in those ESC lines. Histone acetylation status correlated directly with expression of LRP1B and inversely with the methylation status of the CpG island. Methylation of LRP1B was also detected in primary esophageal tumors. Restoration of LRP1B expression in ESC cells reduced colony formation. These results suggest that loss of LRP1B function in esophageal carcinogenesis most often occurs either by homozygous deletion or by transcriptional silencing through hypermethylation of its CpG island.

PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation

“…To determine whether hypermethylation of CpG sites is associated with transcriptional silencing of the PRTFDC1 gene, we tested the promoter activity of this CpG island in OSCC cells (NA and HSC-2) using a 891-bp fragment (Fragment 1) covering the entire CpG island and 482-bp (Fragment 2) and 409-bp (Fragment 3) fragments covering uncommonly and commonly methylated regions, respectively, in cell lines without expression of PRTFDC1 (Figures 3a and b). We observed a remarkable increase in transcriptional activity of the reporter plasmid containing Fragments 1 and 3 compared with the mock reporter plasmid and reporter plasmid containing Fragment 2 in the plasmidcontaining cell lines (Figure 3b), suggesting that the CpG island around exon 1 of PRTFDC1, especially the region commonly methylated in PRTFDC1-nonexpressing cell lines, contains promoter activity, although few studies, including ours, have shown that promoter activity can be observed in fragments, especially CpG islands, not containing a 5 0 sequence around transcriptional start sites (Kolb 2003;Nakagawachi et al, 2003;Sonoda et al, 2004;Misawa et al, 2005).…”

“…We performed colony-formation assays (Sonoda et al, 2004) using the full coding sequence of PRTFDC1 with a Myc-tag cloned into a mammalian expression vector (pCMV-3Tag4A-PRTFDC1) and cells lacking expression of PRTFDC1. As shown in Figure 5, 3 weeks after transfection and subsequent selection of drug-resistant colonies, the numbers of large colonies produced by PRTFDC1-transfected NA and OM-2 cells decreased compared to those produced by empty vector-transfected cells.…”

“…Reporter assay was performed as described elsewhere (Sonoda et al, 2004) using each construct or a control empty vector and an internal control pRL-hTK vector (Promega).…”

“…Array-based CGH (array-CGH) technique allows high-throughput and quantitative analyses of copy-number changes at high-resolution throughout the genome, providing many advantages, including the identification of homozygous loss, over conventional methods (Snijders et al, 2001;Inazawa et al, 2004), although the resolution fully depends on the type of array. Indeed, our previous array-CGH studies using inhouse bacterial artificial chromosome-(BAC-)/P1-derived artificial chromosome-(PAC-) based arrays (BAC/ PAC-arrays) have successfully mapped complete genetic losses, allowing the rapid identification of candidate TSGs inactivated mainly by genetic and/or epigenetic mechanisms (Sonoda et al, 2004;Izumi et al, 2005;Takada et al, 2005bTakada et al, , 2006Imoto et al, 2006).…”

Genetic analysis of multifocal superficial urothelial cancers by array-based comparative genomic hybridisation

“…Array-CGH analysis was performed with the MCG Cancer Array-800, which consists of B800 BACs harbouring 800 known cancerrelated genes, and is intended for diagnosis of cancer-specific copy number aberrations (Sonoda et al, 2004). Array-CGH experiments were carried out as described previously (Takada et al, 2005) with minor modifications.…”