Abstract
Leucine-rich repeat-containing 3B (LRRC3B) is an evolutionarily highly conserved leucine-rich repeat-containing protein, but its biological significance is unknown. Using restriction landmark genomic scanning and pyrosequencing, we found that the promoter region of LRRC3B was aberrantly methylated in gastric cancer. Gastric cancer cell lines displayed epigenetic silencing of LRRC3B, but treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A increased LRRC3B expression in gastric cancer cell lines. Real-time reverse transcription-PCR analysis of 96 paired primary gastric tumors and normal adjacent tissues showed that LRRC3B expression was reduced in 88.5% of gastric tumors compared with normal adjacent tissues. Pyrosequencing analysis of the promoter region revealed that LRRC3B was significantly hypermethylated in gastric tumors. Stable transfection of LRRC3B in SNU-601 cells, a gastric cancer cell line, inhibited anchorage-dependent and anchorage-independent colony formation, and LRRC3B expression suppressed tumorigenesis in nude mice. Microarray analysis of LRRC3B-expressing xenograft tumors showed induction of immune response-related genes and IFN signaling genes. H&E-stained sections of LRRC3B-expressing xenograft tumors showed lymphocyte infiltration in the region. We suggest that LRRC3B is a putative tumor suppressor gene that is silenced in gastric cancers by epigenetic mechanisms and that LRRC3B silencing in cancer may play an important role in tumor escape from immune surveillance.
Highlights
Leucine-rich repeat (LRR)-containing 3B (LRRC3B) is a putative LRR-containing transmembrane protein found by the Secreted Protein Discovery Initiative undertaken to identify new secreted and transmembrane proteins [1]
We report the identification of Leucine-rich repeat-containing 3B (LRRC3B) as a target of aberrant methylation in gastric cancer and show LRRC3B silencing by epigenetic mechanisms in gastric cancer cell lines and primary gastric tumor tissues
restriction landmark genomic scanning (RLGS) assays were performed on gastric cancer cell lines and gastric cancer tissues to look for aberrant methylation of genomic DNA compared with normal gastric mucosal tissue [16]
Summary
Leucine-rich repeat (LRR)-containing 3B (LRRC3B) is a putative LRR-containing transmembrane protein found by the Secreted Protein Discovery Initiative undertaken to identify new secreted and transmembrane proteins [1]. DNA methylation associated with histone modification is a key mechanism to inhibit the expression of tumor suppressor genes in cancer, and DNA methylation markers have been applied in cancer risk assessment, early detection, prognosis, and prediction of response to cancer therapy [13,14,15]. We have used restriction landmark genomic scanning (RLGS) to identify methylation markers in gastric cancer on a whole-genome scale [16]. We previously reported aberrant methylation of LIMS2, a regulator of cell migration [17], DCBLD2, a cell growth repressor [18], and Protein kinase D1 [19] in gastric cancer by RLGS and pyrosequencing, a quantitative methylation analysis of multiple CpG sites. We report the identification of LRRC3B as a target of aberrant methylation in gastric cancer and show LRRC3B silencing by epigenetic mechanisms in gastric cancer cell lines and primary gastric tumor tissues. We examined the function of LRRC3B as a tumor suppressor in vitro and in vivo and provide evidence that loss of LRRC3B function in gastric cancer likely promotes tumor escape from immune surveillance
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