COX-2 (PTGS2) gene methylation in epithelial, subepithelial lymphocyte and stromal tissue compartments in a spectrum of esophageal squamous neoplasia

Abstract

Background-Previous studies have shown important effects of stromal elements in carcinogenesis. To explore the tumor-stromal relationship in esophageal neoplasia, we examined methylation of COX-2 (PTGS2), a gene etiologically associated with the development of gastrointestinal cancers, in adjacent foci of epithelium, subepithelial lymphocytes and nonlymphocytic stromal cells found in sections of normal squamous epithelium, squamous dysplasia and invasive esophageal squamous cell carcinoma.Methods-Adjacent foci of epithelium, subepithelial lymphocytic aggregates and non-lymphocytic stromal tissues were laser microdissected from six fully embedded, ethanol fixed, esophagectomy samples from Shanxi, China, a high-risk region for esophageal cancer. Promoter CpG site-specific hypermethylation status of COX-2 was determined using real-time methylation specific PCR (qMS-PCR) based on Taqman Chemistry. The methylation status of a subset of samples was confirmed by pyrosequencing.Results-Forty-nine microdissected foci were analyzed. COX-2 gene methylation was significantly more common in subepithelial lymphocytes (12/16 (75% of all foci)) than in epithelial foci (3/16 (19%)) or foci of non-lymphocytic stromal tissues (3/17 (18%)) (Fisher's Exact p=0.05). Two of three epithelial samples and all three stromal samples that showed COX-2 methylation were adjacent to foci of methylated subepithelial lymphocytes. Pyrosequencing confirmed the methylation status in a subset of samples.Corresponding authors/reprint requests: Mark J. Roth, MD, Nutritional Epidemiology Branch, DCEG, National Cancer Institute, 6120 Executive Boulevard, Suite 320, MSC 7232, Bethesda, Maryland 20892-7232, USA, +1-301-402-8276T, +1-301-496-6829F, mr166i@nih.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of interestNone. Conclusions-In these esopohageal cancer patients, COX-2 gene methylation was more common in subepithelial lymphocytes than in adjacent epithelial or stromal cells in both grades of dysplasia and in foci of invasive cancer. These findings raise the possibility that methylation of subepithelial lymphocytes may be important for tumorigenesis. Future studies of gene methylation should consider separate evaluation of epithelial and non-epithelial cell populations. NIH Public AccessCondensed abstract-COX2 (PTGS2) gene methylation increases with disease severity and is more common in subepithelial lymphocytes than in adjacent epithelial or stromal cells in dysplastic and early invasive esophageal squamous cell cancer foci.

Genetic alterations and epigenetic alterations of cancer-associated fibroblasts

“…These genes are methylated in breast cancer, but are only slightly or not methylated in non-neoplastic breast cancers (120). Gene methylation is a dynamic process in esophageal squamous cancer and cervical cancer; thus, the level of methylation changes with the progression of cancer (121,122). Apart from locally methylated DNA, global gene hypomethylation was confirmed in CAFs of gastric cancer (123).…”

Enduring epigenetic landmarks define the cancer microenvironment

“…Here we ask if genome-wide differences in the epigenome underpin the persistent phenotypic differences between CAFs and NPFs. Earlier studies using low-resolution techniques found examples of DNA methylation changes in CAFs or tumor stroma from various cancer types (Hu et al 2005;Fiegl et al 2006;Hanson et al 2006;Rodriguez-Canales et al 2007;Dawsey et al 2008;Jiang et al 2008;Banerjee et al 2014;Vizoso et al 2015). However, the full epigenome landscape of CAFs remains uncharted, so the precise epigenetic changes and their contribution to altered transcriptional patterns is unknown.…”

Evaluation of DNA methylation of inflammatory genes following treatment of chronic periodontitis: A pilot case–control study

“…For each gene, CpG positions were selected based on previously published data (Dawsey et al., ; Madrigano et al., ; Cantone et al., ), as they are more consistent in terms of repeatability of the assay. Four CpG islands were analysed for COX‐2 and TNF‐ α genes while two were assessed for IFN‐ γ gene.…”

“…The CpG positions were selected according to data published in literature for the same assessed genes (Dawsey et al., ; Madrigano et al., ; Cantone et al., , which ensures consistency in terms of repeatability of the assay.…”

“…Analysis of DNA methylation was performed using previously published methods (Bollati et al, 2007;Tarantini et al, 2013) The CpG positions were selected according to data published in literature for the same assessed genes (Dawsey et al, 2008;Madrigano et al, 2012;Cantone et al, 2017), which ensures consistency in terms of repeatability of the assay.…”