Abstract B20: Global hypomethylation of esophageal myofibroblasts is associated with accelerated tumor growth.
Authors: Giger Olivier T. , Brown Benjamin , Liloglou Triantafillos , Jithesh Puthen V. , Dockray J Graham , Varro Andrea 
Published: May 2015

Cancer associated myofibroblasts (CAMs) originating from either tissue-resident fibroblast precursors or from bone marrow derived mesenchymal stromal cells, contribute to the tumor microenvironment by the secretion of growth factors, cytokines, proteases and ECM proteins. Exaggerated CAM reactions have been associated with adverse outcomes in various cancers.

The role of altered DNA CpG-island methylation in promoting tumorigenesis has been widely studied in cancer cells; altered DNA methylation in cancer stromal cells including CAMs has also been described but its biological significance remains uncertain. Our present objective was to determine the significance of DNA methylation in influencing the properties of esophageal CAMs. In initial studies we demonstrated different DNA methylation patterns in seven low passage primary myofibroblast lines from esophageal adenocarcinomas (n=3) and squamous cell carcinoma (n=4) patients in comparison to matched myofibroblasts derived from adjacent, macroscopically tumor-free, tissue (ATMs). Further, we found changes in global DNA methylation assessed in LINE1 elements by pyrosequencing analysis between CAMs and matched ATMs. Cluster analysis of the myofibroblast methylome determined by Illumina 450k human DNA methylation array revealed distinct methylation patterns in CAMs and ATMs. In order to generate a model for studying the biological consequences of epigenetic modification of myofibroblasts we treated cultured primary low passage normal esophageal tissue myofibroblasts (NTMs) with the DNA methyl transferase inhibitor, 5'Aza-2'deoxycytidin (DAC). A three-day exposure of NTMs to 0.1μM DAC induced a transient 10% decrease of global DNA methylation assessed by LINE1 element analysis; DAC (0.1 μM) also decreased myofibroblast proliferation by 20 %, and this persisted up to 28 days after treatment, and it stably increased migration in transwell migration assays by 50%. In a nude mouse xenograft model using esophageal squamous carcinoma cells (OE21) inclusion of CAMs stimulated tumor growth more than the inclusion of ATMs; moreover, myofibroblasts pretreated with DAC increased tumor growth compared with xenografts of OE21cells and untreated myofibroblasts.

Our data indicate (a) modification of DNA methylation in esophageal cancer myofibroblasts, and (b) that DNA demethylation induced by DAC stimulates migration of CAMs as well as their capacity to promote xenograft growth in vivo. Understanding the mechanisms and consequences of epigenetic changes in esophageal stromal cells may be useful in developing future anticancer strategies.

Citation Format: Olivier T. Giger, Benjamin Brown, Triantafillos Liloglou, Puthen V. Jithesh, J Graham Dockray, Andrea Varro. Global hypomethylation of esophageal myofibroblasts is associated with accelerated tumor growth.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B20. doi:10.1158/1538-7445.CHTME14-B20