Promoter Hypomethylation of EpCAM-Regulated Bone Morphogenetic Protein Gene Family in Recurrent Endometrial Cancer

Abstract

Purpose Epigenetic regulation by promoter methylation plays a key role in tumorigenesis. Our goal was to investigate whether altered DNA methylation signatures associated with oncogenic signaling delineate biomarkers predictive of endometrial cancer recurrence. Experimental Design Methyl-CpG-capture sequencing was used for global screening of aberrant DNA methylation in our endometrial cancer cohort, followed by validation in an independent The Cancer Genome Atlas (TCGA) cohort. Bioinformatics as well as functional analyses in vitro, using RNA interference (RNAi) knockdown, were performed to examine regulatory mechanisms of candidate gene expression and contribution to aggressive phenotype, such as epithelial–mesenchymal transition (EMT). Results We identified 2,302 hypermethylated loci in endometrial tumors compared with control samples. Bone morphogenetic protein (BMP) family genes, including BMP1, 2, 3, 4, and 7, were among the frequently hypermethylated loci. Interestingly, BMP2, 3, 4, and 7 were less methylated in primary tumors with subsequent recurrence and in patients with shorter diseasefree interval compared with nonrecurrent tumors, which was validated and associated with poor survival in the TCGA cohort (BMP4, P = 0.009; BMP7, P = 0.007). Stimulation of endometrial cancer cells with epidermal growth factor (EGF) induced EMT and transcriptional activation of these genes, which was mediated by the epithelial cell adhesion molecule (EpCAM). EGF signaling was implicated in maintaining the promoters of candidate BMP genes in an active chromatin configuration and thus subject to transcriptional activation. Conclusions Hypomethylation signatures of candidate BMP genes associated with EpCAM-mediated expression present putative biomarkers predictive of poor survival in endometrial cancer.

EpCAM-Regulated Transcription Exerts Influences on Nanomechanical Properties of Endometrial Cancer Cells That Promote Epithelial-to-Mesenchymal Transition

“…Supporting our previous observation that EGF treatment led to changes in an epithelial morphology to a more fibroblastic mesenchymal appearance in RL95-2 cells (16), the AFM results further provide quantitative proof of their elevated softness and reduced adhesiveness, leading to an increase in cell movement. This enhanced cell movement follows EGF stimulation in a time-dependent manner.…”

“…Images were visualized by Nikon Eclipse microscope (Nikon Instruments). Western blot analysis was performed as described previously (16). Antibodies against α-tubulin (membrane/cytoplasmic fraction) and Lamin B1 (nuclear fraction, Abcam) were used as loading controls.…”

Mutual regulation of microRNAs and DNA methylation in human cancers

“…65 Genome-wide analysis of DNA methylation has shown that the epigenetic process can also occur in chromosome regions such as contiguous CpG islands and gene families. [66][67][68] Hypermethylation of HOXA gene clusters was reported in breast and lung cancer. 69,70 Lastly, the protocadherin (PCDH) gene cluster was found to be hypermethylated in Wilms tumor and the region across chromosome 2q14.2 in colorectal cancer.…”

Methyl-binding DNA capture Sequencing for Patient Tissues

“…We have used MBDCap-seq to study DNA methylation alterations in a large number of patients from diverse cancer types including breast 12 , endometrial 13 , prostate 14 , and liver cancers among others. Here we demonstrate some information from the breast cancer study published recently 12 .…”