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Supplementary Figures 1 - 13, Tables 1 - 5 from Promoter Hypomethylation of EpCAM-Regulated Bone Morphogenetic Protein Gene Family in Recurrent Endometrial Cancer

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posted on 2023-03-31, 17:54 authored by Ya-Ting Hsu, Fei Gu, Yi-Wen Huang, Joseph Liu, Jianhua Ruan, Rui-Lan Huang, Chiou-Miin Wang, Chun-Liang Chen, Rohit R. Jadhav, Hung-Cheng Lai, David G. Mutch, Paul J. Goodfellow, Ian M. Thompson, Nameer B. Kirma, Tim Hui-Ming Huang

PDF file - 1049K, Supplementary Figure S1. Summarized sequencing reads and coverage of MBDCap-seq Supplementary Figure S2. Reproducibility test of MBDCap-seq Supplementary Figure S3. Validation of MBDCap-seq by MassARRAY analysis Supplementary Figure S4. Distribution of differentially methylated regions (DMRs) in endometrial tumors relative to normal control Supplementary Figure S5. Network analysis of differential methylation loci by using Steiner-tree analysis Supplementary Figure S6. DNA methylation distribution in early and late stage of the TCGA endometrial cancer cohort Supplementary Figure S7. Relative expression of BMP genes with EGF treatment in the presence or absence of PI3K/AKT and Raf (MAPK) inhibitors in endometrial cancer cells Supplementary Figure S8. Induction of invasion by EGF in AN3CA and HEC1A cell lines Supplementary Figure S9. Knockdown expression of BMP4 and BMP7 in RL95-2 cells Supplementary Figure S10. Relative expression of BMPs and BMPRs in normal endometrial cell and endometrial cancer cell lines Supplementary Figure S11. Microfluidics-based PCR analysis of EMT gene panel in RL95-2 cells with or without EGF treatment Supplementary Figure S12. Knockdown expression of EpCAM by different shRNA sequences in RL95-2 cells Supplementary Figure S13. Proposed model for EpICD mediated regulation of BMP genes by EGF Supplementary Table 1. Characteristics of endometrial cancer patients Supplemental Table 2. Summary of sequencing reads in endometrial tumors an normal controls Supplementary Table 3. Differentially methylated promoter CpG islands in recurrent relative to non-recurrent tumors Supplementary Table 4. List of primer sequences Supplementary Table 5. List of siRNA and shRNA sequences

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ARTICLE 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 disease-free 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. Clin Cancer Res; 19(22); 6272–85. ©2013 AACR.

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