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Supplementary Figure S1. Test of the methylation status of candidate genes used pooled DNA from endometrial tissues and cervical scrapings. Supplementary Figure S2. Verification of 14 candidate genes in the pilot set of cervical scrapings. Supplementary Table S1: Demographics relating to the two data sets of genome-wide DNA methylation. Supplementary Table S2. Demographics related to the clinical samples. Supplementary Table S3: Summary the area under the receiver-operating characteristic curve for distinction between endometrial cancer and noncarcinoma samples in pilot testing. Supplementary Table S4: Summary of cophenetic correlation results for analysis of consensus clustering of DNA methylation patterns. Supplementary Table S5: List of 180 hypermethylated genes in four clusters. Supplementary Table S6. Summary of the methylation levels of four candidate genes in cervical scrapings from endometrial cancer and noncarcinoma samples.

This file contains eight supplementary figures depicting the experiments validated in different cell lines and cohorts, as well as the results supportive to the main text.

Supplementary Figure S1 Expression of EpCAM, LEF1 in human endometrial cancer patients, cancer cell lines and mouse endometrial tissues. Supplementary Figure S2 Quantification of EpEX immunofluorescence staining and EGFR expression in endometrial cancer cell lines. Supplementary Figure S3 Summary of ChIP-seq of EpICD in RL95-2 cells. Supplementary Figure S4 Overview of ChIP-seq of LEF1 in RL95-2 cells. Supplementary Figure S5 Pathway enrichment analysis unique/shared TSS-bound genes in 12 and 24 hours EGF. Supplementary Figure S6 105 EpICD-LEF1-TSS gene expression profiles in UCEC (Uterine Corpus Endometrial Carcinoma) patients and normal control samples in TCGA cohort. Supplementary Figure S7 EpCAM-edited cells showed reduced EGF-stimulated gene activation. Supplementary Figure S8 RL95-2 cells showed no apoptosis and cytoskeleton content induced by EGF treatments in RL95-2 and EpCAM-edited clone. Supplementary Figure S9 EpCAM molecules were detected with recognition AFM only on cells expressing this protein.

Resistance to cancer treatment can be driven by epigenetic reprogramming of specific transcriptomes in favor of the refractory phenotypes. Here we discover that tamoxifen resistance in breast cancer is driven by a regulatory axis consisting of a master transcription factor, its cofactor, and an epigenetic regulator. The oncogenic histone methyltransferase EZH2 conferred tamoxifen resistance by silencing the expression of the estrogen receptor α (ERα) cofactor GREB1. In clinical specimens, induction of DNA methylation of a particular CpG-enriched region at the GREB1 promoter negatively correlated with GREB1 levels and cell sensitivity to endocrine agents. GREB1 also ensured proper cellular reactions to different ligands by recruiting distinct sets of ERα cofactors to cis-regulatory elements, which explains the contradictory biological effects of GREB1 on breast cancer cell growth in response to estrogen or antiestrogen. In refractory cells, EZH2-dependent repression of GREB1 triggered chromatin reallocation of ERα coregulators, converting the antiestrogen into an agonist. In clinical specimens from patients receiving adjuvant tamoxifen treatment, expression levels of EZH2 and GREB1 were correlated negatively, and taken together better predicted patient responses to endocrine therapy. Overall, our work suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program.Significance: This study suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program defined within. Cancer Res; 78(3); 671–84. ©2017 AACR.

Overexpression of epithelial cell adhesion molecule (EpCAM) has been implicated in advanced endometrial cancer, but its roles in this progression remain to be elucidated. In addition to its structural role in modulating cell-surface adhesion, here we demonstrate that EpCAM is a regulatory molecule in which its internalization into the nucleus turns on a transcription program. Activation of EGF/EGFR signal transduction triggered cell-surface cleavage of EpCAM, leading to nuclear internalization of its cytoplasmic domain EpICD. ChIP-seq analysis identified target genes that are coregulated by EpICD and its transcription partner, LEF-1. Network enrichment analysis further uncovered a group of 105 genes encoding functions for tight junction, adherent, and cell migration. Furthermore, nanomechanical analysis by atomic force microscopy revealed increased softness and decreased adhesiveness of EGF-stimulated cancer cells, implicating acquisition of an epithelial–mesenchymal transition (EMT) phenotype. Thus, genome editing of EpCAM could be associated with altering these nanomechanical properties towards a less aggressive phenotype. Using this integrative genomic–biophysical approach, we demonstrate for the first time an intricate relationship between EpCAM-regulated transcription and altered biophysical properties of cells that promote EMT in advanced endometrial cancer. Cancer Res; 76(21); 6171–82. ©2016 AACR.

Purpose: Endometrial cancer is a common gynecologic cancer whose incidence is increasing annually worldwide. Current methods to detect endometrial cancer are unreliable and biomarkers are unsatisfactory for screening. Cervical scrapings were reported as a potential source of material for molecular testing. DNA methylation is a promising cancer biomarker, but limited use for detecting endometrial cancer.Experimental Design: We analyzed two methylomics databases of endometrioid-type endometrial cancer. Using nonnegative matrix factorization algorithm clustered the methylation pattern and reduced the candidate genes. We verified in pools DNA from endometrial cancer tissues and cervical scrapings, and validated in 146 cervical scrapings from patients with endometrioid-type endometrial cancer (n = 50), uterine myoma (n = 40), and healthy controls (n = 56) using quantitative methylation–specific PCR (QMSP). The logistic regression was used to evaluate the performance of methylation signal and gene combination.Results: We filtered out 180 methylated genes, which constituted four consensus clusters. Serial testing of tissues and cervical scrapings detected 14 genes that are hypermethylated in endometrial cancer. Three genes, BHLHE22, CDO1, and CELF4, had the best performance. Individual genes were sensitivity of 83.7%–96.0% and specificity of 78.7%–96.0%. A panel comprising any two of the three hypermethylated genes reached a sensitivity of 91.8%, specificity of 95.5%, and odds ratio of 236.3 (95% confidence interval, 56.4–989.6). These markers were also applied to cervical scrapings of type II endometrial cancer patients, and detected in 13 of 14 patients.Conclusions: This study demonstrates the potential use of methylated BHLHE22/CDO1/CELF4 panel for endometrial cancer screening of cervical scrapings. Clin Cancer Res; 23(1); 263–72. ©2016 AACR.

This file contains the supplementary information including materials and methods, primer sequences, clinicopathological characteristics of patient samples and supplemental figures legends.

Supplementary Figure Legends Fig S1-S9

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

Transgenic mice,Immunofluorescence staining and image quantification, Proliferation and migration assays, ChIP-seq and network-based pathway analysis, TCGA expression analysis, CRISPR/Cas9 genome-editing of EpCAM, Apoptosis assay, Nanomechanical imaging of cells with Atomic Force Microscopy (AFM), Probing of cell surface with molecular recognition AFM

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.

Purpose: To identify novel endometrial cancer-specific methylation markers and to determine their association with clinicopathologic variables and survival outcomes.Experimental Design: Differential methylation hybridization analysis (DMH) was done for 20 endometrioid endometrial cancers using normal endometrial DNA as a reference control. Combined bisulfite restriction analysis (COBRA) was used to verify methylation of sequences identified by DMH. Bisulfite sequencing was undertaken to further define CpG island methylation and to confirm the reliability of the COBRA. The methylation status of newly identified markers and the MLH1 promoter was evaluated by COBRA in a large series of endometrioid (n = 361) and non-endometrioid uterine cancers (n = 23).Results: DMH and COBRA identified two CpG islands methylated in tumors but not in normal DNAs: SESN3 (PY2B4) and TITF1 (SC77F6/154). Bisulfite sequencing showed dense methylation of the CpG islands and confirmed the COBRA assays. SESN3 and TITF1 were methylated in 20% and 70% of endometrioid tumors, respectively. MLH1 methylation was seen in 28% of the tumors. TITF1 and SESN3 methylation was highly associated with MLH1 methylation (P < 0.0001). SESN3 and TITF1 were methylated in endometrioid and non-endometrioid tumors, whereas MLH1 methylation was restricted to endometrioid tumors. Methylation at these markers was not associated with survival outcomes.Conclusions: The 5′ CpG islands for SESN3 and TITF1 are novel cancer-specific methylation markers. Methylation at these loci is strongly associated with aberrant MLH1 methylation in endometrial cancers. SESN3, TITF1 and MLH1 methylation did not predict overall survival or disease-free survival in this large cohort of patients with endometrioid endometrial cancer.

The purpose of this study was to investigate the effect of Huaier extract supernatant(HES) on the proliferation, apoptosis, autophagy, and migration of human gastric cancer HGC-27 and MGC-803 cells and its molecular mechanisms. The main components in HES were preliminarily analyzed by high-performance liquid chromatography-mass spectrometry(HPLC-MS). Methyl thiazolyl tetrazolium(MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine(EdU) staining assay were used to explore the effect of HES on the proliferation of human gastric cancer HGC-27 and MGC-803 cells. Hoechst staining and flow cytometry assay were used to determine the effect of HES on apoptosis of human gastric cancer HGC-27 and MGC-803 cells. Acridine orange staining and cell scratch assay were used to determine the effect of HES on autophagy and migration of human gastric cancer HGC-27 and MGC-803 cells, respectively. Western blot was used to investigate the regulatory effect of HES on the expression levels of proteins related to apoptosis, epithelial-mesenchymal transition(EMT), and signaling pathways in human gastric cancer HGC-27 and MGC-803 cells. The results showed that HES mainly contained some components with high polarities. HES significantly reduced the cell viability of human gastric cancer cells in a dose-and time-dependent manner. The IC_(50 )values after 48 h of HES treatment in human gastric cancer HGC-27 and MGC-803 cells were 7.56 and 10.77 g·L~(-1), respectively. Meanwhile, HES inhibited the colony-forming ability and short-term proliferation of human gastric cancer cells. The apoptosis rates of HGC-27 and MGC-803 cells treated with 8 g·L~(-1) HES for 72 h were 62.13%±8.92% and 54.50%±3.26%, respectively. HES also promoted autophagy in human gastric cancer cells and impaired their migration ability in vitro. Moreover, HES up-regulated the cleavage of the apoptosis marker poly ADP-ribose polymerase(PARP) and the protein expression level of the epithelial cell marker E-cadherin, and down-regulated the protein levels of phosphorylated-mammalian target of rapamycin(p-mTOR), phosphorylated-S6(p-S6), and phosphorylated-extracellular signal-regulated kinase(p-ERK) in human gastric cancer cells. Therefore, HES is one of the effective anti-tumor components of Huaier, which inhibits the proliferation and migration of human gastric cancer cells, and induces apoptosis and autophagy. Moreover, the mTOR signal and ERK signal may be involved in the anti-gastric cancer effect of HES. This study provides novel references for the in-depth research and clinical application of Huaier. It is also of great significance to promote the scientific development and utilization of Huaier.

Background: Obesity is not only associated with cardiovascular diseases but also a primary cause of liver dysfunction and other related diseases. This study’s aim was to determine the impact of a combination of dietary modification and aerobic exercise on liver function in overweight and obese adult males. Methods: 45 overweight or obese men were randomly divided between the control group (n = 22) and intervention group (n = 23). Subjects in the intervention group were provided with dietary modification and aerobic exercise programmes. Dietary modification is a diet which restricts calorie intake and balances nutrients. Before and after 12 weeks of intervention, participants’ anthropometric characteristics and biochemical parameters relating to liver function including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were measured. Results: 12 weeks of aerobic exercise and dietary modification resulted in average weight loss of 10.6%, and body mass index, waist circumference and fat percentage decreased by 10.2%, 9.4% and 14.5% (p < 0.05). AST, ALT, GGT and ALP in the intervention group reduced by 20.6%, 18.1%, 37.7% and 6.1% (p < 0.05). Compared to the control group, AST, ALT, GGT and ALP in the intervention group were markedly lower (p < 0.05). Furthermore, there was a markedly positive relationship between the reduction rates of body weight and GGT (p < 0.05). Conclusion: 12 weeks of aerobic exercise and dietary modification caused significant weight, waist circumference and body fat percentage reduction in overweight and obese men and their liver function was improved. The findings can provide a scientific reference for the improvement of liver function and prevention of liver diseases among overweight and obese people.

SummaryTranscription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that Androgen Receptor (AR), a steroid hormone-regulated transcription factor, forms condensates through multivalent interactions in response to androgen signaling to orchestrate enhancer assembly. We demonstrate that the intrinsically disordered N-terminal domain (NTD) of AR drives 1,6-Hexanediol-sensitive condensate formation and that NTD deletion or aromatic residue mutation reduces AR self-association and abolishes AR transcriptional activity. AR NTD can be substituted by intrinsically disordered regions (IDRs) from selective proteins for AR condensation capacity and transactivation function. Surprisingly, strengthened AR condensation capacity caused by extending the polyQ tract within AR NTD also leads to impaired transcriptional activity without affecting AR binding on enhancers. Furthermore, either NTD deletion or polyQ extension reduces heterotypic multivalent interactions between AR and other enhancer components. These results suggest the importance of an optimal level of AR condensation in mediating AR-AR homotypic and AR-cofactor heterotypic interactions to regulate enhancer assembly in response to signals. Our study supports the notion that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies, thereby providing novel molecular insights for potential therapeutic strategies to treat prostate cancer and other AR-involved diseases by targeting AR multivalent interactions.

Background and objective: Motor performance plays an important role in daily activities for older adults. The purpose of the study was to construct a method for measuring physical fitness age that can be used to assess motor performance in elderly men and to verify its validity. Methods: Four items of physical fitness including grip strength, balancing on one leg with eyes open, 30 s chair stand test and 6 min walk test were selected and measured in a random order. Principal component analysis was employed to build physical fitness age based on motor performance variables from 94 elderly men with a mean age of 71.20 ± 5.05 years. Physical fitness age = 115.516-0.652 × grip strength - 0.142 × balancing on one leg with eyes open - 0.839 × 30 s chair stand - 0.061 × 6 min walk distance + 0.541 × chronological age. Results: Validation test from 13 men with exercise habit and 12 men without exercise habit showed that the physical fitness age in the exercised adults was significantly lower than its chronological age (63.91 ± 5.40 vs. 70.92 ± 4.17, P < 0.001), while no significant difference was observed between physical fitness age and chronological age in the elderly without exercise habits (71.23 ± 7.49 vs. 70.83 ± 5.19, P = 0.646). Conclusion: Physical fitness age is a valid indicator to evaluate and monitor motor performance. Participation in physical exercise may lower the physical fitness age by improving motor performance.

The study aimed to develop a physical fitness age score to assess motor function in community-dwelling elderly women and verify its validity. Principal component analysis was employed to build a physical fitness age score based on motor function variables. Validation test showed that the physical fitness age in the exercised older women was significantly lower than their chronological age, while no significant difference was observed between the physical fitness age and the chronological age in the normal elderly. The findings suggest that physical fitness age score is a valid approach to evaluate motor function in Chinese community-dwelling elderly women.

As a traditional Chinese medicine, Chinese dragon's blood has multiple effects, such as activating blood to remove blood stasis, softening and dispelling stagnation, astringent and hemostasis, clearing swelling and relieving pain, regulating menstruation and rectifying the blood, so it is calledquot;an effective medicine of promoting blood circulationquot;. It has been widely used clinically to treat a variety of diseases. With the further research on Chinese dragon's blood, its anti-tumor medicinal value is gradually emerging. Modern pharmacological studies have shown that Chinese dragon's blood exerts anti-tumor effects mainly by inhibiting cell proliferation, inducing apoptosis, inducing DNA damage and cell cycle arrest, inducing senescence and autophagy of tumor cells, inhibiting metastasis and angiogenesis, as well as reversing multidrug resistance. This article focuses on the research progress on anti-tumor effects of Chinese dragon's blood extract and its chemical components, with a view to provide new references for the in-depth research and reasonable utilization of Chinese dragon's blood.