Jiyeon Yun

CD82/KAI1 Maintains the Dormancy of Long-Term Hematopoietic Stem Cells through Interaction with DARC-Expressing Macrophages

Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 in niche-mediated LT-HSC maintenance. We found that CD82/KAI1 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82(-/-) mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-β1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC(+) BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche.

Gene silencing of EREG mediated by DNA methylation and histone modification in human gastric cancers

Epiregulin (EREG) induces cell growth by binding to the epidermal growth factor receptor (EGFR). Expression of EREG affects sensitivity to cetuximab a chimeric monoclonal antibody that inhibits the EGFR signaling pathway. The mechanism through which EREG is regulated is largely unknown, but a methyl-array study previously performed by our group revealed that EREG is methylated in gastric cancer cells. In this study, we found that EREG gene expression was low in 7 out of 11 gastric cancer cells and this downregulation was mediated by aberrant CpG methylation of the EREG promoter. Treatment with 5-aza-CdR restored EREG expression and demethylated CpG sites in the EREG promoter. Compared with DNA methyltransferase 1 (DNMT1), knock-down of DNA methyltransferase 3b (DNMT3b) significantly increased the expression of EREG and led to the demethylation of specific CpG sites in the EREG promoter, suggesting that DNMT3b primarily regulates CpG methylation and silencing of the EREG gene. EREG methylation was observed in 30% (4/13) of human primary gastric tumor tissues we evaluated. In addition to DNA methylation, results from a chromatin immunoprecipitation assay demonstrated that transcriptional levels of EREG were associated with the enrichment of active histone marks (H3K4me3 and AcH3) and of a repressive mark (H3K27me2). Treatment with 5-aza-CdR dynamically increased the low occupancy of H3K4me3 and AcH3, while decreasing the high enrichment of H3K27me2, indicating that dynamic histone modifications contribute to EREG regulation in addition to DNA methylation. Finally, the combination of 5-aza-CdR and cetuximab exerted a synergistic anti-proliferative effect on gastric cancer cells. Taken together, the results of our study showed for the first time that EREG is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification.

Disruption of CTCF/cohesin-mediated high-order chromatin structures by DNA methylation downregulates PTGS2 expression

The CCCTC-binding factor (CTCF)/cohesin complex regulates gene transcription via high-order chromatin organization of the genome. De novo methylation of CpG islands in the promoter region is an epigenetic hallmark of gene silencing in cancer. Although the CTCF/cohesin complex preferentially targets hypomethylated DNA, it remains unclear whether the CTCF/cohesin-mediated high-order chromatin structure is affected by DNA methylation during tumorigenesis. We found that DNA methylation downregulates the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), which is an inducible, rate-limiting enzyme for prostaglandin synthesis, by disrupting CTCF/cohesin-mediated chromatin looping. We show that the CTCF/cohesin complex is enriched near a CpG island associated with PTGS2 and that the PTGS2 locus forms chromatin loops through methylation-sensitive binding of the CTCF/cohesin complex. DNA methylation abolishes the association of the CTCF/cohesin complex with the PTGS2 CpG island. Disruption of chromatin looping by DNA methylation abrogates the enrichment of transcriptional components, such as positive elongation factor b, at the transcriptional start site of the PTGS2 locus. These alterations result in the downregulation of PTGS2. Our results provide evidence that CTCF/cohesin-mediated chromatin looping of the PTGS2 locus is dynamically influenced by the DNA methylation status.

Association between salivary amylase (AMY1) gene copy numbers and insulin resistance in asymptomatic Korean men

Salivary amylase gene (AMY1) copy number variations (CNVs) correlate directly with salivary amylase activity and serum amylase levels. Previously, individuals with high AMY1 CNVs exhibited low postprandial glucose levels and postprandial early insulin surge, suggesting that high AMY1 gene copy numbers may play a role in lowering the risk of insulin resistance.

Highly angiogenic CXCR4+CD31+ monocyte subset derived from 3D culture of human peripheral blood

Ex vivo expansion of human circulating angiogenic cells is a major challenge in autologous cell therapy for ischemic diseases. Here, we demonstrate that hematosphere-derived CXCR4(+)CD31(+) myeloid cells using peripheral blood possess robust proangiogenic capacity such as formation of vessel-like structures and tip cell-like morphology in Matrigel. We also found that CD31 positive myeloid cells are principal cellular component of hematospheres by magnetic cell sorting. Flow cytometry analysis showed that fresh peripheral blood contained 40.3 ± 15.2% of CXCR4(+)CD31(+) myeloid cells, but at day 5 of hematosphere culture, most of myeloid cells were CXCR4(+)CD31(+) by 86.9 ± 5.4%. Hematosphere culture significantly increased the production of angiogenic niche-supporting cytokines. Moreover, CD31-homophilic interaction and VEGF-VEGF receptor loop signaling were essential for sphere formation and acquisition of angiogenic capacity in hematospheres. Matrigel plug and ischemic hindlimb model provide in vivo evidence that hematosphere-derived myeloid cells have highly vasculogenic capacities, participate in new and mature vessel formation, and exert therapeutic effects on ischemic hindlimb. In conclusion, our strategy for ex vivo expansion of human CXCR4(+)CD31(+) angiogenic cells using hematospheres provides an autologous therapeutic cell source for ischemic diseases and a new model for investigating the microenvironment of angiogenesis.

Erythropoietin priming improves the vasculogenic potential of G-CSF mobilized human peripheral blood mononuclear cells

AIMS From our previous clinical trials, intracoronary infusion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells ((mob)PBMCs) proved to be effective in improving myocardial contractility and reducing infarct volume in acute myocardial infarction. We tested the effect of priming (mob)PBMCs with erythropoietin (EPO) to augment its therapeutic efficacy. METHODS AND RESULTS (mob)PBMCs were obtained from healthy volunteers after a 3-day subcutaneous injection of G-CSF (10 μg/kg). About 40% of (mob)PBMCs were EPO receptor (EPOR) (+) and responded to 6 h EPO-priming (10 IU/mL) by increasing the expression of vasculogenic factors (i.e. IL8, IL10, bFGF, PDGF, MMP9) and adhesion molecules (i.e. integrin αV, β1, β2, β8) through the JAK2 and Akt pathway. These responses were also observed in PBMCs from elderly patients with coronary disease. The conditioned media from EPO-primed (mob)PBMCs contained various cytokines such as IL8, IL10, TNFα, and PDGF, which enhanced the migration and tube formation capability of endothelial cells. EPO-primed (mob)PBMCs also showed increased adhesion on endothelial cells or fibronectin. Augmented vasculogenic potential of EPO-primed (mob)PBMCs was confirmed in a Matrigel plug assay, ischaemic hindlimb, and myocardial infarction models of athymic nude mice. There were two action mechanisms: (i) cellular effects confirmed by direct incorporation of human (mob)PBSCs into mouse vasculature and (ii) indirect humoral effects confirmed by the therapeutic effect of the supernatant of EPO-primed (mob)PBMCs. CONCLUSION Brief ex vivo EPO-priming is a novel method to augment the vasculogenic potential of human (mob)PBMCs, which would help to achieve better results after intracoronary infusion in myocardial infarction patients.

Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

Gene amplification is a hallmark of cancer with chromosomal instability although the underlying mechanism by which altered copy numbers are maintained is largely unclear. Cohesin, involved in sister chromatid cohesion, DNA repair, cell cycle progression and transcriptional regulation of key developmental genes, is frequently overexpressed in human cancer. Here we show that cohesin-dependent change in DNA replication controls the copy numbers of amplified genes in cancer cells with chromosomal instability. We found that the down-regulation of elevated cohesin leads to copy number-associated gene expression changes without disturbing chromosomal segregation. Highly amplified genes form typical long-range chromatin interactions, which are stabilized by enriched cohesin. The spatial proximities among cohesin binding sites within amplified genes are decreased by RAD21-knockdown, resulting in the rapid decline of amplified gene expression. After several passages, cohesin depletion inhibits DNA replication initiation by reducing the recruitment of pre-replication complexes such as minichromosome maintenance subunits 7 (MCM7), DNA polymerase α, and CDC45 at replication origins near the amplified regions, and as a result, decreases the DNA copy numbers of highly amplified genes. Collectively, our data demonstrate that cohesin-mediated chromatin organization and DNA replication are important for stabilizing gene amplification in cancer cells with chromosomal instability.

Cross-cultural adaptation and validation of the Korean Toronto Extremity Salvage Score for extremity sarcoma

A Korean version of Toronto Extremity Salvage Score (TESS), a widely used disease‐specific patient‐reported questionnaire for assessing physical function of sarcoma patients, has not been developed.

Aberrant Epigenetic Modifications of LPHN2 Function as a Potential Cisplatin-Specific Biomarker for Human Gastrointestinal Cancer

Purpose Epigenetic alterations of specific genes have recently been identified as diagnostic biomarkers for human cancers. However, there are currently no standardized epigenetic biomarkers for drug sensitivity in human gastrointestinal cancer. Therefore, the aim of this study is to identify a novel epigenetic biomarker in gastrointestinal cancer. Materials and Methods Using bisulfite sequencing and pyrosequencing analysis, DNA methylation patterns of gastric, colon primary tissues and their cancer cells were analyzed, and histone modifications were analyzed using chromatin immunoprecipitation assay. In addition, cancer cells were exposed to cisplatin and treated with a DNA methyltransferase inhibitor. Results We report that in human gastric and colon cancers, latrophilin 2 (LPHN2) is silenced by epigenetic modifications, including CpG island methylation and aberrant histone modifications. We also confirmed that LPHN2 was silenced by DNA hypermethylation in primary gastric and colon tumor tissues compared to their normal counterparts. Interestingly, we found that cancer cells with methylated LPHN2 showed higher sensitivity to cisplatin. Also, 5-aza- 2′-deoxycytidine combined with cisplatin decreased the cytotoxicity of cisplatin in cancer cells with methylated LPHN2. In addition, LPHN2 knockdown in cancer cells with high LPHN2 expression sensitized these cells to the anti-proliferative effects of cisplatin. Conclusion In human gastrointestinal cancer, we found that LPHN2 is regulated by epigenetic modifications, and that cancer cells with lower LPHN2 expression show higher sensitivity to cisplatin. Therefore, the methylation status of LPHN2 is a potential novel epigenetic biomarker for cisplatin treatment in human gastric and colon cancers.

Identification of Long-Range Epigenetic Silencing on Chromosome 15q25 and Its Clinical Implication in Gastric Cancer

Recent genome-wide epigenomic and transcription profiling studies have demonstrated that epigenetic silencing can encompass multiple neighboring genes, termed as long-range epigenetic silencing (LRES). Herein, we identified a novel LRES region by comparing gene expression of human colon cancer HCT116 cells with their DNA methyltransferase 1 and DNA methyltransferase 3B double-knockout derivative double-knockout cells. Ten consecutive genes spanning 3 Mb of chromosome 15q25 were coordinately silenced, with eight genes showing promoter CpG island hypermethylation and enrichment of repressive histone marks, which were evaluated by bisulfite sequencing analysis and chromatin immunoprecipitation assay. Comparison of primary gastric tumor specimens with normal tissue confirmed that the long-range silencing of this region was tumor specific. Methylation of genes within the LRES region was evaluated in 190 gastric tumor tissues using the MethyLight assay, and their association with clinicopathological features, such as older age, high-grade differentiation, and diffuse or mixed-type histology, was determined. LRES-positive gastric cancer patients (six or more methylated genes) showed lower recurrence and better survival. Our findings emphasize the differential dynamics of DNA methylation and histone modification, indicating the importance of studying the relationship of each epigenetic modification in the context of chromatin domains. Patients with LRES showed lower recurrence and better prognosis, indicating that stratifying patients according to underlying molecular features, such as LRES regions, may better predict recurrence and survival.