Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity

INTRODUCTION: Loss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions. METHODS: Levels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro. RESULTS: H4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro. CONCLUSIONS: H4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner

A novel role for the condensin II complex in cellular senescence

<p>Although cellular senescence is accompanied by global alterations in genome architecture, how the genome is restructured during the senescent processes is not well understood. Here, we show that the hCAP-H2 subunit of the condensin II complex exists as either a full-length protein or an N-terminus truncated variant (ΔN). While the full-length hCAP-H2 associates with mitotic chromosomes, the ΔN variant exists as an insoluble nuclear structure. When overexpressed, both hCAP-H2 isoforms assemble this nuclear architecture and induce senescence-associated heterochromatic foci (SAHF). The hCAP-H2ΔN protein accumulates as cells approach senescence, and hCAP-H2 knockdown inhibits oncogene-induced senescence. This study identifies a novel mechanism whereby condensin drives senescence via nuclear/genomic reorganization.</p

Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors

Abstract Background Transplantation of pancreatic β cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)—PDX1 and NKX6.1—has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. Methods synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. Results Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. Conclusions In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells

Single-Cell Analysis of Circulating Tumor Cells from Patients with Colorectal Cancer Captured with a Dielectrophoresis-Based Micropore System

This study aimed to analyze circulating tumor cells (CTCs) from patients with colorectal cancer (CRC). We designed a dielectrophoresis-based micropore system and tested its cell capture with HT29 colon cancer cells. Then, blood samples were drawn from 24 patients with stages II-IV CRC. Mononuclear cells were isolated and loaded into the micropore system. Single cells were positioned into small pores with dielectrophoresis. After labeling the cells with the appropriate antibodies, tumor-like cells were collected with an automated micromanipulator. We collected 43 CTCs from 15 out of 24 patient samples. The presence of CTC was significantly associated with ling metastasis. We performed whole genome amplification, followed by PCR and Sanger sequencing, to examine the point mutations in the KRAS, BRAF, and PIK3CA genes. This mutation analysis was successfully performed in 35 cells. Among the 14 cytokeratin (CK)-positive cells, we found PIK3CA mutations in three cells (21%) from two patients. Among the 21 CK-negative cells, we found a KRAS mutation in one cell (5%) from one patient and a PIK3CA mutation in one cell (5%) from one patient. It is noteworthy that these mutations were not detected in the corresponding primary tumors. In conclusion, dielectrophoresis-based capture in a micropore system was useful for detecting both CK-positive and CK-negative CTCs. This simple method could be applied to various tumor types

BET Bromodomain Inhibition Promotes Anti-tumor Immunity by Suppressing PD-L1 Expression

Restoration of anti-tumor immunity by blocking PD-L1 signaling through the use of antibodies has proven to be beneficial in cancer therapy. Here, we show that BET bromodomain inhibition suppresses PD-L1 expression and limits tumor progression in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of anti-tumor cytotoxic T cells. The BET inhibitor limited tumor progression in a cytotoxic T-cell-dependent manner. Together, these data demonstrate a small-molecule approach to block PD-L1 signaling. Given the fact that BET inhibitors have been proven to be safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a treatment strategy for targeting PD-L1 expression

Long noncoding RNA 01534 maintains cancer stemness by downregulating endoplasmic reticulum stress response in colorectal cancer

Abstract Background Studies have shown that cancer stemness and the endoplasmic reticulum (ER) stress response are inversely regulated in colorectal cancer (CRC), but the mechanism has not been fully clarified. Long noncoding RNAs (lncRNAs) play key roles in cancer progression and metastasis. In this study we investigated lncRNA 01534 (LINC01534) as a possible modulator between cancer stemness and ER stress response. Methods In vitro experiments using CRC cell lines were performed to explore a possible role of LINC01534. The expression of LINC01534 in clinical CRC samples was assessed by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) and in situ hybridization. Results Silencing LINC01534 led to suppression of cell proliferation, invasiveness, and cell cycle progression at the G2‐M phase, and promoted apoptosis. Moreover, we found that silencing LINC01534 suppressed cancer stemness, while it activated the ER stress response, especially through the PERK/eIF2α signaling pathway. In situ hybridization revealed LINC01534 was expressed in tumor cells and upregulated in CRC tissues compared with normal epithelium. A survival survey indicated that high LINC01534 expression was significantly associated with shorter overall survival in 187 CRC patients. Conclusion This is the first report on LINC01534 in human cancer. Our findings suggest that LINC01534 may be an important modulator of the maintenance of cancer stemness and suppression of the ER stress response, and that it could be a novel prognostic factor in CRC

Super Carbonate Apatite-miR-497a-5p Complex Is a Promising Therapeutic Option against Inflammatory Bowel Disease

The incidence of inflammatory bowel disease (IBD) is increasing worldwide. It is reported that TGF-β/Smad signal pathway is inactivated in patients with Crohn’s disease by overexpression of Smad 7. With expectation of multiple molecular targeting by microRNAs (miRNAs), we currently attempted to identify certain miRNAs that activate TGF-β/Smad signal pathway and aimed to prove in vivo therapeutic efficacy in mouse model. Through Smad binding element (SBE) reporter assays, we focused on miR-497a-5p. This miRNA is common between mouse and human species and enhanced the activity of TGF-β/Smad signal pathway, decreased Smad 7 and/or increased phosphorylated Smad 3 expression in non-tumor cell line HEK293, colorectal cancer cell line HCT116 and mouse macrophage J774a.1 cells. MiR-497a-5p also suppressed the production of inflammatory cytokines TNF-α, IL-12p40, a subunit of IL-23, and IL-6 when J774a.1 cells were stimulated by lipopolysaccharides (LPS). In a long-term therapeutic model for mouse dextran sodium sulfate (DSS)-induced colitis, systemic delivery of miR-497a-5p load on super carbonate apatite (sCA) nanoparticle as a vehicle restored epithelial structure of the colonic mucosa and suppressed bowel inflammation compared with negative control miRNA treatment. Our data suggest that sCA-miR-497a-5p may potentially have a therapeutic ability against IBD although further investigation is essential

Tumor-suppressive role of the musculoaponeurotic fibrosarcoma gene in colorectal cancer

Summary: Somatic cell reprogramming using the microRNAs miR-200c, miR-302s, and miR-369s leads to increased expression of cyclin-dependent kinase inhibitors in human colorectal cancer (CRC) cells and suppressed tumor growth. Here, we investigated whether these microRNAs inhibit colorectal tumorigenesis in CPC;Apc mice, which are prone to colon and rectal polyps. Repeated administration of microRNAs inhibited polyp formation. Microarray analysis indicated that c-MAF, which reportedly shows oncogene-like behavior in multiple myeloma and T cell lymphoma, decreased in tumor samples but increased in microRNA-treated normal mucosa. Immunohistochemistry identified downregulation of c-MAF as an early tumorigenesis event in CRC, with low c-MAF expression associated with poor prognosis. Of note, c-MAF expression and p53 protein levels were inversely correlated in CRC samples. c-MAF knockout led to enhanced tumor formation in azoxymethane/dextran sodium sulfate–treated mice, with activation of cancer-promoting genes. c-MAF may play a tumor-suppressive role in CRC development