Epigenetic Silencing of CHOP Expression by the Histone Methyltransferase EHMT1 Regulates Apoptosis in Colorectal Cancer Cells

Colorectal cancer (CRC) has a high mortality rate among cancers worldwide. To reduce this mortality rate, chemotherapy (5-fluorouracil, oxaliplatin, and irinotecan) or targeted therapy (bevacizumab, cetuximab, and panitumumab) has been used to treat CRC. However, due to various side effects and poor responses to CRC treatment, novel therapeutic targets for drug development are needed. In this study, we identified the overexpression of EHMT1 in CRC using RNA sequencing (RNA-seq) data derived from TCGA, and we observed that knocking down EHMT1 expression suppressed cell growth by inducing cell apoptosis in CRC cell lines. In Gene Ontology (GO) term analysis using RNA-seq data, apoptosis-related terms were enriched after EHMT1 knockdown. Moreover, we identified the CHOP gene as a direct target of EHMT1 using a ChIP (chromatin immunoprecipitation) assay with an anti-histone 3 lysine 9 dimethylation (H3K9me2) antibody. Finally, after cotransfection with siEHMT1 and siCHOP, we again confirmed that CHOP-mediated cell apoptosis was induced by EHMT1 knockdown. Our findings reveal that EHMT1 plays a key role in regulating CRC cell apoptosis, suggesting that EHMT1 may be a therapeutic target for the development of cancer inhibitors.     

Re-Identification on Korean Penicillium Sequences in GenBank Collected by Software GenMine

Penicillium species have been actively studied in various fields, and many new and unrecorded species continue to be reported in Korea. Moreover, unidentified and misidentified Korean Penicillium species still exist in GenBank. Therefore, it is necessary to revise the Korean Penicillium inventory based on accurate identification. We collected Korean Penicillium nucleotide sequence records from GenBank using the newly developed software, GenMine, and re-identified Korean Penicillium based on the maximum likelihood trees. A total of 1681 Korean Penicillium GenBank nucleotide sequence records were collected from GenBank. In these records, 1208 strains with four major genes (Internal Transcribed Spacer rDNA region, β-tubulin, Calmodulin and RNA polymerase II) were selected for Penicillium re-identification. Among 1208 strains, 927 were identified, 82 were identified as other genera, the rest remained undetermined due to low phylogenetic resolution. Identified strains consisted of 206 Penicillium species, including 156 recorded species and 50 new species candidates. However, 37 species recorded in the national list of species in Korea were not found in GenBank. Further studies on the presence or absence of these species are required through literature investigation, additional sampling, and sequencing. Our study can be the basis for updating the Korean Penicillium inventory.     

API-2-Induced Cell Migration Is Overcome by Small Molecular Approaches Inhibiting β-Catenin

Frequent mutation of APC (90%) in advanced colorectal cancer (CRC) results in the simultaneous activation of Wnt/β-catenin and AKT signaling pathways, and the current therapeutic limitations of the AKT inhibitors for treating CRC patients are nuclear β-catenin-induced EMT and bypassing apoptosis. In this study, we discover that the combinatorial treatment of an AKT inhibitor and KY1022, a β-catenin destabilizer, effectively overcomes the current limitations of API-2, an AKT inhibitor, by reducing nuclear β-catenin. Taken together, we demonstrate that the simultaneous suppression of Wnt/β-catenin with the AKT signaling pathways is an ideal strategy for suppressing the AKT-inhibitor-mediated metastasis and for maximizing the therapeutic effects of AKT inhibitors.     

Checkpoint Kinase 1 (CHK1) Functions as Both a Diagnostic Marker and a Regulator of Epithelial-to-Mesenchymal Transition (EMT) in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is more difficult to treat and has a higher mortality rate than other subtypes. Although hormone receptor-targeted therapy is an effective treatment to increase survival rate in breast cancer patients, it is not suitable for TNBC patients. To address the issues, differentially expressed genes (DEGs) in TNBC patients from the Gene Expression Omnibus (GEO) database were analyzed. A total of 170 genes were obtained from three Genomic Spatial Events (GSEs) using the intersection of each GSE dataset and 61 DEGs were identified after validation with the gene enrichment analysis. We combined this with the degree scores from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction (PPI) network, of which 7 genes were correlated with survival rate. Finally, a proteomics database revealed that only the CHK1 protein level was differently expressed in basal-like compared with other subtypes. We demonstrated that CHK1 expression was higher in TNBC cell lines compared with non-TNBC cell lines, and CHK1 promotes epithelial to mesenchymal transition (EMT) as well as migration and invasion ability. Our study provides new insight into the TNBC subnetwork that may be useful in the prognosis and treatment of TNBC patients.     

Selective cytotoxicity of a novel mitochondrial complex I inhibitor,YK-135, against EMT-subtype gastric cancer cell lines due to impaired glycolytic capacity

Epithelial-to-mesenchymal transition (EMT)-subtype gastric cancers have the worst prognosis due to their higher recurrence rate, higher probability of developing metastases and higher chemo-resistance compared to those of other molecular subtypes. Pharmacologically actionable somatic mutations are rarely found in EMT-subtype gastric cancers, limiting the utility of targeted therapies. Here, we conducted a high-throughput chemical screen using 37 gastric cancer cell lines and 48,467 synthetic small-molecule compounds. We identified YK-135, a small-molecule compound that showed higher cytotoxicity toward EMT-subtype gastric cancer cell lines than toward non-EMT-subtype gastric cancer cell lines. YK-135 exerts its cytotoxic effects by inhibiting mitochondrial complex I activity and inducing AMP-activated protein kinase (AMPK)-mediated apoptosis. We found that the lower glycolytic capacity of the EMT-subtype gastric cancer cells confers synthetic lethality to the inhibition of mitochondrial complex I, possibly by failing to maintain energy homeostasis. Other well-known mitochondrial complex I inhibitors (e.g., rotenone and phenformin) mimic the efficacy of YK-135, supporting our results. These findings highlight mitochondrial complex I inhibitors as promising therapeutic agents for EMT-subtype gastric cancers and YK-135 as a novel chemical scaffold for further drug development.     

A novel pathway responsible for lipopolysaccharide-induced translational regulation of TNF-α and IL-6 expression involves protein kinase C and fascin

Fascin, as a substrate of protein kinase C (PKC), is a well-known cytoskeletal regulatory protein required for cell migration, invasion, and adhesion in normal and cancer cells. In an effort to identify the role of fascin in PKC-mediated cellular signaling, its expression was suppressed by stable transfection of specific short hairpin RNAs (shRNAs) in mouse monocytic leukemia RAW264.7 cells. Suppression of fascin expression resulted in impaired cellular migration and invasion through extracellular matrix proteins. Unexpectedly, the specific shRNA transfectants exhibited a marked reduction in LPS-induced expression of TNF-α and IL-6 by blocking the translation of their mRNAs. Transient transfection assay using a luciferase expression construct containing the 3' untranslated region of TNF-α or IL-6 mRNA revealed a significant reduction in both LPS- and PMA- (the direct activator of PKC) induced reporter activity in cells transfected with fascin-specific shRNA, indicating that fascin-mediated translational regulation targeted 3' untranslated region. Furthermore, LPS-induced translational activation of reporter expression was blocked by a pharmacological inhibitor of PKC, and the dominant-negative form of PKCα attenuated LPS-induced translational activation. The same type of regulation was also observed in the human monocytic leukemia cell line THP-1 and in mouse peritoneal macrophages. These data demonstrate the involvement of fascin in the PKC-mediated translational regulation of TNF-α and IL-6 expression during the LPS response.