Epigenetic alterations of TP53INP1 by EHMT2 regulate the cell cycle in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a type of cancer with high incidence and mortality rates. Although various chemical interventions are being developed to treat gastric cancer, there is a constant demand for research into new GC treatment targets and modes of action (MOAs) because of the low effectiveness and side effects of current treatments. METHODS: Using the TCGA data portal, we identified EHMT2 overexpression in GC samples. Using RNA-seq and EHMT2-specific siRNA, we investigated the role of EHMT2 in GC cell proliferation and validated its function with two EHMT2-specific inhibitors. Through the application of 3D spheroid culture, patient-derived gastric cancer organoids (PDOs), and an in vivo model, we confirmed the role of EHMT2 in GC cell proliferation. RESULTS: In this study, we found that EHMT2, a histone 3 lysine 9 (H3K9) methyltransferase, is significantly overexpressed in GC patients compared with healthy individuals. Knockdown of EHMT2 with siRNA induced G1 cell cycle arrest and attenuated GC cell proliferation. Furthermore, we confirmed that TP53INP1 induction by EHMT2 knockdown induced cell cycle arrest and inhibited GC cell proliferation. Moreover, specific EHMT2 inhibitors, BIX01294 and UNC0638, induced cell cycle arrest in GC cell lines through TP53INP1 upregulation. The efficacy of EHMT2 inhibition was further confirmed in a 3D spheroid culture system, PDOs, and a xenograft model. CONCLUSIONS: Our findings suggest that EHMT2 is an attractive therapeutic target for GC treatment.

Maternal nanoplastic ingestion induces an increase in offspring body weight through altered lipid species and microbiota.

The rapidly increasing prevalence of obesity and overweight, especially in children and adolescents, has become a serious societal issue. Although various genetic and environmental risk factors for pediatric obesity and overweight have been identified, the problem has not been solved. In this study, we examined whether environmental nanoplastic (NP) pollutants can act as environmental obesogens using mouse models exposed to NPs derived from polystyrene and polypropylene, which are abundant in the environment. We found abnormal weight gain in the progeny until 6 weeks of age following the oral administration of NPs to the mother during gestation and lactation. Through a series of experiments involving multi-omic analyses, we have demonstrated that NP-induced weight gain is caused by alterations in the lipid composition (lysophosphatidylcholine/phosphatidylcholine ratio) of maternal breast milk and he gut microbiota distribution of the progeny. These data indicate that environmental NPs can act as obesogens in childhood.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis.

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Generation of a human fibroblast-derived induced pluripotent stem cell line KRIBBi009-A from a patient with breast cancer.

Patient-derived human induced pluripotent stem cells (iPSCs) provide a potentially useful resource for studying disease pathology and therapeutics. In this study, we generated the breast cancer patient-derived KRIBBi009-A-iPSC line from normal fibroblasts using the Sendai virus, which expressed pluripotent markers and exhibited differentiation capacity across 3 germ layers through in vitro differentiation and in vivo teratoma assay. A normal karyotype and the absence of cross-contamination of the cell lines were confirmed. Consequently, the developed iPSC line has been confirmed to be suitable for use in various studies.

GPX8 regulates clear cell renal cell carcinoma tumorigenesis through promoting lipogenesis by NNMT.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), with its hallmark phenotype of high cytosolic lipid content, is considered a metabolic cancer. Despite the implication of this lipid-rich phenotype in ccRCC tumorigenesis, the roles and regulators of de novo lipid synthesis (DNL) in ccRCC remain largely unexplained. METHODS: Our bioinformatic screening focused on ccRCC-lipid phenotypes identified glutathione peroxidase 8 (GPX8), as a clinically relevant upstream regulator of DNL. GPX8 genetic silencing was performed with CRISPR-Cas9 or shRNA in ccRCC cell lines to dissect its roles. Untargeted metabolomics, RNA-seq analyses, and other biochemical assays (e.g., lipid droplets staining, fatty acid uptake, cell proliferation, xenograft, etc.) were carried out to investigate the GPX8's involvement in lipid metabolism and tumorigenesis in ccRCC. The lipid metabolic function of GPX8 and its downstream were also measured by isotope-tracing-based DNL flux measurement. RESULTS: GPX8 knockout or downregulation substantially reduced lipid droplet levels (independent of lipid uptake), fatty acid de novo synthesis, triglyceride esterification in vitro, and tumor growth in vivo. The downstream regulator was identified as nicotinamide N-methyltransferase (NNMT): its knockdown phenocopied, and its expression rescued, GPX8 silencing both in vitro and in vivo. Mechanically, GPX8 regulated NNMT via IL6-STAT3 signaling, and blocking this axis suppressed ccRCC survival by activating AMPK. Notably, neither the GPX8-NNMT axis nor the DNL flux was affected by the von Hippel Lindau (VHL) status, the conventional regulator of ccRCC high lipid content. CONCLUSIONS: Taken together, our findings unravel the roles of the VHL-independent GPX8-NNMT axis in ccRCC lipid metabolism as related to the phenotypes and growth of ccRCC, which may be targeted for therapeutic purposes.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer.

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

EHMT1 knockdown induces apoptosis and cell cycle arrest in lung cancer cells by increasing CDKN1A expression.

Dozens of histone methyltransferases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human diseases such as cancer remain largely unclear. Here, we demonstrate the involvement of EHMT1, a histone lysine methyltransferase, in lung cancer. Immunohistochemical analysis indicated that the expression levels of EHMT1 are significantly elevated in human lung carcinomas compared with non-neoplastic lung tissues. Through gene ontology analysis of RNA-seq results, we showed that EHMT1 is clearly associated with apoptosis and the cell cycle process. Moreover, FACS analysis and cell growth assays showed that knockdown of EHMT1 induced apoptosis and G1 cell cycle arrest via upregulation of CDKN1A in A549 and H1299 cell lines. Finally, in 3D spheroid culture, compared to control cells, EHMT1 knockdown cells exhibited reduced aggregation of 3D spheroids and clear upregulation of CDKN1A and downregulation of E-cadherin. Therefore, the results of the present study suggest that EHMT1 plays a critical role in the regulation of cancer cell apoptosis and the cell cycle by modulating CDKN1A expression. Further functional analyses of EHMT1 in the context of human tumorigenesis may aid in the development of novel therapeutic strategies for cancer.

The development of a functional human small intestinal epithelium model for drug absorption.

Advanced technologies are required for generating human intestinal epithelial cells (hIECs) harboring cellular diversity and functionalities to predict oral drug absorption in humans and study normal intestinal epithelial physiology. We developed a reproducible two-step protocol to induce human pluripotent stem cells to differentiate into highly expandable hIEC progenitors and a functional hIEC monolayer exhibiting intestinal molecular features, cell type diversity, and high activities of intestinal transporters and metabolic enzymes such as cytochrome P450 3A4 (CYP3A4). Functional hIECs are more suitable for predicting compounds metabolized by CYP3A4 and absorbed in the intestine than Caco-2 cells. This system is a step toward the transition from three-dimensional (3D) intestinal organoids to 2D hIEC monolayers without compromising cellular diversity and function. A physiologically relevant hIEC model offers a novel platform for creating patient-specific assays and support translational applications, thereby bridging the gap between 3D and 2D culture models of the intestine.

Coiled-coil domain containing 50-V2 protein positively regulates neurite outgrowth.

The coiled-coil domain containing 50 (CCDC50) protein is a phosphotyrosine-dependent signalling protein stimulated by epidermal growth factor. It is highly expressed in neuronal cells in the central nervous system; however, the roles of CCDC50 in neuronal development are largely unknown. In this study, we showed that the depletion of CCDC50-V2 impeded the neuronal development process, including arbor formation, spine density development, and axonal outgrowth, in primary neurons. Mechanistic studies revealed that CCDC50-V2 positively regulated the nerve growth factor receptor, while it downregulated the epidermal growth factor receptor pathway. Importantly, JNK/c-Jun activation was found to be induced by the CCDC50-V2 overexpression, in which the interaction between CCDC50-V2 and JNK2 was also observed. Overall, the present study demonstrates a novel mechanism of CCDC50 function in neuronal development and provides new insight into the link between CCDC50 function and the aetiology of neurological disorders.

Vitrification for cryopreservation of 2D and 3D stem cells culture using high concentration of cryoprotective agents.

BACKGROUND: Vitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation. However, high concentrations (up to ~ 6-8 M) of cryoprotective agents (CPAs) used in stem cell induce osmotic and metabolic injuries. Moreover, the application of conventional slow-freezing methods to cultures of 3-D organoids of stem cells in various studies, is limited by their size. RESULTS: In this study, we evaluated the effect of high concentrations of CPAs including cytotoxicity and characterized human mesenchymal stem cell (MSC) at single cell level. The cell viability, cellular damage, and apoptotic mechanisms as well as the proliferation capacity and multipotency of cells subjected to vitrification were similar to those in the slow-freezing group. Furthermore, we identified the possibility of vitrification of size-controlled 3-D spheroids for cryopreservation of organoid with high survivability. CONCLUSIONS: Our results demonstrate successful vitrification of both single cell and spheroid using high concentration of CPAs in vitro without cytotoxicity.

Ubiquitination of PPAR-gamma by pVHL inhibits ACLY expression and lipid metabolism, is implicated in tumor progression.

BACKGROUND: Intracellular lipid accumulation is associated with various diseases, particularly cancer. Mitochondrial dysfunction is considered as a cause of lipid accumulation; however, the related underlying mechanism remains unclear. FINDINGS: We found that Von Hippel-Lindau (VHL)-deficiency led to lipid accumulation and mitochondrial dysfunction in renal cell carcinoma cells. Moreover, VHL downregulated ATP-citrate lyase (ACLY), a key enzyme in de novo lipid synthesis, at the transcriptional level, which inhibited intracellular lipid accumulation in human renal carcinoma tissues. We identified PPARγ as the transcription factor regulating ACLY expression by binding to the cis-regulatory site PPRE on its promoter. VHL directly interacted with and promoted ubiquitination of PPARγ, leading to its degradation both in vitro and in vivo, resulting in the downregulation of ACLY. Furthermore, adenovirus-mediated VHL overexpression substantially ameliorated hepatic steatosis induced by a high-fat diet in db/db mice. Importantly, low VHL expression was associated with high ACLY expression and poor prognosis in human liver carcinoma in a dataset in The Cancer Genome Atlas. CONCLUSIONS: VHL plays role in cellular lipid metabolism via regulating mitochondria and targeting PPARγ, a transcription factor for ACLY independent of hypoxia-inducible factor 1α. A novel VHL-PPARγ-ACLY axis and its implication in fatty liver disease and cancer were uncovered.

Low-dose interleukin-2 alleviates dextran sodium sulfate-induced colitis in mice by recovering intestinal integrity and inhibiting AKT-dependent pathways.

Several phase 1/2 clinical trials showed that low-dose interleukin-2 (IL-2) treatment is a safe and effective strategy for the treatment of chronic graft-versus-host disease, hepatitis C virus-induced vasculitis, and type 1 diabetes. Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that lacks satisfactory treatment. In this study, we aimed to determine the effects of low-dose IL-2 as a therapeutic for UC on dextran sulfate sodium (DSS)-induced colitis. Methods: Mice with DSS-induced colitis were intraperitoneally injected with low-dose IL-2. Survival, body weight, disease activity index, colon length, histopathological score, myeloperoxidase activity and inflammatory cytokine levels as well as intestinal barrier integrity were examined. Differential gene expression after low-dose IL-2 treatment was analyzed by RNA-sequencing. Results: Low-dose IL-2 significantly improved the symptoms of DSS-induced colitis in mice and attenuated pro-inflammatory cytokine production and immune cell infiltration. The most effective dose range of IL-2 was 16K-32K IU/day. Importantly, low-dose IL-2 was effective in ameliorating the disruption of epithelial barrier integrity in DSS-induced colitis tissues by restoring tight junction proteins and mucin production and suppressing apoptosis. The colon tissue of DSS-induced mice exposed to low-dose IL-2 mimic gene expression patterns in the colons of control mice. Furthermore, we identified the crucial role of the PI3K-AKT pathway in exerting the therapeutic effect of low-dose IL-2. Conclusions: The results of our study suggest that low-dose IL-2 has therapeutic effects on DSS-induced colitis and potential clinical value in treating UC.

A three-dimensional hyaluronic acid-based niche enhances the therapeutic efficacy of human natural killer cell-based cancer immunotherapy.

Adoptive transfer of natural killer (NK) cells is becoming one of the most important parts of cancer immunotherapy. However, recent accomplishments have focused on the improvement of the targeting effects based on the engineering of chimeric antigen receptors (CARs) on cell surfaces. Despite the large quantity of therapeutic cells required for clinical applications, the technology for ex vivo expansion is not well developed. Herein, a three-dimensional (3D) engineered hyaluronic acid-based niche for cell expansion (3D-ENHANCE) is introduced. Compared with the conventional two-dimensional (2D) method, NK-92 cell lines and human EGFR-specific (CAR)-NK cells cultured in 3D-ENHANCE yield favorable mRNA expressions, elevated cytokine release, upregulated proliferative and tumor-lytic abilities, and result in enhanced antitumor efficacy. Furthermore, controllable degradation rates can be realized by tuning the formulation of 3D-ENHANCE so that it can be applied as an implantable cell reservoir at surgical sites. In vivo results with the incompletely resected MDA-MB-231 model confirm that the peri-operative implantation of 3D-ENHANCE prevents the relapse and metastases after surgery. Overall, 3D-ENHANCE presents an effective cytokine-free niche for ex vivo expansion and postsurgical treatment that enhances the low-therapeutic efficacy of human NK cells.

RNA-seq based transcriptome analysis of EHMT2 functions in breast cancer.

For breast cancer treatment, hormone therapy is effective for hormone receptor-positive breast cancer but not for TNBC (triple-negative breast cancer). Thus, many researchers have attempted to identify more effective therapeutic candidates for all subtypes of breast cancer. In this study, we established an RNA-seq analytical pipeline to analyze the subtype-specific functions of EHMT2 in the MB231 and MCF7 cell lines. After EHMT2 knockdown, we identified subtype-specific DEGs (differentially expressed genes) and overlapping DEGs. Through GO (Gene Ontology) analysis, GSEA (gene set enrichment analysis), and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis using the DEGs, we identified the subtype-specific functions of EHMT2 in the MB231 and MCF7 cell lines. Therefore, herein, we suggest that EHMT2 is an attractive therapeutic target for the treatment of all types of breast cancer.

TIPRL potentiates survival of lung cancer by inducing autophagy through the eIF2α-ATF4 pathway.

Autophagy, an intracellular system of degrading damaged organelles and misfolded proteins, is essential for cancer cell survival. Despite the progress made towards understanding the mechanism, identification of novel autophagy regulators presents a major obstacle in developing anticancer therapies. Here, we examine the association between the TOR signaling pathway regulator-like (TIPRL) protein and autophagy in malignant transformation of tumors. We show that TIPRL upregulation in non-small cell lung cancer (NSCLC) potentiated autophagy activity and enabled autophagic clearance of metabolic and cellular stress, conferring a survival advantage to cancer cells. Importantly, the interaction of TIPRL with eukaryotic initiation factor 2α (eIF2α) led to eIF2α phosphorylation and activation of the eIF2α-ATF4 pathway, thereby inducing autophagy. Conversely, TIPRL depletion increased apoptosis by reducing autophagic clearance, which was markedly enhanced in TIPRL-depleted A549 xenografts treated with 2-deoxy-D-glucose. Overall, the study indicated that TIPRL is a potential regulator of autophagy and an important drug target for lung cancer therapy.

Effective reconstruction of functional organotypic kidney spheroid for in vitro nephrotoxicity studies.

Stable and reproducible kidney cellular models could accelerate our understanding of diseases, help therapeutics development, and improve nephrotoxicity screenings. Generation of a reproducible in vitro kidney models has been challenging owing to the cellular heterogeneity and structural complexity of the kidney. We generated mixed immortalized cell lines that stably maintained their characteristic expression of renal epithelial progenitor markers for the different lineages of kidney cellular compartments via the BMP7 signaling pathway from a mouse and a human whole kidney. These cells were used to generate functional and matured kidney spheroids containing multiple renal lineages, such as the proximal tubule, loop of Henle, distal tubules, and podocytes, using extracellular matrix and physiological force, named spheroid-forming unit (SFU). They expressed all apical and basolateral transporters that are important for drug metabolism and displayed key functional aspects of the proximal tubule, including protein endocytosis and increased gamma-glutamyltransferase activity, and cyclic AMP responded to external cues, such as parathyroid hormone. Following exposure, cells fluxed and took up drugs via proximal tubule-specific apical or basolateral transporters, and displayed increased cell death and expression of renal injury marker. Here, we developed a new differentiation method to generate kidney spheroids that structurally recapitulate important features of the kidney effectively and reproducibly using mixed immortalized renal cells, and showed their application for renal toxicity studies.

Propionate of a microbiota metabolite induces cell apoptosis and cell cycle arrest in lung cancer.

Short­chain fatty acids (SCFAs; butyrate, propionate and acetate) are metabolites derived from the gut microbiota via dietary fiber fermentation. In colon cancer, treatment with SCFAs, mainly butyrate and propionate, suppresses cell proliferation, migration and invasion. Furthermore, although sodium butyrate is known to induce cell apoptosis in lung cancer, the anticancer effects of sodium propionate (SP) on lung cancer are not well understood. In the present study, SP treatment induced cell cycle arrest, especially in the G2/M phase, and cell apoptosis in the H1299 and H1703 lung cancer cell lines. As determined by reverse transcription­quantitative PCR and western blotting, Survivin and p21 expression levels were significantly affected by SP treatment, suggesting that SP treatment suppressed cell proliferation in these lung cancer cell lines. Thus, it was proposed that the SP­mediated regulation of Survivin and p21 in lung cancer may be applicable to lung cancer therapy.

Blockade of STAT3 Causes Severe In Vitro and In Vivo Maturation Defects in Intestinal Organoids Derived from Human Embryonic Stem Cells.

Human intestinal organoids (hIOs), which resemble the human intestine structurally and physiologically, have emerged as a new modality for the study of the molecular and cellular biology of the intestine in vitro. We recently developed an in vitro maturation technique for generating functional hIOs from human pluripotent stem cells (hPSCs). Here, we investigated the function of STAT3 for inducing in vitro maturation of hIOs. This was accompanied by the tyrosine phosphorylation of STAT3, whereas treatment with pharmacological inhibitors of STAT3 suppressed the phosphorylation of STAT3 and the expression of intestinal maturation markers. We generated and characterized STAT3 knockout (KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9-mediated gene editing. We found that STAT3 KO does not affect the differentiation of hESCs into hIOs but rather affects the in vitro maturation of hIOs. STAT3 KO hIOs displayed immature morphologies with decreased size and reduced budding in hIOs even after in vitro maturation. STAT3 KO hIOs showed markedly different profiles from hIOs matured in vitro and human small intestine. Additionally, STAT3 KO hIOs failed to maintain upon in vivo transplantation. This study reveals a core signaling pathway consisting of STAT3 controlling the in vitro maturation of hIOs derived from hPSCs.

Ubiquitination of MAP1LC3B by pVHL is associated with autophagy and cell death in renal cell carcinoma.

Von Hippel Lindau (VHL) expression is significantly decreased in high-grade RCC, and autophagy, which is involved in tumor growth, invasion, differentiation, and metastasis, is activated in various human cancers. However, the relationship of autophagy and VHL in tumor progression remains controversial. Here, we showed that the expression levels of VHL and microtubule-associated protein 1 light chain 3B (MAP1LC3B, LC3B) were inversely correlated with various tumor grades of RCC tissues. pVHL was found to possess the LIR motif within a beta domain that interacted with MAP1LC3B and ubiquitinated it. The L101A VHL mutant failed to interact with MAP1LC3B, thereby failing to induce ubiquitination. MAP1LC3B-mediated autophagy was inhibited by functional pVHL and the ubiquitination of MAPLC3B was implicated in autophagy-induced cell death. We screened various autophagy inducers to determine the physiological function of the inhibition of LC3B-mediated autophagy by pVHL using VHL-deficient and VHL-expressing cell lines. MLN9708, a proteasome inhibitor, potently induced autophagy via the induction of MAP1LC3B and sensitized the cell to autophagy-mediated cell death in VHL-deficient and VHL-mutant (L101A) cells. In conclusion, our results showed that pVHL interacts with MAPL1LC3B and inhibits LC3B-mediated autophagy via MAP1LC3B ubiquitination. Furthermore, the activation of autophagy by the proteasome inhibitor MLN9708 induced cell death, indicating that MLN9708 can be used for VHL-deficient RCC therapy.

Stabilization of E2-EPF UCP protein is implicated in hepatitis B virus-associated hepatocellular carcinoma progression.

Hepatitis B virus (HBV) X protein (HBx) is associated with hepatocarcinogenesis. E2-EPF ubiquitin carrier protein (UCP) catalyzes ubiquitination of itself and von Hippel-Lindau protein (pVHL) for degradation and associates with tumor growth and metastasis. However, it remains unknown whether HBx modulates the enzyme activity of UCP and thereby influences hepatocarcinogenesis. Here, we show that UCP is highly expressed in liver tissues of HBx-transgenic mice, but not non-transgenic mice. UCP was more frequently expressed in HBV-positive liver cancers than in HBV-negative liver cancers. HBx binds to UCP specifically and serotype independently, and forms a ternary complex with UCP and pVHL. HBx inhibits self-ubiquitination of UCP, but enhances UCP-mediated pVHL ubiquitination, resulting in stabilization of hypoxia-inducible factor-1α and -2α. HBx and UCP stabilize each other by mutually inhibiting their ubiquitination. HBx promotes cellular proliferation and metastasis via UCP. Our findings suggest that UCP plays a key role in HBV-related hepatocarcinogenesis.