Txnip regulates the Oct4-mediated pluripotency circuitry via metabolic changes upon differentiation.

Thioredoxin interacting protein (Txnip) is a stress-responsive factor regulating Trx1 for redox balance and involved in diverse cellular processes including proliferation, differentiation, apoptosis, inflammation, and metabolism. However, the biological role of Txnip function in stem cell pluripotency has yet to be investigated. Here, we reveal the novel functions of mouse Txnip in cellular reprogramming and differentiation onset by involving in glucose-mediated histone acetylation and the regulation of Oct4, which is a fundamental component of the molecular circuitry underlying pluripotency. During reprogramming or PSC differentiation process, cellular metabolic and chromatin remodeling occur in order to change its cellular fate. Txnip knockout promotes induced pluripotency but hinders initial differentiation by activating pluripotency factors and promoting glycolysis. This alteration affects the intracellular levels of acetyl-coA, a final product of enhanced glycolysis, resulting in sustained histone acetylation on active PSC gene regions. Moreover, Txnip directly interacts with Oct4, thereby repressing its activity and consequently deregulating Oct4 target gene transcriptions. Our work suggests that control of Txnip expression is crucial for cell fate transitions by modulating the entry and exit of pluripotency.

Immunotherapy in Hematologic Malignancies: Emerging Therapies and Novel Approaches.

Immunotherapy is extensively investigated for almost all types of hematologic tumors, from preleukemic to relapse/refractory malignancies. Due to the emergence of technologies for target cell characterization, antibody design and manufacturing, as well as genome editing, immunotherapies including gene and cell therapies are becoming increasingly elaborate and diversified. Understanding the tumor immune microenvironment of the target disease is critical, as is reducing toxicity. Although there have been many successes and newly FDA-approved immunotherapies for hematologic malignancies, we have learned that insufficient efficacy due to disease relapse following treatment is one of the key obstacles for developing successful therapeutic regimens. Thus, combination therapies are also being explored. In this review, immunotherapies for each type of hematologic malignancy will be introduced, and novel targets that are under investigation will be described.

Regulation of Hematopoietic Stem Cell Fate and Malignancy.

The regulation of hematopoietic stem cell (HSC) fate decision, whether they keep quiescence, self-renew, or differentiate into blood lineage cells, is critical for maintaining the immune system throughout one's lifetime. As HSCs are exposed to age-related stress, they gradually lose their self-renewal and regenerative capacity. Recently, many reports have implicated signaling pathways in the regulation of HSC fate determination and malignancies under aging stress or pathophysiological conditions. In this review, we focus on the current understanding of signaling pathways that regulate HSC fate including quiescence, self-renewal, and differentiation during aging, and additionally introduce pharmacological approaches to rescue defects of HSC fate determination or hematopoietic malignancies by kinase signaling pathways.

TXNIP Regulates Natural Killer Cell-Mediated Innate Immunity by Inhibiting IFN-γ Production during Bacterial Infection.

The function of natural killer (NK) cell-derived interferon-γ (IFN-γ) expands to remove pathogens by increasing the ability of innate immune cells. Here, we identified the critical role of thioredoxin-interacting protein (TXNIP) in the production of IFN-γ in NK cells during bacterial infection. TXNIP inhibited the production of IFN-γ and the activation of transforming growth factor ß-activated kinase 1 (TAK1) activity in primary mouse and human NK cells. TXNIP directly interacted with TAK1 and inhibited TAK1 activity by interfering with the complex formation between TAK1 and TAK1 binding protein 1 (TAB1). Txnip-/- (KO) NK cells enhanced the activation of macrophages by inducing IFN-γ production during Pam3CSK4 stimulation or Staphylococcus aureus (S. aureus) infection and contributed to expedite the bacterial clearance. Our findings suggest that NK cell-derived IFN-γ is critical for host defense and that TXNIP plays an important role as an inhibitor of NK cell-mediated macrophage activation by inhibiting the production of IFN-γ during bacterial infection.

TSPYL5-mediated inhibition of p53 promotes human endothelial cell function.

Testis-specific protein, Y-encoded like (TSPYL) family proteins (TSPYL1-6), which are members of the nucleosome assembly protein superfamily, have been determined to be involved in the regulation of various cellular functions. However, the potential role of TSPYL family proteins in endothelial cells (ECs) has not been determined. Here, we demonstrated that the expression of TSPYL5 is highly enriched in human ECs such as human umbilical vein endothelial cells (HUVECs) and human pluripotent stem cell-differentiated ECs (hPSC-ECs). Importantly, TSPYL5 overexpression was shown to promote EC proliferation and functions, such as migration and tube formation, by downregulating p53 expression. Adriamycin-induced senescence was markedly blocked by TSPYL5 overexpression. In addition, the TSPYL5 depletion-mediated loss of EC functions was blocked by p53 inhibition. Significantly, TSPYL5 overexpression promoted angiogenesis in Matrigel plug and wound repair in a mouse skin wound healing model in vivo. Our results suggest that TSPYL5, a novel angiogenic regulator, plays a key role in maintaining endothelial integrity and function. These findings extend the understanding of TSPYL5-dependent mechanisms underlying the regulation of p53-related functions in ECs.

ESRP1-Induced CD44 v3 Is Important for Controlling Pluripotency in Human Pluripotent Stem Cells.

The importance of alternative splicing (AS) events in pluripotency regulation has been highlighted by the determination of different roles and contributions of different splice isoforms of pluripotency-related genes and by the identification of distinct pluripotency-related splicing factors. In particular, epithelial splicing regulatory protein 1 (ESRP1) has been characterized as an essential splicing factor required for the regulation of human pluripotency and differentiation. Nevertheless, a detailed molecular characterization of ESRP1 (mRNA splice variants 1-6) in human pluripotency is lacking. In this study, we determined that ESRP1 splice variants are differentially expressed in undifferentiated and differentiated human pluripotent stem cells (PSCs). Undifferentiated human PSCs predominantly expressed the ESRP1 v1, v4, and v5, and their expression was downregulated upon differentiation. Ectopic expression of ESRP1 v1, v4, or v5 enhanced the pluripotent reprogramming of human fibroblasts and restored the ESRP1 knockdown-mediated reduction of reprogramming efficiency. Notably, undifferentiated human PSCs expressed the cell surface protein CD44 variant 3 (CD44 v3), and isoform switching from CD44 v3 to CD44 variant 6 (CD44 v6) occurred upon differentiation. Importantly, the human PSC-specific ESRP1 variants influenced CD44 v3 expression. CD44 knockdown or inhibition of binding of CD44 with its major ligand, hyaluronan, significantly induced the loss of human PSC pluripotency and the reduction of reprogramming efficiency. Our results demonstrate that the effect of ESRP1 and CD44 on human PSC pluripotency is isoform-dependent and that ESRP1-induced CD44 v3 is functionally associated with human PSC pluripotency control. Stem Cells 2018;36:1525-1534.

Causes and Mechanisms of Hematopoietic Stem Cell Aging.

Many elderly people suffer from hematological diseases known to be highly age-dependent. Hematopoietic stem cells (HSCs) maintain the immune system by producing all blood cells throughout the lifetime of an organism. Recent reports have suggested that HSCs are susceptible to age-related stress and gradually lose their self-renewal and regeneration capacity with aging. HSC aging is driven by cell-intrinsic and -extrinsic factors that result in the disruption of the immune system. Thus, the study of HSC aging is important to our understanding of age-related immune diseases and can also provide potential strategies to improve quality of life in the elderly. In this review, we delineate our understanding of the phenotypes, causes, and molecular mechanisms involved in HSC aging.

TLR4/NF-κB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells.

Overcoming drug resistance is one of key issues in treating refractory acute myeloid leukemia (AML). The Toll-like receptor 4 (TLR4) signaling pathway is involved in many aspects of biological functions of AML cells, including the regulation of pro-inflammatory cytokine products, myeloid differentiation, and survival of AML cells. Thus, targeting TLR4 of AML patients for therapeutic purposes should be carefully addressed. In this regard, we investigated the possible role of TLR4 as a regulatory factor against fludarabine (FA) cytotoxicity activity. Here, we identified the differential expression of TLR4 and CD14 receptors in AML cell lines and examined their relationship to FA sensitivity. We found that the stimulation of TLR4 with lipopolysaccharide (LPS) in a TLR4-expressing cell line, THP-1, increased cell viability under FA treatment condition and showed that TLR4 stimulation overcame FA sensitivity through the activation of NF-κB, which subsequently upregulated several anti-apoptotic genes. The inhibition of TLR4/NF-κB signaling could partially or completely reverse LPS-induced cell survival under FA treatment conditions. Interestingly, we found that the expression of thioredoxin-interacting protein (TXNIP), a well-known tumor suppressor, was induced by FA treatment; however, it was suppressed by LPS treatment. Furthermore, the expression level of TXNIP was critical for FA-induced cytotoxicity or LPS-induced FA resistance of THP-1 cells. Our data suggest that TXNIP plays an important role in FA-induced cytotoxicity and TLR4/NF-κB-mediated FA resistance of AML cells. Therefore, TXNIP may be a potential therapeutic target for AML treatment.

Kinetic Mechanism of Formation of Hyperactive Embryonic Ras in Cells.

Embryonic Ras (ERas)--a new subset of Ras proteins--are characterized by a unique p-loop residue, unique Switch II residues, and an unusual extended N-terminus. When expressed, both murine and human ERas are highly populated in their GTP-bound forms. The expression of murine ERas is linked to the development of murine embryonic cells, and the expression of human ERas is correlated to certain human cancers. Mutation-based kinetic analyses, in combination with assessments of the kinetic parameter-based calculation of the fraction of the GTP-bound active form of ERas proteins, explain the kinetic mechanism that produces the unprecedented hyperactive ERas. The ERas-specific p-loop residue contributes ERas proteins to intrinsically populate their GTP-bound form in cells. Furthermore, the ERas-specific Switch II residues block the catalytic action of p120GAP on ERas proteins. This blockage sustains the previously mentioned GTP-bound ERas proteins. In essence, the combined work of the ERas-specific p-loop and Switch II residues populates the exceedingly high GTP-bound form of ERas in cells. This study also rules out any kinetic function of the unique ERas-specific N-terminus in the production of the hyperactive GTP-bound ERas in cells. The biological role of this N-terminus remains uninvestigated. Intriguingly, the ERas-specific p-loop residue matches the mutated Ser residue of the Costello Syndrome G12S HRas mutant that also intrinsically populates its GTP-bound form in cells. However, because the effector protein of ERas differs from that of G12S HRas, this kinetic similarity does not confer on ERas biological and/or pathophysiological similarity to G12S HRas.

Development of pluripotent stem cell-derived epidermal organoids that generate effective extracellular vesicles in skin regeneration.

Cellular skin substitutes such as epidermal constructs have been developed for various applications, including wound healing and skin regeneration. These cellular models are mostly derived from primary cells such as keratinocytes and fibroblasts in a two-dimensional (2D) state, and further development of three-dimensional (3D) cultured organoids is needed to provide insight into the in vivo epidermal phenotype and physiology. Here, we report the development of epidermal organoids (EpiOs) generated from induced pluripotent stem cells (iPSCs) as a novel epidermal construct and its application as a source of secreted biomolecules recovered by extracellular vesicles (EVs) that can be utilized for cell-free therapy of regenerative medicine. Differentiated iPSC-derived epidermal organoids (iEpiOs) are easily cultured and expanded through multiple organoid passages, while retaining molecular and functional features similar to in vivo epidermis. These mature iEpiOs contain epidermal stem cell populations and retain the ability to further differentiate into other skin compartment lineages, such as hair follicle stem cells. By closely recapitulating the epidermal structure, iEpiOs are expected to provide a more relevant microenvironment to influence cellular processes and therapeutic response. Indeed, iEpiOs can generate high-performance EVs containing high levels of the angiogenic growth factor VEGF and miRNAs predicted to regulate cellular processes such as proliferation, migration, differentiation, and angiogenesis. These EVs contribute to target cell proliferation, migration, and angiogenesis, providing a promising therapeutic tool for in vivo wound healing. Overall, the newly developed iEpiOs strategy as an organoid-based approach provides a powerful model for studying basic and translational skin research and may also lead to future therapeutic applications using iEpiOs-secreted EVs.

Kinetic mechanisms of mutation-dependent Harvey Ras activation and their relevance for the development of Costello syndrome.

Costello syndrome is linked to activating mutations of a residue in the p-loop or the NKCD/SAK motifs of Harvey Ras (HRas). More than 10 HRas mutants that induce Costello syndrome have been identified; G12S HRas is the most prevalent of these. However, certain HRas p-loop mutations also are linked to cancer formation that are exemplified with G12V HRas. Despite these relations, specific links between types of HRas mutations and diseases evade definition because some Costello syndrome HRas p-loop mutations, such as G12S HRas, also often cause cancer. This study established novel kinetic parameter-based equations that estimate the value of the cellular fractions of the GTP-bound active form of HRas mutant proteins. Such calculations differentiate between two basic kinetic mechanisms that populate the GTP-bound form of Ras in cells. (i) The increase in the level of GTP-bound Ras is caused by the HRas mutation-mediated perturbation of the intrinsic kinetic characteristics of Ras. This generates a broad spectrum of the population of the GTP-bound form of HRas that typically causes Costello syndrome. The upper end of this spectrum of HRas mutants, as exemplified by G12S HRas, can also cause cancer. (ii) The increase in the level of GTP-bound Ras occurs because the HRas mutations perturb the action of p120GAP on Ras. This causes production of a significantly high population of the only GTP-bound form of HRas linked merely to cancer formation. HRas mutant G12V belongs to this category.

A novel small molecule facilitates the reprogramming of human somatic cells into a pluripotent state and supports the maintenance of an undifferentiated state of human pluripotent stem cells.

Booster of pluripotency: RSC133, a new synthetic derivative of indoleacrylic acid/indolepropionic acid, exhibits dual activity by inhibiting histone deacetylase and DNA methyltransferase. Furthermore it potently improves the reprogramming of human somatic cells into a pluripotent state and aids the growth and maintenance of human pluripotent stem cells (hPSCs).

Direct Laser Interference Ink Printing Using Copper Metal-Organic Decomposition Ink for Nanofabrication.

In this study, we developed an effective and rapid process for nanoscale ink printing, direct laser interference ink printing (DLIIP), which involves the photothermal reaction of a copper-based metal-organic decomposition ink. A periodically lined copper pattern with a width of 500 nm was printed on a 240 µm-wide line at a fabrication speed of 17 mm/s under an ambient environment and without any pre- or post-processing steps. This pattern had a resistivity of 3.5 µΩ∙cm, and it was found to exhibit a low oxidation state that was twice as high as that of bulk copper. These results demonstrate the feasibility of DLIIP for nanoscale copper printing with fine electrical characteristics.

Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host.

Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) are the major virulence factors responsible for hemorrhagic colitis, which can lead to life-threatening systemic complications including acute renal failure (hemolytic uremic syndrome) and neuropathy. Here, we report that O-GlcNAcylation, a type of post-translational modification, was acutely increased upon induction of endoplasmic reticulum (ER) stress in host cells by Stxs. Suppression of the abnormal Stx-mediated increase in O-GlcNAcylation effectively inhibited apoptotic and inflammatory responses in Stx-susceptible cells. The protective effect of O-GlcNAc inhibition for Stx-mediated pathogenic responses was also verified using three-dimensional (3D)-cultured spheroids or organoids mimicking the human kidney. Treatment with an O-GlcNAcylation inhibitor remarkably improved the major disease symptoms and survival rate for mice intraperitoneally injected with a lethal dose of Stx. In conclusion, this study elucidates O-GlcNAcylation-dependent pathogenic mechanisms of Stxs and demonstrates that inhibition of aberrant O-GlcNAcylation is a potential approach to treat Stx-mediated diseases.

Synaptotagmin 11 scaffolds MKK7-JNK signaling process to promote stem-like molecular subtype gastric cancer oncogenesis.

BACKGROUND: Identifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC. METHODS: In vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient's survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation. SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis. RESULTS: SYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover, SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC. SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells. CONCLUSION: SYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

Oct-4 controls cell-cycle progression of embryonic stem cells.

Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the 'stemness' of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation.

Wnt/ß-catenin Signaling Inhibitors suppress the Tumor-initiating properties of a CD44+CD133+ subpopulation of Caco-2 cells.

Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44-CD133-, CD44-CD133+, and CD44+CD133+ subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44+CD133+ cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44+CD133+ Caco-2 cells contained mixed populations of CD44+CD133+ and non-CD44+CD133+ Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44+CD133+ subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44+CD133+ cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/ß-catenin pathway was over-activated in CD44+CD133+ cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/ß-catenin signaling inhibitors. Our findings suggest that the CD44+CD133+ subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development.

Altered Gene Expression Profiles in Neural Stem Cells Derived from Duchenne Muscular Dystrophy Patients with Intellectual Disability.

Intellectual disability (ID) is a neurodevelopmental disorder defined by below-average intelligence (intelligence quotient of <70) accompanied by adaptive behavior deficits. Defects in the functions of neural stem cells during brain development are closely linked to the pathogenesis of ID. To understand the molecular etiology of ID, we examined neural stem cells from individuals with Duchenne muscular dystrophy (DMD), a genetic disorder in which approximately one-third of the patients exhibit ID. In this study, we generated induced pluripotent stem cells from peripheral blood mononuclear cells from a normal individual and DMD patients with and without ID to identify ID-specific functional and molecular abnormalities. We found defects in neural ectoderm formation in the group of DMD patients with ID. Our transcriptome analysis of patient-derived neural stem cells revealed altered expression of genes related to the hippo signaling pathway and neuroactive ligand-receptor interaction, implicating these in the pathogenesis of ID in patients with DMD.

Mutation in the DNA-binding domain of the EWS-Oct-4 oncogene results in dominant negative activity that interferes with EWS-Oct-4-mediated transactivation.

The EWS-Oct-4 protein is a chimeric molecule in which the amino terminal domain (NTD) of the EWS becomes fused to the carboxy terminal domain (CTD) of the Oct-4 transcription factor. It was identified in human bone and soft-tissue tumors associated with t(6;22)(p21;q12). Using in vitro and in vivo systems, we found that the EWS-Oct-4 protein self-associates. The major domains required for self-association mapped to the EWS NTD (amino acids 70-163) and the POU DNA-binding domain. EWS-Oct-4 protein also associated with EWS-Oct-4 (V351P), which contains a mutation in the POU DNA-binding domain. Using electrophoretic mobility shift assays, we found that the EWS-Oct-4 (V351P) mutant interfered with wild-type EWS-Oct-4 DNA-binding activity. In addition, we found that EWS-Oct-4-mediated transcriptional activation was inhibited by EWS-Oct-4 (V351P) protein in vivo. Thus, this mutation in the POU DNA-binding domain results in a dominant negative protein. These findings suggest that the biological functions of the EWS-Oct-4 oncogene can be modulated by the dominant negative mutant EWS-Oct-4 (V351P).