The Effects of Busulfan on Xenogeneic Transplantation of Human Peripheral Blood Mononuclear Cells in Recipient Mice.

BACKGROUND: Humanized mouse models with engraftment of human peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs) are effective tools for the study of human immunity. Busulfan has been used as a substitute for irradiation in human hematopoietic stem cell (HSC) transplantation models, but it has not been tested in human peripheral blood mononuclear cell (PBMC) transplantation models. METHODS: This study evaluated PBMC engraftment using cytometry and enzyme-linked immunosorbent assay (ELISA) in female NOD.CB17/Prkdcscid/JKrb/ IL2 receptor γ-/- (NIG) mice treated with busulfan. RESULTS: In this model, the percentage of human CD3+ T cell engraftment in the blood was 28.2%, with dominant infiltration of CD8+ cells in the spleen 3 weeks post PBMC transplantation. Production of human cytokines, including Interleukin (IL)-12p70, IL-4, IL-5, IFN-γ, IL-6, IL-8, IL-22, Tumor Necrosis Factor alpha, and IL-10, was determined in mice treated with busulfan. CONCLUSIONS: Our findings demonstrate that busulfan treatment is a beneficial alternative for simple and efficient PBMC engraftment in a rodent model, possibly helping to evaluate human immunity in preclinical studies.

AK2 is an AMP-sensing negative regulator of BRAF in tumorigenesis.

The RAS-BRAF signaling is a major pathway of cell proliferation and their mutations are frequently found in human cancers. Adenylate kinase 2 (AK2), which modulates balance of adenine nucleotide pool, has been implicated in cell death and cell proliferation independently of its enzyme activity. Recently, the role of AK2 in tumorigenesis was in part elucidated in some cancer types including lung adenocarcinoma and breast cancer, but the underlying mechanism is not clear. Here, we show that AK2 is a BRAF-suppressor. In in vitro assays and cell model, AK2 interacted with BRAF and inhibited BRAF activity and downstream ERK phosphorylation. Energy-deprived conditions in cell model and the addition of AMP to cell lysates strengthened the AK2-BRAF interaction, suggesting that AK2 is involved in the regulation of BRAF activity in response to cell metabolic state. AMP facilitated the AK2-BRAF complex formation through binding to AK2. In a panel of HCC cell lines, AK2 expression was inversely correlated with ERK/MAPK activation, and AK2-knockdown or -knockout increased BRAF activity and promoted cell proliferation. Tumors from HCC patients showed low-AK2 protein expression and increased ERK activation compared to non-tumor tissues and the downregulation of AK2 was also verified by two microarray datasets (TCGA-LIHC and GSE14520). Moreover, AK2/BRAF interaction was abrogated by RAS activation in in vitro assay and cell model and in a mouse model of HRASG12V-driven HCC, and AK2 ablation promoted tumor growth and BRAF activity. AK2 also bound to BRAF inhibitor-insensitive BRAF mutants and attenuated their activities. These findings indicate that AK2 monitoring cellular AMP levels is indeed a negative regulator of BRAF, linking the metabolic status to tumor growth.

Leukotriene B4 receptor-2 contributes to KRAS-driven lung tumor formation by promoting interleukin-6-mediated inflammation.

Although lung cancer is the leading cause of cancer-related deaths worldwide and KRAS is the most frequently mutated oncogene in lung cancer cases, the mechanism by which KRAS mutation drives lung cancer has not been fully elucidated. Here, we report that the expression levels of leukotriene B4 receptor-2 (BLT2) and its ligand-producing enzymes (5-LOX, 12-LOX) were highly increased by mutant KRAS and that BLT2 or 5-/12-LOX blockade attenuated KRAS-driven lung cell proliferation and production of interleukin-6 (IL-6), a principal proinflammatory mediator of lung cancer development. Next, we explored the roles of BLT2 and 5-/12-LOX in transgenic mice with lung-specific expression of mutant KRAS (KrasG12D) and observed that BLT2 or 5-/12-LOX inhibition decreased IL-6 production and tumor formation. To further determine whether BLT2 is involved in KRAS-driven lung tumor formation, we established a KrasG12D/BLT2-KO double-mutant mouse model. In the double-mutant mice, we observed significantly suppressed IL-6 production and lung tumor formation. Additionally, we observed high BLT2 expression in tissue samples from patients with KrasG12D-expressing lung adenocarcinoma, supporting the contributory role of BLT2 in KRAS-driven human lung cancer. Collectively, our results suggest that BLT2 is a potential contributor to KRAS-driven lung cancer and identify an attractive therapeutic target for KRAS-driven lung cancer.

Expression of Organic Anion Transporting Polypeptides in an H-Ras 12V Transgenic Mouse Model of Spontaneous Hepatocellular Carcinoma.

PURPOSE: Expression of organic anion transporting polypeptides (OATPs) 1B1/1B3 in hepatocellular carcinoma (HCC) induces a paradoxical enhancement of gadoxetic acid on liver magnetic resonance imaging (MRI). We examined the expression profile of OATPs with regard to tumor differentiation in a genetically modified H-Ras 12V mouse model of spontaneous HCC that undergoes multistep hepatocarcinogenesis with minimal inter-individual variation. MATERIALS AND METHODS: Tumor nodules were harvested from transgenic H-Ras 12V mice. Hematoxylin and eosin-stained slides were examined for tumor differentiation and high-grade pathological components (tumor necrosis, thickened trabeculae, or vascular invasion). Immunohistochemistry of OATP 1B1/1B3 was performed, and OATP expression was assessed. RESULTS: We examined well-differentiated HCCs (n=59) in which high-grade pathological components were absent (n=49) or present (n=10). Among the well-differentiated HCCs without high-grade pathological components (n=49), OATP expression was negative, weak positive, and moderate positive in 23, 17, and nine cases, respectively. Among the well-differentiated HCCs with high-grade pathological components (n=10), OATP expression was negative, weak positive, and moderate positive in one, two, and seven cases, respectively. The ratio of positive OATP 1B1/1B3 expressing tumors was higher in HCCs with high-grade pathological components than in those without high-grade pathological components (p=0.004). CONCLUSION: Our findings support those of previous clinical studies that have reported the frequent appearance of gadoxetic acid-enhanced MRI in moderately differentiated HCC.

Regulation of habenular G-protein gamma 8 on learning and memory via modulation of the central acetylcholine system.

Guanine nucleotide binding protein (G protein) gamma 8 (Gng8) is a subunit of G proteins and expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies have demonstrated that Gng8 is involved in brain development; however, the roles of Gng8 on cognitive function have not yet been addressed. In the present study, we investigated the expression of Gng8 in the brain and found that Gng8 was predominantly expressed in the MHb-IPN circuit of the mouse brain. We generated Gng8 knockout (KO) mice by CRISPR/Cas9 system in order to assess the role of Gng8 on cognitive function. Gng8 KO mice exhibited deficiency in learning and memory in passive avoidance and Morris water maze tests. In addition, Gng8 KO mice significantly reduced long-term potentiation (LTP) in the hippocampus compared to that of wild-type (WT) mice. Furthermore, we observed that levels of acetylcholine (ACh) and choline acetyltransferase (ChAT) in the MHb and IPN of Gng8 KO mice were significantly decreased, compared to WT mice. The administration of nAChR α4ß2 agonist A85380 rescued memory impairment in the Gng8 KO mice, suggesting that Gng8 regulates cognitive function via modulation of cholinergic activity. Taken together, Gng8 is a potential therapeutic target for memory-related diseases and/or neurodevelopmental diseases.

Sestrin2 Attenuates Cellular Senescence by Inhibiting NADPH Oxidase 4 Expression.

BACKGROUND: Sestrin2 (Sesn2) is involved in the maintenance of metabolic homeostasis and aging via modulation of the 5' AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) pathway. METHODS: Wild-type and Sesn2 knockout (KO) mice of the 129/SvJ background were maintained in a pathogen-free authorized facility under a 12-hour dark/light cycle at 20°C-22°C and 50%-60% humidity. Mouse embryonic fibroblasts (MEFs) were prepared from 13.5-day-old embryos derived from Sesn2-KO mice mated with each other. RESULTS: The MEFs from Sesn2-KO mice showed enlarged and flattened morphologies and senescence-associated ß-galactosidase activity, accompanied by an elevated level of reactive oxygen species. These senescence phenotypes recovered following treatment with N-acetyl-cysteine. Notably, the mRNA levels of NADPH oxidase 4 (NOX4) and transforming growth factor (TGF)-ß were markedly increased in Sesn2-KO MEFs. Treatment of Sesn2-KO MEFs with the NOX inhibitor diphenyleneiodonium and the TGF-ß inhibitor SB431542 restored cell growth inhibited by Sesn2-KO. CONCLUSION: Sesn2 attenuates cellular senescence via suppression of TGF-ß- and NOX4-induced reactive oxygen species generation and subsequent inhibition of AMPK.

Aglycosylated antibody-producing mice for aglycosylated antibody-lectin coupled immunoassay for the quantification of tumor markers (ALIQUAT).

Targeting aberrant glycoforms has been validated for in vitro cancer diagnostic development, and several assays are currently in routine clinical use. Because N-glycans in Fc region of antibodies show cross-reactivity with various lectins, high-quality aglycosylated antibodies are exceptionally important for immunoassay platform-based quantitative measurements. Previously, aglycosylated antibody acquisition relied on incomplete, uneconomical and onerous enzymatic and chemical methods. Here, we edited four murine immunoglobulin G genes using adenine base-editing and homology-directed recombination (HDR)-mediated gene editing methods to generate aglycosylated antibody-producing mice. Resulting aglycosylated antibodies showed required analytical performances without compromised protein stability. Thus, this aglycosylated monoclonal antibody-lectin coupled immunoassay for the quantification of tumour markers (ALIQUAT) method can provide a robust, versatile and accessible immunoassay platform to quantify specific glycoforms in precision cancer diagnostics. Moreover, the engineered mice can be used as a host to produce various aglycosylated antibodies in a convenient and robust fashion, thereby expanding in vitro diagnostic development opportunities that utilize glycoforms as a disease-specific biomarkers.

Depression-like behaviors induced by defective PTPRT activity through dysregulated synaptic functions and neurogenesis.

PTPRT has been known to regulate synaptic formation and dendritic arborization of hippocampal neurons. PTPRT-/- null and PTPRT-D401A mutant mice displayed enhanced depression-like behaviors compared with wild-type mice. Transient knockdown of PTPRT in the dentate gyrus enhanced the depression-like behaviors of wild-type mice, whereas rescued expression of PTPRT ameliorated the behaviors of PTPRT-null mice. Chronic stress exposure reduced expression of PTPRT in the hippocampus of mice. In PTPRT-deficient mice the expression of GluR2 (also known as GRIA2) was attenuated as a consequence of dysregulated tyrosine phosphorylation, and the long-term potentiation at perforant-dentate gyrus synapses was augmented. The inhibitory synaptic transmission of the dentate gyrus and hippocampal GABA concentration were reduced in PTPRT-deficient mice. In addition, the hippocampal expression of GABA transporter GAT3 (also known as SLC6A11) was decreased, and its tyrosine phosphorylation was increased in PTPRT-deficient mice. PTPRT-deficient mice displayed reduced numbers and neurite length of newborn granule cells in the dentate gyrus and had attenuated neurogenic ability of embryonic hippocampal neural stem cells. In conclusion, our findings show that the physiological roles of PTPRT in hippocampal neurogenesis, as well as synaptic functions, are involved in the pathogenesis of depressive disorder.

Two base pair deletion in IL2 receptor γ gene in NOD/SCID mice induces a highly severe immunodeficiency.

Genome editing has recently emerged as a powerful tool for generating mutant mice. Small deletions of nucleotides in the target genes are frequently found in CRISPR/Cas9 mediated mutant mice. However, there are very few reports analyzing the phenotypes in small deleted mutant mice generated by CRISPR/Cas9. In this study, we generated a mutant by microinjecting sgRNAs targeting the IL2 receptor γ gene and Cas9 protein, into the cytoplasm of IVF-derived NOD.CB17/Prkdcscid/JKrb (NOD/SCID) mice embryos, and further investigated whether a 2 bp deletion of the IL2 receptor γ gene affects severe deficiency of immune cells as seen in NOD/LtSz-scid IL2 receptor γ-/- (NSG) mice. Our results show that the thymus weight of mutant mice is significantly less than that of NOD/SCID mice, whereas the spleen weight was marginally less. T and B cells in the mutant mice were severely deficient, and NK cells were almost absent. In addition, tumor growth was exceedingly increased in the mutant mice transplanted with HepG2, Raji and A549 cells, but not in nude and NOD/SCID mice. These results suggest that the NOD/SCID mice with deletion of 2 bp in the IL2 receptor γ gene shows same phenotype as NSG mice. Taken together, our data indicates that small deletions by genome editing is sufficient to generate null mutant mice.

Bufalin inhibits hepatitis B virus-associated hepatocellular carcinoma development through androgen receptor dephosphorylation and cell cycle-related kinase degradation.

PURPOSE: Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), which has a male predominance, lacks effective therapeutic options. Previously, the cardiac glycoside analogue bufalin has been found to inhibit HBV infection and HCC development. As yet, however, its molecular role in HBV-associated HCC has remained obscure. METHODS: Colony formation and soft agar assays, xenograft and orthotopic mouse models and HBV X protein (HBx) transgenic mice with exposure to diethylnitrosamine were used to evaluate the effect of bufalin on HBV-associated HCC growth and tumorigenicity. HBx-induced oncogenic signaling regulated by bufalin was assessed using PCR array, chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, transcription and protein expression assays. Synergistic HCC therapeutic effects were examined using combinations of bufalin and sorafenib. RESULTS: We found that bufalin exerted a more profound effect on inhibiting the proliferation of HBV-associated HCC cells than of non HBV-associated HCC cells. Bufalin significantly inhibited HBx-induced malignant transfromation in vitro and tumorigenicity in vivo. Androgen receptor (AR) signaling was found to be a target of bufalin resistance to HBV-associated hepatocarcinogenesis. We also found that bufalin induced both AR dephosphorylation and cell cycle-related kinase (CCRK) degradation to inhibit ß-catenin/TCF signaling, which subsequently led to cell cycle arrest via cyclin D1 down-regulation and p21 up-regulation, resulting in HCC regression. Furthermore, we found that bufalin reduced > 60% diethylnitrosamine-induced hepatocarcinogenesis in HBx transgenic mice, and improved the sensitivity of refractory HBV-associated HCC cells to sorafenib treatment. CONCLUSION: Our results indicate that bufalin acts as a potential anti-HCC therapeutic candidate to block HBx-induced AR/CCRK/ß-catenin signaling by targeting AR and CCRK, which may provide a novel strategy for the treatment of HBV-associated HCC.

Relevance of reactive oxygen species in liver disease observed in transgenic mice expressing the hepatitis B virus X protein.

The hepatitis B virus (HBV) infects approximately 240 million people worldwide, causing chronic liver disease (CLD) and liver cancer. Although numerous studies have been performed to date, unfortunately there is no conclusive drug or treatment for HBV induced liver disease. The hepatitis B virus X (HBx) is considered a key player in inducing CLD and hepatocellular carcinoma (HCC). We generated transgenic (Tg) mice expressing HBx protein, inducing HCC at the age of 11-18 months. The incidence of histological phenotype, including liver tumor, differed depending on the genetic background of HBx Tg mice. Fatty change and tumor generation were observed much earlier in livers of HBx Tg hybrid (C57BL/6 and CBA) (HBx-Tg hybrid) mice than in HBx Tg C57BL/6 (HBx-Tg B6) mice. Inflammation was also enhanced in the HBx-Tg B6 mice as compared to HBx-Tg hybrid mice. HBx may be involved in inducing and promoting hepatic steatosis, glycemia, hepatic fibrosis, and liver cancer. Reactive oxygen species (ROS) generation was remarkably increased in livers of HBx Tg young mice compared to young wild type control mice. Previous studies on HBx Tg mice indicate that the HBx-induced ROS plays a role in inducing and promoting CLD and HCC.

In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens.

Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5' target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.

Serum anti-EIF3A autoantibody as a potential diagnostic marker for hepatocellular carcinoma.

Tumor-associated autoantibodies are promising diagnostic biomarkers for early detection of tumors. We have screened a novel tumor-associated autoantibody in hepatocellular carcinoma (HCC) model mice. Its target antigen was identified as eukaryotic translation initiation factor 3 subunit A (EIF3A) by proteomic analysis, and the elevated expression of EIF3A in HCC tissues of tumor model mice as well as human patients was shown. Also, its existence in tumor-derived exosomes was revealed, which seem to be the cause of tumor-associated autoantibody production. To use serum anti-EIF3A autoantibody as biomarker, ELISA detecting anti-EIF3A autoantibody in human serum was performed using autoantibody-specific epitope. For the sensitive detection of serum autoantibodies its specific conformational epitopes were screened from the random cyclic peptide library, and a streptavidin antigen displaying anti-EIF3A autoantibody-specific epitope, XC90p2(-CPVRSGFPC-), was used as capture antigen. It distinguished patients with HCC (n = 102) from healthy controls (n = 0285) with a sensitivity of 79.4% and specificity of 83.5% (AUC = 0.87). Also, by simultaneously detecting with other HCC biomarkers, including alpha-fetoprotein, HCC diagnostic sensitivity improved from 79.4% to 85%. Collectively, we suggest that serum anti-EIF3A autoantibody is a useful biomarker for the diagnosis of HCC and the combinational detection of related biomarkers can enhance the accuracy of the cancer diagnosis.

The Cxadr-Adam10 complex plays pivotal roles in tight junction integrity and early trophoblast development in mice.

Understanding preimplantation embryo development has important implications for assisted reproductive technologies (ARTs) after the introduction of in vitro fertilisation and embryo transfer because most embryonic losses occur during pre/peri-implantation. Recent studies have shown that tight junctions (TJs) are important components for embryos to develop to the blastocyst stage. However, their biological function after cavitation has not been extensively studied. We examined TJ assembly focusing on coxsackievirus and adenovirus receptor (Cxadr) and A disintegrin and metalloproteinase 10 (Adam10) using siRNA and/or an Adam10-specific inhibitor (GI254023X). TJ-associated genes, including occludin and tight junction protein 1 (Tjp1), were downregulated in the Cxadr knockdown (KD) embryos but were unaltered in Adam10 KD embryos. However, Adam10 KD or chemical inhibition affected subcellular localisation of Adam10, Cxadr, and Tjp1, leading to disrupted TJ assembly. Furthermore, Cxadr KD or GI254023X-treated blastocysts showed a relatively smaller outgrowth area and aberrant expression of transcription factor AP-2γ, a trophoblast-specific marker in the in vitro embryo outgrowth assay. In summary, we demonstrated that the Cxadr-Adam10 complex might moderate TJ integrity/stability and play pivotal roles during early embryonic development. Collectively, understanding the establishment of the TJ complex and its integrity will provide insight into translational research for predicting and selecting developmental competency for ART.

Protective Role of Peroxiredoxin I in Heat-Killed Staphylococcus Aureus-infected Mice.

BACKGROUND/AIM: Staphylococcus aureus (S. aureus) is a major gram-positive pathogen, which can cause toxic and immunogenic injuries both in nosocomial and community-acquired infections. Peroxiredoxin (Prx) I plays crucial roles in cellular apoptosis, proliferation, and signal transduction as well as in immunoregulation. The present study aimed to investigate whether Prx I protects mice from death caused by the heat-killed Staphylococcus aureus. MATERIALS AND METHODS: In the present study, we challenged the wild-type and Prx I-deficient mice with heat-killed S. aureus (HKSA). The effects of Prx I were evaluated by a series of in vitro and in vivo experiments including western blot, Haematoxylin and Eosin staining, splenocyte analysis and cytokines analysis. RESULTS: Intra-peritoneal (ip) inoculation of HKSA resulted in increased mortality of Prx I-knockout (KO) mice with severe liver damage and highly populated spleens with lymphocytes. Furthermore, HKSA infections also bursted the production of both pro-inflammatory and anti-inflammatory serum cytokines in Prx I KO compared to wild-type mice. CONCLUSION: Enhanced mortality of S. aureus-infected mice with Prx I deficiency suggested that Prx I may protect against the infection-associated lethality of mice.

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.

Aberrant enhancer hypomethylation contributes to hepatic carcinogenesis through global transcriptional reprogramming.

Hepatocellular carcinomas (HCC) exhibit distinct promoter hypermethylation patterns, but the epigenetic regulation and function of transcriptional enhancers remain unclear. Here, our affinity- and bisulfite-based whole-genome sequencing analyses reveal global enhancer hypomethylation in human HCCs. Integrative epigenomic characterization further pinpoints a recurrent hypomethylated enhancer of CCAAT/enhancer-binding protein-beta (C/EBPß) which correlates with C/EBPß over-expression and poorer prognosis of patients. Demethylation of C/EBPß enhancer reactivates a self-reinforcing enhancer-target loop via direct transcriptional up-regulation of enhancer RNA. Conversely, deletion of this enhancer via CRISPR/Cas9 reduces C/EBPß expression and its genome-wide co-occupancy with BRD4 at H3K27ac-marked enhancers and super-enhancers, leading to drastic suppression of driver oncogenes and HCC tumorigenicity. Hepatitis B X protein transgenic mouse model of HCC recapitulates this paradigm, as C/ebpß enhancer hypomethylation associates with oncogenic activation in early tumorigenesis. These results support a causal link between aberrant enhancer hypomethylation and C/EBPß over-expression, thereby contributing to hepatocarcinogenesis through global transcriptional reprogramming.

MicroRNA-122 negatively associates with peroxiredoxin-II expression in human gefitinib-resistant lung cancer stem cells.

Previously, we demonstrated that Prx II is important for survival of the gefitinib-resistant A549 (A549/GR) cell line, an NSCLC cell line derived by repeated exposure to gefitinib. Therefore, in this study, we used A549/GR cells to investigate the role of Prx II in GR NSCLC stemness. Initially, to explore the stemness characteristics and investigate the association of Prx II with those stemness characteristics, we successfully isolated a stem cell-like population from A549/GR cells. A549/GR CD133+ cells possessed important cancer stemness characteristics, including the abilities to undergo metastasis, angiogenesis, self-renewal, and to express stemness genes and epithelial-mesenchymal transition (EMT) markers. However, those characteristics were abolished by knocking down Prx II expression. MicroRNA 122 (miR-122) targets Prx II in A549/GR cancer stem cells (CSCs), thereby inhibiting the stemness characteristics in vitro and in vivo. Next, we investigate whether miR-122 overexpression was associated with Prx II expression and Prx-II-induced stemness characteristics, we transfected miR-122 into A549/GR CSCs. MiR-122 inhibited A549/GR stemness by downregulating the Hedgehog, Notch, and Wnt/ß-catenin pathways. Taken together, our data suggest that Prx II promotes A549/GR stemness, and that targeting Prx II and miR-122 is a potentially viable strategy for anti-cancer-stem cell therapy in GR NSCLCs.

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle.

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or ßcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.

Loss of tumor suppressor IGFBP4 drives epigenetic reprogramming in hepatic carcinogenesis.

Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.