Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing ...expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH.
We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632.
Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced β-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA.
We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.
Mutations and epigenetic aberrant signaling of growth factors pathways contribute to carcinogenesis. Recent studies reveal that non-coding RNAs are controllers of gene expression. H19 is an imprinted ...gene that demonstrates maternal monoallelic expression without a protein product; although its expression is shut off in most tissues postnatally, it is re-activated during adult tissue regeneration and tumorigenesis. Moreover, H19 is highly expressed in liver metastasis derived from a range of carcinomas. The objective of this study is to explore the role of H19 in carcinogenesis, and to determine its identification as an anti-tumor target.
By controlling oxygen pressure during tumor cell growth and H19 expression levels, we investigated the role of H19 expression in vitro and in vivo in hepatocellular (HCC) and bladder carcinoma. Hypoxia upregulates the level of H19 RNA. Ablations of tumorigenicity of HCC and bladder carcinomas in vivo are seen by H19 knockdown which also significantly abrogates anchorage-independent growth after hypoxia recovery, while ectopic H19 expression enhances tumorigenic potential of carcinoma cells in vivo. Knocking-down H19 message in hypoxic stress severely diminishes p57(kip2) induction. We identified a number of potential downstream targets of H19 RNA, including angiogenin and FGF18.
H19 RNA harbors pro-tumorigenic properties, thus the H19 gene behaves as an oncogene and may serve as a potential new target for anti-tumor therapy.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like ...C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors.
We used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles.
We report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs.
Our results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.
CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. ...We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.
These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.
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Nodding syndrome (NS) is a catastrophic and enigmatic childhood epilepsy, accompanied by multiple neurological impairments and neuroinflammation. Of all the infectious, environmental and ...psychological factors associated with NS, the major culprit is Onchocerca Volvulus (Ov)-a parasitic worm transmitted to human by blackflies. NS seems to be an 'Autoimmune Epilepsy' in light of the recent findings of deleterious autoimmune antibodies to Glutamate receptors and to Leiomodin-I in NS patients. Moreover, we recently found immunogenetic fingerprints in HLA peptide-binding grooves associate with protection or susceptibility to NS. Macrophage migration inhibitory factor (MIF) is an immune-regulatory cytokine playing a central role in modulating innate and adaptive immunity. MIF is also involved in various pathologies: infectious, autoimmune and neurodegenerative diseases, epilepsy and others. Herein, two functional polymorphisms in the MIF gene, a -794 CATT5-8 microsatellite repeat and a -173 G/C single-nucleotide polymorphism, were assessed in 49 NS patients and 51 healthy controls from South Sudan. We also measured MIF plasma levels in established NS patients and healthy controls. We discovered that the frequency of the high-expression MIF -173C containing genotype was significantly lower in NS patients compared to healthy controls. Interestingly however, MIF plasma levels were significantly elevated in NS patients than in healthy controls. We further demonstrated that the HLA protective and susceptibility associations are dominant over the MIF association with NS. Our findings suggest that MIF might have a dual role in NS. Genetically controlled high-expression MIF genotype is associated with disease protection. However, elevated MIF in the plasma may contribute to the detrimental autoimmunity, neuroinflammation and epilepsy.
While systemic tumor-stimulating effects can occur following ablation of normal liver linked to the IL-6/HGF/VEGF cytokinetic pathway, the potential for tumor cells themselves to produce these ...unwanted effects is currently unknown. Here, we study whether partially treated tumors induce increased tumor growth post-radiofrequency thermal ablation (RFA).
Tumor growth was measured in three immunocompetent, syngeneic tumor models following partial RFA of the target tumor (in subcutaneous CT26 and MC38 mouse colorectal adenocarcinoma, N = 14 each); and in a distant untreated tumor following partial RFA of target subcutaneous R3230 rat breast adenocarcinoma (N = 12). Tumor cell proliferation (ki-67) and microvascular density (CD34) was assessed. In R3230 tumors, in vivo mechanism of action was assessed following partial RFA by measuring IL-6, HGF, and VEGF expression (ELISA) and c-Met protein (Western blot). Finally, RFA was performed in R3230 tumors with adjuvant c-Met kinase inhibitor or VEGF receptor inhibitor (at 3 days post-RFA, N = 3/arm, total N = 12).
RFA stimulated tumor growth in vivo in residual, incompletely treated surrounding CT26 and MC38 tumor at 3-6 days (p < 0.01). In R3230, RFA increased tumor growth in distant tumor 7 days post treatment compared to controls (p < 0.001). For all models, Ki-67 and CD34 were elevated (p < 0.01, all comparisons). IL-6, HGF, and VEGF were also upregulated post incomplete tumor RFA (p < 0.01). These markers were suppressed to baseline levels with adjuvant c-MET kinase or VEGF receptor inhibition.
Incomplete RFA of a target tumor can sufficiently stimulate residual tumor cells to induce accelerated growth of distant tumors via the IL-6/c-Met/HGF pathway and VEGF production.
The potential of the high-affinity CXCR4 antagonist BL-8040 as a monotherapy-mobilizing agent and its derived graft composition and quality were evaluated in a phase I clinical study in healthy ...volunteers (NCT02073019).
The first part of the study was a randomized, double-blind, placebo-controlled dose escalation phase. The second part of the study was an open-label phase, in which 8 subjects received a single injection of BL-8040 (1 mg/kg) and approximately 4 hours later underwent a standard leukapheresis procedure. The engraftment potential of the purified mobilized CD34
cells was further evaluated by transplanting the cells into NSG immunodeficient mice.
BL-8040 was found safe and well tolerated at all doses tested (0.5-1 mg/kg). The main treatment-related adverse events were mild to moderate. Transient injection site and systemic reactions were mitigated by methylprednisolone, paracetamol, and promethazine pretreatment. In the first part of the study, BL-8040 triggered rapid and substantial mobilization of WBCs and CD34
cells in all tested doses. Four hours postdose, the count rose to a mean of 8, 37, 31, and 35 cells/μL (placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysis revealed substantial mobilization of immature dendritic, T, B, and NK cells. In the second part, the mean CD34
cells/kg collected were 11.6 × 10
cells/kg. The graft composition was rich in immune cells.
The current data demonstrate that BL-8040 is a safe and effective monotherapy strategy for the collection of large amounts of CD34
cells and immune cells in a one-day procedure for allogeneic HSPC transplantation.
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Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses ...oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53
wt) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53
null cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-α as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-α abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53
null hypoxic cells. In p53
wt hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-α are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-α activity
. In vivo a significant increase in H19 expression occurred in tumors derived from p53
null cells but not in p53
wt cells. Taken together, our results indicate that a functional link exists between p53, HIF1-α and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.
The tumor suppressor p53 is a central regulator of signaling pathways that controls the cell cycle and maintains the integrity of the human genome. p53 level is regulated by mouse double minute 2 ...homolog (Mdm2), which marks p53 for proteasomal degradation. The p53‐Mdm2 circuitry is subjected to complex regulation by a variety of mechanisms, including microRNAs (miRNAs). We found a novel effector of this regulatory circuit, namely, miR‐122*, the passenger strand of the abundantly expressed liver‐specific miR‐122. Here, we demonstrate that miR‐122* levels are reduced in human hepatocellular carcinoma (HCC). We found that miR‐122* targets Mdm2, thus participating as an important player in the p53‐Mdm2 circuitry. Moreover, we observed significant negative correlation between levels of miR‐122* and Mdm2 in a large set of human HCC samples. In vivo tumorigenicity assays demonstrate that miR‐122* is capable of inhibiting tumor growth, emphasizing the tumor‐suppressor characteristics of this miRNA. Furthermore, we show that blocking miR‐122 in murine livers with an antagomiR‐122 (miRNA inhibitor) results in miR‐122* accumulation, leading to Mdm2 repression followed by elevated p53 protein levels. Conclusion: miR‐122*, the passenger strand of miR‐122, regulates the activity of p53 by targeting Mdm2. Importantly, similarly to miR‐122, miR‐122* is significantly down‐regulated in human HCC. We therefore propose that miR‐122* is an important contributor to the tumor suppression activity previously attributed solely to miR‐122. (Hepatology 2016;64:1623‐1636)
Image-guided radiofrequency ablation (RFA) is used to treat focal tumors in the liver and other organs. Despite potential advantages over surgery, hepatic RFA can promote local and distant tumor ...growth by activating pro-tumorigenic growth factor and cytokines. Thus, strategies to identify and suppress pro-oncogenic effects of RFA are urgently required to further improve the therapeutic effect. Here, the proliferative effect of plasma of Hepatocellular carcinoma or colorectal carcinoma patients 90 min post-RFA was tested on HCC cell lines, demonstrating significant cellular proliferation compared to baseline plasma. Multiplex ELISA screening demonstrated increased plasma pro-tumorigenic growth factors and cytokines including the FGF protein family which uniquely and selectively activated HepG2. Primary mouse and immortalized human hepatocytes were then subjected to moderate hyperthermia in-vitro, mimicking thermal stress induced during ablation in the peri-ablational normal tissue. Resultant culture medium induced proliferation of multiple cancer cell lines. Subsequent non-biased protein array revealed that these hepatocytes subjected to moderate hyperthermia also excrete a similar wide spectrum of growth factors. Recombinant FGF-2 activated multiple cell lines. FGFR inhibitor significantly reduced liver tumor load post-RFA in MDR2-KO inflammation-induced HCC mouse model. Thus, Liver RFA can induce tumorigenesis via the FGF signaling pathway, and its inhibition suppresses HCC development.
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