[GENE THERAPY: FROM TECHNOLOGY TO REALITY].

INTRODUCTION: Gene therapy has made major achievements in the last few decades. These were in numerous medical disciplines, including metabolic, oncologic, infectious and regenerative. As of today, regulatory agencies, both in the USA and Europe, approved for clinical usage numerous gene therapy treatments. However, we are still facing a number of significant obstacles including: 1. Efficient delivery systems, 2. Immunological responses, and 3. recently we have learned that many gene therapy approaches are very expensive. The COVID-19 pandemic was a period in which genetic vaccination had proved its efficacy, by which RNA in a synthetic delivery system was very effective. This review will focus on three fast developing technologies in gene therapy: 1. CAR-T cells, 2. CRISPR-Cas and 3. RNAi.

Postexposure-vaccine-prophylaxis against COVID-19.

During the COVID-19 pandemic, postexposure-vaccine-prophylaxis is not a practice. Following exposure, only patient isolation is imposed. Moreover, no therapeutic prevention approach is applied. We asked whether evidence exists for reduced mortality rate following postexposure-vaccine-prophylaxis. To estimate the effectiveness of postexposure-vaccine-prophylaxis, we obtained data from the Israeli Ministry of Health registry. The study population consisted of Israeli residents aged 12 years and older, identified for the first time as PCR-positive for SARS-CoV-2, between December 20th, 2020 (the beginning of the vaccination campaign) and October 7th, 2021. We compared "recently injected" patients-that proved PCR-positive on the same day or on 1 of the 5 consecutive days after first vaccination (representing an unintended postexposure-vaccine-prophylaxis)s-to unvaccinated control group. Among Israeli residents identified PCR-positive for SARS-CoV-2, 11 687 were found positive on the day they received their first vaccine injection (BNT162b2) or on 1 of the 5 days thereafter. In patients over 65 years, 143 deaths occurred among 1412 recently injected (10.13%) compared to 255 deaths among the 1412 unvaccinated (18.06%), odd ratio (OR) 0.51 (95% confidence interval [CI]: 0.41-0.64; p < 0.001). A significant reduction in the death toll was observed among the 55-64 age group, with 8 deaths occurring among the 1320 recently injected (0.61%) compared to 24 deaths among the 1320 unvaccinated control (1.82%), OR 0.33 (95% CI: 0.13-0.76; p = 0.007). Postexposure-vaccine-prophylaxis is effective against death in COVID-19 infection.

Prediction of Protumorigenic Effects after Image-Guided Radiofrequency Ablation of Hepatocellular Carcinoma Using Biomarkers.

PURPOSE: To perform radiofrequency (RF) ablation of hepatocellular carcinoma (HCC) and to assess serological and histopathological markers of tumorigenesis in distant untreated tumors to determine whether these were associated with unfavorable outcomes such as early relapse and increased biological aggressiveness. MATERIALS AND METHODS: The study cohort comprised 13 patients from a prospective single-arm study. All patients underwent 2 ablation sessions of multifocal HCC nodules 14 days apart. Core biopsy samples of untreated tumors were acquired at baseline and at the time of the second ablation session. Samples were stained immunohistochemically with Ki-67 (proliferation) and CD34 (microvasculature). Blood plasma was obtained at baseline and 2 days after the initial ablation session and analyzed for hepatocyte growth factor (HGF), vascular endothelial growth factor C, and angiopoietin-2 using an enzyme-linked immunosorbent assay. The clinical follow-up period ranged from 7 to 25 months. Patients were stratified as responders (complete remission or limited and delayed recurrence at >6 months; n = 6) or nonresponders (any recurrence within 6 months or >3 new tumors or any new tumor of >3 cm thereafter; n = 7). RESULTS: In 3 of 7 nonresponders, the Ki-67 index markedly increased in untreated tumors, whereas Ki-67 was stable in all responders. Microvascular density strongly increased in a single nonresponder only. HGF and angiopoietin-2 increased by >30% in 3 of 7 and 4 of 7 nonresponders, respectively, whereas they were stable or decreased in responders. Overall, ≥2 biomarkers were elevated in 6 of 7 (85.7%) nonresponders, whereas 4 of 6 responders demonstrated no increased biomarker and 2 patients demonstrated increase in 1 biomarker only (P = .002). CONCLUSIONS: RF ablation of HCC can produce protumorigenic factors that induce effects in distant untreated tumors. These may potentially function as biomarkers of clinical outcome.

Radiofrequency Ablation-Induced Tumor Growth Is Suppressed by MicroRNA-21 Inhibition in Murine Models of Intrahepatic Colorectal Carcinoma.

PURPOSE: To investigate the role of microRNA-21 (miR21) in radiofrequency (RF) ablation-induced tumor growth and whether miR21 inhibition suppresses tumorigenesis. MATERIAL AND METHODS: Standardized liver RF ablation was applied to 35 C57/BL6 mice. miR21 and target proteins pSTAT3, PDCD4, and PTEN were assayed 3 hours, 24 hours, and 3 days after ablation. Next, 53 Balb/c and 44 C57BL/6 mice received Antago-miR21 or scrambled Antago-nc control, followed by intrasplenic injection of 10,000 CT26 or MC38 colorectal tumor cells, respectively. Hepatic RF ablation or sham ablation was performed 24 hours later. Metastases were quantified and tumor microvascular density (MVD) and cellular proliferation were assessed at 14 or 21 days after the procedures, respectively. RESULTS: RF ablation significantly increased miR21 levels in plasma and hepatic tissue at 3 and 24 hours as well as target proteins at 3 days after ablation (P < .05, all comparisons). RF ablation nearly doubled tumor growth (CT26, 2.0 SD ± 1.0 fold change [fc]; MC38, 1.9 SD ± 0.9 fc) and increased MVD (CT26, 1.9 SD ± 1.0 fc; MC38, 1.5 ± 0.5 fc) and cellular proliferation (CT26, 1.7 SD ± 0.7 fc; MC38, 1.4 SD ± 0.5 fc) compared with sham ablation (P < .05, all comparisons). RF ablation-induced tumor growth was suppressed when Antago-miR21 was administered (CT26, 1.0 SD ± 0.7 fc; MC38, 0.9 SD ± 0.4 fc) (P < .01, both comparisons). Likewise, Antago-miR21 decreased MVD (CT26, 1.0 SD ± 0.3 fc; MC38, 1.0 SD ± 0.2 fc) and cellular proliferation (CT26, 0.9 SD ± 0.3 fc; MC38, 0.8 SD ± 0.3 fc) compared with baseline (P < .05, all comparisons). CONCLUSIONS: RF ablation upregulates protumorigenic miR21, which subsequently influences downstream tumor-promoting protein pathways. This effect can potentially be suppressed by specific inhibition of miR21, rendering this microRNA a pivotal and targetable driver of tumorigenesis after hepatic thermal ablation.

Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL-6 trans-signaling.

BACKGROUND & AIMS: Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA) and combined HCC-CCA tumors (cHCC-CCA). It has been suggested, but not unequivocally proven, that hepatic progenitor cells (HPCs) can contribute to hepatocarcinogenesis. We aimed to determine whether HPCs contribute to HCC, cHCC-CCA or both types of tumors. METHODS: To trace progenitor cells during hepatocarcinogenesis, we generated Mdr2-KO mice that harbor a yellow fluorescent protein (YFP) reporter gene driven by the Foxl1 promoter which is expressed specifically in progenitor cells. These mice (Mdr2-KOFoxl1-CRE;RosaYFP) develop chronic inflammation and HCCs by the age of 14-16 months, followed by cHCC-CCA tumors at the age of 18 months. RESULTS: In this Mdr2-KOFoxl1-CRE;RosaYFP mouse model, liver progenitor cells are the source of cHCC-CCA tumors, but not the source of HCC. Ablating the progenitors, caused reduction of cHCC-CCA tumors but did not affect HCCs. RNA-sequencing revealed enrichment of the IL-6 signaling pathway in cHCC-CCA tumors compared to HCC tumors. Single-cell RNA-sequencing (scRNA-seq) analysis revealed that IL-6 is expressed by immune and parenchymal cells during senescence, and that IL-6 is part of the senescence-associated secretory phenotype. Administration of an anti-IL-6 antibody to Mdr2-KOFoxl1-CRE;RosaYFP mice inhibited the development of cHCC-CCA tumors. Blocking IL-6 trans-signaling led to a decrease in the number and size of cHCC-CCA tumors, indicating their dependence on this pathway. Furthermore, the administration of a senolytic agent inhibited IL-6 and the development of cHCC-CCA tumors. CONCLUSION: Our results demonstrate that cHCC-CCA, but not HCC tumors, originate from HPCs, and that IL-6, which derives in part from cells in senescence, plays an important role in this process via IL-6 trans-signaling. These findings could be applied to develop new therapeutic approaches for cHCC-CCA tumors. LAY SUMMARY: Combined hepatocellular carcinoma-cholangiocarcinoma is the third most prevalent type of primary liver cancer (i.e. a cancer that originates in the liver). Herein, we show that this type of cancer originates in stem cells in the liver and that it depends on inflammatory signaling. Specifically, we identify a cytokine called IL-6 that appears to be important in the development of these tumors. Our results could be used for the development of novel treatments for these aggressive tumors.

Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA 122.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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 ß-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. CONCLUSIONS: 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.

Incomplete thermal ablation of tumors promotes increased tumorigenesis.

PURPOSE: 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). METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Gut microbiota shape 'inflamm-ageing' cytokines and account for age-dependent decline in DNA damage repair.

OBJECTIVE: Failing to properly repair damaged DNA drives the ageing process. Furthermore, age-related inflammation contributes to the manifestation of ageing. Recently, we demonstrated that the efficiency of repair of diethylnitrosamine (DEN)-induced double-strand breaks (DSBs) rapidly declines with age. We therefore hypothesised that with age, the decline in DNA damage repair stems from age-related inflammation. DESIGN: We used DEN-induced DNA damage in mouse livers and compared the efficiency of their resolution in different ages and following various permutations aimed at manipulating the liver age-related inflammation. RESULTS: We found that age-related deregulation of innate immunity was linked to altered gut microbiota. Consequently, antibiotic treatment, MyD88 ablation or germ-free mice had reduced cytokine expression and improved DSBs rejoining in 6-month-old mice. In contrast, feeding young mice with a high-fat diet enhanced inflammation and facilitated the decline in DSBs repair. This latter effect was reversed by antibiotic treatment. Kupffer cell replenishment or their inactivation with gadolinium chloride reduced proinflammatory cytokine expression and reversed the decline in DSBs repair. The addition of proinflammatory cytokines ablated DSBs rejoining mediated by macrophage-derived heparin-binding epidermal growth factor-like growth factor. CONCLUSIONS: Taken together, our results reveal a previously unrecognised link between commensal bacteria-induced inflammation that results in age-dependent decline in DNA damage repair. Importantly, the present study support the notion of a cell non-autonomous mechanism for age-related decline in DNA damage repair that is based on the presence of 'inflamm-ageing' cytokines in the tissue microenvironment, rather than an intrinsic cellular deficiency in the DNA repair machinery.

Thermal Ablation Induces Transitory Metastatic Growth by Means of the STAT3/c-Met Molecular Pathway in an Intrahepatic Colorectal Cancer Mouse Model.

Background Systemic protumorigenic effects have been noted after radiofrequency ablation (RFA) of normal liver and have been linked to an interleukin 6/signal transducer and activator of transcription 3 (STAT3)/hepatocyte growth factor (HGF)/tyrosine-protein kinase Met (c-Met)/vascular endothelial growth factor (VEGF) cytokinetic pathway. Purpose To elucidate kinetics of RFA protumorigenic effects on intrahepatic metastatic implantation and growth and determine potential molecular targets for pharmacologic suppression of these effects. Materials and Methods An intrahepatic metastasis model was established by implanting CT26 and MC38 tumor cells into 216 7-8-week-old male Balb/C and C57BL6 mice, respectively, by means of splenic injection. Between June 2017 and March 2019, mice underwent tumor injection, followed 24 hours later by either standardized RFA (70°C ± 1, 5 minutes, 1-cm tip) or a sham procedure (needle placement without heating) (12 animals per arm, n = 48). Next, RFA or sham procedures were performed, followed by splenic tumor cell injection at 1 day, 3 days, or 7 days later (six animals per arm, n = 72). Finally, PHA-665752 and S3I-201 were used to block c-Met or STAT3, respectively, prior to either RFA or sham treatment (six animals per arm, n = 96). Livers were harvested at 14 days for CT26 and 21days for MC38 for tumor quantification. Ki-67 and CD34 immunohistochemistry measured proliferative indexes and microvascular density, respectively. Data were compared with analysis of variance and the two-tailed Student t test. Results RFA performed after tumor cell injection induced increased metastatic tumor number (103 ± 45 vs 52 ± 44 [CT26], P = .009 and 87 ± 51 vs 39 ± 20 [MC38], P = .007), cellular proliferation (P < .001 for both), and intratumoral neovascularization (P < .001 for both), compared with the sham procedure. Tumor cell injection performed 1 day and 3 days after RFA also increased these indexes (P < .05), while no difference was demonstrated for cell injection 7 days after RFA (P > .05). Adjuvant c-Met or STAT3 inhibition reduced intrahepatic metastatic parameters after RFA to baseline (P < .03), equivalent to the sham group (P > .05). Conclusion Radiofrequency ablation of normal liver promotes intrahepatic metastatic implantation and increased growth over a short-lived (1-3 days) temporal window in animal models. This phenomenon can be potentially neutralized with specific inhibition of pathways including hepatocyte growth factor/tyrosine-protein kinase Met and signal transducer and activator of transcription 3. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Nikolic in this issue.

Dual-Targeted Autoimmune Sword in Fatal Epilepsy: Patient's glutamate receptor AMPA GluR3B peptide autoimmune antibodies bind, induce Reactive Oxygen Species (ROS) in, and kill both human neural cells and T cells.

Nodding Syndrome (NS) is a fatal pediatric epilepsy of unknown etiology, accompanied by multiple neurological impairments, and associated with Onchocerca volvulus (Ov), malnutrition, war-induced trauma, and other insults. NS patients have neuroinflammation, and ~50% have cross-reactive Ov/Leiomodin-1 neurotoxic autoimmune antibodies. RESULTS: Studying 30 South Sudanese NS patients and a similar number of healthy subjects from the same geographical region, revealed autoimmune antibodies to 3 extracellular peptides of ionotropic glutamate receptors in NS patients: AMPA-GluR3B peptide antibodies (86%), NMDA-NR1 peptide antibodies (77%) and NMDA-NR2 peptide antibodies (87%) (in either 1:10, 1:100 or 1:1000 serum dilution). In contrast, NS patients did not have 26 other well-known autoantibodies that target the nervous system in several autoimmune-mediated neurological diseases. We demonstrated high expression of both AMPA-GluR3 and NMDA-NR1 in human neural cells, and also in normal human CD3+ T cells of both helper CD4+ and cytotoxic CD8+ types. Patient's GluR3B peptide antibodies were affinity-purified, and by themselves precipitated short 70 kDa neuronal GluR3. NS patient's affinity-purified GluR3B peptide antibodies also bound to, induced Reactive Oxygen Species (ROS) in, and killed both human neural cells and T cells within 1-2 hours only. NS patient's purified IgGs, or serum (1:10 or 1:30), induced similar effects. In vivo video EEG experiments in normal mice, revealed that when NS patient's purified IgGs were released continuously (24/7 for 1 week) in normal mouse brain, they induced all the following: 1.Seizures, 2. Cerebellar Purkinje cell loss, 3. Degeneration in the hippocampus and cerebral cortex, and 4. Elevation of CD3+ T cells, and of activated Mac-2+microglia and GFAP+astrocytes in both the gray and white matter of the cerebral cortex, hippocampus, corpus calossum and cerebellum of mice. NS patient's serum cytokines: IL-1ß, IL-2, IL-6, IL-8, TNFα, IFNγ, are reduced by 85-99% compared to healthy subjects, suggesting severe immunodeficiency in NS patients. This suspected immunodeficiency could be caused by combined effects of the: 1. Chronic Ov infection, 2. Malnutrition, 3. Killing of NS patient's T cells by patient's own GluR3B peptide autoimmune antibodies (alike the killing of normal human T cells by the NS patient's GluR3B peptide antibodies found herein in vitro). CONCLUSIONS: Regardless of NS etiology, NS patients suffer from 'Dual-targeted Autoimmune Sword': autoimmune AMPA GluR3B peptide antibodies that bind, induce ROS in, and kill both neural cells and T cells. These neurotoxic and immunotoxic GluR3B peptide autoimmune antibodies, and also NS patient's NMDA-NR1/NR2A and Ov/Leiomodin-1 autoimmune antibodies, must be silenced or removed. Moreover, the findings of this study are relevant not only to NS, but also to many more patients with other types of epilepsy, which have GluR3B peptide antibodies in serum and/or CSF. This claim is based on the following facts: 1. The GluR3 subunit is expressed in neural cells in crucial brains regions, in motor neurons in the spinal cord, and also in other cells in the body, among them T cells of the immune system, 2. The GluR3 subunit has diverse neurophysiological role, and its deletion or abnormal function can: disrupt oscillatory networks of both sleep and breathing, impair motor coordination and exploratory activity, and increase the susceptibility to generate seizures, 3. GluR3B peptide antibodies were found so far in ~27% of >300 epilepsy patients worldwide, which suffer from various other types of severe, intractable and enigmatic epilepsy, and which turned out to be 'Autoimmune Epilepsy'. Furthermore, the findings of this study could be relevant to different neurological diseases besides epilepsy, since other neurotransmitter-receptors autoantibodies are present in other neurological and psychiatric diseases, e.g. autoimmune antibodies against other GluRs, Dopamine receptors, GABA receptors, Acetylcholine receptors and others. These neurotransmitter-receptors autoimmune autoantibodies might also act as 'Dual-targeted Autoimmune Sword' and damage both neural cells and T cells (as the AMPA-GluR3B peptide antibodies induced in the present study), since T cells, alike neural cells, express most if not all these neurotransmitter receptors, and respond functionally to the respective neurotransmitters - a scientific and clinical topic we coined 'Nerve-Driven Immunity'.

Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

Chronic liver inflammation (CLI) is a risk factor for development of hepatocellular carcinoma (HCC). Galectin-1 (Gal1) is involved in the regulation of inflammation, angiogenesis, and tumorigenesis, exhibiting multiple anti-inflammatory and protumorigenic activities. We aimed to explore its regulatory role in CLI and HCC progression using an established model of CLI-mediated HCC development, Abcb4 [multidrug-resistance 2 (Mdr2)]-knockout (KO) mice, which express high levels of Gal1 in the liver. We generated double-KO (dKO) Gal1-KO/Mdr2-KO mice on C57BL/6 and FVB/N genetic backgrounds and compared HCC development in the generated strains with their parental Mdr2-KO strains. Loss of Gal1 increased liver injury, inflammation, fibrosis, and ductular reaction in dKO mice of both strains starting from an early age. Aged dKO mutants displayed earlier hepatocarcinogenesis and increased tumor size compared with control Mdr2-KO mice. We found that osteopontin, a well-known modulator of HCC development, and oncogenic proteins Ntrk2 (TrkB) and S100A4 were overexpressed in dKO compared with Mdr2-KO livers. Our results demonstrate that in Mdr2-KO mice, a model of CLI-mediated HCC, Gal1-mediated protection from hepatitis, liver fibrosis, and HCC initiation dominates over its known procarcinogenic activities at later stages of HCC development. These findings suggest that anti-Gal1 treatments may not be applicable at all stages of CLI-mediated HCC.-Potikha, T., Pappo, O., Mizrahi, L., Olam, D., Maller, S. M., Rabinovich, G. A., Galun, E., Goldenberg, D. S. Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

Moderate hyperthermic heating encountered during thermal ablation increases tumor cell activity.

Purpose: The aim of this study was to determine whether moderate hyperthermic doses, routinely encountered in the periablational zone during thermal ablation, activate tumor cells sufficiently to secrete pro-tumorigenic factors that can induce increased proliferation.Material and methods: R3230 rat mammary tumor cells and human cancer cell lines, MCF7 breast adenocarcinoma, HepG2 and Huh7 HCC, and HT-29 and SW480 colon adenocarcinoma, were heated in to 45 ± 1 °C or 43 ± 1 °C in vitro for 5-10 min and incubated thereafter at 37 °C for 1.5, 3 or 8 hr (n = 3 trials each; total N = 135). mRNA expression profiles of cytokines implicated in RF-induced tumorigenesis including IL-6, TNFα, STAT3, HGF, and VEGF, were evaluated by relative quantitative real-time PCR. HSP70 was used as control. c-Met and STAT3 levels were assessed by Western blot. Finally, naïve cancer cells were incubated with medium from R3230 and human cancer cells that were subjected to 43-45 °C for 5 or 10 min and incubated for 3 or 8 h at 37 °C in an xCELLigence or incuCyte detection system.Results: Cell-line-specific dose and time-dependent elevations of at least a doubling in HSP70, IL-6, TNFα, STAT3, and HGF gene expression were observed in R3230 and human cancer cells subjected to moderate hyperthermia. R3230 and several human cell lines showed increased phosphorylation of STAT3 3 h post-heating and increased c-Met following heating. Medium of cancer cells subject to moderate hyperthermia induced statistically significant accelerated cell growth of all cell lines compared to non-heated media (p < 0.01, all comparisons).Conclusion: Heat-damaged human tumor cells by themselves can induce proliferation of tumor by releasing pro-tumorigenic factors.

A Protective Function of IL-22BP in Ischemia Reperfusion and Acetaminophen-Induced Liver Injury.

Acute liver injury can be secondary to a variety of causes, including infections, intoxication, and ischemia. All of these insults induce hepatocyte death and subsequent inflammation, which can make acute liver injury a life-threatening event. IL-22 is a dual natured cytokine which has context-dependent protective and pathogenic properties during tissue damage. Accordingly, IL-22 was shown to promote liver regeneration upon acute liver damage. However, other studies suggest pathogenic properties of IL-22 during chronic liver injury. IL-22 binding protein (IL-22BP, IL-22Ra2) is a soluble inhibitor of IL-22 that regulates IL-22 activity. However, the significance of endogenous IL-22BP in acute liver injury is unknown. We hypothesized that IL-22BP may play a role in acute liver injury. To test this hypothesis, we used Il22bp-deficient mice and murine models of acute liver damage induced by ischemia reperfusion and N-acetyl-p-aminophenol (acetaminophen) administration. We found that Il22bp-deficient mice were more susceptible to acute liver damage in both models. We used Il22 × Il22bp double-deficient mice to show that this effect is indeed due to uncontrolled IL-22 activity. We could demonstrate mechanistically increased expression of Cxcl10 by hepatocytes, and consequently increased infiltration of inflammatory CD11b+Ly6C+ monocytes into the liver in Il22bp-deficient mice upon liver damage. Accordingly, neutralization of CXCL10 reversed the increased disease susceptibility of Il22bp-deficient mice. In conclusion, our data indicate that IL-22BP plays a protective role in acute liver damage, via controlling IL-22-induced Cxcl10 expression.

Metabolic Circuit Involving Free Fatty Acids, microRNA 122, and Triglyceride Synthesis in Liver and Muscle Tissues.

BACKGROUND & AIMS: Effective treatments are needed for hepatic steatosis characterized by accumulation of triglycerides in hepatocytes, which leads to hepatocellular carcinoma. MicroRNA 122 (MIR122) is expressed only in the liver, where it regulates lipid metabolism. We investigated the mechanism by which free fatty acids (FFAs) regulate MIR122 expression and the effect of MIR122 on triglyceride synthesis. METHODS: We analyzed MIR122 promoter activity and validated its target mRNAs by transfection of Luciferase reporter plasmids into Huh7, BNL-1ME, and HEK293 cultured cell lines. We measured levels of microRNAs and mRNAs by quantitative real-time PCR analysis of RNA extracted from plasma, liver, muscle, and adipose tissues of C57BL/6 mice given the FFA-inducer CL316243. MIR122 was inhibited using an inhibitor of MIR122. Metabolic profiles of mice were determined using metabolic chambers and by histologic analyses of liver tissues. We performed RNA sequence analyses to identify metabolic pathways involving MIR122. RESULTS: We validated human Agpat1 and Dgat1 mRNAs, involved in triglyceride synthesis, as targets of MIR122. FFAs increased MIR122 expression in livers of mice by activating the retinoic acid-related orphan receptor alpha, and induced secretion of MIR122 from liver to blood. Circulating MIR122 entered muscle and adipose tissues of mice, reducing mRNA levels of genes involved in triglyceride synthesis. Mice injected with an inhibitor of MIR122 and then given CL316243, accumulated triglycerides in liver and muscle tissues, and had reduced rates of ß-oxidation. There was a positive correlation between level of FFAs and level of MIR122 in plasma samples from 6 healthy individuals, collected before and during fasting. CONCLUSIONS: In biochemical and histologic studies of plasma, liver, muscle, and adipose tissues from mice, we found that FFAs increase hepatic expression and secretion of MIR122, which regulates energy storage vs expenditure in liver and peripheral tissues. Strategies to reduce triglyceride levels, by increasing MIR122, might be developed for treatment of metabolic syndrome.

Targeting STAT3 to Suppress Systemic Pro-Oncogenic Effects from Hepatic Radiofrequency Ablation.

Purpose To (a) identify key expressed genes in the periablational rim after radiofrequency ablation (RFA) and their role in driving the stimulation of distant tumor growth and (b) use adjuvant drug therapies to block key identified mediator(s) to suppress off-target tumorigenic effects of hepatic RFA. Materials and Methods This institutional animal care and use committee-approved study was performed in C57BL6 mice (n = 20) and F344 rats (n = 124). First, gene expression analysis was performed in mice after hepatic RFA or sham procedure; mice were sacrificed 24 hours to 7 days after treatment. Data were analyzed for differentially expressed genes (greater than twofold change) and their functional annotations. Next, animals were allocated to hepatic RFA or sham treatment with or without STAT3 (signal transducer and activator of transcription 3) inhibitor S3I-201 for periablational phosphorylated STAT3 immunohistochemistry analysis at 24 hours. Finally, animals with subcutaneous R3230 adenocarcinoma tumors were allocated to RFA or sham treatment with or without a STAT3 inhibitor (S3I-201 or micellar curcumin, eight arms). Outcomes included distant tumor growth, proliferation (Ki-67 percentage), and microvascular density. Results At 24 hours, 217 genes had altered expression (107 upregulated and 110 downregulated), decreasing to 55 genes (27 upregulated and 28 downregulated) and 18 genes (four upregulated, 14 downregulated) at 72 hours and 7 days, respectively. At 24 hours, STAT3 occurred in four of seven activated pathways associated with pro-oncogenic genes at network analysis. Immunohistochemistry analysis confirmed elevated periablational phosphorylated STAT3 24 hours after RFA, which was suppressed with S3I-201 (percentage of positive cells per field: 31.7% ± 3.4 vs 3.8% ± 1.7; P < .001). Combined RFA plus S3I-201 reduced systemic distant tumor growth at 7 days (end diameter: 11.8 mm ± 0.5 with RFA plus S3I-201, 19.8 mm ± 0.7 with RFA alone, and 15 mm ± 0.7 with sham procedure; P < .001). STAT3 inhibition with micellar curcumin also suppressed postablation stimulation of distant tumor growth, proliferation, and microvascular density (P < .01). Conclusion Gene expression analysis identified multiple pathways upregulated in the periablational rim after hepatic RFA, of which STAT3 was active in four of seven. Postablation STAT3 activation is linked to increased distant tumor stimulation and can be suppressed with adjuvant STAT3 inhibitors. © RSNA, 2017.

Interleukin 6-dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis.

Liver cancer, which typically develops on a background of chronic liver inflammation, is now the second leading cause of cancer mortality worldwide. For patients with liver cancer, surgical resection is a principal treatment modality that offers a chance of prolonged survival. However, tumor recurrence after resection, the mechanisms of which remain obscure, markedly limits the long-term survival of these patients. We have shown that partial hepatectomy in multidrug resistance 2 knockout (Mdr2-/- ) mice, a model of chronic inflammation-associated liver cancer, significantly accelerates hepatocarcinogenesis. Here, we explore the postsurgical mechanisms that drive accelerated hepatocarcinogenesis in Mdr2-/- mice by perioperative pharmacological inhibition of interleukin-6 (IL6), which is a crucial liver regeneration priming cytokine. We demonstrate that inhibition of IL6 signaling dramatically impedes tumorigenesis following partial hepatectomy without compromising survival or liver mass recovery. IL6 blockade significantly inhibited hepatocyte cell cycle progression while promoting a hypertrophic regenerative response, without increasing apoptosis. Mdr2-/- mice contain hepatocytes with a notable persistent DNA damage response (γH2AX, 53BP1) due to chronic inflammation. We show that liver regeneration in this microenvironment leads to a striking increase in hepatocytes bearing micronuclei, a marker of genomic instability, which is suppressed by IL6 blockade. CONCLUSION: Our findings indicate that genomic instability derived during the IL6-mediated liver regenerative response within a milieu of chronic inflammation links partial hepatectomy to accelerated hepatocarcinogenesis; this suggests a new therapeutic approach through the usage of an anti-IL6 treatment to extend the tumor-free survival of patients undergoing surgical resection. (Hepatology 2017;65:1600-1611).

Radiofrequency ablation (RFA)-induced systemic tumor growth can be reduced by suppression of resultant heat shock proteins.

PURPOSE: To determine the role of hepatic radiofrequency ablation (RFA) heating parameters and their activation of heat shock proteins (HSPs) in modulating distant tumor growth. METHODS AND MATERIALS: First, to study the effects of RFA dose on distant tumor growth, rats with subcutaneous R3230 adenocarcinoma (10 ± 1 mm) were assigned to 3 different hepatic RF doses (60 °C × 10 min, 70 °C × 5 min or 90 °C × 2 min) that induced identical sized ablation or sham (n = 6/arm). Post-RFA tumor growth rates, cellular proliferation (Ki-67) and microvascular density (MVD) were compared at 7d. Next, the effect of low and high power doses on local HSP70 expression and cellular infiltration (α-SMA + myofibroblasts and CD68 + macrophages), cytokine (IL-6) and growth factor (HGF and VEGF) expression was assessed. Finally, 60 °C × 10 min and 90 °C × 2 min RFA were combined with anti-HSP micellar quercetin (MicQ, 2 mg/ml). A total of 150 animals were used. RESULTS: Lower RF heating (70 °C × 5 min and 60 °C × 10 min) resulted in larger distant tumors at 7d (19.2 ± 0.8 mm for both) while higher RF heating (90 °C × 2) led to less distant tumor growth (16.7 ± 1.5 mm, p < .01 for both), though increased over sham (13.5 ± 0.5 mm, p < .01). Ki-67 and MVD correlated with tumor growth (p < .01 for all). Additionally, lower dose 60 °C × 10 min hepatic RFA had more periablational HSP70 compared to 90 °C × 2 min (rim: 1.106 ± 163 µm vs. 360 ± 18 µm, p < .001), with similar trends for periablational α-SMA, CD68 and CDC47 (p < .01 for all). Anti-HSP70 MicQ blocked distant tumor growth for lower dose (60 °C × 10: RF/MicQ 14.6 ± 0.4 mm vs. RF alone: 18.1 ± 0.4 mm, p < .01) and higher dose RFA (90 °C × 2 min: RF/MicQ 14.6 ± 0.5 mm vs. RF alone: 16.4 ± 0.7 mm, p < .01). CONCLUSION: Hepatic RF heating parameters alter periablational HSP70, which can influence and stimulate distant tumor growth. Modulation of RF heating parameters alone or in combination with adjuvant HSP inhibition can reduce unwanted, off-target systemic tumorigenic effects.

Inflammation-Induced Expression and Secretion of MicroRNA 122 Leads to Reduced Blood Levels of Kidney-Derived Erythropoietin and Anemia.

BACKGROUND & AIMS: Anemia is associated commonly with acute and chronic inflammation, but the mechanisms of their interaction are not clear. We investigated whether microRNA 122 (MIR122), which is generated in the liver and is secreted into the blood, is involved in the development of anemia associated with inflammation. METHODS: We characterized the primary transcript of the human liver-specific MIR122 using Northern blot, quantitative real-time polymerase chain reaction, and 3' and 5' rapid amplification of cDNA ends analyses. We studied regulation of MIR122 in human hepatocellular carcinoma cell lines (Huh7 and HepG2) as well as in C57BL/6 and mice with disruption of the tumor necrosis factor (Tnf) gene. Liver tissues were collected and analyzed by bioluminescence imaging or immunofluorescence. Inflammation in mice was induced by lipopolysaccharide (LPS) or by cerulein injections. Mice were given 4 successive injections of LPS, leading to inflammation-induced anemia. Steatohepatitis was induced with a choline-deficient, high-fat diet. Hemolytic anemia was stimulated by phenylhydrazine injection. MIR122 was inhibited in mice by tail-vein injection of an oligonucleotide antagonist of MIR122. MicroRNA and messenger RNA levels were determined by quantitative real-time polymerase chain reaction. RESULTS: The primary transcript of MIR122 spanned 5 kb, comprising 3 exons; the third encodes MIR122. Within the MIR122 promoter region we identified a nuclear factor-κB binding site and showed that RELA (NF-κB p65 subunit), as well as activators of NF-κB (TNF and LPS), increased promoter activity of MIR122. Administration of LPS to mice induced secretion of MIR122 into blood, which required TNF. Secreted MIR122 reached the kidney and reduced expression of erythropoietin (Epo), which we identified as a MIR122 target gene. Injection of mice with an oligonucleotide antagonist of MIR122 increased blood levels of EPO, reticulocytes, and hemoglobin. We found an inverse relationship between blood levels of MIR122 and EPO in mice with acute pancreatitis or steatohepatitis, and also in patients with acute inflammation. CONCLUSION: In mice, we found that LPS-induced inflammation increases blood levels of MIR122, which reduces expression of Epo in the kidney; this is a mechanism of inflammation-induced anemia. Strategies to block MIR122 in patients with inflammation could reduce the development or progression of anemia.

The liver-specific microRNA-122*, the complementary strand of microRNA-122, acts as a tumor suppressor by modulating the p53/mouse double minute 2 homolog circuitry.

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).