Epigenetic activation of SOX11 is associated with recurrence and progression of ductal carcinoma in situ to invasive breast cancer.

BACKGROUND: Risk of recurrence and progression of ductal carcinoma in situ (DCIS) to invasive cancer remains uncertain, emphasizing the need for developing predictive biomarkers of aggressive DCIS. METHODS: Human cell lines and mouse models of disease progression were analyzed for candidate risk predictive biomarkers identified and validated in two independent DCIS cohorts. RESULTS: RNA profiling of normal mammary and DCIS tissues (n = 48) revealed that elevated SOX11 expression correlates with MKI67, EZH2, and DCIS recurrence score. The 21T human cell line model of DCIS progression to invasive cancer and two mouse models developing mammary intraepithelial neoplasia confirmed the findings. AKT activation correlated with chromatin accessibility and EZH2 enrichment upregulating SOX11 expression. AKT and HER2 inhibitors decreased SOX11 expression along with diminished mammosphere formation. SOX11 was upregulated in HER2+ and basal-like subtypes (P < 0.001). Longitudinal DCIS cohort (n = 194) revealed shorter recurrence-free survival in SOX11+ than SOX11- patients (P = 0.0056 in all DCIS; P < 0.0001 in HER2+ subtype) associated with increased risk of ipsilateral breast event/IBE (HR = 1.9, 95%CI = 1.2-2.9; P = 0.003). DISCUSSION: Epigenetic activation of SOX11 drives recurrence of DCIS and progression to invasive cancer, suggesting SOX11 as a predictive biomarker of IBE.

Targeting CXCR4 abrogates resistance to trastuzumab by blocking cell cycle progression and synergizes with docetaxel in breast cancer treatment.

BACKGROUND: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. METHODS: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. RESULTS: Using a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. CONCLUSIONS: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.

GWAS in Mice Maps Susceptibility to HIV-Associated Nephropathy to the Ssbp2 Locus.

BACKGROUND: To gain insight into the pathogenesis of collapsing glomerulopathy, a rare form of FSGS that often arises in the setting of viral infections, we performed a genome-wide association study (GWAS) among inbred mouse strains using a murine model of HIV-1 associated nephropathy (HIVAN). METHODS: We first generated F1 hybrids between HIV-1 transgenic mice on the FVB/NJ background and 20 inbred laboratory strains. Analysis of histology, BUN, and urinary NGAL demonstrated marked phenotypic variation among the transgenic F1 hybrids, providing strong evidence for host genetic factors in the predisposition to nephropathy. A GWAS in 365 transgenic F1 hybrids generated from these 20 inbred strains was performed. RESULTS: We identified a genome-wide significant locus on chromosome 13-C3 and multiple additional suggestive loci. Crossannotation of the Chr. 13 locus, including single-cell transcriptomic analysis of wildtype and HIV-1 transgenic mouse kidneys, nominated Ssbp2 as the most likely candidate gene. Ssbp2 is highly expressed in podocytes, encodes a transcriptional cofactor that interacts with LDB1 and LMX1B, which are both previously implicated in FSGS. Consistent with these data, older Ssbp2 null mice spontaneously develop glomerulosclerosis, tubular casts, interstitial fibrosis, and inflammation, similar to the HIVAN mouse model. CONCLUSIONS: These findings demonstrate the utility of GWAS in mice to uncover host genetic factors for rare kidney traits and suggest Ssbp2 as susceptibility gene for HIVAN, potentially acting via the LDB1-LMX1B transcriptional network.

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

Cancer-associated rs6983267 SNP and its accompanying long noncoding RNA CCAT2 induce myeloid malignancies via unique SNP-specific RNA mutations.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

Novel antimicrobial ointment for infected wound healing in an in vitro and in vivo porcine model.

Microbial contamination of wounds is a significant problem that delays healing, particularly when bacterial biofilms are present. A novel combination of pectinic acid (PG) + caprylic acid (CAP) was previously found in vitro to be highly effective in eradicating various pathogens in biofilms with minimal cytotoxicity. In this study, a novel wound ointment was formulated with PG + CAP and first assessed in vitro using a well-established biofilm eradication model. In vitro, the PG + CAP ointment was shown to be efficacious in reducing the microbial biofilms. This ointment was then tested in vivo in two pilot porcine wound healing models, with and without Staphylococcus aureus microbial challenge. Ointments were applied to each wound daily, and healing by wound closure area measurement was assessed weekly over 4 weeks. After 4 weeks, pigs were sacrificed and wounds were scored for reepithelialization, inflammation, granulation tissue, and collagen deposition. We compared PG + CAP to hydroxyethylcellulose + glycerol ointment base (control) and MediHoney (comparator). In the porcine microbial challenge model, the novel antimicrobial PG + CAP wound ointment rapidly eradicated bacterial organisms embedded in wounds, was safe and well-tolerated, and was associated with enhanced healing compared to ointment base and MediHoney. Specifically, the cumulative histopathology, reepithelialization of epidermis, and mature granulation tissue in the wound bed was significantly better with PG + CAP than with control and MediHoney treatments. This ointment warrants further study as a non-antibiotic ointment for use in treating a wide array of infected wounds.

MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques.

Over the past two decades, 33 cases of colonic adenocarcinomas have been diagnosed in rhesus macaques (Macaca mulatta) at the nonhuman primate colony of the Keeling Center for Comparative Medicine and Research at The University of Texas MD Anderson Cancer Center. The distinctive feature in these cases, based on PET/computed tomography (CT) imaging, was the presence of two or three tumor lesions in different locations, including proximal to the ileocecal juncture, proximal to the hepatic flexure, and/or in the sigmoid colon. These colon carcinoma lesions selectively accumulated [18F]fluorodeoxyglucose ([18F]FDG) and [18F]fluoroacetate ([18F]FACE) at high levels, reflecting elevated carbohydrate and fatty acid metabolism in these tumors. In contrast, the accumulation of [18F]fluorothymidine ([18F]FLT) was less significant, reflecting slow proliferative activity in these tumors. The diagnoses of colon carcinomas were confirmed by endoscopy. The expression of MLH1, MSH2, and MSH6 proteins and the degree of microsatellite instability (MSI) was assessed in colon carcinomas. The loss of MLH1 protein expression was observed in all tumors and was associated with a deletion mutation in the MLH1 promoter region and/or multiple single-nucleotide polymorphism (SNP) mutations in the MLH1 gene. All tumors exhibited various degrees of MSI. The pedigree analysis of this rhesus macaque population revealed several clusters of affected animals related to each other over several generations, suggesting an autosomal dominant transmission of susceptibility for colon cancer. The newly discovered hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques, termed MLH1-rheMac, may serve as a model for development of novel approaches to diagnosis and therapy of Lynch syndrome in humans.

PPARD and Interferon Gamma Promote Transformation of Gastric Progenitor Cells and Tumorigenesis in Mice.

BACKGROUND & AIMS: The peroxisome proliferator-activated receptor delta (PPARD) regulates cell metabolism, proliferation, and inflammation and has been associated with gastric and other cancers. Villin-positive epithelial cells are a small population of quiescent gastric progenitor cells. We expressed PPARD from a villin promoter to investigate the role of these cells and PPARD in development of gastric cancer. METHODS: We analyzed gastric tissues from mice that express the Ppard (PPARD1 and PPARD2 mice) from a villin promoter, and mice that did not carry this transgene (controls), by histology and immunohistochemistry. We performed cell lineage-tracing experiments and analyzed the microbiomes, chemokine and cytokine production, and immune cells and transcriptomes of stomachs of these mice. We also performed immunohistochemical analysis of PPARD levels in 2 sets of human gastric tissue microarrays. RESULTS: Thirty-eight percent of PPARD mice developed spontaneous, invasive gastric adenocarcinomas, with severe chronic inflammation. Levels of PPARD were increased in human gastric cancer tissues, compared with nontumor tissues, and associated with gastric cancer stage and grade. We found an inverse correlation between level of PPARD in tumor tissue and patient survival time. Gastric microbiomes from PPARD and control mice did not differ significantly. Lineage-tracing experiments identified villin-expressing gastric progenitor cells (VGPCs) as the origin of gastric tumors in PPARD mice. In these mice, PPARD up-regulated CCL20 and CXCL1, which increased infiltration of the gastric mucosa by immune cells. Immune cell production of inflammatory cytokines promoted chronic gastric inflammation and expansion and transformation of VGPCs, leading to tumorigenesis. We identified a positive-feedback loop between PPARD and interferon gamma signaling that sustained gastric inflammation to induce VGPC transformation and gastric carcinogenesis. CONCLUSIONS: We found PPARD overexpression in VPGCs to result in inflammation, dysplasia, and tumor formation. PPARD and VGPCs might be therapeutic targets for stomach cancer.

Sulindac plus a phospholipid is effective for polyp reduction and safer than sulindac alone in a mouse model of colorectal cancer development.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and sulindac are effective for colorectal cancer prevention in humans and some animal models, but concerns over gastro-intestinal (GI) ulceration and bleeding limit their potential for chemopreventive use in broader populations. Recently, the combination of aspirin with a phospholipid, packaged as PL-ASA, was shown to reduce GI toxicity in a small clinical trial. However, these studies were done for relatively short periods of time. Since prolonged, regular use is needed for chemopreventive benefit, it is important to know whether GI safety is maintained over longer use periods and whether cancer prevention efficacy is preserved when an NSAID is combined with a phospholipid. METHODS: As a first step to answering these questions, we treated seven to eight-week-old, male and female C57B/6 Apcmin/+ mice with the NSAID sulindac, with and without phosphatidylcholine (PC) for 3-weeks. At the end of the treatment period, we evaluated polyp burden, gastric toxicity, urinary prostaglandins (as a marker of sulindac target engagement), and blood chemistries. RESULTS: Both sulindac and sulindac-PC treatments resulted in significantly reduced polyp burden, and decreased urinary prostaglandins, but sulindac-PC treatment also resulted in the reduction of gastric lesions compared to sulindac alone. CONCLUSIONS: Together these data provide pre-clinical support for combining NSAIDs with a phospholipid, such as phosphatidylcholine to reduce GI toxicity while maintaining chemopreventive efficacy.

A Design-Based Stereologic Method to Quantify the Tissue Changes Associated with a Novel Drug-Eluting Tracheobronchial Stent.

BACKGROUND: Granulation tissue is a common complication of airway stenting, but no published methods can quantify the volume and type of tissue that develops. OBJECTIVE: To use design-based stereology to quantify changes in tissue volume and type associated with airway stenting. METHODS: We compared drug-eluting stents (DES) filled with gendine to standard silicone stents in pigs in an assessor-blinded randomized trial. Tracheal stents were placed via rigid bronchoscopy. After 1 month, animals were euthanized and necropsies were performed. Antimicrobial effects of the DES were assessed in trachea tissue samples, on the DES surface, and with residual gel from the DES reservoir. Tracheal thickness was measured using orthogonal intercepts. Design-based stereology was used to quantify the volume density of tissues using a point-counting method. The volume of each tissue was normalized to cartilage volume, which is unaffected by stenting. RESULTS: Pigs were randomized to DES (n = 36) or control stents (n = 9). The drug was successfully eluted from the DES, and the stent surface showed antibacterial activity. DES and controls did not differ in tissue microbiology, tracheal thickness, or granulation tissue volume. Compared to nonstented controls, stented airways demonstrated a 110% increase in soft-tissue volume (p = 0.005). Submucosal connective tissue (118%; p < 0.0001), epithelium (70%; p < 0.0001), submucosal glands (47%; p = 0.001), and smooth muscle (41%; p < 0.0001) increased in volume. CONCLUSION: Stenting doubles the volume of soft tissue in the trachea. Design-based stereology can quantify the tissue changes associated with airway stenting.

Development of an Electroporation and Nanoparticle-based Therapeutic Platform for Bone Metastases.

Purpose To assess for nanopore formation in bone marrow cells after irreversible electroporation (IRE) and to evaluate the antitumoral effect of IRE, used alone or in combination with doxorubicin (DOX)-loaded superparamagnetic iron oxide (SPIO) nanoparticles (SPIO-DOX), in a VX2 rabbit tibial tumor model. Materials and Methods All experiments were approved by the institutional animal care and use committee. Five porcine vertebral bodies in one pig underwent intervention (IRE electrode placement without ablation [n = 1], nanoparticle injection only [n = 1], and nanoparticle injection followed by IRE [n = 3]). The animal was euthanized and the vertebrae were harvested and evaluated with scanning electron microscopy. Twelve rabbit VX2 tibial tumors were treated, three with IRE, three with SPIO-DOX, and six with SPIO-DOX plus IRE; five rabbit VX2 tibial tumors were untreated (control group). Dynamic T2*-weighted 4.7-T magnetic resonance (MR) images were obtained 9 days after inoculation and 2 hours and 5 days after treatment. Antitumor effect was expressed as the tumor growth ratio at T2*-weighted MR imaging and percentage necrosis at histologic examination. Mixed-effects linear models were used to analyze the data. Results Scanning electron microscopy demonstrated nanopores in bone marrow cells only after IRE (P , .01). Average volume of total tumor before treatment (503.1 mm3 ± 204.6) was not significantly different from those after treatment (P = .7). SPIO-DOX was identified as a reduction in signal intensity within the tumor on T2*-weighted images for up to 5 days after treatment and was related to the presence of iron. Average tumor growth ratios were 103.0% ± 75.8 with control treatment, 154.3% ± 79.7 with SPIO-DOX, 77% ± 30.8 with IRE, and -38.5% ± 24.8 with a combination of SPIO-DOX and IRE (P = .02). The percentage residual viable tumor in bone was significantly less for combination therapy compared with control (P = .02), SPIO-DOX (P , .001), and IRE (P = .03) treatment. The percentage residual viable tumor in soft tissue was significantly less with IRE (P = .005) and SPIO-DOX plus IRE (P = .005) than with SPIO-DOX. Conclusion IRE can induce nanopore formation in bone marrow cells. Tibial VX2 tumors treated with a combination of SPIO-DOX and IRE demonstrate enhanced antitumor effect as compared with individual treatments alone. © RSNA, 2017 Online supplemental material is available for this article.

A new HIF-1α/RANTES-driven pathway to hepatocellular carcinoma mediated by germline haploinsufficiency of SART1/HAF in mice.

UNLABELLED: The hypoxia-inducible factor (HIF), HIF-1, is a central regulator of the response to low oxygen or inflammatory stress and plays an essential role in survival and function of immune cells. However, the mechanisms regulating nonhypoxic induction of HIF-1 remain unclear. Here, we assess the impact of germline heterozygosity of a novel, oxygen-independent ubiquitin ligase for HIF-1α: hypoxia-associated factor (HAF; encoded by SART1). SART1(-/-) mice were embryonic lethal, whereas male SART1(+/-) mice spontaneously recapitulated key features of nonalcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC), including steatosis, fibrosis, and inflammatory cytokine production. Male, but not female, SART1(+/-) mice showed significant up-regulation of HIF-1α in circulating and liver-infiltrating immune cells, but not in hepatocytes, before development of malignancy. Additionally, Kupffer cells derived from male, but not female, SART1(+/-) mice produced increased levels of the HIF-1-dependent chemokine, regulated on activation, normal T-cell expressed and secreted (RANTES), compared to wild type. This was associated with increased liver-neutrophilic infiltration, whereas infiltration of lymphocytes and macrophages were not significantly different. Neutralization of circulating RANTES decreased liver neutrophilic infiltration and attenuated HCC tumor initiation/growth in SART1(+/-) mice. CONCLUSION: This work establishes a new tumor-suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in male mice and identifies RANTES as a novel therapeutic target for NASH and NASH-driven HCC.

Differentiation therapy for hepatocellular carcinoma: Multifaceted effects of miR-148a on tumor growth and phenotype and liver fibrosis.

UNLABELLED: The death rate from hepatocellular carcinoma (HCC) is increasing, and liver cancer is the second leading cause of cancer-related mortality worldwide. Most patients with HCC have underlying liver cirrhosis and compromised liver function, limiting treatment options. Cirrhosis is associated with cell dedifferentiation and expansion of hepatocholangiolar progenitor cells. We identified a microRNA signature associated with HCC and hepatocytic differentiation of progenitor cells. We further identified miR-148a as an inducer of hepatocytic differentiation that is down-regulated in HCC. MiR-148a-mimetic treatment in vivo suppressed tumor growth, reduced tumor malignancy and liver fibrosis, and prevented tumor development. These effects were associated with an increased differentiated phenotype and mediated by IκB kinase alpha/NUMB/NOTCH signaling. CONCLUSION: miR-148a is an inhibitor of the IκB kinase alpha/NUMB/NOTCH pathway and an inducer of hepatocytic differentiation that when deregulated promotes HCC initiation and progression. Differentiation-targeted therapy may be a promising strategy to treat and prevent HCC.

Aurora kinase-A overexpression in mouse mammary epithelium induces mammary adenocarcinomas harboring genetic alterations shared with human breast cancer.

Recent data from The Cancer Genome Atlas analysis have revealed that Aurora kinase A (AURKA) amplification and overexpression characterize a distinct subset of human tumors across multiple cancer types. Although elevated expression of AURKA has been shown to induce oncogenic phenotypes in cells in vitro, findings from transgenic mouse models of Aurora-A overexpression in mammary glands have been distinct depending on the models generated. In the present study, we report that prolonged overexpression of AURKA transgene in mammary epithelium driven by ovine ß-lactoglobulin promoter, activated through multiple pregnancy and lactation cycles, results in the development of mammary adenocarcinomas with alterations in cancer-relevant genes and epithelial-to-mesenchymal transition. The tumor incidence was 38.9% (7/18) in Aurora-A transgenic mice at 16 months of age following 4-5 pregnancy cycles. Aurora-A overexpression in the tumor tissues accompanied activation of Akt, elevation of Cyclin D1, Tpx2 and Plk1 along with downregulation of ERα and p53 proteins, albeit at varying levels. Microarray comparative genomic hybridization (CGH) analyses of transgenic mouse mammary adenocarcinomas revealed copy gain of Glp1r and losses of Ercc5, Pten and Tcf7l2 loci. Review of human breast tumor transcriptomic data sets showed association of these genes at varying levels with Aurora-A gain of function alterations. Whole exome sequencing of the mouse tumors also identified gene mutations detected in Aurora-A overexpressing human breast cancers. Our findings demonstrate that prolonged overexpression of Aurora-A can be a driver somatic genetic event in mammary adenocarcinomas associated with deregulated tumor-relevant pathways in the Aurora-A subset of human breast cancer.

Interleukin-30 (IL27p28) alleviates experimental sepsis by modulating cytokine profile in NKT cells.

BACKGROUND & AIMS: Sepsis is an acute systemic inflammatory response to infection associated with high patient mortality (28-40%). We hypothesized that interleukin (IL)-30, a novel cytokine protecting mice against liver injury resulting from inflammation, would generate a protective effect against systemic inflammation and sepsis-induced death. METHODS: Sepsis was induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). The inhibitory effects of IL-30 on septic inflammation and associated therapeutic effects were determined in wild-type, IL30 (p28)(-/-), IL10(-/-), and CD1d(-/-) mice. RESULTS: Mice treated with pIL30 gene therapy or recombinant IL-30 protein (rIL30) were protected from LPS-induced septic shock or CLP-induced polymicrobial sepsis and showed markedly less liver damage and lymphocyte apoptosis than control septic mice. The resulting reduction in mortality was mediated through attenuation of the systemic pro-inflammatory response and augmentation of bacterial clearance. Mice lacking IL-30 were more sensitive to LPS-induced sepsis. Natural killer-like T cells (NKT) produced much higher levels of IL-10 and lower levels of interferon-gamma and tumor necrosis factor-alpha in IL-30-treated septic mice than in control septic mice. Likewise, deficiency in IL-10 or NKT cells abolished the protective role of IL-30 against sepsis. Furthermore, IL-30 induced IL-10 production in purified and LPS-stimulated NKT cells. Blocking IL-6R or gp130 inhibited IL-30 mediated IL-10 production. CONCLUSIONS: IL-30 is important in modulating production of NKT cytokines and subsequent NKT cell-mediated immune regulation of other cells. Therefore, IL-30 has a role in prevention and treatment of sepsis via modulation of cytokine production by NKT.

Irreversible Electroporation in the Epidural Space of the Porcine Spine: Effects on Adjacent Structures.

Purpose To determine the effects of irreversible electroporation (IRE) on the neural tissues after ablation in the epidural space of the porcine spine. Materials and Methods The institutional animal care and use committee approved this study. With the IRE electrode positioned in the right lateral recess of the spinal epidural space, 20 IRE ablations were performed with computed tomographic (CT) guidance by using different applied voltages in four animals that were euthanized immediately after magnetic resonance (MR) imaging of the spine, performed 6 hours after IRE (terminal group). Histopathologic characteristics of the neural tissues were assessed and used to select a voltage for a survival study. Sixteen CT-guided IRE ablations in the epidural space were performed by using 667 V in four animals that were survived for 7 days (survival group). Clinical characteristics, MR imaging findings (obtained 6 hours after IRE and before euthanasia), histopathologic characteristics, and simulated electric field strengths were assessed. A one-way analysis of variance was used to compare the simulated electric field strength to histologic findings. Results The mean distance between the IRE electrode and the spinal cord and nerve root was 1.71 mm ± 0.90 and 8.47 mm + 3.44, respectively. There was no clinical evidence of paraplegia after IRE ablation. MR imaging and histopathologic examination showed no neural tissue lesions within the spinal cord; however, five of 16 nerve roots (31.2%) demonstrated moderate wallerian degeneration in the survival group. The severity of histopathologic injury in the survival group was not significantly related to either the simulated electric field strength or the distance between the IRE electrode and the neural structure (P > .05). Conclusion Although the spinal cord appears resistant to the toxic effects of IRE, injury to the nerve roots may be a limiting factor for the use of IRE ablation in the epidural space. © RSNA, 2016 Online supplemental material is available for this article.

TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Aberrantly high expression of TRIM24 occurs in human cancers, including hepatocellular carcinoma. In contrast, TRIM24 in the mouse is reportedly a liver-specific tumour suppressor. To address this dichotomy and to uncover direct regulatory functions of TRIM24 in vivo, we developed a new mouse model that lacks expression of all Trim24 isoforms, as the previous model expressed normal levels of Trim24 lacking only exon 4. METHODS: To produce germline-deleted Trim24(dlE1) mice, deletion of the promoter and exon 1 of Trim24 was induced in Trim24(LoxP) mice by crossing with a zona pellucida 3-Cre line for global deletion. Liver-specific deletion (Trim24(hep)) was achieved by crossing with an albumin-Cre line. Phenotypic analyses were complemented by protein, gene-specific and global RNA expression analyses and quantitative chromatin immunoprecipitation. RESULTS: Global loss of Trim24 disrupted hepatic homeostasis in 100% of mice with highly significant, decreased expression of oxidation/reduction, steroid, fatty acid, and lipid metabolism genes, as well as increased expression of genes involved in unfolded protein response, endoplasmic reticulum stress and cell cycle pathways. Trim24(dlE1/dlE1) mice have markedly depleted visceral fat and, like Trim24(hep/hep) mice, spontaneously develop hepatic lipid-filled lesions, steatosis, hepatic injury, fibrosis and hepatocellular carcinoma. CONCLUSIONS: TRIM24, an epigenetic co-regulator of transcription, directly and indirectly represses hepatic lipid accumulation, inflammation, fibrosis and damage in the murine liver. Complete loss of Trim24 offers a model of human non-alcoholic fatty liver disease, steatosis, fibrosis and development of hepatocellular carcinoma in the absence of high-fat diet or obesity.

IL-30 (IL27p28) attenuates liver fibrosis through inducing NKG2D-rae1 interaction between NKT and activated hepatic stellate cells in mice.

UNLABELLED: Chronic hepatic diseases, such as cirrhosis, hepatocellular carcinoma, and virus-mediated immunopathogenic infections, affect billions of people worldwide. These diseases commonly initiate with fibrosis. Owing to the various side effects of antifibrotic therapy and the difficulty of diagnosing asymptomatic patients, suitable medication remains a major concern. To overcome this drawback, the use of cytokine-based sustained therapy might be a suitable alternative with minimal side effects. Here, we studied the therapeutic efficacy and potential mechanisms of interleukin (IL)-30 as antifibrosis therapy in murine liver fibrosis models. CCl4 or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) 0.1% (wt/wt) Purina 5015 Chow (LabDiet, St. Louis, MO) was fed for 3 weeks to induce liver fibrosis. Either control vector (pCtr) or pIL30 was injected hydrodynamically once per week. A significant decrease in collagen deposition and reduced expression of alpha-smooth muscle actin (α-SMA) protein indicated that IL-30-based gene therapy dramatically reduced bridging fibrosis that was induced by CCl4 or DDC. Immunophenotyping and knockout studies showed that IL-30 recruits natural-killer-like T (NKT) cells to the liver to remove activated hepatic stellate cells (HSCs) significantly and ameliorate liver fibrosis. Both flow cytometric and antibody-mediated neutralization studies showed that liver NKT cells up-regulate the natural killer group 2, member D (NKG2D) ligand and bind with the NKG2D ligand, retinoic acid early inducible 1 (Rae1), and positively activated HSCs to ameliorate liver fibrosis. Furthermore, adoptive transfer of liver NKT cells in T-cell-deficient mice showed reduction of fibrosis upon IL-30 administration. CONCLUSIONS: Highly target-specific liver NKT cells selectively remove activated HSCs through an NKG2D-Rae1 interaction to ameliorate liver fibrosis after IL-30 treatment.

PBI-05204, a supercritical CO2 extract of Nerium oleander, inhibits growth of human pancreatic cancer via targeting the PI3K/mTOR pathway.

Introduction Oleandrin, a cardiac glycoside, exerts strong anti-proliferative activity against various human malignancies in in vitro cells. Here, we report the antitumor efficacy of PBI-05204, a supercritical C02 extract of Nerium oleander containing oleandrin, in a human pancreatic cancer Panc-1 orthotopic model. Results While all the control mice exhibited tumors by the end of treatment, only 2 of 8 mice (25%) treated for 6 weeks with PBI-05204 (40 mg/kg) showed dissectible tumor at the end of the treatment period. The average tumor weight (222.9 ± 116.9 mg) in mice treated with PBI-05204 (20 mg/kg) was significantly reduced from that in controls (920.0 ± 430.0 mg) (p < 0.05). Histopathologic examination of serial sections from each pancreas with no dissectible tumor in the PBI-05204 (40 mg/kg) treated group showed that the pancreatic tissues of 5/6 mice were normal while the remaining mouse had a tumor the largest diameter of which was less than 2.3 mm. In contrast, while gemcitabine alone did not significantly reduce tumor growth, PBI-05204 markedly enhanced the antitumor efficacy of gemcitabine in this particular model. Ki-67 staining was reduced in pancreatic tumors from mice treated with PBI-05204 (20 mg/kg) compared to that of control, suggesting that PBI-05204 inhibited the proliferation of the Panc-1 tumor cells. PBI-05204 suppressed expression of pAkt, pS6, and p4EPB1 in a concentration-dependent manner in both Panc-1 tumor tissues and human pancreatic cancer cell lines, implying that this novel botanical drug exerts its potent antitumor activity, at least in part, through down-regulation of PI3k/Akt and mTOR pathways.