Project description:Background & Aims: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. Methods: We analyzed gene expression profiles of non-tumor liver tissues from 392 patients with early-stage HCC (training set, n=167 and validation set, n=225) and liver tissue from patients with cirrhosis without HCC (n=216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of N-nitrosodiethylamine followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then given orally the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, given orally aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. Results: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of non-tumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor beta signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% 1.21–4.80). Mice with chemically-induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel downregulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. Conclusions: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that associates with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and developed fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.

Project description:Background & Aims: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. Methods: We analyzed gene expression profiles of non-tumor liver tissues from 392 patients with early-stage HCC (training set, n=167 and validation set, n=225) and liver tissue from patients with cirrhosis without HCC (n=216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of N-nitrosodiethylamine followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then given orally the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, given orally aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. Results: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of non-tumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor beta signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% 1.21–4.80). Mice with chemically-induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel downregulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. Conclusions: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that associates with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and developed fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis. To assess the impact of sulindac or aspirin/clopidogrel treatment on the immune-mediated cancer field, we collected samples from a choline-deficient high-fat diet fed mouse model developing NASH, HCC and fibrosis, previously published (Malehmir M et al, Nat Med, 2019) . We analyzed gene expression profiling of non-tumor liver samples from mice fed a CD-HFD for 12 weeks treated with vehicle that presented HCC and fibrosis (considered as high-risk group) (n=4); as well as mice fed a CD-HFD for 12 weeks treated with sulindac (n=10) or aspirin/clopidogrel (n=6). As controls, we analyzed samples from mice fed a chow-diet for 12 weeks (considered as healthy group) (n=5).

Project description:Chlorpyrifos oxon (CPO), the toxic metabolite of the organophosphorus (OP) insecticide chlorpyrifos, causes developmental neurotoxicity in humans and rodents. CPO is hydrolyzed by paraoxonase-1 (PON1), with protection determined by PON1 levels and the human Q192R polymorphism. To examine how the Q192R polymorphism influences fetal toxicity associated with gestational CPO exposure, we measured biomarker inhibition and fetal-brain gene expression in wild-type (PON1+/+), PON1-knockout (PON1-/-), and tgHuPON1R192 and tgHuPON1Q192 transgenic mice. Pregnant mice exposed dermally to 0, 0.50, 0.75 or 0.85 mg/kg/d CPO from gestational days (GD) 6 through 17 were sacrificed on GD18. Biomarkers of CPO exposure inhibited in maternal tissues included brain acetylcholinesterase (AChE), RBC acylpeptide hydrolase (APH), plasma butyrylcholinesterase (BChE) and carboxylesterase (CES). Fetal plasma BChE was inhibited in PON1-/- and tgHuPON1Q192, but not PON1+/+ or tgHuPON1R192 mice. Fetal brain AChE and plasma CES were inhibited in PON1-/- mice, but not in other genotypes. Pregnant mice (wild type (WT), PON1-knockout (KO), tgHuPON1R192 (R-tg) and tgHuPON1Q192 (Q-tg)) were exposed to various amounts of CPO (0, 0.5, 0.75 and 0.85 mg/kg/d) for 12 days (gestational days 6-17). On gestational day 18, dams were sacrificed and fetal brains were collected. A total of 264 fetal brains from 80 dams were processed to extract total RNA using TRIZOL and the QIAamp Tissue kit from QIAGEN. Microarray analysis was performed using the fetuses of 5 dams per experimental group (total RNA was pooled from individual fetal brains from each dam). The dams used for fetal-brain microarray analysis were selected using a random-number generator, after first eliminating dams with brain AChE activities > 1.5 SD compared to the mean for their treatment group. RNA samples isolated from individual fetal brains from each dam were combined, then labeled and hybridized to Affymetrix Mouse Gene 1.0 ST microarrays.

Project description:Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater. Saltwater acclimation requires salmonids to make various adjustments in color, shape, size, metabolism, catabolism, and osmotic and ion regulation. The molecular mechanisms underlying this transition are largely unknown. The present study acclimated coho salmon (Oncorhynchus kisutch) to four different salinities (<0.5, 8, 16, and 32 ppth) and assessed gene expression through microarray analysis of gill, liver and olfactory tissues. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory tissues are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1,616. Gills had 1,074 differentially expressed genes and olfactory tissue had 924. The difference in the number of differentially expressed genes may be due to the higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy to fuel other metabolic and osmoregulatory tissues like gills. Differentially expressed genes were tissue and salinity treatment dependent. There were no genes differentially expressed in all salinity treatments and all three tissues. Five genes were targeted for microarray confirmation by qPCR and included CCAAT/enhancer binding protein ? (CEBPB), calpain 1 (CAPN1), proto-oncogene, serine/threonine kinase (Pim1), aldolase B, fructose-bisphosphate (aldob), and complement component 3 (c3). qPCR expression profiles of these genes matched array outputs. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significant. Most terms were tissue dependent. For liver, oxygen binding and transport terms were highlighted, suggesting possible impacts on metabolism. For gills, muscle and cytoskeleton related terms were emphasized and for olfactory tissues, immune response related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosenors and signal transduction cascades. Overall this study suggests that Pacific salmonids share many salinity acclimation molecular mechanisms with other species, with a few new genes identified, and that although the three tissues shared certain underlying mechanism, many of the differentially expressed genes were tissue-specific. To assess how salinity acclimation in coho salmon (Oncorhynchus kisutch) impacted gene expression in gills, liver, and olfactory tissues.

Project description:Gene-expression profiles of hepatitis C-related, early-stage liver cirrhosis Background & Aims: Liver cirrhosis affects 1%M-bM-^HM-^R2% of population and is the major risk factor of hepatocellular carcinoma (HCC). Hepatitis C cirrhosis-related HCC is the most rapidly increasing cause of cancer death in the US. Non-invasive methods have been developed to identify patients with asymptomatic, early-stage cirrhosis, increasing the burden of HCC surveillance, but biomarkers are needed to identify patients with cirrhosis who are most in need of surveillance. We investigated whether a liver-derived 186-gene signature previously associated with outcomes of patients with HCC is prognostic for patients newly diagnosed with cirrhosis but without HCC. Methods: We performed gene expression profile analysis of formalin-fixed needle biopsies from the livers of 216 patients with hepatitis C-related early-stage (Child-Pugh class A) cirrhosis who were prospectively followed for a median of 10 years at an Italian center. We evaluated whether the 186-gene signature was associated with death, progression of cirrhosis, and development of HCC. Results: Fifty-five (25%), 101 (47%), and 60 (28%) patients were classified as having poor-, intermediate-, and good-prognosis signatures, respectively. In multivariable Cox regression modeling, the poor-prognosis signature was significantly associated with death (P=.004), progression to advanced cirrhosis (P<.001), and development of HCC (P=.009). The 10-year rates of survival were 63%, 74%, and 85% and the annual incidences of HCC were 5.8%, 2.2%, and 1.5% for patients with poor-, intermediate-, and good-prognosis signatures, respectively. Conclusions: A 186-gene signature used to predict outcomes of patients with HCC is also associated with outcomes of patients with hepatitis C-related early-stage cirrhosis. This signature might be used to identify patients with cirrhosis in most need of surveillance and strategies to prevent their development of HCC. 216 liver biopsy specimens

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Induction of hepatocellular carcinoma through activation of stromal cells in Pdgf-c transgenic mice

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Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: evidence for stromal induction of hepatocellular carcinoma.

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