Comprehensive tissue deconvolution of cell-free DNA by deep learning for disease diagnosis and monitoring.

Plasma cell-free DNA (cfDNA) is a noninvasive biomarker for cell death of all organs. Deciphering the tissue origin of cfDNA can reveal abnormal cell death because of diseases, which has great clinical potential in disease detection and monitoring. Despite the great promise, the sensitive and accurate quantification of tissue-derived cfDNA remains challenging to existing methods due to the limited characterization of tissue methylation and the reliance on unsupervised methods. To fully exploit the clinical potential of tissue-derived cfDNA, here we present one of the largest comprehensive and high-resolution methylation atlas based on 521 noncancer tissue samples spanning 29 major types of human tissues. We systematically identified fragment-level tissue-specific methylation patterns and extensively validated them in orthogonal datasets. Based on the rich tissue methylation atlas, we develop the first supervised tissue deconvolution approach, a deep-learning-powered model, cfSort, for sensitive and accurate tissue deconvolution in cfDNA. On the benchmarking data, cfSort showed superior sensitivity and accuracy compared to the existing methods. We further demonstrated the clinical utilities of cfSort with two potential applications: aiding disease diagnosis and monitoring treatment side effects. The tissue-derived cfDNA fraction estimated from cfSort reflected the clinical outcomes of the patients. In summary, the tissue methylation atlas and cfSort enhanced the performance of tissue deconvolution in cfDNA, thus facilitating cfDNA-based disease detection and longitudinal treatment monitoring.

Hippo signaling pathway activation during SARS-CoV-2 infection contributes to host antiviral response.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19-associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin, a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.

PNPASE regulates RNA import into mitochondria.

RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3' --> 5' exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE-imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria.

HCC EV ECG score: An extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma.

BACKGROUND AND AIMS: The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC. APPROACH AND RESULTS: Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n  = 106) and an independent validation cohort ( n  = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99). CONCLUSION: HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

The Major Histocompatibility Complex Class II-CD4 Immunologic Synapse in Alcoholic Hepatitis and Autoimmune Liver Pathology: The Role of Aberrant Major Histocompatibility Complex Class II in Hepatocytes.

The major histocompatibility complex class II (MHC II)-CD4 immunologic synapse is classically described between the T-cell receptor of CD4-positive lymphocytes and MHC II on antigen-presenting cells. This interaction and others between surrounding costimulatory and checkpoint molecules promote differentiation of naïve CD4 T lymphocytes into helper T cells subtypes, including types 1, 2, and 17 helper T cells, that have more tailored immunologic responses. Although MHC II is mainly produced by professional antigen-presenting cells, it can be aberrantly produced by other cell types, including hepatocytes in various liver pathologies, such as autoimmune hepatitis and alcoholic hepatitis. This can lead to direct targeting of hepatocytes by CD4-positive lymphocytes, which form an immunologic synapse with the hepatocyte. The lymphocytes internalize the MHC II-CD4 complexes in a phagocytosis-like mechanism and in the process eat the hepatocyte piece by piece. We review the evidence for this mechanism and the role of these autoimmune responses in various liver diseases, including alcoholic hepatitis, autoimmune hepatitis, and primary biliary cirrhosis. The role of aberrant MHC II in malignancy, including hepatocellular carcinoma, is also reviewed. Further understanding of this mechanism can lead to better understanding of the immune mechanisms involved in these liver pathologies, with potential diagnostic and therapeutic applications.

Hippo Signaling Pathway Has a Critical Role in Zika Virus Replication and in the Pathogenesis of Neuroinflammation.

Zika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities, including microcephaly and eye disease. The cellular/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Herein, we noted that the Hippo/Salvador-Warts-Hippo signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated after ZIKV infection. In human fetal retinal pigment epithelial cells, there is an early induction of transcriptional coactivator, Yes-associated protein (YAP), which is later degraded with a corresponding activation of the TANK binding kinase 1/interferon regulatory factor 3 type I interferon pathway. YAP/transcriptional co-activator with a PDZ-binding domain (TAZ) silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnar1-/- knockout mouse model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing phosphorylated YAP Ser127 in the retina and brain. Hippo pathway is activated in major cellular components of the blood-brain barrier, including endothelial cells and astrocytes. In addition, this result suggests AMP-activated protein kinase signaling pathway's role in regulating YAP/TAZ in ZIKV-infected cells. These data demonstrate that ZIKV infection might initiate a cross talk among AMP-activated protein kinase-Hippo-TBK1 pathways, which could regulate antiviral and energy stress responses during oculoneuronal inflammation.

Development of Capsular Fibrosis Beneath the Liver Surface in Humans and Mice.

Glisson's capsule is the connective tissue present in the portal triad as well as beneath the liver surface. Little is known about how Glisson's capsule changes its structure in capsular fibrosis (CF), which is characterized by fibrogenesis beneath the liver surface. In this study, we found that the human liver surface exhibits multilayered capsular fibroblasts and that the bile duct is present beneath the mesothelium, whereas capsular fibroblasts are scarce and no bile ducts are present beneath the mouse liver surface. Patients with cirrhosis caused by alcohol abuse or hepatitis C virus infection show development of massive CF. To examine the effect of alcohol on CF in mice, we first injected chlorhexidine gluconate (CG) intraperitoneally and then fed alcohol for 1 month. The CG injection induces CF consisting of myofibroblasts beneath the mesothelium. One month after CG injection, the fibrotic area returns to the normal structure. In contrast, additional alcohol feeding sustains the presence of myofibroblasts in CF. Cell lineage tracing revealed that mesothelial cells give rise to myofibroblasts in CF, but these myofibroblasts disappear 1 month after recovery with or without alcohol feeding. Capsular fibroblasts isolated from the mouse liver spontaneously differentiated into myofibroblasts and their differentiation was induced by transforming growth factor beta 1 (TGF-ß1) or acetaldehyde in culture. In alcohol-fed mice, infiltrating CD11b+ Ly-6CLow/- monocytes had reduced mRNA expression of matrix metalloproteinase 13 and matrix metalloproteinase 9 and increased expression of tissue inhibitor of matrix metalloproteinase 1, Tgfb1, and interleukin-10 during resolution of CF. Conclusion: The present study revealed that the structure of Glisson's capsule is different between human and mouse livers and that alcohol impairs the resolution of CF by changing the phenotype of Ly-6CLow/- monocytes.

Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection.

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization's and National Institutes of Health's high-priority MDR pathogens for targeted development of new therapies. Here, we show that stabilizing the host's vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii, P. aeruginosa, and CPKP. We show that the pharmacological inhibition of ARF6-GTP phenocopies endothelium-specific Arf6 disruption in enhancing the survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small-molecule inhibitors acts by the restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of the TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host's vasculature with small-molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and reemerging pathogens.

Moderate alcohol intake promotes pancreatic ductal adenocarcinoma development in mice expressing oncogenic Kras.

Kras mutations are associated with pancreatic ductal adenocarcinoma (PDAC). Although tobacco smoking, pancreatitis, and obesity are known environmental risk factors for PDAC, the contribution of moderate alcohol intake to PDAC remains elusive. In the present study, we tested whether a combination of risk factors or moderate alcohol intake induces PDAC development in mice. Control Pdx1Cre and Pdx1Cre;LSL-KrasG12D mutant mice were fed a Western alcohol diet containing high levels of cholesterol and saturated fat, 3.5% alcohol, and lipopolysaccharide for 5 mo. In addition, mice were treated with cerulein, for induction of pancreatitis, and nicotine every month. Treatment with all of these risk factors promoted development of advanced pancreatic neoplasia and PDAC in the Pdx1Cre;LSL-KrasG12D mice but not in the control Pdx1Cre mice. Moderate alcohol intake or Western diet feeding also significantly promoted advanced neoplasia and PDAC development in Pdx1Cre;LSL-KrasG12D mice compared with mice fed a regular chow. Alcohol, but not Western diet, increased tumor development in the liver in the Pdx1Cre;LSL-KrasG12D mice, but its origin remained elusive due to leakiness of Pdx1Cre in hepatocytes. RNA-seq analysis revealed that alcohol feeding increases expression of markers for tumors (Epcam, Krt19, Prom1, Wt1, and Wwtr1), stroma (Dcn, Fn1, and Tnc), and cytokines (Tgfb1 and Tnf) and decreases expression of Fgf21 and Il6 in the pancreatic tumor tissues. Immunostaining showed heterogeneous expression of nephronectin, S100 calcium-binding protein A6, and vascular cell adhesion molecule 1 in pancreatic tumors surrounded by podoplanin-positive stromal cells. Our data indicate that moderate alcohol drinking is a risk factor for development of PDAC.NEW & NOTEWORTHY Heavy alcohol intake has been suspected to be a risk factor of pancreatic ductal adenocarcinoma (PDAC) in humans. However, the contribution of moderate alcohol intake to PDAC development remains elusive. In the present study, we experimentally show that moderate alcohol feeding significantly induces advanced stages of pancreatic intraepithelial neoplasia development and invasive PDAC in Pdx1Cre;LSL-KrasG12D mutant mice. Our data indicate that moderate alcohol drinking is a risk factor for PDAC.

RARß acts as both an upstream regulator and downstream effector of miR-22, which epigenetically regulates NUR77 to induce apoptosis of colon cancer cells.

This study investigates the mechanism and consequences of microRNA-22 ( miR-22) induction. Our data revealed for the first time that retinoic acid (RA) and histone deacetylase (HDAC) inhibitors, including short-chain fatty acids and suberanilohydroxamic acid (SAHA), could individually or in combination induce miR-22. This induction was mediated via RA receptor ß (RARß) binding to a direct repeat 5 (DR5) motif. In addition, we uncovered HDAC1 as a novel miR-22 target. In an miR-22-dependent manner, HDAC inhibitors and RA reduced HDAC1, HDAC4, and sirtuin 1 (SIRT1), which were involved in chromatin remodeling of the RARß and nerve growth factor IB ( NUR77). Thus, HDAC inhibitors and RA-induced miR-22 resulted in simultaneous induction of cytoplasmic RARß and NUR77, leading to apoptosis of colon cancer cells. In mice, miR-22 and its inducers inhibited the growth of xenograft colon cancer. Moreover, tumor size reduction was accompanied by elevated miR-22, NUR77, and RARß and by reduced HDACs. In human colon polyps and adenocarcinomas, miR-22 and RARß were consistently reduced, which was associated with elevated HDAC1, HDAC4, and SIRT1 in colon adenocarcinomas. Results from this study revealed a novel anticancer mechanism of RARß via miR-22 induction to epigenetically regulate itself and NUR77, providing a promising cancer treatment modality using miR-22 and its inducers.-Hu, Y., French, S. W., Chau, T., Liu, H.-X., Sheng, L., Wei, F., Stondell, J., Garcia, J. C., Du, Y., Bowlus, C. L., Wan, Y.-J. Y. RARß acts as both an upstream regulator and downstream effector of miR-22, which epigenetically regulates NUR77 to induce apoptosis of colon cancer cells.

Altered regulation of LncRNA analysis of human alcoholic hepatitis with Mallory-Denk Bodies (MDBs) is revealed by RNA sequencing.

Mallory-Denk Bodies (MDBs) are prevalent in a variety of liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Long noncoding RNAs (lncRNAs) are considered as emerging new gene regulators, which participates in many functional activities through diverse mechanisms. We previously reported the mechanisms involved in the formation of liver MDBs in mouse model and in AH livers where MDBs had formed. To investigate the regulation of mRNAs expression and the probable role of lncRNAs in AH livers with MDBs, RNA-Seq analyses was further conducted to determine the mRNA and lncRNA expression profiles of the AH livers compared with the normal livers. It showed that different lncRNAs have different information contribution degrees by principal component analysis, and the integrated analysis of lncRNA-mRNA co-expression networks were linked to endocytosis, cell cycle, p53 signaling pathways in the human. Based on the co-expression networks, we identify 36 mRNAs that could be as potential biomarkers of alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). To our knowledge, this is the first report on the regulatory network of lncRNAs associated with liver MDB formation in human, and these results might offer new insights into the molecular mechanisms of liver MDB formation and the progression of AH to HCC.

Pregnane X receptor promotes ethanol-induced hepatosteatosis in mice.

The pregnane X receptor (PXR, NR1I2) is a xenobiotic-sensing nuclear receptor that modulates the metabolic response to drugs and toxic agents. Both PXR activation and deficiency promote hepatic triglyceride accumulation, a hallmark feature of alcoholic liver disease. However, the molecular mechanism of PXR-mediated activation of ethanol (EtOH)-induced steatosis is unclear. Here, using male wildtype (WT) and Pxr-null mice, we examined PXR-mediated regulation of chronic EtOH-induced hepatic lipid accumulation and hepatotoxicity. EtOH ingestion for 8 weeks significantly (1.8-fold) up-regulated Pxr mRNA levels in WT mice. The EtOH exposure also increased mRNAs encoding hepatic constitutive androstane receptor (3-fold) and its target, Cyp2b10 (220-fold), in a PXR-dependent manner. Furthermore, WT mice had higher serum EtOH levels and developed hepatic steatosis characterized by micro- and macrovesicular lipid accumulation. Consistent with the development of steatosis, lipogenic gene induction was significantly increased in WT mice, including sterol regulatory element-binding protein 1c target gene fatty-acid synthase (3.0-fold), early growth response-1 (3.2-fold), and TNFα (3.0-fold), whereas the expression of peroxisome proliferator-activated receptor α target genes was suppressed. Of note, PXR deficiency suppressed these changes and steatosis. Protein levels, but not mRNAs levels, of EtOH-metabolizing enzymes, including alcohol dehydrogenase 1, aldehyde dehydrogenase 1A1, and catalase, as well as the microsomal triglyceride transfer protein, involved in regulating lipid output were higher in Pxr-null than in WT mice. These findings establish that PXR signaling contributes to ALD development and suggest that PXR antagonists may provide a new approach for ALD therapy.

Hepatic Tm6sf2 overexpression affects cellular ApoB-trafficking, plasma lipid levels, hepatic steatosis and atherosclerosis.

The human transmembrane 6 superfamily member 2 (TM6SF2) gene has been implicated in plasma lipoprotein metabolism, alcoholic and non-alcoholic fatty liver disease and myocardial infarction in multiple genome-wide association studies. To investigate the role of Tm6sf2 in metabolic homeostasis, we generated mice with elevated expression using adeno-associated virus (AAV)-mediated gene delivery. Hepatic overexpression of mouse Tm6sf2 resulted in phenotypes previously observed in Tm6sf2-deficient mice including reduced plasma lipid levels, diminished hepatic triglycerides secretion and increased hepatosteatosis. Furthermore, increased hepatic Tm6sf2 expression protected against the development of atherosclerosis in LDL-receptor/ApoB48-deficient mice. In cultured human hepatocytes, Tm6sf2 overexpression reduced apolipoprotein B secretion and resulted in its accumulation within the endoplasmic reticulum (ER) suggesting impaired ER-to-Golgi trafficking of pre-very low-density lipoprotein (VLDL) particles. Analysis of two metabolic trait-associated coding polymorphisms in the human TM6SF2 gene (rs58542926 and rs187429064) revealed that both variants impact TM6SF2 expression by affecting the rate of protein turnover. These data demonstrate that rs58542926 (E167K) and rs187429064 (L156P) are functional variants and suggest that they influence metabolic traits through altered TM6SF2 protein stability. Taken together, our results indicate that cellular Tm6sf2 level is an important determinant of VLDL metabolism and further implicate TM6SF2 as a causative gene underlying metabolic disease and trait associations at the 19p13.11 locus.

Mitochondrial Dysfunction, Through Impaired Autophagy, Leads to Endoplasmic Reticulum Stress, Deregulated Lipid Metabolism, and Pancreatitis in Animal Models.

BACKGROUND & AIMS: Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca2+, mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats. METHODS: Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence. RESULTS: Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca2+ overload or through a Ca2+ overload-independent pathway that involved reduced activity of ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas. CONCLUSIONS: In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.

Baicalein Targets GTPase-Mediated Autophagy to Eliminate Liver Tumor-Initiating Stem Cell-Like Cells Resistant to mTORC1 Inhibition.

Drug resistance is a major problem in the treatment of liver cancer. Mammalian Target of Rapamycin 1 (mTORC1) inhibitors have been tested for the treatment of liver cancer based on hyperactive mTOR in this malignancy. However, their clinical trials showed poor outcome, most likely due to their ability to upregulate CD133 and promote chemoresistance. The CD133+ tumor-initiating stem cell-like cells (TICs) isolated from mouse and human liver tumors are chemoresistant, and identification of an approach to abrogate this resistance is desired. In search of a compound that rescinds resistance of TICs to mTORC1 inhibition and improves chemotherapy, we identified baicalein (BC), which selectively chemosensitizes TICs and the human hepatocellular carcinoma (HCC) cell line Huh7 cells but not mouse and human primary hepatocytes. Nanobead pull-down and mass-spectrometric analysis, biochemical binding assay, and three-dimensional computational modeling studies reveal BC's ability to competitively inhibit guanosine triphosphate binding of SAR1B guanosine triphosphatase, which is essential for autophagy. Indeed, BC suppresses autophagy induced by an mTORC1 inhibitor and synergizes cell death caused by mTORC1 inhibition in TIC and Huh7 spheroid formation and in the patient-derived xenograft model of HCC. The BC-induced chemosensitization is rescued by SAR1B expression and phenocopied by SAR1B knockdown in cancer cells treated with a mTORC1 inhibitor. Conclusion: These results identify SAR1B as a target in liver TICs and HCC cells resistant to mTORC1 inhibition.

Pyroptosis by caspase11/4-gasdermin-D pathway in alcoholic hepatitis in mice and patients.

Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH is largely unknown. To address this question, unbiased RNA sequencing and proteomic analyses were performed on livers of the recently developed AH mouse model, which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared to gene expression profiling data from AH patients. This cross-analysis has identified Casp11 (CASP4 in humans) as a commonly up-regulated gene known to be involved in the noncanonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients, but not in chronic ASH mice and healthy human livers. Gasdermin-D (GSDMD), which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and severity of AH in the mouse model. Conversely, the deficiency of interleukin-18, the key antimicrobial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Furthermore, hepatocyte-specific expression of constitutively active GSDMD worsens hepatocellular lytic death and polymorphonuclear leukocyte inflammation. CONCLUSION: These results implicate pyroptosis induced by the CASP11/4-GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737-1753).

The Genetic Architecture of Diet-Induced Hepatic Fibrosis in Mice.

We report the genetic analysis of a "humanized" hyperlipidemic mouse model for progressive nonalcoholic steatohepatitis (NASH) and fibrosis. Mice carrying transgenes for human apolipoprotein E*3-Leiden and cholesteryl ester transfer protein and fed a "Western" diet were studied on the genetic backgrounds of over 100 inbred mouse strains. The mice developed hepatic inflammation and fibrosis that was highly dependent on genetic background, with vast differences in the degree of fibrosis. Histological analysis showed features characteristic of human NASH, including macrovesicular steatosis, hepatocellular ballooning, inflammatory foci, and pericellular collagen deposition. Time course experiments indicated that while hepatic triglyceride levels increased steadily on the diet, hepatic fibrosis occurred at about 12 weeks. We found that the genetic variation predisposing to NASH and fibrosis differs markedly from that predisposing to simple steatosis, consistent with a multistep model in which distinct genetic factors are involved. Moreover, genome-wide association identified distinct genetic loci contributing to steatosis and NASH. Finally, we used hepatic expression data from the mouse panel and from 68 bariatric surgery patients with normal liver, steatosis, or NASH to identify enriched biological pathways. Conclusion: The pathways showed substantial overlap between our mouse model and the human disease.

The different expression of tumor suppressors, RASSF1A, RUNX3, and GSTP1, in patients with alcoholic steatohepatitis (ASH) vs non-alcoholic steatohepatitis (NASH).

As the fifth most common cancer and the second leading cause of cancer related deaths worldwide, hepatocellular carcinoma (HCC) causes up to one million deaths annually. Alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) are becoming the two major risk factors because both may develop liver fibrosis and hepatocellular carcinoma (HCC) if left untreated. However, compared with 3-10% of patients with ASH may progress to HCC annually, about only 0.5% NASH patients may progress to HCC annually. The present study is to clarify the protein expression differences of tumor suppressor genes (TSGs) between ASH and NASH. In liver biopsied specimens from NASH and ASH patients, using an immunofluorescence method and morphometrically quantitating the fluorescence intensity, we studied the protein expression within hepatocytes cytoplasm of candidate TSGs including RUNX3, GSTP1, and RASSF1A. Compared with the control group of patients, the expression levels of all three proteins were upregulated in the ASH group of patients (p < .001 in all molecules). While RUNX3 was upregulated, GSTP1 and RASSF1 did not change in the NASH group of patients. The most important finding is that compared with the ASH group of patients, the expression levels of all three TSG proteins, RUNX3, GSTP1, and RASSF1, were significantly lower in the NASH group of patients (p < .001 in all three molecules). These results confirmed our previous finding that there are significant differences of many molecules including TSGs that changed in NASH compared to ASH. Thus, we conclude that there are significantly different TSGs and pathways involved during the pathogenesis of HCC development in NASH compared to ASH that may help to develop different strategies for prevention and treatment of NASH and ASH patients.

miR-186-5p promotes cell growth, migration and invasion of lung adenocarcinoma by targeting PTEN.

OBJECTIVE: To explore the expression of miR-186-5p in lung adenocarcinoma (LUAD) and its possible function associated with cancer cell proliferation, migration and invasion. METHODS: MiR-186-5p expression levels in LUAD samples, human LUAD cell lines H1299 and NCI-H1975, and normal human lung epithelial cell line BEAS-IB were assessed by quantitative real-time PCR (qRT-PCR). H1299 and NCI-H1975 cells were transfected with miR-186-5p mimic or miRNA negative control. CCK-8 assay was performed to evaluate the cell proliferation. Transwell assay and transwell-matrigel™ invasion assay were applied to assess the migration and invasion abilities of H1299 and NCI-H1975 cells. RESULTS: miR-186-5p expression was significantly up-regulated in LUAD tumor tissues and LUAD cell lines as compared with tumor-adjacent tissues and normal human lung epithelial cells, respectively. MiR-186-5p overexpression remarkably promoted the proliferation, migration and invasion of LUAD cells. Furthermore, phosphatase and tensin homolog (PTEN) was a direct target of miR-186-5p verified by luciferase reporter assay. Overexpression of PTEN significantly suppressed LUAD cells to proliferate, migrate and invade. MiR-186-5p overexpression-induced LUAD cell phenotype could be partially rescued by co-overexpression of miR-186-5p and PTEN. CONCLUSION: This study demonstrated that miR-186-5p is up-regulated in LUAD, and functionally associated with cell proliferation, migration and invasion. MiR-186-5p promotes the proliferation, migration and invasion of LUAD cells by targeting PTEN. MiR-186-5p may be utilized as a novel molecular marker and therapeutic target of LUAD.

TKS5-positive invadopodia-like structures in human tumor surgical specimens.

Invadopodia, cancer cell protrusions with proteolytic activity, are functionally associated with active remodeling of the extracellular matrix. Here, we show that the invadopodia-related protein TKS5 is expressed in human pancreatic adenocarcinoma lines, and demonstrate that pancreatic cancer cells depend on TKS5 for invadopodia formation and function. Immunofluorescence staining of human pancreatic cancer cells reveals that TKS5 is a marker of mature and immature invadopodia. We also analyze the co-staining patterns of TKS5 and the commonly used invadopodia marker Cortactin, and find only partial co-localization of these two proteins at invadopodia, with a large fraction of TKS5-positive invadopodia lacking detectable levels of Cortactin. Whereas compelling evidence exist on the role of invadopodia as mediators of invasive migration in cultured cells and in animal models of cancer, these structures have never been detected inside human tumors. Here, using antibodies against TKS5 and Cortactin, we describe for the first time structures strongly resembling invadopodia in various paraffin-embedded human tumor surgical specimens from pancreas and other organs. Our results strongly suggest that invadopodia are present inside human tumors, and warrants further investigation on their regulation and occurrence in surgical specimens, and on the value of TKS5 antibodies as pathological research and diagnostic tools.