Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

HNF1B-driven three-dimensional chromatin structure for molecular classification in pancreatic cancers.

The molecular subtypes of pancreatic cancer (PC), either classical/progenitor-like or basal/squamous-like, are currently a major topic of research because of their direct association with clinical outcomes. Some transcription factors (TFs) have been reported to be associated with these subtypes. However, the mechanisms by which these molecular signatures of PCs are established remain unknown. Epigenetic regulatory processes, supported by dynamic changes in the chromatin structure, are essential for transcriptional profiles. Previously, we reported the importance of open chromatin profiles in the biological features and transcriptional status of PCs. Here, we aimed to analyze the relationships between three-dimensional (3D) genome structures and the molecular subtypes of human PCs using Hi-C analysis. We observed a correlation of the specific elements of 3D genome modules, including compartments, topologically associating domains, and enhancer-promoter loops, with the expression of related genes. We focused on HNF1B, a TF that is implicated in the progenitor subtype. Forced expression of HNF1B in squamous-type PC organoids induced the upregulation and downregulation of genes associated with progenitor and squamous subtypes, respectively. Long-range genomic interactions induced by HNF1B were accompanied by compartment modulation and H3K27ac redistribution. We also found that these HNF1B-induced changes in subtype-related gene expression required an intrinsically disordered region, suggesting a possible involvement of phase separation in compartment modulation. Thus, mapping of 3D structural changes induced by TFs, such as HNF1B, may become a useful resource for further understanding the molecular features of PCs.

WWP1 Gain-of-Function Inactivation of PTEN in Cancer Predisposition.

BACKGROUND: Patients with PTEN hamartoma tumor syndrome (PHTS) have germline mutations in the tumor-suppressor gene encoding phosphatase and tensin homologue (PTEN). Such mutations have been associated with a hereditary predisposition to multiple types of cancer, including the Cowden syndrome. However, a majority of patients who have PHTS-related phenotypes have tested negative for PTEN mutations. In a previous study, we found that the E3 ubiquitin ligase WWP1 negatively regulates the function of PTEN. METHODS: In a prospective cohort study conducted from 2005 through 2015, we enrolled 431 patients with wild-type PTEN who met at least the relaxed diagnostic criteria of the International Cowden Consortium. Patients were scanned for WWP1 germline variants. We used the Cancer Genome Atlas (TCGA) data set as representative of apparently sporadic cancers and the Exome Aggregation Consortium data set excluding TCGA (non-TCGA ExAC) and the noncancer Genome Aggregation Database (gnomAD) as representative of population controls without a reported cancer diagnosis. We established both in vitro and murine in vivo models to functionally characterize representative WWP1 variants. RESULTS: The existence of germline WWP1 variants was first established in a family with wild-type PTEN who had oligopolyposis and early-onset colon cancers. A validation series indicated that WWP1 germline variants occurred in 5 of 126 unrelated patients (4%) with oligopolyposis as a predominant phenotype. Germline WWP1 variants, particularly the WWP1 K740N and N745S alleles, were enriched in patients who did not have PHTS but had prevalent sporadic cancers, including PTEN-related cancer types in TCGA (odds ratio, 1.5; 95% confidence interval, 1.1 to 2.1; P = 0.01). The prioritized WWP1 variants resulted in gain-of-function effects, which led to aberrant enzymatic activation with consequent PTEN inactivation, thereby triggering hyperactive growth-promoting PI3K signaling in cellular and murine models. CONCLUSIONS: In this study involving patients with disorders resulting in a predisposition to the development of multiple malignant neoplasms without PTEN germline mutations, we confirmed the function of WWP1 as a cancer-susceptibility gene through direct aberrant regulation of the PTEN-PI3K signaling axis. (Funded by the National Institutes of Health and others.).

MNX1-HNF1B Axis Is Indispensable for Intraductal Papillary Mucinous Neoplasm Lineages.

BACKGROUND & AIMS: Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMN) with an associated invasive carcinoma (IPMNinv) arise from 2 distinct precursors, and their fundamental differences remain obscure. Here, we aimed to assess the difference of chromatin architecture regulating the transcriptional signatures or biological features in pancreatic cancers. METHODS: We established 28 human organoids from distinct subtypes of pancreatic tumors, including IPMN, IPMNinv, and PDAC. We performed exome sequencing (seq), RNA-seq, assay for transposase-accessible chromatin-seq, chromatin immunoprecipitation-seq, high-throughput chromosome conformation capture, and phenotypic analyses with short hairpin RNA or clustered regularly interspaced short palindromic repeats interference. RESULTS: Established organoids successfully reproduced the histology of primary tumors. IPMN and IPMNinv organoids harbored GNAS, RNF43, or KLF4 mutations and showed the distinct expression profiles compared with PDAC. Chromatin accessibility profiles revealed the gain of stomach-specific open regions in IPMN and the pattern of diverse gastrointestinal tissues in IPMNinv. In contrast, PDAC presented an impressive loss of accessible regions compared with normal pancreatic ducts. Transcription factor footprint analysis and functional assays identified that MNX1 and HNF1B were biologically indispensable for IPMN lineages. The upregulation of MNX1 was specifically marked in the human IPMN lineage tissues. The MNX1-HNF1B axis governed a set of genes, including MYC, SOX9, and OLFM4, which are known to be essential for gastrointestinal stem cells. High-throughput chromosome conformation capture analysis suggested the HNF1B target genes to be 3-dimensionally connected in the genome of IPMNinv. CONCLUSIONS: Our organoid analyses identified the MNX1-HNF1B axis to be biologically significant in IPMN lineages.

Hepatitis B virus-associated hepatocellular carcinoma with Smc5/6 complex deficiency is susceptible to PARP inhibitors.

DNA repair processes represent attractive synthetic lethal targets because many cancers exhibit impaired DNA repair pathways, which leads to dependence on specific repair proteins. The finding that poly (ADP-ribose) polymerase (PARP)-1 inhibitors are highly effective against cancers with deficient homologous recombination highlights the potential of this approach. In hepatitis B viral (HBV) infection, degradation of the structural maintenance of the chromosome 5/6 (Smc5/6) complex, which plays a key role in repairing double-stranded DNA breaks by homologous recombination, is induced by HBV regulatory protein X (HBx). Here, we hypothesized that a deficiency in the Smc5/6 complex in HBV-associated hepatocellular carcinoma (HCC) increases susceptibility to PARP inhibitors via a deficiency in homologous recombination. We confirmed impaired double-stranded DNA break repair in HBx-expressing HCC cells using a sensitive reporter to monitor homologous recombination. Treatment with a PARP inhibitor was significantly more effective against HBx-expressing HCC cells, and overexpression of Smc5/6 prevented these effects. Overall, our results suggest that homologous recombination deficiency in HBV-associated HCC leads to increased susceptibility to PARP inhibitors.

Smaller extracellular vesicles are released from pancreatic cancer cells by the alteration of the lipid composition under low glucose conditions.

Extracellular vesicles (EVs) released from cells into the blood facilitate intercellular communication and serve as new biomarkers to understand the pathophysiology of several conditions. Although the importance of the cargo inside EVs has been extensively studied, the sizes of EVs that vary with different types of cancers are relatively poorly explored. Here, we show that pancreatic cancer cell-derived EVs are significantly smaller than non-cancer cell-derived EVs. The smaller size distribution of these EVs was confirmed by specifically isolating and examining tumor-derived EVs from the heterogeneous EV population isolated from the sera of patients with pancreatic ductal adenocarcinoma. In vitro analyses mimicking tumor microenvironment conditions revealed that low glucose conditions reduced the size distribution and increased the level of unsaturated fatty acids in the tumor-derived EVs. Because the lipid composition defines the fluidity of the membrane, the results suggest that the alterations in the size of EVs could be due to the alteration of the fluidity and stability of the membrane covering the EVs. Furthermore, the uptake of smaller EVs by recipient cells was increased, which may lead to enhanced functional results. These results provide fundamental insights into the factors defining the size of EVs, which may be important for developing cancer screening methods and understanding cancer-related pathophysiology.

Pevonedistat, a Neuronal Precursor Cell-Expressed Developmentally Down-Regulated Protein 8-Activating Enzyme Inhibitor, Is a Potent Inhibitor of Hepatitis B Virus.

Hepatitis B virus (HBV) infection is a major health concern worldwide. To prevent HBV-related mortality, elimination of viral proteins is considered the ultimate goal of HBV treatment; however, currently available nucleos(t)ide analogs rarely achieve this goal, as viral transcription from episomal viral covalently closed circular DNA (cccDNA) is not prevented. HBV regulatory protein X was recently found to target the protein structural maintenance of chromosomes 5/6 (Smc5/6) for ubiquitination and degradation by DDB1-CUL4-ROC1 E3 ligase, resulting in enhanced viral transcription from cccDNA. This ubiquitin-dependent proteasomal pathway requires an additional ubiquitin-like protein for activation, neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8). Here, we show that pevonedistat, a NEDD8-activating enzyme inhibitor, works efficiently as an antiviral agent. Pevonedistat significantly restored Smc5/6 protein levels and suppressed viral transcription and protein production in the HBV minicircle system in in vitro HBV replication models and in human primary hepatocytes infected naturally with HBV. Conclusion: These results indicate that pevonedistat is a promising compound to treat chronic HBV infection.

A phase II trial of gemcitabine, S-1 and LV combination (GSL) therapy in patients with advanced pancreatic cancer.

Purpose Our previous phase I trial suggested feasibility of addition of leucovorin (LV) to S-1 and gemcitabine therapy in advanced pancreatic cancer. The aim of this phase II trial was to assess the efficacy and toxicity of gemcitabine, S-1 and LV (GSL) combination therapy for advanced pancreatic cancer. Methods Chemotherapy-naïve patients with histologically or cytologically proven advanced pancreatic cancer were enrolled. Gemcitabine was administered at a dose of 1000 mg/m2 by 30 min infusion on days 1, S-1 40 mg/m2 orally twice daily and LV 25 mg orally twice daily on days 1 to 7 every 2 weeks. Primary end point was progression free survival (PFS). Results A total of 49 patients with advanced pancreatic cancer (19 locally advanced and 30 metastatic) were enrolled. Overall response rate and disease control rate were 32.7% and 87.8%. The median PFS and overall survival (OS) were 10.8 (95% confidence interval [CI], 7.4-13.5) and 20.7 (95% CI 13.0-NA) months with 1-year survival rate of 73.4%. Major Grade 3-4 toxicities were neutropenia (22.4%) and stomatitis (14.3%). No toxicity related death was observed. Conclusions In this single center, phase II trial, gemcitabine, S-1 and LV combination therapy was tolerable and can potentially be a treatment option for advanced pancreatic cancer.

Repression of MicroRNA Function Mediates Inflammation-associated Colon Tumorigenesis.

BACKGROUND & AIMS: Little is known about the mechanisms by which chronic inflammation contributes to carcinogenesis, such as the development of colon tumors in patients with inflammatory bowel diseases. Specific microRNA (miRNAs) can function as suppressors or oncogenes, and widespread alterations in miRNA expression have been associated with tumorigenesis. We studied whether alterations in miRNA function contribute to inflammation-associated colon carcinogenesis. METHODS: We studied the effects of inflammatory cytokines, such as tumor necrosis factor, interleukin-1α (IL1A), and IL1ß (IL1B), on miRNA function, measured by activity of reporter constructs containing miRNA-binding sites in their 3' untranslated regions, in human 293T embryonic kidney, Caco-2, HT29, and HCT116 colon carcinoma cells, as well as dicer+/+ and dicer-/-, and Apobec3+/+ and Apobec3-/- mouse embryonic fibroblasts. Cells were analyzed by immunoblots, immunohistochemistry, and flow cytometry. We generated transgenic mice expressing reporter constructs regulated by LET7B, MIR122, and MIR29b response elements; some mice were given injections of miRNA inhibitors (anti-MIR122 or anti-LET7B), a negative control, or tumor necrosis factor. Liver tissues were collected and analyzed by immunoblotting. Reporter mice were given azoxymethane followed by dextran sulfate sodium to induce colitis and colon tumors; some mice were given the ROCK inhibitor fasudil along with these agents (ROCK inhibitors increase miRNA function). Colon tissues were collected and analyzed by immunohistochemistry, immunoblots, and fluorescence microscopy. RESULTS: Incubation of cell lines with inflammatory cytokines reduced the ability of miRNAs to down-regulate expression from reporter constructs; dicer was required for this effect, so these cytokines relieve miRNA-dependent reductions in expression. The cytokines promoted degradation of APOBEC3G, which normally promotes miRNA loading into argonaute 2-related complexes. Mice with colitis had reduced miRNA function, based on increased expression of reporter genes. Administration of fasudil to mice did not reduce the severity of colitis that developed but greatly reduced the numbers of colon tumors formed (mean 2 tumors/colon in mice given fasudil vs 9 tumors/colon in mice given control agent). We made similar observations in IL10-deficient mice. CONCLUSIONS: We found inflammatory cytokines to reduce the activities of miRNAs. In mice with colitis, activities of miRNAs are reduced; administration of an agent that increases miRNA function prevents colon tumor formation in these mice. This pathway might be targeted to prevent colon carcinogenesis in patients with inflammatory bowel diseases.

Transcriptional activation of the MICA gene with an engineered CRISPR-Cas9 system.

Major histocompatibility complex class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. Because immune cells, such as natural killer (NK) cells, recognize virally infected or transformed cells and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells, MICA expression levels are associated with NK cell-mediated immunity. Here, we report that an engineered clustered regularly interspaced short palindromic repeats-Cas9-related complex targeting MICA gene promoter sequences activates transcription of the MICA gene from its endogenous locus. Inhibiting microRNA function, which targets the 3' untranslated region of the MICA gene, enhances this activation. These results demonstrate that the combination of Cas9-based transcriptional activators and simultaneous modulation of microRNA function may be a powerful tool for enhancing MICA protein expression and efficient anti-pathogenic cell immunity.

MicroRNAs and liver disease.

The biological roles of microRNAs (miRNAs) have been extensively studied. miRNA122 represents more than half of the miRNAs expressed in the liver and has various physiological and pathological functions, which include enhancing hepatitis virus replication, regulating lipid metabolism and suppressing hepatocellular carcinoma. miRNAs, whether globally or individually, have been linked with hepatocarcinogenesis. Furthermore, some miRNAs have been shown to be involved in the pathogenesis of nonalcoholic steatohepatitis. Using nucleotide-based strategies, these miRNAs may be developed as potential therapeutic targets. Because changes in miRNA expression can be measured in sera, they may be used as non-invasive biomarkers if they correctly reflect the pathological state of the liver. In this review, we show the biological roles of representative miRNAs in liver disease and discuss the current issues that remain to be clarified for future clinical applications.

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

Development of a screening method to identify regulators of MICA shedding.

Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding.

MicroRNA-140 acts as a liver tumor suppressor by controlling NF-κB activity by directly targeting DNA methyltransferase 1 (Dnmt1) expression.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that regulate the expression of specific target genes. While deregulated miRNA expression levels have been detected in many tumors, whether miRNA functional impairment is also involved in carcinogenesis remains unknown. We investigated whether deregulation of miRNA machinery components and subsequent functional impairment of miRNAs are involved in hepatocarcinogenesis. Among miRNA-containing ribonucleoprotein complex components, reduced expression of DDX20 was frequently observed in human hepatocellular carcinomas, in which enhanced nuclear factor-κB (NF-κB) activity is believed to be closely linked to carcinogenesis. Because DDX20 normally suppresses NF-κB activity by preferentially regulating the function of the NF-κB-suppressing miRNA-140, we hypothesized that impairment of miRNA-140 function may be involved in hepatocarcinogenesis. DNA methyltransferase 1 (Dnmt1) was identified as a direct target of miRNA-140, and increased Dnmt1 expression in DDX20-deficient cells hypermethylated the promoters of metallothionein genes, resulting in decreased metallothionein expression leading to enhanced NF-κB activity. MiRNA-140-knockout mice were prone to hepatocarcinogenesis and had a phenotype similar to that of DDX20 deficiency, suggesting that miRNA-140 plays a central role in DDX20 deficiency-related pathogenesis. CONCLUSION: These results indicate that miRNA-140 acts as a liver tumor suppressor, and that impairment of miRNA-140 function due to a deficiency of DDX20, a miRNA machinery component, could lead to hepatocarcinogenesis.

Increased expression of TNFRSF14 and LIGHT in biliary epithelial cells of patients with primary sclerosing cholangitis.

BACKGROUND AND AIMS: There is a lack of biliary epithelial molecular markers for primary sclerosing cholangitis (PSC). We analyzed candidates from disease susceptibility genes identified in recent genome-wide association studies (GWAS). METHODS: Expression levels of GWAS genes were analyzed in archival liver tissues of patients with PSC and controls. Immunohistochemical analysis was performed to evaluate expression levels in the biliary epithelia of PSC (N = 45) and controls (N = 12). Samples from patients with primary biliary cholangitis (PBC) were used as disease controls (N = 20). RESULTS: Hepatic expression levels of ATXN2, HHEX, PRDX5, MST1, and TNFRSF14 were significantly altered in the PSC group. We focused on the immune-related receptor, TNFRSF14. Immunohistochemistry revealed that high expression of TNFRSF14 in biliary epithelial cells was observed only in the PSC group. In addition, the expression of LIGHT, which encodes a TNFRSF14-activating ligand, was increased in PSC liver. Immunohistochemistry showed that high expression of LIGHT was more common in PSC biliary epithelia (53%) than in the PBC (15%) or control (0%) groups; moreover, it was positively associated with fibrotic progression, although it was not an independent prognostic factor. CONCLUSIONS: TNFRSF14 and LIGHT are promising candidate markers for PSC.

Gut Bacteria-derived Membrane Vesicles Induce Colonic Dysplasia by Inducing DNA Damage in Colon Epithelial Cells.

BACKGROUND & AIMS: Colorectal cancer (CRC) is the third most common cancer in the world. Gut microbiota has recently been implicated in the development of CRC. Actinomyces odontolyticus is one of the most abundant bacteria in the gut of patients with very early stages of CRC. A odontolyticus is an anaerobic bacterium existing principally in the oral cavity, similar to Fusobacterium nucleatum, which is known as a colon carcinogenic bacterium. Here we newly determined the biological functions of A odontolyticus on colonic oncogenesis. METHODS: We examined the induction of intracellular signaling by A odontolyticus in human colonic epithelial cells (CECs). DNA damage levels in CECs were confirmed using the human induced pluripotent stem cell-derived gut organoid model and mouse colon tissues in vivo. RESULTS: A odontolyticus secretes membrane vesicles (MVs), which induce nuclear factor kappa B signaling and also produce excessive reactive oxygen species (ROS) in colon epithelial cells. We found that A odontolyticus secretes lipoteichoic acid-rich MVs, promoting inflammatory signaling via TLR2. Simultaneously, those MVs are internalized into the colon epithelial cells, co-localize with the mitochondria, and cause mitochondrial dysfunction, resulting in excessive ROS production and DNA damage. Induction of excessive DNA damage in colonic cells by A odontolyticus-derived MVs was confirmed in the gut organoid model and also in mouse colon tissues. CONCLUSIONS: A odontolyticus secretes MVs, which cause chronic inflammation and ROS production in colonic epithelial cells, leading to the initiation of CRC.

Inhibition of microRNA122 decreases SREBP1 expression by modulating suppressor of cytokine signaling 3 expression.

While inhibition of microRNA122 (miR122) function in vivo results in reduced serum cholesterol and fatty acid levels, the molecular mechanisms underlying the link between miR122 function and lipid metabolism remains unclear. Because the expression of SREBP1, a central transcription factor involved in lipid metabolism, is known to be increased by suppressor of cytokine signaling 3 (SOCS3) expression, and because we previously found that SOCS3 expression is regulated by miR122, in this study, we examined the correlation between miR122 status and the expression levels of SOCS3 and SREBP1. SREBP1 expression decreased when SOCS3 expression was reduced by miR122 silencing in vitro. Conversely, SREBP1 expression in miR122-silenced cells was restored by enforced expression of SOCS3. Such correlations were observed in human liver tissues with different miR122 expression levels. These signaling links may explain one of the molecular mechanisms linking inhibition of miR122 function or decreased expression of miR122 to decreased fatty acid and cholesterol levels, in the inhibition of miR122 function, or in pathological status in chronic liver diseases.

Extracellular vesicle­mediated RNA editing may underlie the heterogeneity and spread of hepatocellular carcinoma in human tissue and in vitro.

Extracellular vesicles (EVs) produced by various cells, including tumor cells, carry biomolecules to neighboring cells. In hepatocellular carcinoma (HCC), adenosine to inosine RNA editing of antizyme inhibitor 1 (AZIN1), specifically regulated by adenosine deaminase acting on RNA­1 (ADAR1), promotes carcinogenesis. The present study examined if EVs and ADAR1 in the EVs released from HCC cells are transferred to neighboring cells in co­culture systems and reporter assay. Distribution of the ADAR1 expression in human tissues were examined by immunohistochemistry. EVs released from HCC cells containing ADAR1 were delivered to neighboring HCC cells and non­cancerous hepatocytes. The increased ADAR1 protein levels resulted in serine to glycine substitution at residue 367 of AZIN1, which augmented transformation potential and increased aggressive behavior of cancer cells. In clinically resected samples, ADAR1 distribution was highly heterogeneous within the tumor specimen and denser in non­cancerous tissue surrounding the HCC tissue. These observations suggested that ADAR1 protein may be delivered from HCC cells to neighboring cells via EVs and that EV­mediated RNA editing may serve a pivotal role in determining HCC heterogeneity and spread.

Cabozantinib inhibits HBV-RNA transcription by decreasing STAT3 binding to the enhancer region of cccDNA.

BACKGROUND: Precision medicine and customized therapeutics based on the features of each patient are important for maximizing therapeutic effects. Because most cases of HCC occur in the damaged liver through various etiologies, such as hepatitis virus infection, steatohepatitis, and autoimmune hepatitis, there should be a rationale for the choice of therapeutic options based on these etiologies. Although cabozantinib, an oral multikinase inhibitor, has demonstrated clinical effectiveness in advanced HCC, subgroup analyses showed a lower HR for death in HBV-related HCC. This study aimed to determine the therapeutic effects of cabozantinib in HBV-related HCC. METHODS: Using HBV infection models and gene knockout cells, we determined the crucial signaling axis responsible for the effects of cabozantinib on HBV. A chromatin immunoprecipitation assay was performed to determine the interaction between the signaling molecules and HBV DNA. Agonists and inhibitors were used for confirmation. RESULTS: Cabozantinib inhibited HBV replication through the HGF-mesenchymal-epithelial transition factor-signal transducer and activator of transcription 3 (MET-STAT3) signaling axis. The importance of STAT3 in viral replication has been confirmed using gene-edited STAT3 knockout cells. The chromatin immunoprecipitation assay revealed that the binding levels of phosphorylated STAT3 to enhancer region 1 of HBV covalently closed circular DNA were significantly increased by HGF stimulation. CONCLUSIONS: Cabozantinib has favorable therapeutic effects on HBV-related HCC because it inhibits HCC not only directly but also indirectly by means of inhibitory effects on HBV.

Combination of serum human satellite RNA and miR-21-5p levels as a biomarker for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis due to the difficulty of its diagnosis. Because human satellite II (HSATII) RNA, a satellite repeat RNA, is highly and specifically expressed in human PDAC, the serum HSATII RNA level may be a biomarker of PDAC. To measure the serum HSATII RNA level with high sensitivity and reproducibility, we previously developed a convenient method, tandem repeat amplification by nuclease protection (TRAP) combined with droplet digital PCR (ddPCR). Here, we refined the original method by simultaneously measuring the serum miR-21-5p level to enhance the detection of PDAC. The resulting PDAC-Index, constructed using serum HSATII RNA and miR-21-5p levels, discriminated patients with PDAC with high accuracy. We verified the clinical usefulness of the PDAC-Index as a supportive test in difficult-to-diagnose cases. The PDAC-Index has satisfactory diagnostic performance and may routinely be applied for detecting PDAC.