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.

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.

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.

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.

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.

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.

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.

Long-term survival after palliative argon plasma coagulation for intraductal papillary mucinous neoplasm of the bile duct.

Intraductal papillary mucinous neoplasm of the bile duct (IPNB) is an epithelial tumor that can cause obstructive jaundice and cholangitis due to mucin production. Although the effectiveness of argon plasma coagulation in IPNB treatment has been demonstrated, the long-term effect of the therapy is largely unknown. Here, we have presented a patient with IPNB who underwent argon plasma coagulation with a follow-up period of more than 2 years. A 74-year-old woman was referred to our department for treatment of obstructive jaundice. Endoscopic retrograde cholangiopancreatography revealed marked dilation of intrahepatic and extrahepatic bile ducts and thick mucin drainage from the ampulla of Vater. IPNB was diagnosed pathologically from biopsy specimens. Surgery was not recommended because of the extensive intrahepatic spread of the lesion. Endoscopic sphincterotomy, endoscopic papillary large balloon dilation, and insertion of a metallic stent could not resolve the obstructive jaundice. Finally, argon plasma coagulation with percutaneous cholangioscopy was performed 3 times over 1 month. After treatment, obstructive jaundice was resolved and the patient's clinical condition has been stable for more than 2 years, except for a single episode of transient cholangitis. In conclusion, argon plasma coagulation may be an alternative to surgery for the palliation of jaundice with IPNB.

Aberrant expression of a novel circular RNA in pancreatic cancer.

Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are produced from pre-mRNAs through a process known as back-splicing. Although circRNAs are expressed under specific conditions, current understanding of their comprehensive expression status is still limited. Here, we performed a large-scale circRNA profiling analysis in human pancreatic ductal adenocarcinoma (PDAC) tissues, using circular RNA-specific RNA sequencing. We identified more than 40,000 previously unknown circRNAs, some of which were upregulated in PDAC tissues, compared with normal pancreatic tissues. We determined the full-length sequence of a circRNA upregulated in PDAC, which was derived from two noncoding RNA loci on chromosome 12. The novel circRNA, named circPDAC RNA, was not expressed in normal human cells, but was expressed in PDAC and other carcinoma cells. While postulated biological functions, such as peptide production from the circPDAC RNA, were not detected, its aberrant expression was confirmed in other PDAC tissues and in serum from a PDAC patient. These results demonstrate that comprehensive studies are necessary to reveal the expression status of circRNAs and that the circPDAC RNA identified here might serve as a novel biomarker for cancers, including PDAC.

Emerging Roles of Exosomal Circular RNAs in Cancer.

Circular RNA (circRNA) is a type of non-coding RNA that forms a covalently closed continuous loop. The expression pattern of circRNA varies among cell types and tissues, and many circRNAs are aberrantly expressed in various cancers. Aberrantly expressed circRNAs have been shown to play crucial roles in carcinogenesis, functioning as microRNA sponges or new templates for protein translation. Recent research has shown that circRNAs are enriched in exosomes. Exosomes are secretory vesicles that mediate intercellular communication through the delivery of cargo, including proteins, lipids, DNA, and RNA. Exosome-mediated crosstalk between cancer cells and the tumor microenvironment promotes the epithelial-mesenchymal transition, angiogenesis, and immune escape, and thus may contribute to cancer invasion and metastasis. In this review, we discuss the biological functions of exosomal circRNAs and their significance in cancer progression. Additionally, we discuss the potential clinical applications of exosomal circRNAs as biomarkers and in cancer therapy.

Hepatitis C virus relapse after successful treatment with direct-acting antivirals, followed by sarcomatous changes in hepatocellular carcinoma: a case report.

BACKGROUND: Combination therapy of interferon and ribavirin has traditionally been used to eradicate hepatitis C virus. The sustained virologic response achieved with interferon-related therapy is persistent, and late relapses after achieving sustained virologic response at 24 weeks using this therapy are reportedly rare (< 1%). In 2014, interferon-free therapy with direct-acting antivirals was developed, and the rate of sustained virologic response was improved. However, the persistence thereof remains uncertain, and the appropriate follow-up period for hepatitis C virus-positive patients is under discussion. CASE PRESENTATION: A 74-year-old Japanese man who had hepatitis C virus-related hepatocellular carcinoma and was successfully treated with radiofrequency ablation four times underwent direct-acting antiviral therapy with daclatasvir and asunaprevir; sustained virologic response at 24 weeks was confirmed. However, although he had no high risk factors for reinfection, hepatitis C virus ribonucleic acid was detected again 6 months after achieving sustained virologic response at 24 weeks. Moreover, he developed active hepatitis with an increased viral load. Five months after development of hepatitis, recurrent hepatocellular carcinoma emerged in segment II, where we had performed radiofrequency ablation 17 months previously. The recurrent hepatocellular carcinoma enlarged quite rapidly and induced multiple peritoneal disseminations and lung metastases. He died 3 months after the abrupt recurrence. A sarcomatous change in the hepatocellular carcinoma was identified during the autopsy. CONCLUSIONS: Although sustained virologic response at 24 weeks has generally been regarded to denote complete eradication of hepatitis C virus, we present a patient in whom hepatitis C virus recurred 6 months after achieving sustained virologic response at 24 weeks with direct-acting antiviral therapy. In addition, a sarcomatous change in hepatocellular carcinoma emerged 5 months after active hepatitis developed due to late hepatitis C virus relapse in this case. The sarcomatous change in hepatocellular carcinoma is generally thought to be related to anticancer therapies, such as radiofrequency ablation. However, in this case, late viral relapse and active hepatitis in addition to the previous radiofrequency ablation could have been the trigger. There may be a need for follow-up of hepatitis C virus ribonucleic acid beyond sustained virologic response at 24 weeks with direct-acting antiviral therapy, owing to the possibility of late viral relapse and tumorigenesis.

Paraneoplastic dermatomyositis appearing after nivolumab therapy for gastric cancer: a case report.

BACKGROUND: While dermatomyositis is often associated with malignancy, several autoimmune diseases like myositis can be caused by immune checkpoint inhibitors. Differentially diagnosing malignancy-associated dermatomyositis or myositis caused by immune checkpoint inhibitors is sometimes difficult, particularly when a patient with malignancy shows the symptoms of myositis after checkpoint inhibitor administration. We experienced such a case in which we had difficulties in diagnosing paraneoplastic dermatomyositis or drug-associated myositis. In this case, all of our team initially assumed that the diagnosis was myositis caused by immune checkpoint inhibitors. However, it turned out finally that the diagnosis was paraneoplastic dermatomyositis. Because the diagnosis was unexpected, we report here. CASE PRESENTATION: We report the case of a 71-year-old Japanese man who developed clinical symptoms of myositis, such as muscle aches and weakness, after initiation of nivolumab therapy for his gastric cancer. He was initially diagnosed with nivolumab-induced myositis, because the myositis symptoms appeared after nivolumab administration, and nivolumab is known to trigger various drug-associated autoimmune diseases. However, according to his characteristic skin lesions, the type of muscle weakness, his serum marker profiles, electromyography of his deltoid muscle, and magnetic resonance imaging, he was finally diagnosed as having paraneoplastic dermatomyositis. Accordingly, treatment with intravenously administered corticosteroid pulse treatment, immunoglobulin injection, and tacrolimus was applied; his symptoms subsequently improved. However, to our regret, at day 142 after administration, he died due to rapid worsening of his gastric cancer. CONCLUSION: Differentially diagnosing paraneoplastic dermatomyositis or drug-associated myositis caused by immune checkpoint inhibitors is difficult in some cases. The differential diagnosis is crucial because it influences the decision regarding the appropriateness of the use of immunosuppressive treatment against the autoimmune diseases as well as the decision regarding the appropriateness of the continuous use of immune checkpoint inhibitors against the primary cancers. Because subclinical autoimmune disease may become overt after administering immune checkpoint inhibitors, non-apparent autoimmune diseases, which have already existed, should also be considered to avoid the delay of appropriate treatment, when symptoms of autoimmune diseases are recognized.

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.

Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine.

Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

Diagnostic and therapeutic application of noncoding RNAs for hepatocellular carcinoma.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression posttranscriptionally, targeting thousands of messenger RNAs. Long noncoding RNAs (lncRNAs), another class of noncoding RNAs, have been determined to be also involved in transcription regulation and translation of target genes. Since deregulated expression levels or functions of miRNAs and lncRNAs in hepatocellular carcinoma (HCC) are frequently observed, clinical use of noncoding RNAs for novel diagnostic and therapeutic applications in the management of HCCs is highly and emergently expected. Here, we summarize recent findings regarding deregulated miRNAs and lncRNAs for their potential clinical use as diagnostic and prognostic biomarkers of HCC. Specifically, we emphasize the deregulated expression levels of such noncoding RNAs in patients' sera as noninvasive biomarkers, a field that requires urgent improvement in the clinical surveillance of HCC. Since nucleotide-based strategies are being applied to clinical therapeutics, we further summarize clinical and preclinical trials using oligonucleotides involving the use of miRNAs and small interfering RNAs against HCC as novel therapeutics. Finally, we discuss current open questions, which must be clarified in the near future for realistic clinical applications of these new strategies.

The flavonoid apigenin inhibits hepatitis C virus replication by decreasing mature microRNA122 levels.

Despite recent progress in the development of direct-acting antivirals against hepatitis C virus (HCV), chronic HCV infection remains an important health burden worldwide. MicroRNA122 (miR122), a liver-specific microRNA (miRNA), positively regulates HCV replication, and systemic application of antisense oligonucleotides against miR122 led to the long-lasting suppression of HCV viremia in human clinical trials. Here, we report that apigenin, a flavonoid and an inhibitor of maturation of a subset of miRNAs, inhibits HCV replication in vitro. Apigenin decreased the expression levels of mature miR122 without significantly affecting cell growth. Because supplementation of synthesized miR122 oligonucleotides or overexpression of constitutively active TRBP blocked these effects, the inhibitory effects of apigenin on HCV replication seemed to be dependent on the reduction of mature miR122 expression levels through inhibition of TRBP phosphorylation. Thus, apigenin intake, either through regular diet or supplements, may decrease HCV replication in chronically infected patients.

Specific delivery of microRNA93 into HBV-replicating hepatocytes downregulates protein expression of liver cancer susceptible gene MICA.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). To date, the lack of efficient in vitro systems supporting HBV infection and replication has been a major limitation of HBV research. Although primary human hepatocytes support the complete HBV life cycle, their limited availability and difficulties with gene transduction remain problematic. Here, we used human primary hepatocytes isolated from humanized chimeric uPA/SCID mice as efficient sources. These hepatocytes supported HBV replication in vitro. Based on analyses of mRNA and microRNA (miRNA) expression levels in HBV-infected hepatocytes, miRNA93 was significantly downregulated during HBV infection. MiRNA93 is critical for regulating the expression levels of MICA protein, which is a determinant for HBV-induced HCC susceptibility. Exogenous addition of miRNA93 in HBV-infected hepatocytes using bionanocapsules consisted of HBV envelope L proteins restored MICA protein expression levels in the supernatant. These results suggest that the rescued suppression of soluble MICA protein levels by miRNA93 targeted to HBV-infected hepatocytes using bionanocapsules may be useful for the prevention of HBV-induced HCC by altering deregulated miRNA93 expression.