Whole-transcriptome analyses have revealed that a large proportion of the human genome is transcribed in non-protein-coding transcripts, designated as long non-coding RNAs (lncRNAs). Rather than ...being "transcriptional noise", increasing evidence indicates that lncRNAs are key players in the regulation of many biological processes, including transcription, post-translational modification and inhibition and chromatin remodeling. Indeed, lncRNAs are widely dysregulated in human cancers, including hepatocellular carcinoma (HCC). Functional studies are beginning to provide insights into the role of oncogenic and tumor suppressive lncRNAs in the regulation of cell proliferation and motility, as well as oncogenic and metastatic potential in HCC. A better understanding of the molecular mechanisms and the complex network of interactions in which lncRNAs are involved could reveal novel diagnostic and prognostic biomarkers. Crucially, it may provide novel therapeutic opportunities to add to the currently limited number of therapeutic options for HCC patients. In this review, we summarize the current status of the field, with a focus on the best characterized dysregulated lncRNAs in HCC.
Loss of serum HBsAg is a hallmark of spontaneous and therapy induced resolution of HBV infection, since it generally reflects a profound decrease in viral replication. However, integrated HBV DNA can ...contribute to HBsAg expression independent of viral replication. The relative contributions of these sources of HBsAg are not well understood. Specifically, it is not known whether actively transcribed HBV integration could spread throughout the entire liver.
The relative distribution of HBsAg and HBV RNA in liver biopsy tissue from HBeAg-negative (HBe−) patients was analyzed by immunohistochemistry and in situ hybridization (ISH), respectively. Frozen biopsy tissue was used for molecular analysis of intrahepatic viral RNA, virus-host chimeric transcripts and viral DNA.
Immunohistochemistry and ISH analysis revealed HBsAg and HBV RNA positivity in virtually all hepatocytes in the liver of some HBe− patients despite very low viremia. Reverse transcription quantitative PCR and RNA-sequencing analysis confirmed high expression levels of HBV envelope-encoding RNAs. However, the amount of viral transcriptional template (covalently closed circular (ccc)DNA) was too low to support this ubiquitous HBV RNA expression. In contrast, levels of total cellular HBV DNA were consistent with ubiquitous HBV integration. Finally, RNA-sequencing revealed the presence of many HBV-host chimeric transcripts with the potential for HBsAg expression.
Transcriptionally active HBV integration can extend to the entire liver in some HBe− patients. This can lead to ubiquitous HBsAg expression independent of HBV replication. In such patients, HBsAg is probably not a clinically useful surrogate marker for viral resolution or functional cure.
Loss of serum hepatitis B surface antigen (HBsAg) indicates resolution of HBV infection. However, integrated HBV DNA can contribute to HBsAg production independently of viral replication. We investigated the extent of HBsAg-producing viral integration in the livers of patients with low serum viral loads. Our findings suggest that transcriptionally active HBV integration can extend to the entire liver in some patients, questioning the clinical utility of HBsAg as a surrogate marker for viral replication.
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•A unique set of liver biopsies from patients with HBeAg-negative chronic hepatitis B infection was studied.•HBV RNA and antigen expression are ubiquitous in the liver despite low viremia.•Levels of the viral transcriptional template cccDNA are not sufficient for ubiquitous HBV RNA and antigen expression.•Ubiquitous viral antigen expression is associated with virus-host chimeric transcription.•Intrahepatic viral DNA and RNA levels are consistent with widespread HBV integration.
Head and neck squamous cell carcinoma (HNSCC) has the potential for early metastasis and is associated with poor survival. Ano1 (Dog1) is an established and sensitive marker for the diagnosis of ...gastrointestinal stromal tumors (GIST) and has recently been identified as a Ca(2+) activated Cl(-) channel. Although the ANO1 gene is located on the 11q13 locus, a region which is known to be amplified in different types of human carcinomas, a detailed analysis of Ano1 amplification and expression in HNSCC has not been performed. It is thus still unclear how Ano1 contributes to malignancy in HNSCC. We analyzed genomic amplification of the 11q13 locus and Ano1 together with Ano1-protein expression in a large collection of HNSCC samples. We detected a highly significant correlation between amplification and expression of Ano1 and showed that HNSCC patients with Ano1 protein expression have a poor overall survival. We further analyzed the expression of the Ano1 protein in more than 4'000 human samples from 80 different tumor types and 76 normal tissue types and detected that besides HNSCC and GISTs, Ano1 was rarely expressed in other tumor samples or healthy human tissues. In HNSCC cell lines, expression of Ano1 caused Ca(2+) activated Cl(-) currents, which induced cell motility and cell migration in wound healing and in real time migration assays, respectively. In contrast, knockdown of Ano1 did not affect intracellular Ca(2+) signaling and surprisingly did not reduce cell proliferation in BHY cells. Further, expression and activity of Ano1 strongly correlated with the ability of HNSCC cells to regulate their volume. Thus, poor survival in HNSCC patients is correlated with the presence of Ano1. Our results further suggest that Ano1 facilitates regulation of the cell volume and causes cell migration, which both can contribute to metastatic progression in HNSCC.
Background Allergic diseases are considered a health burden because of their high and constantly increasing prevalence, high direct and indirect costs, and undesirable effects on quality of life. ...Probiotics have been suggested as an intervention to prevent allergic diseases. Objective We sought to synthesize the evidence supporting use of probiotics for the prevention of allergies and inform World Allergy Organization guidelines on probiotic use. Methods We performed a systematic review of randomized trials assessing the effects of any probiotic administered to pregnant women, breast-feeding mothers, and/or infants. Results Of 2403 articles published until December 2014 identified in Cochrane Central Register of Controlled Trials, MEDLINE, and Embase, 29 studies fulfilled a priori specified inclusion criteria for the analyses. Probiotics reduced the risk of eczema when used by women during the last trimester of pregnancy (relative risk RR, 0.71; 95% CI, 0.60-0.84), when used by breast-feeding mothers (RR, 0.57; 95% CI, 0.47-0.69), or when given to infants (RR, 0.80; 95% CI, 0.68-0.94). Evidence did not support an effect on other allergies, nutrition status, or incidence of adverse effects. The certainty in the evidence according to the Grading of Recommendation Assessment Development and Evaluation approach is low or very low because of the risk of bias, inconsistency and imprecision of results, and indirectness of available research. Conclusion Probiotics used by pregnant women or breast-feeding mothers and/or given to infants reduced the risk of eczema in infants; however, the certainty in the evidence is low. No effect was observed for the prevention of other allergic conditions.
The Yes‐associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show ...that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down‐regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug‐induced apoptosis. We further show that the proangiogenic microfibrillar‐associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31+ vasculature. Conclusions: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins. (Hepatology 2015;62:1497–1510)
Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only ...treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies.
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•Organoids can be derived from tumor needle biopsies of liver cancers•Organoids retain the morphology and tumor marker expression of the original tumors•Tumor organoids preserve the genetic heterogeneity of the originating tumors•Tumor organoids provide a tool for developing tailored therapies
Nuciforo et al. report the generation of patient-derived liver cancer organoids. Tissue was collected from all disease stages and major etiologies using an ultrasound-guided coaxial needle biopsy technique. Tumor organoids preserve the morphology and genetic heterogeneity of their originating tumors and provide opportunities to develop targeted therapies for hepatocellular carcinoma (HCC).
LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic ...zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.
Oncogenic gene fusions involving the 3' region of ROS1 kinase have been identified in various human cancers. In this study, we sought to characterize ROS1 fusion genes in non-small cell lung cancer ...(NSCLC) and establish the fusion proteins as drug targets.
An NSCLC tissue microarray (TMA) panel containing 447 samples was screened for ROS1 rearrangement by FISH. This assay was also used to screen patients with NSCLC. In positive samples, the identity of the fusion partner was determined through inverse PCR and reverse transcriptase PCR. In addition, the clinical efficacy of ROS1 inhibition was assessed by treating a ROS1-positive patient with crizotinib. The HCC78 cell line, which expresses the SLC34A2-ROS1 fusion, was treated with kinase inhibitors that have activity against ROS1. The effects of ROS1 inhibition on proliferation, cell-cycle progression, and cell signaling pathways were analyzed by MTS assay, flow cytometry, and Western blotting.
In the TMA panel, 5 of 428 (1.2%) evaluable samples were found to be positive for ROS1 rearrangement. In addition, 1 of 48 patients tested positive for rearrangement, and this patient showed tumor shrinkage upon treatment with crizotinib. The patient and one TMA sample displayed expression of the recently identified SDC4-ROS1 fusion, whereas two TMA samples expressed the CD74-ROS1 fusion and two others expressed the SLC34A2-ROS1 fusion. In HCC78 cells, treatment with ROS1 inhibitors was antiproliferative and downregulated signaling pathways that are critical for growth and survival.
ROS1 inhibition may be an effective treatment strategy for the subset of patients with NSCLC whose tumors express ROS1 fusion genes.
Hepatocellular carcinoma (HCC) is among the leading causes of cancer‐related death. Despite the advances in diagnosis and management of HCC, the biology of this tumor remains poorly understood. ...Recent evidence highlighted long noncoding RNAs (lncRNAs) as crucial determinants of HCC development. In this study we report the lncRNA HOXA transcript at the distal tip (HOTTIP) as significantly up‐regulated in HCC specimens. The HOTTIP gene is located in physical contiguity with HOXA13 and directly controls the HOXA locus gene expression by way of interaction with the WDR5/MLL complex. HOX genes encode transcription factors regulating embryonic development and cell fate. We previously described HOX genes deregulation to be involved in hepatocarcinogenesis. Indeed, we observed the marked up‐regulation of HOXA13 in HCC. Here, by correlating clinicopathological and expression data, we demonstrate that the levels of HOTTIP and HOXA13 are associated with HCC patients' clinical progression and predict disease outcome. In contrast to the majority of similar studies, our data were obtained from snap‐frozen needle HCC biopsies (n = 52) matched with their nonneoplastic counterparts collected from patients who had not yet received any HCC‐tailored therapeutic treatments at the time of biopsy. In addition, taking advantage of gain and loss of function experiments in liver cancer‐derived cell lines (HuH‐6 and HuH‐7), we uncover a novel bidirectional regulatory loop between HOTTIP/HOXA13. Conclusion: Our study highlights the key role of HOTTIP and HOXA13 in HCC development by associating their expression with metastasis and survival in HCC patients, provides novel insights on the function of lncRNA‐driven hepatocarcinogenesis, and paves the way for further investigation about the possible role of HOTTIP as a predictive biomarker of HCC. (Hepatology 2014;59:911–923)
The existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in ...BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)– hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.
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•Hepatocytes throughout the liver contribute to liver homeostasis and repair•AXIN2+ pericentral hepatocytes do not comprise bona fide liver stem cells•Hepatocytes upregulate AXIN2 and LGR5 during regeneration•Ablation of AXIN2+ hepatocytes only transiently disrupts the pericentral zone
The existence of specialized liver stem cells, like AXIN2+ pericentral hepatocytes, is controversial. Sun and colleagues now show that hepatocytes throughout the liver contribute to liver homeostasis and upregulate the tissue stem cell markers AXIN2 and LGR5 during regeneration from injury, with no superior proliferative capacity of AXIN2+ pericentral hepatocytes.
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