Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. ...Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET protooncogene, S100 family members and the caveolins. Of interest, we found that exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis.
Alternative splicing (AS) allows generation of cell type–specific mRNA transcripts and contributes to hallmarks of cancer. Genome‐wide analysis for AS in human hepatocellular carcinoma (HCC), ...however, is limited. We sought to obtain a comprehensive AS landscape in HCC and define tumor‐associated variants. Single‐molecule real‐time long‐read RNA sequencing was performed on patient‐derived HCC cells, and presence of splice junctions was defined by SpliceMap‐LSC‐IDP algorithm. We obtained an all‐inclusive map of annotated AS variants and further discovered 362 alternative spliced variants that are not previously reported in any database (neither RefSeq nor GENCODE). They were mostly derived from intron retention and early termination codon with an in‐frame open reading frame in 81.5%. We corroborated many of these predicted unannotated and annotated variants to be tumor specific in an independent cohort of primary HCC tumors and matching nontumoral liver. Using the combined Sanger sequencing and TaqMan junction assays, unique and common expressions of spliced variants including enzyme regulators (ARHGEF2, SERPINH1), chromatin modifiers (DEK, CDK9, RBBP7), RNA‐binding proteins (SRSF3, RBM27, MATR3, YBX1), and receptors (ADRM1, CD44v8‐10, vitamin D receptor, ROR1) were determined in HCC tumors. We further focused functional investigations on ARHGEF2 variants (v1 and v3) that arise from the common amplified site chr.1q22 of HCC. Their biological significance underscores two major cancer hallmarks, namely cancer stemness and epithelial‐to‐mesenchymal transition–mediated cell invasion and migration, although v3 is consistently more potent than v1. Conclusion: Alternative isoforms and tumor‐specific isoforms that arise from aberrant splicing are common during the liver tumorigenesis. Our results highlight insights gained from the analysis of AS in HCC.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Oncoviral infection is responsible for 12%-15% of cancer in humans. Convergent evidence from epidemiology, pathology, and oncology suggests that new viral etiologies for cancers remain to be ...discovered. Oncoviral profiles can be obtained from cancer genome sequencing data; however, widespread viral sequence contamination and noncausal viruses complicate the process of identifying genuine oncoviruses. Here, we propose a novel strategy to address these challenges by performing virome-wide screening of early-stage clonal viral integrations. To implement this strategy, we developed VIcaller, a novel platform for identifying viral integrations that are derived from any characterized viruses and shared by a large proportion of tumor cells using whole-genome sequencing (WGS) data. The sensitivity and precision were confirmed with simulated and benchmark cancer data sets. By applying this platform to cancer WGS data sets with proven or speculated viral etiology, we newly identified or confirmed clonal integrations of hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), and BK Virus (BKV), suggesting the involvement of these viruses in early stages of tumorigenesis in affected tumors, such as HBV in
and
(also known as
) gene loci in liver cancer, HPV and BKV in bladder cancer, and EBV in non-Hodgkin's lymphoma. We also showed the capacity of VIcaller to identify integrations from some uncharacterized viruses. This is the first study to systematically investigate the strategy and method of virome-wide screening of clonal integrations to identify oncoviruses. Searching clonal viral integrations with our platform has the capacity to identify virus-caused cancers and discover cancer viral etiologies.
The mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC ...cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects β-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.
•HBV integration into the host genome can cause transcription of viral-human chimeras•HBx-LINE1 exerts oncogenic functions as a long noncoding RNA-like transcript•HBx-LINE1 promotes tumorigenicity via activation of Wnt/β-catenin signaling•HBx-LINE1 increases risk of HCC development
Lau et al. discover a viral-human fusion transcript that is detectable in a quarter of hepatitis B virus-associated hepatocellular carcinomas (HCCs). This chimeric transcript promotes HCC by functioning like a long noncoding RNA to activate the Wnt pathway.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Purpose: This study aims to profile the expressions of 156 microRNAs (miRNA) in hepatocellular carcinoma (HCC) and to characterize
the functions of miR-222, the most significantly upregulated ...candidate identified.
Experimental Design: miRNA expression profile in HCC tumors, matching adjacent cirrhotic livers, and cell lines was conducted using quantitative
PCR. Common miR-222 upregulations were further validated in a larger cohort of tumors. The functional effects of miR-222 inhibition
on HCC cell lines were examined. The downstream modulated pathways and target of miR-222 were investigated by coupling gene
expression profiling and pathway analysis, and by in silico prediction, respectively. Luciferase reporter assay was done to confirm target interaction.
Results: We identified a 40-miRNA signature that could discriminate tumors from adjacent cirrhotic liver tissue, and further corroborated
common miR-222 overexpression in tumors relative to its premalignant counterpart (55.3%; P < 0.0001). Increased miR-222 expression correlated significantly with advanced stage HCC and with the shorter disease-free
survival of patients ( P ≤ 0.01). Inhibition of miR-222 in Hep3B and HKCI-9 significantly retarded cell motility ( P < 0.05). Further investigations suggested that AKT signaling was the major pathway influenced by miR-222. A consistent reduction
of AKT phosphorylation in Hep3B and HKCI-9 was shown following miR-222 suppression. The protein phosphatase 2A subunit B (PPP2R2A)
was predicted as a putative miR-222 target in silico . We found that miR-222 inhibition could augment the tumor protein level and restore luciferase activity in reporter construct
containing the PPP2R2A 3′ untranslated region ( P = 0.0066).
Conclusions: Our study showed that miR-222 overexpression is common in HCC and could confer metastatic potentials in HCC cells, possibly
through activating AKT signaling. Clin Cancer Res; 16(3); 867–75
Background & Aims: Recent studies have emphasized causative links between microRNA (miRNA) deregulations and cancer development. In hepatocellular carcinoma (HCC), information on differentially ...expressed miRNA remained largely undefined. Methods: Array-based miRNA profiling was performed on HCC cells that were derived from chronic carriers of hepatitis B virus (HBV) and hepatitis C virus (HCV), and nonviral-associated patients. Specific microRNA (miR)-223 and miR-222 deregulations were verified in an independent series of tumors. The functional effect of miR-223 was examined further. An integrative analysis of messenger RNA (mRNA) array with in silico predictions defined potential downstream targets of miR-223. A luciferase reporter assay was conducted to confirm target association. Results: Distinct up-regulations of miR-222, miR-221, and miR-31, and down-regulations of miR-223, miR-126, and miR-122a were identified. Further investigations suggested the highly deregulated miR-223 and miR-222 could unequivocally distinguish HCC from adjacent nontumoral liver, irrespective of viral associations ( P ≤ .0002). Re-expression of miR-223 in HBV, HCV, and non-HBV non-HCV–related HCC cell lines revealed a consistent inhibitory effect on cell viability ( P < .01). Integrative analysis further implicated Stathmin 1 ( STMN1 ) as a downstream target of miR-223. A strong inverse relationship between STMN1 mRNA and miR-223 expressions was shown ( P = .006). A substantial reduction in STMN1 protein was further demonstrated upon restoration of miR-223 expression in HCC cell lines. We further showed that miR-223 readily could suppress the luciferase activity in reporter construct containing the STMN1 3' untranslated region ( P = .02). Conclusions: Our study revealed specific miRNA differential expressions in HCC and underscores the potential importance of miR-223 down-regulations in the development of HCC.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for SARS-CoV-2. The full-length membrane form of ...ACE2 (memACE2) undergoes ectodomain shedding to generate a shed soluble form (solACE2) that mediates SARS-CoV-2 entry via receptor-mediated endocytosis. Currently, it is not known how the physiological regulation of ACE2 shedding contributes to the etiology of COVID-19 in vivo. The present study identifies Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) as a critical host protease for solACE2-mediated SARS-CoV-2 infection. SARS-CoV-2 infection leads to increased activation of MT1-MMP that is colocalized with ACE2 in human lung epithelium. Mechanistically, MT1-MMP directly cleaves memACE2 at M706-S to release solACE2
that binds to the SARS-CoV-2 spike proteins (S), thus facilitating cell entry of SARS-CoV-2. Human solACE2
enables SARS-CoV-2 infection in both non-permissive cells and naturally insusceptible C57BL/6 mice. Inhibition of MT1-MMP activities suppresses solACE2-directed entry of SARS-CoV-2 in human organoids and aged mice. Both solACE2 and circulating MT1-MMP are positively correlated in plasma of aged mice and humans. Our findings provide in vivo evidence demonstrating the contribution of ACE2 shedding to the etiology of COVID-19.
A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the ...identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%–13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133
− counterparts, CD133
+ cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133
+ cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133
+ tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133
+ and CD133
− cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133
+ TICs. Functional studies on miR-130b lentiviral-transduced CD133
− cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133
+ TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133
− cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133
+ liver TICs, in part, via silencing TP53INP1.
► CD133 identifies liver TICs/CSCs and is a prognostic marker for HC ► miR-130b promotes CD133
+ liver TICs growth and self renewal ► miR-130b regulates CD133
+ liver TICs via silencing TP53INP1
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC ...prognostication.
To identify recurrent somatic mutations with prognostic significance in patients with CRC.
Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases.
Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6-14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study.
The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging.
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