Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

ChIPseek, a web-based analysis tool for ChIP data.

BACKGROUND: Chromatin is a dynamic but highly regulated structure. DNA-binding proteins such as transcription factors, epigenetic and chromatin modifiers are responsible for regulating specific gene expression pattern and may result in different phenotypes. To reveal the identity of the proteins associated with the specific region on DNA, chromatin immunoprecipitation (ChIP) is the most widely used technique. ChIP assay followed by next generation sequencing (ChIP-seq) or microarray (ChIP-chip) is often used to study patterns of protein-binding profiles in different cell types and in cancer samples on a genome-wide scale. However, only a limited number of bioinformatics tools are available for ChIP datasets analysis. RESULTS: We present ChIPseek, a web-based tool for ChIP data analysis providing summary statistics in graphs and offering several commonly demanded analyses. ChIPseek can provide statistical summary of the dataset including histogram of peak length distribution, histogram of distances to the nearest transcription start site (TSS), and pie chart (or bar chart) of genomic locations for users to have a comprehensive view on the dataset for further analysis. For examining the potential functions of peaks, ChIPseek provides peak annotation, visualization of peak genomic location, motif identification, sequence extraction, and comparison between datasets. Beyond that, ChIPseek also offers users the flexibility to filter peaks and re-analyze the filtered subset of peaks. ChIPseek supports 20 different genome assemblies for 12 model organisms including human, mouse, rat, worm, fly, frog, zebrafish, chicken, yeast, fission yeast, Arabidopsis, and rice. We use demo datasets to demonstrate the usage and intuitive user interface of ChIPseek. CONCLUSIONS: ChIPseek provides a user-friendly interface for biologists to analyze large-scale ChIP data without requiring any programing skills. All the results and figures produced by ChIPseek can be downloaded for further analysis. The analysis tools built into ChIPseek, especially the ones for selecting and examine a subset of peaks from ChIP data, provides invaluable helps for exploring the high through-put data from either ChIP-seq or ChIP-chip. ChIPseek is freely available at http://chipseek.cgu.edu.tw.

Matrix metalloproteinase 12 is induced by heterogeneous nuclear ribonucleoprotein K and promotes migration and invasion in nasopharyngeal carcinoma.

BACKGROUND: Overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a DNA/RNA binding protein, is associated with metastasis in nasopharyngeal carcinoma (NPC). However, the mechanisms underlying hnRNP K-mediated metastasis is unclear. The aim of the present study was to determine the role of matrix metalloproteinase (MMP) in hnRNP K-mediated metastasis in NPC. METHODS: We studied hnRNP K-regulated MMPs by analyzing the expression profiles of MMP family genes in NPC tissues and hnRNP K-knockdown NPC cells using Affymetrix microarray analysis and quantitative RT-PCR. The association of hnRNP K and MMP12 expression in 82 clinically proven NPC cases was determined by immunohistochemical analysis. The hnRNP K-mediated MMP12 regulation was determined by zymography and Western blot, as well as by promoter, DNA pull-down and chromatin immunoprecipitation (ChIP) assays. The functional role of MMP12 in cell migration and invasion was demonstrated by MMP12-knockdown and the treatment of MMP12-specific inhibitor, PF-356231. RESULTS: MMP12 was overexpressed in NPC tissues, and this high level of expression was significantly correlated with high-level expression of hnRNP K (P = 0.026). The levels of mRNA, protein and enzyme activity of MMP12 were reduced in hnRNP K-knockdown NPC cells. HnRNP K interacting with the region spanning -42 to -33 bp of the transcription start site triggered transcriptional activation of the MMP12 promoter. Furthermore, inhibiting MMP12 by MMP12 knockdown and MMP12-specific inhibitor, PF-356231, significantly reduced the migration and invasion of NPC cells. CONCLUSIONS: Overexpression of MMP12 was significantly correlated with hnRNP K in NPC tissues. HnRNP K can induce MMP12 expression and enzyme activity through activating MMP12 promoter, which promotes cell migration and invasion in NPC cells. In vitro experiments suggest that NPC metastasis with high MMP12 expression may be treated with PF-356231. HnRNP K and MMP12 may be potential therapeutic markers for NPC, but additional validation studies are warranted.

Molecular Diagnosis of Nasopharyngeal Carcinoma: Past and Future.

Nasopharyngeal carcinoma (NPC) is a malignant tumor originated from the nasopharynx epithelial cells and has been linked with Epstein-Barr virus (EBV) infection, dietary habits, environmental and genetic factors. It is a common malignancy in Southeast Asia, especially with gender preference among men. Due to its non-specific symptoms, NPC is often diagnosed at a late stage. Thus, the molecular diagnosis of NPC plays a crucial role in early detection, treatment selection, disease monitoring, and prognosis prediction. This review aims to provide a summary of the current state and the latest emerging molecular diagnostic techniques for NPC, including EBV-related biomarkers, gene mutations, liquid biopsy, and DNA methylation. Challenges and potential future directions of NPC molecular diagnosis will be discussed.

Nasopharyngeal carcinoma patient-derived xenograft mouse models reveal potential drugs targeting cell cycle, mTOR, and autophagy pathways.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) infection. To test preclinical NPC drugs, we established two patient-derived xenograft (PDX) mouse models, EBV-positive PDX-B13 and EBV-negative PDX-Li41, for drug screening. METHODS: Based on next generation sequencing (NGS) studies, PDX-B13 had CCND1 copy number (CN) gain but CDKN2A CN loss, whereas PDX-Li41 had CDKN2A and RB1 CN loss, TSC1 (negative regulator of mTOR) frameshift deletion mutation, and increased activation of mTOR, a serine/threonine kinase that governs metabolism, autophagy, and apoptosis. Increased mTOR was also associated with poor NPC prognosis. RESULTS: Everolimus, an mTOR inhibitor, suppressed tumor growth in the two PDX NPC models and had an additive antitumor effect with palbociclib, a CDK4/6 inhibitor. PDX tumors treated with various drugs or untreated were subjected to RNA sequencing, transcriptome profile analysis, and selective Western blotting to understand the interactions between these drugs and gene expression profiles. Palbociclib also suppressed EB viral nuclear antigen (EBNA1) expression in PDX-B13. Everolimus together with autophagy inhibitor, hydroxychloroquine, had additive anti-tumor effect on PDX-B13 tumor. Immunohistochemistry revealed that high mTOR levels were correlated with poor overall survival in patients with metastatic NPC (N = 90). CONCLUSIONS: High mTOR levels are a poor prognostic factor in NPC, and cell cycle, mTOR and autophagy pathways may serve as therapeutic targets in NPC. In addition, PDX models can be used for efficiently testing potential NPC drugs.

Aberrant methylation impairs low density lipoprotein receptor-related protein 1B tumor suppressor function in gastric cancer.

DNA methylation plays a significant role in tumor progression. In this study, we used CpG microarray and differential methylation hybridization approaches to identify low density lipoprotein receptor-related protein 1B (LRP1B) as a novel epigenetic target in gastric cancer. LRP1B was hypermethylated in four gastric cancer cell lines, and low LRP1B mRNA expression was associated with high methylation levels in gastric cancer cell lines. Addition of a DNA methylation inhibitor (5-Aza-dC) restored the mRNA expression of LRP1B in these cell lines, indicating that DNA methylation is involved in regulating LRP1B expression. In 45 out of 74 (61%) clinical samples, LRP1B was highly methylated; LRP1B mRNA expression was significantly lower in 15 out of 19 (79%, P < 0.001) gastric tumor tissues than in corresponding adjacent normal tissues. In addition, ectopic expression of mLRP1B4 in gastric cancer cell lines suppressed cell growth, colony formation and tumor formation in nude mice. These results collectively indicate that LRP1B is a functional tumor suppressor gene in gastric cancer and that is regulated by DNA methylation.

A Patient-Derived Xenograft Model of Dedifferentiated Endometrial Carcinoma: A Proof-of-Concept Study for the Identification of New Molecularly Informed Treatment Approaches.

Conventional treatment of dedifferentiated endometrial carcinoma (DEC)-an uncommon and highly aggressive uterine malignancy-is beset by high failure rates. A line of research that holds promise to overcome these limitations is tailored treatments targeted on specific molecular alterations. However, suitable preclinical platforms to allow a reliable implementation of this approach are still lacking. Here, we developed a patient-derived xenograft (PDX) model for preclinical testing of investigational drugs informed by molecular data. The model-termed PDX-mLung was established in mice implanted with lung metastatic lesions obtained from a patient with DEC. Histologic and whole-exome genetic analyses revealed a high degree of identity between PDX-mLung and the patient's parental lesions (both primary DEC and lung metastases). Interestingly, molecular analyses revealed that PDX-mLung harbored druggable alterations including a FGFR2 mutation and CCNE2 amplification. Targeted combined treatment with the FGFR inhibitor lenvatinib and the cell cycle inhibitor palbociclib was found to exert synergistic therapeutic effects against in vivo tumor growth. Based on the results of RNA sequencing, lenvatinib and palbociclib were found to exert anti-tumor effects by interfering interferon signaling and activating hormonal pathways, respectively. Collectively, these data provide proof-of-concept evidence on the value of PDX models for preclinical testing of molecularly informed drug therapy in difficult-to-treat human malignancies. Further clinical research is needed to examine more rigorously the potential usefulness of the lenvatinib and palbociclib combination in patients with DEC.

Combination of Epithelial Growth Factor Receptor Blockers and CDK4/6 Inhibitor for Nasopharyngeal Carcinoma Treatment.

BACKGROUND: Nasopharyngeal carcinoma (NPC) involves host genetics, environmental and viral factors. In clinical observations, patients of young and old ages were found to have higher recurrence and metastatic rates. METHODS: Cytokine array was employed to screen druggable target(s). The candidate target(s) were confirmed through patient-derived xenografts (PDXs) and a new EBV-positive cell line, NPC-B13. RESULTS: Overexpression of epithelial growth factor (EGF) and EGF receptor (EGFR) was detected in young patients than in older patients. The growth of NPC PDX tumors and cell lines was inhibited by EGFR inhibitors (EGFRi) cetuximab and afatinib when used separately or in combination with the cell cycle blocker palbociclib. Western blot analysis of these drug-treated PDXs demonstrated that the blockade of the EGF signaling pathway was associated with a decrease in the p-EGFR level and reduction in PDX tumor size. RNA sequencing results of PDX tumors elucidated that cell cycle-related pathways were suppressed in response to drug treatments. High EGFR expression (IHC score ≥ grade 3) was correlated with poor survival in metastatic patients (p = 0.008). CONCLUSIONS: Our results provide encouraging preliminary data related to the combination treatment of EGFRi and palbociclib in patients with NPC.

Glutamate receptor, ionotropic, kainate 2 silencing by DNA hypermethylation possesses tumor suppressor function in gastric cancer.

Aberrant DNA methylation is considered a major mechanism for silencing tumor suppressor genes in gastric cancer. We used CpG microarray and differential methylation hybridization strategies to identify potential tumor suppressor genes and recovered glutamate receptor, ionotropic, kainate 2 (GRIK2) as a novel epigenetic target in gastric cancer. Additional experiments showed that the promoter region of GRIK2 was hypermethylated in 3 of the 4 tested gastric cancer cell lines, and its expression was restored by treatment of cells with the DNA methylation inhibitor, 5'-aza-dC. In clinical samples, the GRIK2 promoter was differentially hypermethylated in tumor tissues compared with adjacent normal tissues (p < 0.001), and this methylation was inversely correlated with the expression level of GRIK2 mRNA (r = -0.44). Functional studies further showed that GRIK2-expressing gastric cancer cell lines showed decreased colony formation and cell migration. Taken together, these results suggest that GRIK2 may play a tumor-suppressor role in gastric cancer. Future studies are warranted to examine whether DNA hypermethylation of the GRIK2 promoter can be used as a potential tumor marker for gastric cancer.

Targeted methylation of CMV and E1A viral promoters.

DNA methylation is a gene-silencing and host defense system that can down-regulate viral gene expression in mammalian cells. An established targeted DNA methylation method was used to demonstrate that genome-integrated CMV and adenovirus type 5 E1A promoters were hypermethylated after MCF7 and HEK293 cells were transfected with in vitro methylated viral promoter fragments. In both cases, the targeted methylation-induced gene silencing could be reversed by addition of 5-aza-2'-deoxycytidine, confirming that the CMV and E1A promoters are regulated by DNA methylation. The kinetics of the targeted DNA methylation was determined using a reporter system in live cells. In conclusion, targeted DNA methylation is able to efficiently silence susceptible viral promoters and provides an alternative strategy to study the impact of loci-specific DNA methylation in viral gene expression.

ASC modulates HIF-1α stability and induces cell mobility in OSCC.

High-level expression of ASC (Apoptosis-associated speck-like protein containing a CARD) leads to lymph node metastasis in OSCC, but the underlying mechanism remains unclear. Here, we show that HIF-1α participates in ASC-induced metastasis. We identified 195 cell-motion-associated genes that were highly activated in ASC-overexpressed SAS_ASC cells; of them, 14 representative genes were found to be overexpressed in OSCC tissues in our previously reported RNA-seq dataset, OSCC-Taiwan. Nine of the 14 genes were also upregulated in OSCC-TCGA samples. Among the nine genes, RRAS2, PDGFA, and VEGFA, were correlated with poor overall survival of patients in OSCC-TCGA dataset. We further demonstrated that the promoters of these 14 ASC-induced genes contained binding motifs for the transcription-regulating factor, HIF-1α. We observed that ASC interacted with and stabilized HIF-1α in both the cytoplasm and the nucleus under normoxia. Molecules involved in the HIF-1α pathway, such as VHL and PHD2, showed no difference in their gene and protein levels in the presence or absence of ASC, but the expression of HIF-1α-OH, and the ubiquitination of HIF-1α were both decreased in SAS_ASC cells versus SAS_con cells. The migration and invasion activities of SAS_ASC cells were reduced when cells were treated with the HIF-1α synthesis inhibitor, digoxin. Taken together, our results demonstrate that the novel ASC-HIF-1α regulatory pathway contributes to lymph node metastasis in OSCC, potentially suggesting a new treatment strategy for OSCC.

Structural Pattern Differences in Unbranched Rod-like RNA of Hepatitis Delta Virus affect RNA Editing.

Hepatitis delta virus (HDV) RNA forms an unbranched rod-like structure and complexes with the delta antigen (HDAg). Host ADAR1-catalyzed RNA editing at the amber/W site of the small HDAg leads to the production of the large HDAg, which inhibits replication and is required for virion assembly. For HDV genotype 1, amber/W editing is controlled by HDAg and the RNA structure immediate vicinity and downstream of the editing site. Here, the effects of 20 mutants carrying an increased length of consecutive base-pairing at various sites in HDV RNA on amber/W site editing were examined. All nine mutants carrying genomic regions that formed up to 15 consecutive base pairs, which is also the maximum length observed in 41 naturally occurring HDV genomes, showed normal editing rate. However, mutants carrying a 16 or 17 consecutive base-paired antigenomic segment located as far as 114 nt upstream could increase editing efficiency, possibly by interfering with HDAg binding. These data show for the first time that extended base-pairing upstream of the amber/W site could increase HDV RNA editing efficiency. Furthermore, it appears that the naturally occurring HDV RNA structures have been selected for suboptimal amber/W RNA editing, which favors the HDV replication cycle.

Targeted sequencing of cancer-related genes in nasopharyngeal carcinoma identifies mutations in the TGF-ß pathway.

Approximately, 25% of nasopharyngeal carcinoma (NPC) patients develop recurrent disease. NPC may involve relatively few genomic alterations compared to other cancers due to its association with Epstein-Barr virus (EBV). We envisioned that in-depth sequencing of tumor tissues might provide new insights into the genetic alterations of this cancer. Thirty-three NPC paired tumor/adjacent normal or peripheral blood mononuclear cell samples were deep-sequenced (>1000×) with respect to a panel of 409 cancer-related genes. Newly identified mutations and its correlation with clinical outcomes were evaluated. Profiling of somatic mutations and copy number variations (CNV) in NPC tumors identified alterations in RTK/RAS/PI3K, NOTCH, DNA repair, chromatin remodeling, cell cycle, NF-κB, and TGF-ß pathways. In addition, patients harbored CNV among 409 cancer-related genes and missense mutations in TGF-ß/SMAD signaling were associated with poor overall survival and poor recurrence-free survival, respectively. The CNV events were correlated with plasma EBV copies, while mutations in TGFBR2 and SMAD4 abrogate SMAD-dependent TGF-ß signaling. Functional analysis revealed that the new TGFBR2 kinase domain mutants were incapable of transducing the signal, leading to failure of phosphorylation of SMAD2/3 and activation of downstream TGF-ß-mediated cell growth arrest. This study provides evidence supporting CNV and dysregulated TGF-ß signaling contributes to exacerbating the NPC pathogenesis.

MCP-1 Promoter Polymorphism at 2518 is associated with metastasis of nasopharyngeal carcinoma after treatment.

PURPOSE: We herein examined whether the single nucleotide polymorphism (SNP) at -2518 of the MCP-1 gene promoter region influences clinical outcomes among nasopharyngeal carcinoma (NPC) patients. EXPERIMENTAL DESIGN: The study population consisted of 411 NPC patients without metastasis at diagnosis. All patients were treated at the Chang Gung Memorial Hospital from March 1994 to November 2004. The MCP-1 SNP-2518 genotype of each patient was determined by TaqMan genotyping kit. Statistical analyses were conducted to compare disease-specific survival (DSS), progression-free survival (PFS), local recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) of patients according to genotype. MCP-1 expression in tumor biopsies was examined by immunohistochemistry. RESULTS: Among 411 NPC patients, carriers of AA and AG genotypes were prone to distant metastasis than that of GG genotype (hazard ratio, 2.21; P = 0.017, and hazard ratio, 2.23; P = 0.005, for AA and AG genotype, respectively) after initial radiotherapy. No genotype-specific significant difference was found in DSS, PFS, and LRFS. Furthermore, immunohistochemistry revealed that MCP-1 expression level was higher in NPC tumor cells from GG carriers compared with those from AA and AG carriers. CONCLUSIONS: MCP-1 SNP-2518 may be a valuable genetic marker for assessing the risk of developing distant metastasis after the radiotherapy in NPC patients. Carriers of A allele may require more aggressive chemotherapy implicating a potential marker for personalized medicine. We speculate that a regulatory SNP may be associated with the distant metastasis of NPC. Validation studies are warranted.

Activation of DNA methyltransferase 1 by EBV LMP1 Involves c-Jun NH(2)-terminal kinase signaling.

EBV latent membrane protein 1 (LMP1) activates cellular DNA methyltransferases, resulting in hypermethylation and silencing of E-cadherin. However, the underlying mechanism remains to be elucidated. In this study, we show that LMP1 directly induces the dnmt1 promoter activity through its COOH-terminal activation region-2 YYD domain. Using (i) LMP1 mutants, (ii) dominant negative mutants c-jun NH(2)-terminal kinase (JNK)-DN, p38-DN, and constitutive active mutant IkappaB, as well as (iii) dsRNAs targeting c-Jun, JNK, and tumor necrosis factor receptor-associated death domain protein, and (iv) signal transduction inhibitors, we show that LMP1-mediated DNA methyltransferase-1 (DNMT1) activation involves JNK but not nuclear factor kappaB and p38/mitogen-activated protein kinase signaling. In addition, LMP1 is unable to activate dnmt1-P1 promoter with activator protein-1 (AP-1) site mutation. Chromatin immunoprecipitation assay results also confirm that LMP1 activates P1 promoter via the JNK-AP-1 pathway. Furthermore, chromatin immunoprecipitation assay data in LMP1-inducible cells disclose that LMP1 induces formation of a transcriptional repression complex, composed of DNMT1 and histone deacetylase, which locates on E-cadherin gene promoter. Treatment with JNK inhibitor, SP600125, prevents the formation of this repression complex. Statistical analyses of the immunohistochemical staining of 32 nasopharyngeal carcinoma (NPC) biopsies show LMP1 expression (18 of 32, 56.25%), DNMT1 expression (31 of 32, 97%), and phospho-c-Jun (27 of 32, 84.38%), suggesting that overexpression of these proteins is observed in NPC tumor. Overall, these results support a mechanistic link between JNK-AP-1 signaling and DNA methylation induced by the EBV oncogene product LMP1.

Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinoma.

BACKGROUND: Aberrant hypermethylation of cellular genes is a common phenomenon to inactivate genes and promote tumorigenesis in nasopharyngeal carcinoma (NPC). METHODS: Methyl binding domain (MBD)-ChIP sequencing of NPC cells, microarray data of NPC biopsies and gene ontology analysis were conducted to identify a potential tumor suppressor gene CLDN11 that was both hypermethylated and downregulated in NPC. Bisulfite sequencing, qRT-PCR, immunohistochemistry staining of the NPC clinical samples and addition of methylation inhibitor, 5'azacytidine, in NPC cells were performed to verify the correlation between DNA hypermethylation and expression of CLDN11. Promoter reporter and EMSA assays were used to dissect the DNA region responsible for transcription activator binding and to confirm whether DNA methylation could affect activator's binding, respectively. CLDN11 was transiently overexpressed in NPC cells followed by cell proliferation, migration, invasion assays to characterize its biological roles. Co-immunoprecipitation experiments and proteomic approach were carried out to identify novel interacting protein(s) and the binding domain of CLDN11. Anti-tumor activity of the CLDN11 was elucidated by in vitro functional assay. RESULTS: A tight junction gene, CLDN11, was identified as differentially hypermethylated gene in NPC. High methylation percentage of CLDN11 promoter in paired NPC clinical samples was correlated with low mRNA expression level. Immunohistochemistry staining of NPC paired samples tissue array demonstrated that CLDN11 protein expression was relatively low in NPC tumors. Transcription activator GATA1 bound to CLDN11 promoter region - 62 to - 53 and its DNA binding activity was inhibited by DNA methylation. Re-expression of CLDN11 reduced cell migration and invasion abilities in NPC cells. By co-immunoprecipitation and liquid chromatography-tandem mass spectrometry LC-MS/MS, tubulin alpha-1b (TUBA1B) and beta-3 (TUBB3), were identified as the novel CLDN11-interacting proteins. CLDN11 interacted with these two tubulins through its intracellular loop and C-terminus. Furthermore, these domains were required for CLDN11-mediated cell migration inhibition. Treatment with a tubulin polymerization inhibitor, nocodazole, blocked NPC cell migration. CONCLUSIONS: CLDN11 is a hypermethylated and downregulated gene in NPC. Through interacting with microtubules TUBA1B and TUBB3, CLDN11 blocks the polymerization of tubulins and cell migration activity. Thus, CLDN11 functions as a potential tumor suppressor gene and silencing of CLDN11 by DNA hypermethylation promotes NPC progression.

Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma.

BACKGROUND: Patient-derived xenograft (PDX) tumor model has become a new approach in identifying druggable tumor mutations, screening and evaluating personalized cancer drugs based on the mutated targets. METHODS: We established five nasopharyngeal carcinoma (NPC) PDXs in mouse model. Subsequently, whole-exome sequencing (WES) and genomic mutation analyses were performed to search for genetic alterations for new drug targets. Potential drugs were applied in two NPC PDX mice model to assess their anti-cancer activities. RNA sequencing and transcriptomic analysis were performed in one NPC PDX mice to correlate with the efficacy of the anti-cancer drugs. RESULTS: A relative high incident rate of copy number variations (CNVs) of cell cycle-associated genes. Among the five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Furthermore, CCND1 overexpression was observed in > 90% FFPE clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients' plasma and could serve as a screening test to select potential CDK4/6 inhibitor treatment candidates. Based on our NPC PDX model and RNA sequencing, Palbociclib, a cyclin-dependent kinase inhibitor, proved to have anti-tumor effects by inducing G1 arrest. One NPC patient with liver metastatic was treated with Palbociclib, had stable disease response and a drop in Epstein Barr virus (EBV) EBV titer. CONCLUSIONS: Our integrated information of sequencing-based genomic studies and tumor transcriptomes with drug treatment in NPC-PDX models provided guidelines for personalized precision treatments and revealed a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for NPC.

RNA recombination in Hepatitis delta virus: Identification of a novel naturally occurring recombinant.

BACKGROUND/PURPOSE: Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity. It replicates in the nucleus by host RNA polymerase via a rolling circle mechanism. Similar to many RNA viruses encoding their own RNA-dependent RNA polymerases, homologous recombination of HDV occurs in mixed-genotype infections and in cultured cells cotransfected with two HDV sequences, as demonstrated by molecular analyses. METHODS: Among 237 published complete genomic sequences, 34 sequences were reported from the small and isolated Miyako Island, Japan, and belonged to the Asia-specific genotypes, HDV-2 and HDV-4 (the majority of them belonged to the known Miyako Island-specific subgroup, HDV-4M). We investigated the presence of naturally occurring HDV recombinant in Miyako Island using phylogenetic and recombination analyses. RESULTS: We identified a two-switch HDV-4/4M intersubtype recombinant with an unbranched rod-like RNA genome. CONCLUSION: Our data suggest that RNA recombination plays an important role in the rapid evolution of HDV, allowing the production of new HDV strains with correct genomic structures.

APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism.

Oral squamous cell carcinoma is a prominent cancer worldwide, particularly in Taiwan. By integrating omics analyses in 50 matched samples, we uncover in Taiwanese patients a predominant mutation signature associated with cytidine deaminase APOBEC, which correlates with the upregulation of APOBEC3A expression in the APOBEC3 gene cluster at 22q13. APOBEC3A expression is significantly higher in tumors carrying APOBEC3B-deletion allele(s). High-level APOBEC3A expression is associated with better overall survival, especially among patients carrying APOBEC3B-deletion alleles, as examined in a second cohort (n = 188; p = 0.004). The frequency of APOBEC3B-deletion alleles is ~50% in 143 genotyped oral squamous cell carcinoma -Taiwan samples (27A3B -/-:89A3B +/-:27A3B +/+), compared to the 5.8% found in 314 OSCC-TCGA samples. We thus report a frequent APOBEC mutational profile, which relates to a APOBEC3B-deletion germline polymorphism in Taiwanese oral squamous cell carcinoma that impacts expression of APOBEC3A, and is shown to be of clinical prognostic relevance. Our finding might be recapitulated by genomic studies in other cancer types.Oral squamous cell carcinoma is a prevalent malignancy in Taiwan. Here, the authors show that OSCC in Taiwanese show a frequent deletion polymorphism in the cytidine deaminases gene cluster APOBEC3 resulting in increased expression of A3A, which is shown to be of clinical prognostic relevance.

Pyk2 activates the NLRP3 inflammasome by directly phosphorylating ASC and contributes to inflammasome-dependent peritonitis.

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1ß secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1ß secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.