Somatic A-to-I RNA-edited RHOA isoform 2 specific-R176G mutation promotes tumor progression in lung adenocarcinoma.

Adenosine-to-inosine (A-to-I) RNA editing is the most common posttranscriptional editing to create somatic mutations and increase proteomic diversity. However, the functions of the edited mutations are largely underexplored. To identify novel targets in lung adenocarcinoma (LUAD), we conducted a genome-wide somatic A-to-I RNA editing analysis of 23 paired adjacent normal and LUAD transcriptomes and identified 26,280 events, including known nonsynonymous AZIN1-S367G and novel RHOAiso2 (RHOA isoform 2)-R176G, tubulin gamma complex associated protein 2 (TUBGCP2)-N211S, and RBMXL1-I40 M mutations. We validated the edited mutations in silico in multiple databases and in newly collected LUAD tissue pairs with the SEQUENOM MassARRAY® and TaqMan PCR Systems. We selected RHOAiso2-R176G due to its significant level, isoform-specificity, and being the most common somatic edited nonsynonymous mutation of RHOAiso2 to investigate its roles in LUAD tumorigenesis. RHOAiso2 is a ubiquitous but low-expression alternative spliced isoform received a unique Alu-rich exon at the 3' RHOA mRNA to become an editing RNA target, leading to somatic hypermutation and protein diversity. Interestingly, LUAD patients harboring the RHOAiso2-R176G mutation were associated with aberrant RHOA functions, cancer cell proliferation and migration, and poor clinical outcomes in transcriptome analysis. Mechanistically, RHOAiso2-R176G mutation-expressing LUAD cells potentiate RHOA-guanosine triphosphate (GTP) activity to phosphorylate ROCK1/2 effectors and enhance cell proliferation and migration in vitro and increase tumor growth in xenograft and systemic metastasis models in vivo. Taken together, the RHOAiso2-R176G mutation is a common somatic A-to-I edited mutation of the hypermutated RHOA isoform 2. It is an oncogenic and isoform-specific theranostic target that activates RHOA-GTP/p-ROCK1/2 signaling to promote tumor progression.

Prognostic comparative genes predict targets for sorafenib combination therapies in hepatocellular carcinoma.

With the increasing incidence and mortality of human hepatocellular carcinoma (HCC) worldwide, revealing innovative targets to improve therapeutic strategies is crucial for prolonging the lives of patients. To identify innovative targets, we conducted a comprehensive comparative transcriptome analysis of 5,410 human HCCs and 974 mouse liver cancers to identify concordantly expressed genes associated with patient survival. Among the 664 identified prognostic comparative HCC (pcHCC) genes, upregulated pcHCC genes were associated with prognostic clinical features, including large tumor size, vascular invasion and late HCC stages. Interestingly, after validating HCC patient prognoses in multiple independent datasets, we matched the 664 aberrant pcHCC genes with the sorafenib-altered genes in TCGA_LIHC patients and found these 664 pcHCC genes were enriched in sorafenib-related functions, such as downregulated xenobiotic and lipid metabolism and upregulated cell proliferation. Therapeutic agents targeting aberrant pcHCC genes presented divergent molecular mechanisms, including suppression of sorafenib-unrelated oncogenic pathways, induction of sorafenib-unrelated ferroptosis, and modulation of sorafenib transportation and metabolism, to potentiate sorafenib therapeutic effects in HCC combination therapy. Moreover, the pcHCC genes NCAPG and CENPW, which have not been targeted in combination with sorafenib treatment, were knocked down and combined with sorafenib treatment, which reduced HCC cell viability based on disruption to the p38/STAT3 axis, thereby hypersensitizing HCC cells. Together, our results provide important resources and reveal that 664 pcHCC genes represent innovative targets suitable for developing therapeutic strategies in combination with sorafenib based on the divergent synergistic mechanisms for HCC tumor suppression.

A novel HIF1α-STIL-FOXM1 axis regulates tumor metastasis.

BACKGROUND: Metastasis is the major cause of morbidity and mortality in cancer that involves in multiple steps including epithelial-mesenchymal transition (EMT) process. Centrosome is an organelle that functions as the major microtubule organizing center (MTOC), and centrosome abnormalities are commonly correlated with tumor aggressiveness. However, the conclusive mechanisms indicating specific centrosomal proteins participated in tumor progression and metastasis remain largely unknown. METHODS: The expression levels of centriolar/centrosomal genes in various types of cancers were first examined by in silico analysis of the data derived from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and European Bioinformatics Institute (EBI) datasets. The expression of STIL (SCL/TAL1-interrupting locus) protein in clinical specimens was further assessed by Immunohistochemistry (IHC) analysis and the oncogenic roles of STIL in tumorigenesis were analyzed using in vitro and in vivo assays, including cell migration, invasion, xenograft tumor formation, and metastasis assays. The transcriptome differences between low- and high-STIL expression cells were analyzed by RNA-seq to uncover candidate genes involved in oncogenic pathways. The quantitative polymerase chain reaction (qPCR) and reporter assays were performed to confirm the results. The chromatin immunoprecipitation (ChIP)-qPCR assay was applied to demonstrate the binding of transcriptional factors to the promoter. RESULTS: The expression of STIL shows the most significant increase in lung and various other types of cancers, and is highly associated with patients' survival rate. Depletion of STIL inhibits tumor growth and metastasis. Interestingly, excess STIL activates the EMT pathway, and subsequently enhances cancer cell migration and invasion. Importantly, we reveal an unexpected role of STIL in tumor metastasis. A subset of STIL translocate into nucleus and associate with FOXM1 (Forkhead box protein M1) to promote tumor metastasis and stemness via FOXM1-mediated downstream target genes. Furthermore, we demonstrate that hypoxia-inducible factor 1α (HIF1α) directly binds to the STIL promoter and upregulates STIL expression under hypoxic condition. CONCLUSIONS: Our findings indicate that STIL promotes tumor metastasis through the HIF1α-STIL-FOXM1 axis, and highlight the importance of STIL as a promising therapeutic target for lung cancer treatment.

PSPC1 Potentiates IGF1R Expression to Augment Cell Adhesion and Motility.

Paraspeckle protein 1 (PSPC1) overexpression in cancers is known to be the pro-metastatic switch of tumor progression associated with poor prognosis of cancer patients. However, the detail molecular mechanisms to facilitate cancer cell migration remain elusive. Here, we conducted integrated analysis of human phospho-kinase antibody array, transcriptome analysis with RNA-seq, and proteomic analysis of protein pulldown to study the molecular detail of PSPC1-potentiated phenotypical transformation, adhesion, and motility in human hepatocellular carcinoma (HCC) cells. We found that PSPC1 overexpression re-assembles and augments stress fiber formations to promote recruitment of focal adhesion contacts at the protruding edge to facilitate cell migration. PSPC1 activated focal adhesion-associated kinases especially FAK/Src signaling to enhance cell adhesion and motility toward extracellular matrix (ECM). Integrated transcriptome and gene set enrichment analysis indicated that PSPC1 modulated receptor tyrosine kinase IGF1R involved in the focal adhesion pathway and induction of diverse integrins expression. Knockdown IGF1R expression and treatment of IGF1R inhibitor suppressed PSPC1-induced cell motility. Interestingly, knockdown PSPC1-interacted paraspeckle components including NONO, FUS, and the lncRNA Neat1 abolished PSPC1-activated IGF1R expression. Together, PSPC1 overexpression induced focal adhesion formation and facilitated cell motility via activation of IGF1R signaling. PSPC1 overexpression in tumors could be a potential biomarker of target therapy with IGF1R inhibitor for improvement of HCC therapy.

PSPC1 mediates TGF-ß1 autocrine signalling and Smad2/3 target switching to promote EMT, stemness and metastasis.

Activation of metastatic reprogramming is critical for tumour metastasis. However, more detailed knowledge of the underlying mechanism is needed to enable targeted intervention. Here, we show that paraspeckle component 1 (PSPC1), identified in an aberrant 13q12.11 locus, is upregulated and associated with poor survival in patients with cancer. PSPC1 promotes tumorigenesis, epithelial-to-mesenchymal transition (EMT), stemness and metastasis in multiple cell types and in spontaneous mouse cancer models. PSPC1 is the master activator for transcription factors of EMT and stemness and accompanies c-Myc activation to facilitate tumour growth. PSPC1 increases transforming growth factor-ß1 (TGF-ß1) secretion through an interaction with phosphorylated and nuclear Smad2/3 to potentiate TGF-ß1 autocrine signalling. Moreover, PSPC1 acts as a contextual determinant of the TGF-ß1 pro-metastatic switch to alter Smad2/3 binding preference from tumour-suppressor to pro-metastatic genes. Having validated the PSPC1-Smads-TGF-ß1 axis in various cancers, we conclude that PSPC1 is a master activator of pro-metastatic switches and a potential target for anti-metastasis drugs.

Overexpressed-eIF3I interacted and activated oncogenic Akt1 is a theranostic target in human hepatocellular carcinoma.

UNLABELLED: Eukaryotic translation initiation factor 3 subunit I (eIF3I) with transforming capability is often overexpressed in human hepatocellular carcinoma (HCC) but its oncogenic mechanisms remain unknown. We demonstrate that eIF3I is overexpressed in various cancers along with activated Akt1 phosphorylation and kinase activity in an eIF3I dose-dependent manner. A novel eIF3I and Akt1 protein interaction was identified in HCC cell lines and tissues and was required for eIF3I-mediated activation of Akt1 signaling. Expression of either antisense eIF3I or dominant negative Akt1 mutant suppressed eIF3I-mediated Akt1 oncogenic signaling and various other tumorigenic effects. Oncogenic domain mapping of the eIF3I and Akt1 interaction suggested that the C-terminal eIF3I interacted with the Akt1 kinase domain and conferred the majority of oncogenic functions. In addition, eIF3I interaction with Akt1 prevented PP2A dephosphorylation of Akt1 and resulted in constitutively active Akt1 oncogenic signaling. Importantly, concordant expression of endogenous eIF3I and phospho-Akt1 was detected in HCC cell lines and tissues. Treatment of eIF3I overexpressing HCC cells with the Akt1 specific inhibitor API-2 suppressed eIF3I-mediated tumorigenesis in vitro and in vivo. CONCLUSION: We describe a constitutive Akt1 oncogenic mechanism resulting from interaction of overexpressed eIF3I with Akt1 that prevents PP2A-mediated dephosphorylation. Overexpression of eIF3I in HCC is oncogenic and is a surrogate marker and therapeutic target for treatment with Akt1 inhibitors.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/ß-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

IGDB.NSCLC: integrated genomic database of non-small cell lung cancer.

Lung cancer is the most common cause of cancer-related mortality with more than 1.4 million deaths per year worldwide. To search for significant somatic alterations in lung cancer, we analyzed, integrated and manually curated various data sets and literatures to present an integrated genomic database of non-small cell lung cancer (IGDB.NSCLC, http://igdb.nsclc.ibms.sinica.edu.tw). We collected data sets derived from hundreds of human NSCLC (lung adenocarcinomas and/or squamous cell carcinomas) to illustrate genomic alterations [chromosomal regions with copy number alterations (CNAs), gain/loss and loss of heterozygosity], aberrant expressed genes and microRNAs, somatic mutations and experimental evidence and clinical information of alterations retrieved from literatures. IGDB.NSCLC provides user friendly interfaces and searching functions to display multiple layers of evidence especially emphasizing on concordant alterations of CNAs with co-localized altered gene expression, aberrant microRNAs expression, somatic mutations or genes with associated clinicopathological features. These significant concordant alterations in NSCLC are graphically or tabularly presented to facilitate and prioritize as the putative cancer targets for pathological and mechanistic studies of lung tumorigenesis and for developing new strategies in clinical interventions.

A Cullin3-KLHL20 Ubiquitin ligase-dependent pathway targets PML to potentiate HIF-1 signaling and prostate cancer progression.

Tumor hypoxia is associated with disease progression and treatment failure, but the hypoxia signaling mechanism is not fully understood. Here, we show that KLHL20, a Cullin3 (Cul3) substrate adaptor induced by HIF-1, coordinates with the actions of CDK1/2 and Pin1 to mediate hypoxia-induced PML proteasomal degradation. Furthermore, this PML destruction pathway participates in a feedback mechanism to maximize HIF-1α induction, thereby potentiating multiple tumor hypoxia responses, including metabolic reprogramming, epithelial-mesenchymal transition, migration, tumor growth, angiogenesis, and chemoresistance. In human prostate cancer, overexpression of HIF-1α, KLHL20, and Pin1 correlates with PML down-regulation, and hyperactivation of the PML destruction pathway is associated with disease progression. Our study indicates that the KLHL20-mediated PML degradation and HIF-1α autoregulation play key roles in tumor progression.

IGRhCellID: integrated genomic resources of human cell lines for identification.

Cell line identification is emerging as an essential method for every cell line user in research community to avoid using misidentified cell lines for experiments and publications. IGRhCellID (http://igrcid.ibms.sinica.edu.tw) is designed to integrate eight cell identification methods including seven methods (STR profile, gender, immunotypes, karyotype, isoenzyme profile, TP53 mutation and mutations of cancer genes) available in various public databases and our method of profiling genome alterations of human cell lines. With data validation of 11 small deleted genes in human cancer cell lines, profiles of genomic alterations further allow users to search for human cell lines with deleted gene to serve as indigenous knock-out cell model (such as SMAD4 in gene view), with amplified gene to be the cell models for testing therapeutic efficacy (such as ERBB2 in gene view) and with overlapped aberrant chromosomal loci for revealing common cancer genes (such as 9p21.3 homozygous deletion with co-deleted CDKN2A, CDKN2B and MTAP in chromosome view). IGRhCellID provides not only available methods for cell identification to help eradicating concerns of using misidentified cells but also designated genetic features of human cell lines for experiments.

Overlapping high-resolution copy number alterations in cancer genomes identified putative cancer genes in hepatocellular carcinoma.

UNLABELLED: Recurrent cancer genome aberrations are indicators of residing crucial cancer genes. Although recent advances in genomic technologies have led to a global view of cancer genome aberrations, the identification of target genes and biomarkers from the aberrant loci remains difficult. To facilitate searches of cancer genes in human hepatocellular carcinoma (HCC), we established a comprehensive protocol to analyze copy number alterations (CNAs) in cancer genomes using high-density single nucleotide polymorphism arrays with unpaired reference genomes. We identified common HCC genes by overlapping the shared aberrant loci in multiple cell lines with functional validation and clinical implications. A total of 653 amplicons and 57 homozygous deletions (HDs) were revealed in 23 cell lines. To search for novel HCC genes, we overlapped aberrant loci to uncover 6 HDs and 126 amplicons shared by at least two cell lines. We selected two novel genes, fibronectin type III domain containing 3B (FNDC3B) at the 3q26.3 overlapped amplicon and solute carrier family 29 member 2 (SLC29A2) at the 11q13.2 overlapped amplicon, to investigate their aberrations in HCC tumorigenesis. Aberrant up-regulation of FNDC3B and SLC29A2 occurred in multiple HCC data sets. Knockdown of these genes in amplified cells decreased cell proliferation, anchorage-independent growth, and tumor formation in xenograft models. Importantly, up-regulation of SLC29A2 in HCC tissues was significantly associated with advanced stages (P = 0.0031), vascular invasion (P = 0.0353), and poor patient survival (P = 0.0325). Overexpression of FNDC3B or SLC29A2 in unamplified HCC cells promoted cell proliferation through activation of the signal transducer and activator of transcription 3 signaling pathway. CONCLUSION: A standardized genome-wide CNA analysis protocol using data from user-generated or public domains normalized with unpaired reference genomes has been established to facilitate high-throughput detection of cancer genes as significant target genes and biomarkers for cancer diagnosis and therapy.

An introduction to mitochondrial informatics.

In this chapter, we review the public resources available for human mitochondrial DNA and protein related bioinformatics, with a special focus on mitochondrial single nucleotide polymorphisms (mtSNPs). We also review our own freeware tool V-MitoSNP, giving an overview of its implementation and program workflow. Apart from these, we review several protocols for the graphic input of genes, keywords, gene searching by sequence, mtSNP searching by sequence, restriction enzyme mining, primer design, and virtual electrophoresis for PCR-RFLP genotyping. Some databases with similar function are integrated and compared.

Methyl-Typing: an improved and visualized COBRA software for epigenomic studies.

Combined bisulfite restriction analysis (COBRA) is one of the most commonly used methylation quantification methods. However, it focuses on relatively few restriction enzymes. Here, we present Methyl-Typing, a web-based software that provides restriction enzyme mining data for methyl-cytosine-containing sequences following bisulfite-conversion. Gene names, accession numbers, sequences, PCR primers, and file upload are accessible for input. Promoter sequences and restriction enzymes for CpG- and GpC-containing recognition sites are retrieved. Four representative enzymes were tested successfully by COBRA on the experimental work. Therefore, the Methyl-Typing tool provides a comprehensive COBRA-restriction enzyme mining. It is freely available at http://bio.kuas.edu.tw/methyl-typing.

Prim-SNPing: a primer designer for cost-effective SNP genotyping.

Many kinds of primer design (PD) software tools have been developed, but most of them lack a single nucleotide polymorphism (SNP) genotyping service. Here, we introduce the web-based freeware "Prim-SNPing," which, in addition to general PD, provides three kinds of primer design functions for cost-effective SNP genotyping: natural PD, mutagenic PD, and confronting two-pair primers (CTPP) PD. The natural PD and mutagenic PD provide primers and restriction enzyme mining for polymerase chain reaction-restriction fragment of length polymorphism (PCR-RFLP), while CTPP PD provides primers for restriction enzyme-free SNP genotyping. The PCR specificity and efficiency of the designed primers are improved by BLAST searching and evaluating secondary structure (such as GC clamps, dimers, and hairpins), respectively. The length pattern of PCR-RFLP using natural PD is user-adjustable, and the restriction sites of the RFLP enzymes provided by Prim-SNPing are confirmed to be absent within the generated PCR product. In CTPP PD, the need for a separate digestion step in RFLP is eliminated, thus making it faster and cheaper. The output of Prim-SNPing includes the primer list, melting temperature (Tm) value, GC percentage, and amplicon size with enzyme digestion information. The reference SNP (refSNP, or rs) clusters from the Single Nucleotide Polymorphism database (dbSNP) at the National Center for Biotechnology Information (NCBI), and multiple other formats of human, mouse, and rat SNP sequences are acceptable input. In summary, Prim-SNPing provides interactive, user-friendly and cost-effective primer design for SNP genotyping. It is freely available at http://bio.kuas.edu.tw/prim-snping.

An improved PCR method for gender identification of eagles.

Eagles are sexually monomorphic and therefore it is difficult to determine their gender, which is a crucial need for management purposes. In this study, we have developed an improved gender identification method by exploiting length differences between the Chromo-Helicase-DNA binding protein (CHD)-Z and CHD-W genes of Spilornis cheela hoya. By comparing DNA sequences for CHD-W and CHD-Z from 10 species of Falconiformes eagles we designed universal gender identification PCR primers that exploit differences in product size. Standard agarose gels were shown to easily distinguish between the 148-bp CHD-ZW and the 258-bp CHD-W PCR products. When used with 28 samples of S. cheela hoya, our improved universal primers provided a fast and precise gender identification assay.

High-throughput avian molecular sexing by SYBR green-based real-time PCR combined with melting curve analysis.

BACKGROUND: Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved. RESULTS: Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished. CONCLUSION: In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.

V-MitoSNP: visualization of human mitochondrial SNPs.

BACKGROUND: Mitochondrial single nucleotide polymorphisms (mtSNPs) constitute important data when trying to shed some light on human diseases and cancers. Unfortunately, providing relevant mtSNP genotyping information in mtDNA databases in a neatly organized and transparent visual manner still remains a challenge. Amongst the many methods reported for SNP genotyping, determining the restriction fragment length polymorphisms (RFLPs) is still one of the most convenient and cost-saving methods. In this study, we prepared the visualization of the mtDNA genome in a way, which integrates the RFLP genotyping information with mitochondria related cancers and diseases in a user-friendly, intuitive and interactive manner. The inherent problem associated with mtDNA sequences in BLAST of the NCBI database was also solved. DESCRIPTION: V-MitoSNP provides complete mtSNP information for four different kinds of inputs: (1) color-coded visual input by selecting genes of interest on the genome graph, (2) keyword search by locus, disease and mtSNP rs# ID, (3) visualized input of nucleotide range by clicking the selected region of the mtDNA sequence, and (4) sequences mtBLAST. The V-MitoSNP output provides 500 bp (base pairs) flanking sequences for each SNP coupled with the RFLP enzyme and the corresponding natural or mismatched primer sets. The output format enables users to see the SNP genotype pattern of the RFLP by virtual electrophoresis of each mtSNP. The rate of successful design of enzymes and primers for RFLPs in all mtSNPs was 99.1%. The RFLP information was validated by actual agarose electrophoresis and showed successful results for all mtSNPs tested. The mtBLAST function in V-MitoSNP provides the gene information within the input sequence rather than providing the complete mitochondrial chromosome as in the NCBI BLAST database. All mtSNPs with rs number entries in NCBI are integrated in the corresponding SNP in V-MitoSNP. CONCLUSION: V-MitoSNP is a web-based software platform that provides a user-friendly and interactive interface for mtSNP information, especially with regard to RFLP genotyping. Visual input and output coupled with integrated mtSNP information from MITOMAP and NCBI make V-MitoSNP an ideal and complete visualization interface for human mtSNPs association studies.