Zinc Alpha-2-Glycoprotein (ZAG/AZGP1) secreted by triple-negative breast cancer promotes tumor microenvironment fibrosis.

Obesity is a predisposition factor for breast cancer, suggesting a localized, reciprocal interaction between breast cancer cells and the surrounding mammary white adipose tissue. To investigate how breast cancer cells alter the composition and function of adipose tissue, we screened the secretomes of ten human breast cancer cell lines for the ability to modulate the differentiation of adipocyte stem and progenitor cells (ASPC). The screen identified a key adipogenic modulator, Zinc Alpha-2-Glycoprotein (ZAG/AZGP1), secreted by triple-negative breast cancer (TNBC) cells. TNBC-secreted ZAG inhibits adipogenesis and instead induces the expression of fibrotic genes. Accordingly, depletion of ZAG in TNBC cells attenuates fibrosis in white adipose tissue and inhibits tumor growth. Further, high expression of ZAG in TNBC patients, but not other clinical subtypes of breast cancer, is linked to poor prognosis. Our findings suggest a role of TNBC-secreted ZAG in promoting the transdifferentiation of ASPCs into cancer-associated fibroblasts to support tumorigenesis.

Hypermedia-based software architecture enables Test-Driven Development.

Objectives: Using agile software development practices, develop and evaluate an architecture and implementation for reliable and user-friendly self-service management of bioinformatic data stored in the cloud. Materials and methods: Comprehensive Oncology Research Environment (CORE) Browser is a new open-source web application for cancer researchers to manage sequencing data organized in a flexible format in Amazon Simple Storage Service (S3) buckets. It has a microservices- and hypermedia-based architecture, which we integrated with Test-Driven Development (TDD), the iterative writing of computable specifications for how software should work prior to development. Relying on repeating patterns found in hypermedia-based architectures, we hypothesized that hypermedia would permit developing test "templates" that can be parameterized and executed for each microservice, maximizing code coverage while minimizing effort. Results: After one-and-a-half years of development, the CORE Browser backend had 121 test templates and 875 custom tests that were parameterized and executed 3031 times, providing 78% code coverage. Discussion: Architecting to permit test reuse through a hypermedia approach was a key success factor for our testing efforts. CORE Browser's application of hypermedia and TDD illustrates one way to integrate software engineering methods into data-intensive networked applications. Separating bioinformatic data management from analysis distinguishes this platform from others in bioinformatics and may provide stable data management while permitting analysis methods to advance more rapidly. Conclusion: Software engineering practices are underutilized in informatics. Similar informatics projects will more likely succeed through application of good architecture and automated testing. Our approach is broadly applicable to data management tools involving cloud data storage.

Replicative Instability Drives Cancer Progression.

In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.

Detection of circulating tumor DNA without a tumor-informed search using next-generation sequencing is a prognostic biomarker in pancreatic ductal adenocarcinoma.

The confounding effects of next-generation sequencing (NGS) noise on detection of low frequency circulating tumor DNA (ctDNA) without a priori knowledge of solid tumor mutations has limited the applications of circulating cell-free DNA (ccfDNA) in clinical oncology. Here, we use a 118 gene panel and leverage ccfDNA technical replicates to eliminate NGS-associated errors while also enhancing detection of ctDNA from pancreatic ductal adenocarcinomas (PDACs). Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III). Post-operative ccfDNA was also collected from 11 of the patients within 100 days of surgery. ctDNA detection was restricted to variants corresponding to pathogenic mutations in PDAC present in both replicates. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients. Of the nine ctDNA variants detected (variant allele frequency: 0.08%-1.59%), five had a corresponding mutation in tumor DNA. Pre-operative detection of ctDNA was associated with shorter survival (312 vs. 826 days; χ2=5.4, P = 0.021). Guiding ctDNA detection in pre-operative ccfDNA based on mutations present in tumor DNA yielded a similar survival analysis. Detection of ctDNA in the post-operative ccfDNA with or without tumor-informed guidance was not associated with outcomes. Therefore, the detection of PDAC-derived ctDNA during a broad and untargeted survey of ccfDNA with NGS may be a valuable, non-invasive, prognostic biomarker to integrate into the clinical assessment and management of patients prior to surgery.

The stochastic nature of errors in next-generation sequencing of circulating cell-free DNA.

Challenges with distinguishing circulating tumor DNA (ctDNA) from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Here we show early and random PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding, removal of artifacts due to clonal hematopoiesis of indeterminate potential, and suppression of patterned errors. We also demonstrate sample duplicates are necessary to eliminate the stochastic noise associated with NGS. Integration of sample duplicates into NGS analytics may broaden ctDNA applications by removing NGS-related errors that confound identification of true very low frequency variants during searches for ctDNA without a priori knowledge of specific mutations to target.

Automated size selection for short cell-free DNA fragments enriches for circulating tumor DNA and improves error correction during next generation sequencing.

Circulating tumor-derived cell-free DNA (ctDNA) enables non-invasive diagnosis, monitoring, and treatment susceptibility testing in human cancers. However, accurate detection of variant alleles, particularly during untargeted searches, remains a principal obstacle to widespread application of cell-free DNA in clinical oncology. In this study, isolation of short cell-free DNA fragments is shown to enrich for tumor variants and improve correction of PCR- and sequencing-associated errors. Subfractions of the mononucleosome of circulating cell-free DNA (ccfDNA) were isolated from patients with melanoma, pancreatic ductal adenocarcinoma, and colorectal adenocarcinoma using a high-throughput-capable automated gel-extraction platform. Using a 128-gene (128 kb) custom next-generation sequencing panel, variant alleles were on average 2-fold enriched in the short fraction (median insert size: ~142 bp) compared to the original ccfDNA sample, while 0.7-fold reduced in the fraction corresponding to the principal peak of the mononucleosome (median insert size: ~167 bp). Size-selected short fractions compared to the original ccfDNA yielded significantly larger family sizes (i.e., PCR duplicates) during in silico consensus sequence interpretation via unique molecular identifiers. Increments in family size were associated with a progressive reduction of PCR and sequencing errors. Although consensus read depth also decreased at larger family sizes, the variant allele frequency in the short ccfDNA fraction remained consistent, while variant detection in the original ccfDNA was commonly lost at family sizes necessary to minimize errors. These collective findings support the automated extraction of short ccfDNA fragments to enrich for ctDNA while concomitantly reducing false positives through in silico error correction.

Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants.

Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs-the "integrated evaluation"-that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at http://hci-exlovd.hci.utah.edu, with 77 re-evaluable variants.

Genome-wide profiling of the C. elegans dsRNAome.

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3' UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.

DNA methylation profiling in zebrafish.

DNA methylation on cytosine in vertebrates such as zebrafish serves to silence gene expression by interfering with the binding of certain transcription factors and through the recruitment of repressive chromatin machinery. Cytosine DNA methylation is chemically stable and heritable through the germline - but also reversible through many modes, making it a useful and dynamic epigenetic modification. Virtually all of the enzymes and factors involved in the deposition, binding, and removal of cytosine methylation are conserved in zebrafish, and therefore the organism an excellent model for understanding the use of DNA methylation in the control of gene regulation and other processes. Here, we discuss the main approaches to quantifying DNA methylation levels genome-wide in zebrafish: one is an established method for revealing regional methylation (methylated DNA immunoprecipitation (MeDIP)), and the other is an emerging method that reveals DNA methylation at base-pair resolution (shotgun bisulphite sequencing). We also introduce some of the analytical methods that are useful for identifying regions of hypo- or hyper-methylation, and ways to identify differentially methylated regions.

ENCODE tiling array analysis identifies differentially expressed annotated and novel 5' capped RNAs in hepatitis C infected liver.

Microarray studies of chronic hepatitis C infection have provided valuable information regarding the host response to viral infection. However, recent studies of the human transcriptome indicate pervasive transcription in previously unannotated regions of the genome and that many RNA transcripts have short or lack 3' poly(A) ends. We hypothesized that using ENCODE tiling arrays (1% of the genome) in combination with affinity purifying Pol II RNAs by their unique 5' m7GpppN cap would identify previously undescribed annotated and unannotated genes that are differentially expressed in liver during hepatitis C virus (HCV) infection. Both 5'-capped and poly(A)+ populations of RNA were analyzed using ENCODE tiling arrays. Sixty-four annotated genes were significantly increased in HCV cirrhotic as compared to control liver; twenty-seven (42%) of these genes were identified only by analyzing 5' capped RNA. Thirty-one annotated genes were significantly decreased; sixteen (50%) of these were identified only by analyzing 5' capped RNA. Bioinformatic analysis showed that capped RNA produced more consistent results, provided a more extensive expression profile of intronic regions and identified upregulated Pol II transcriptionally active regions in unannotated areas of the genome in HCV cirrhotic liver. Two of these regions were verified by PCR and RACE analysis. qPCR analysis of liver biopsy specimens demonstrated that these unannotated transcripts, as well as IRF1, TRIM22 and MET, were also upregulated in hepatitis C with mild inflammation and no fibrosis. The analysis of 5' capped RNA in combination with ENCODE tiling arrays provides additional gene expression information and identifies novel upregulated Pol II transcripts not previously described in HCV infected liver. This approach, particularly when combined with new RNA sequencing technologies, should also be useful in further defining Pol II transcripts differentially regulated in specific disease states and in studying RNAs regulated by changes in pre-mRNA splicing or 3' polyadenylation status.

Empirical methods for controlling false positives and estimating confidence in ChIP-Seq peaks.

BACKGROUND: High throughput signature sequencing holds many promises, one of which is the ready identification of in vivo transcription factor binding sites, histone modifications, changes in chromatin structure and patterns of DNA methylation across entire genomes. In these experiments, chromatin immunoprecipitation is used to enrich for particular DNA sequences of interest and signature sequencing is used to map the regions to the genome (ChIP-Seq). Elucidation of these sites of DNA-protein binding/modification are proving instrumental in reconstructing networks of gene regulation and chromatin remodelling that direct development, response to cellular perturbation, and neoplastic transformation. RESULTS: Here we present a package of algorithms and software that makes use of control input data to reduce false positives and estimate confidence in ChIP-Seq peaks. Several different methods were compared using two simulated spike-in datasets. Use of control input data and a normalized difference score were found to more than double the recovery of ChIP-Seq peaks at a 5% false discovery rate (FDR). Moreover, both a binomial p-value/q-value and an empirical FDR were found to predict the true FDR within 2-3 fold and are more reliable estimators of confidence than a global Poisson p-value. These methods were then used to reanalyze Johnson et al.'s neuron-restrictive silencer factor (NRSF) ChIP-Seq data without relying on extensive qPCR validated NRSF sites and the presence of NRSF binding motifs for setting thresholds. CONCLUSION: The methods developed and tested here show considerable promise for reducing false positives and estimating confidence in ChIP-Seq data without any prior knowledge of the chIP target. They are part of a larger open source package freely available from http://useq.sourceforge.net/.

RNA maps reveal new RNA classes and a possible function for pervasive transcription.

Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.

Targeted disruption of the murine zyxin gene.

Zyxin is an evolutionarily conserved protein that is concentrated at sites of cell adhesion, where it associates with members of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family of cytoskeletal regulators and is postulated to play a role in cytoskeletal dynamics and signaling. Zyxin transcripts are detected throughout murine embryonic development, and the protein is widely expressed in adults. Here we used a reverse genetic approach to examine the consequences of loss of zyxin function in the mouse. Mice that lack zyxin function are viable and fertile and display no obvious histological abnormalities in any of the organs examined. Because zyxin contributes to the localization of Ena/VASP family members at certain subcellular locations, we carefully examined the zyxin(-/-) mice for evidence of defects that have been observed when Ena/VASP proteins are compromised in the mouse. Specifically, we evaluated blood platelet function, nervous system development, and skin architecture but did not detect any defects in these systems. Zyxin is the founding member of a family of proteins that also includes the lipoma preferred partner (LPP) and thyroid receptor-interacting protein 6 (TRIP6). These zyxin family members display patterns of expression that significantly overlap that of zyxin. Western blot analysis indicates that there is no detectable upregulation of either LPP or TRIP6 expression in tissues derived from zyxin-null mice. Because zyxin family members may have overlapping functions, a comprehensive understanding of the role of these proteins in the mouse will require the generation of compound mutations in which multiple zyxin family members are simultaneously compromised.