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Modeling of genomic profiles from the Cancer Genome Atlas (TCGA) by using recently developed mathematical frameworks has associated a genome-wide pattern of DNA copy-number alterations with a shorter, roughly one-year, median survival time in glioblastoma (GBM) patients. Here, to experimentally test this relationship, we whole-genome sequenced DNA from tumor samples of patients. We show that the patients represent the U.S. adult GBM population in terms of most normal and disease phenotypes. Intratumor heterogeneity affects ≈ 11 % and profiling technology and reference human genome specifics affect 30%. With a 2.25-year Kaplan–Meier median survival difference, a 3.5 univariate Cox hazard ratio, and a 0.78 concordance index, i.e., accuracy, the pattern predicts survival better than and independent of age at diagnosis, which has been the best indicator since 1950. The prognostic classification by the pattern may, therefore, help to manage GBM pseudoprogression. The diagnostic classification may help drugs progress to regulatory approval. The therapeutic predictions, of previously unrecognized targets that are correlated with survival, may lead to new drugs. Other methods missed this relationship in the roughly 3B-nucleotide genomes of the small, order of magnitude of 100, patient cohorts, e.g., from TCGA. Previous attempts to associate GBM genotypes with patient phenotypes were unsuccessful. This is a proof of principle that the frameworks are uniquely suitable for discovering clinically actionable genotype–phenotype relationships.

Arteriovenous hemodialysis graft (AVG) stenosis results in thrombosis and AVG failure, but prevention of stenosis has been unsuccessful due in large part to our limited understanding of the molecular processes involved in neointimal hyperplasia (NH) formation. AVG stenosis develops chiefly as a consequence of highly localized NH formation in the vein-graft anastomosis region. Surprisingly, the vein region just downstream of the vein-graft anastomosis (herein termed proximal vein region) is relatively resistant to NH. We hypothesized that the gene expression profiles of the NH-prone and NH-resistant regions will be different from each other after graft placement, and analysis of their genomic profiles may yield potential therapeutic targets to prevent AVG stenosis. To test this, we evaluated the vein-graft anastomosis (NH-prone) and proximal vein (NH-resistant) regions in a porcine model of AVG stenosis with a porcine microarray. Gene expression changes in these two distinct vein regions, relative to the gene expression in unoperated control veins, were examined at early (5 days) and later (14 days) time points following graft placement. Global genomic changes were much greater in the NH-prone region than in the NH-resistant region at both time points. In the NH-prone region, genes related to regulation of cell proliferation and osteo-/chondrogenic vascular remodeling were most enriched among the significantly upregulated genes, and genes related to smooth muscle phenotype were significantly downregulated. These results provide insights into the spatial and temporal genomic modulation underlying NH formation in AVG and suggest potential therapeutic strategies to prevent and/or limit AVG stenosis.

Background: Preclinical studies indicate that disruption of the transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) leads to pathological oxidative stress in several tissues. However, the transcriptional role of Nrf2 in the heart, a highly aerobic organ sensitive to redox disturbances and aberrant microRNA (miRNA) activity, remains elusive. Here, we tested the hypothesis that Nrf2 ablation disrupts cardiac miRNA abundance leading to impaired transcriptional regulation of the myocardial defense system. Methods: Age-matched wild-type (WT) and Nrf2 knockout (Nrf2 -/- ) mice (n=3-6/group) were used in this study. Independent next-generation sequencing experiments were conducted to evaluate differential expression of mRNA and miRNA in the heart. Real-time qPCR was used to validate RNA sequencing (RNAseq) data. Results: RNAseq for mRNA uncovered 152 differentially expressed genes (DEGs) in Nrf2 -/- hearts, of which 129 were downregulated relative to WT. Novel DEGs previously unreported as Nrf2 targets were detected and Gene Ontology analysis revealed the enrichment (p-/- DEG signatures. Next, small RNAseq detected a total of 27 miRNAs (11 up and 16 downregulated) altered in Nrf2 -/- mice. Validation by qPCR revealed a significant (p-/- hearts as 22 DEGs matched with 2 or more miRNAs. Conclusion: Our results highlight novel mRNA targets and indicate that Nrf2 regulates a subset of cardiac miRNAs. Biological changes distinct from canonical Nrf2 function suggest that regulatory cross-talk with miRNAs contribute to unique roles for Nrf2 in the myocardium.

Background Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) senses oxidative environments and/or stress and initiates a cytoprotective response through transcriptional activation of antioxidant and detoxification genes. Several preclinical studies suggest that Nrf2 combats oxidative stress underlying a variety of pathologies. Despite Nrf2 deficits linked to functional abnormalities in many organ systems, the transcriptional network resulting from Nrf2 deficiency in the heart has remained elusive. Moreover, cross-talk between microRNAs (miRNAs) and cardiac Nrf2 signaling is unknown. Here, we utilized next generation RNA sequencing (RNAseq) to unbiasedly profile basal mRNA and miRNA expression in Nrf2 knockout (Nrf2−/−) hearts. Results RNAseq of mRNA revealed 152 differentially expressed genes (DEGs) in the Nrf2−/−myocardium, of which 129 were downregulated. Grouping of DEGs based on biological function and real-time qPCR validation indicated that DEGs were enriched for; mitochondrial genome and bioenergetics, oxidoreductase capacity, cardiac development, and chaperone activity. Interestingly, RNAseq analysis uncovered 27 significantly altered miRNAs, of which 11 were upregulated and 16 were downregulated in Nrf2−/− hearts. Expression changes were validated for 12 miRNAs using specific primer assays in real-time and revealed a significant decrease in miR-10b-5p, miR-674-3p, miR-3535, and miR-378c while miR-30b-5p, miR-208a-5p, miR-350-3p, and miR-582-5p, and miR-1249-3p levels were increased. High throughput data were integrated using prediction algorithms, and these in silico analyses discovered potential recognition elements within 39 repressed mRNAs which matched the seed sequence for 4 upregulated miRNAs; miR-30b-5p, miR-208a-5p, miR-350-3p, and miR-582-5p. Conclusion These high-throughput data reveal transcriptome-wide effects of myocardial Nrf2 deficiency. Further, our results suggest that Nrf2 may directly or indirectly regulate a sub-set of cardiac miRNAs in the basal setting. This comprehensive analysis is the first evidence to demonstrate a plausible regulatory cross-talk among cardiac miRNAs and the Nrf2 transcriptional network, and provides valuable candidates to examine in future mechanistic and preclinical studies.

In yeast, Adh1 (alcohol dehydrogenase 1) is an abundant zinc-binding protein that is required for the conversion of acetaldehyde to ethanol. Through transcriptome profiling of the Schizosaccharomyces pombe genome, we identified a natural antisense transcript at the adh1 locus that is induced in response to zinc limitation. This antisense transcript (adh1AS) shows a reciprocal expression pattern to that of the adh1 mRNA partner. In this study, we show that increased expression of the adh1AS transcript in zinc-limited cells is necessary for the repression of adh1 gene expression and that the increased level of the adh1AS transcript in zinc-limited cells is a result of two mechanisms. At the transcriptional level, the adh1AS transcript is expressed at a high level in zinc-limited cells. In addition to this transcriptional control, adh1AS transcripts preferentially accumulate in zinc-limited cells when the adh1AS transcript is expressed from a constitutive promoter. This secondary mechanism requires the simultaneous expression of adh1. Our studies reveal how multiple mechanisms can synergistically control the ratio of sense to antisense transcripts and highlight a novel mechanism by which adh1 gene expression can be controlled by cellular zinc availability.

Human breast cancer is a heterogeneous disease composed of different histologies and molecular subtypes, many of which are not replicated in animal models. Here, we report a mouse model of breast cancer that generates unique tumor histologies including tubular, adenosquamous, and lipid-rich carcinomas. Utilizing a nononcogenic variant of polyoma middle T oncogene (PyMT) that requires a spontaneous base-pair deletion to transform cells, in conjunction with lentiviral transduction and orthotopic transplantation of primary mammary epithelial cells, this model sporadically induces oncogene expression in both the luminal and myoepithelial cell lineages of the normal mouse mammary epithelium. Microarray and hierarchical analyses using an intrinsic subtype gene set revealed that lentiviral PyMT generates both luminal and basal-like tumors. Cumulatively, these results show that low-level expression of PyMT in a broad range of cell types significantly increases tumor heterogeneity and establishes a mouse model of several rare human breast cancer subtypes.

The venom peptides (i.e., conotoxins or conopeptides) that species in the genus Conus collectively produce are remarkably diverse, estimated to be around 50,000 to 140,000, but the pace of discovery and characterization of these peptides have been rather slow. To date, only a minor fraction have been identified and studied. However, the advent of next-generation DNA sequencing technologies has opened up opportunities for expediting the exploration of this diversity. The whole transcriptome of a venom duct from the vermivorous marine snail C. pulicarius was sequenced using the 454 sequencing platform. Analysis of the data set resulted in the identification of over eighty unique putative conopeptide sequences, the highest number discovered so far from a Conus venom duct transcriptome. More importantly, majority of the sequences were potentially novel, many with unexpected structural features, hinting at the vastness of the diversity of Conus venom peptides that remains to be explored. The sequences represented at least 14 major superfamilies/types (disulfide- and non-disulfide-rich), indicating the structural and functional diversity of conotoxins in the venom of C. pulicarius . In addition, the contryphans were surprisingly more diverse than what is currently known. Comparative analysis of the O-superfamily sequences also revealed insights into the complexity of the processes that drive the evolution and diversification of conotoxins.

The detection of chromosomal translocations has important implications in the diagnosis, prognosis and treatment of patients with cancer. Current approaches to translocation detection have significant shortcomings, including limited sensitivity and/or specificity, and difficulty in application to formalin-fixed paraffin-embedded (FFPE) clinical samples. We developed a new approach called antibody detection of translocations (ADOT) that avoids the shortcomings of current techniques. ADOT combines a transcriptional microarray-based approach with a novel antibody-based detection method. ADOT allows for the accurate and sensitive identification of translocations and provides exon-level information about the fusion transcript. ADOT can detect translocations in poor-quality unprocessed total ribonucleic acid (RNA). Furthermore, the technique is readily generalizable to detect any potential fusion transcript, including previously undescribed fusions. We demonstrate the feasibility of ADOT by examples in which both known and unknown Ewing sarcoma translocations are identified from cell lines, tumour xenografts and FFPE primary tumours. These results demonstrate that ADOT may be an effective approach for translocation analysis in clinical specimens with significant RNA degradation and may offer a novel diagnostic tool for translocation-based cancers.

Large case-control studies using genome-wide association markers have been published identifying genes related to obesity (1). At least 17 genetic regions have been suggested by such studies. The best replicated of these genes are the FTO and MC4R genes (2–8). There is a long history of candidate gene studies relating common genetic variants to various measures of obesity (9). The gene with the most frequent prevalence of multiple mutations or polymorphisms related to obesity is the MC4R gene (10). Because obesity is an appetite-related condition, the genes related to appetite control have been studied in various populations, particularly the NPY and NPY receptor genes. Generally, there has been poor replication of variants in NPY, PYY, NPY1R and NPY5R being associated with obesity, while more positive studies have implicated NPY2R, a well-known candidate gene for obesity development and control of food intake (11–20). However, even the NPY2R studies have not been compelling because of their marginal significance levels and findings that occur most often in men rather than women (12–14). In addition, most of these studies have genotyped SNPs in the 5’ region of the NPY2R gene or one of the three synonymous change variants in exon 2 of the gene. The NPY2R gene has two exons, but the start codons for transcription occur at exon 2, not exon 1. Intron 1 is a region with low linkage disequilibrium (LD) that is not well tagged by the 5’ SNPs, which are located in one LD block, or the downstream end of intron 1 and exon 2 SNPs, which are in a second LD block. Therefore, we typed multiple markers in intron 1, along with some previously studied 5’ and exon 2 SNPs. The independence of the NPY2R association with BMI was assessed by also typing one or more SNPs in the NPY, NPY1R, NPFFR2 (a G-protein receptor that binds neuropeptides), PPARGC1A (a PPAR-γ coactivator of gene transcription including UCP1), OR7D4 (an olfactory receptor gene), and MC4R genes (14, 21–24). Four SNPs were genotyped in the NPFFR2 gene that formed a haplotype previously shown to be associated with leanness (25). The SNP rs9939609 in the FTO gene was previously genotyped in these subjects and was significantly associated with BMI (5). In addition to testing the independence of each of the above genes on BMI, the ability of haplotypes to improve the associations with BMI was assessed.

MicroRNAs are thought to have an impact on cell proliferation, apoptosis, stress responses, maintenance of stem cell potency, and metabolism and are, therefore, important in the carcinogenic process. In this study, we examined 40 colon tumors, 30 rectal tumors, and 30 normal tissue samples (10 proximal colon, 10 distal colon, and 10 rectal paired with cancer cases) to examine miRNA expression profiles in colon and rectal tumors. MiRNA expression levels were adjusted for multiple comparisons; tumor tissue was compared with noncancerous tissue from the same site. A comparison of normal tissue showed 287 unique miRNAs that were significantly differentially expressed at the 1.5-fold level and 73 with over a two-fold difference in expression between colon and rectal tissue. Examination of miRNAs that were significantly differentially expressed at the 1.5-fold level by tumor phenotype showed 143 unique miRNAs differentially expression for microsatellite instability positive (MSI+) colon tumors; 129 unique miRNAs differentially expressed for CpG Island Methylator Phenotype positive (CIMP+) colon tumors; 135 miRNAs were differentially expressed for KRAS2-mutated colon tumors, and 139 miRNAs were differentially expressed for TP53-mutated colon tumors. Similar numbers of differentially expressed miRNAs were observed for rectal tumors, although the miRNAs differentially expressed differed. There were 129 unique miRNAs for CIMP+, 143 unique miRNAs for KRAS2-mutated, and 136 unique miRNAs for TP53-mutated rectal tumors. These results suggest the importance of miRNAs in colorectal cancer and the need for studies that can confirm these results and provide insight into the diet, lifestyle, and genetic factors that influence miRNA expression.

The deleterious effects of hydrocephalus, a disorder that primarily affects children, include reactive astrocytosis, microgliosis and inflammatory responses; however, the roles that these mechanisms play in the pathophysiology of hydrocephalus are still not clear in terms of cytopathology and gene expression. Therefore we have examined neuroinflammation at both the cellular and the molecular levels in an experimental model of neonatal obstructive hydrocephalus. On post-natal day 1, rats received an intracisternal injection of kaolin to induce hydrocephalus; control animals received saline injections. Prior to sacrifice on post-natal day 22, animals underwent magnetic resonance imaging to quantify ventricular enlargement, and the parietal cortex was harvested for analysis. Immunohistochemistry and light microscopy were performed on 5 hydrocephalic and 5 control animals; another set of 5 hydrocephalic and 5 control animals underwent molecular testing with Western blots and a gene microarray. Scoring of immunoreactivity on a 4-point ranking scale for GFAP and Iba-1 demonstrated an increase in reactive astrocytes and reactive microglia respectively in the hydrocephalic animals compared to controls (2.90±0.11 vs. 0.28±0.26; 2.91±0.11 vs. 0.58±0.23, respectively). Western blots confirmed these results. Microarray analysis identified significant (1.5-fold) changes in 1729 of 33,951 genes, including 26 genes out of 185 genes (26/185) in the cytokine-cytokine receptor interaction pathway, antigen processing and presentation pathways (15/66), and the apoptosis pathway (10/69). Collectively, these results demonstrate alterations in normal physiology and an up-regulation of the inflammatory response. These findings lead to a better understanding of neonatal hydrocephalus and begin to form a baseline for future treatments that may reverse these effects.

We have determined the high-resolution strand-specific transcriptome of the fission yeast S. pombe under multiple growth conditions using a novel RNA-DNA hybridization mapping (HybMap) technique. HybMap uses an antibody against an RNA-DNA hybrid to detect RNA molecules hybridized to a high-density DNA oligonucleotide tiling microarray. HybMap showed exceptional dynamic range and reproducibility, and allowed us to identify strand-specific coding, noncoding and structural RNAs, as well as previously unknown RNAs conserved in distant yeast species. Notably, we found that virtually the entire euchromatic genome (including intergenics) is transcribed, with heterochromatin dampening intergenic transcription. We identified features including large numbers of condition-specific noncoding RNAs, extensive antisense transcription, new properties of antisense transcripts and induced divergent transcription. Furthermore, our HybMap data informed the efficiency and locations of RNA splicing genome-wide. Finally, we observed strand-specific transcription islands around tRNAs at heterochromatin boundaries inside centromeres. Here, we discuss these new features in terms of organism fitness and transcriptome evolution.

Few studies have explored the mechanistic basis for the apparent paradoxical effects of nitric oxide and its interrelated redox species (NO(X)) in cancer biology. Our aim was to determine the differential effects of the redox state and kinetics of nitrosative species on the key cancer processes of apoptosis. Therefore, a murine lung adenocarcinoma cell line was exposed to various NO(X) donor compounds differing in redox state and delivery kinetics. DNA strand breaks (DSBs) were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay) and correlated with cell viability by the MTT and soft agar colony assays, while caspase enzymatic activity was measured using an in vitro fluorogenic caspase assay. Finally, cDNA microarrays defined apoptosis-related gene expression alterations resultant from these NO(X) donors. Exogenous NO(X) differentially influences DSBs, and apoptosis-related cell death and expression based on the redox state and kinetics of NO(X) delivery. In our murine lung adenocarcinoma model we have demonstrated differential effects of NO(X) based on the mode of delivery and redox state. These data suggest that the development of NO(X)-based cancer chemotherapy must consider the redox state and kinetics of delivery into their logical design.

The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC) degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF)/Brown Norwegian (BN) F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm). In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R) to cysteine (C) change at codon 35 of the ATPase, Ca2+ transporting, plasma membrane 3 (Atp2b3) gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT) replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3R35C function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes., Summary: The shaker rat mutant: a new model for essential tremors and ataxia characterized by Purkinje cell degeneration.

Prostate cancer remains the most common male malignancy in Western countries, yet limited information exists regarding genetic changes and clinical correlations. The advent of comparative genomic hybridization microarray (GM) technology has recently allowed for precise screening of DNAs for genetic copy number changes; this offers an advantage over previous techniques, including conventional cytogenetics. A problem with cytogenetic prostate cancer analysis has been the study of the appropriate cell types because this is a highly heterogeneous tumor. We have performed GM using the Spectral Genomics Inc. dye reversal platform on 20 primary prostate tumors. These tumor samples were from frozen tissue collected over the last 10 years and multiple clinical parameters, including follow-up were collected on these patients; cytogenetic analysis was previously attempted on all patients. Eighty percent (16/20) of specimens showed copy number changes, 65% of which were losses and 35% were gains of genetic material. The most common changes observed were loss of an interstitial region of 2q (8 cases, 40%), followed by loss of interstitial 6q (6 cases, 30%), loss at 8p and 13q (5 cases each, 25%), gain at 3p and loss at 5q, 16q, and Xq (4 cases each, 20%), and gain at 8p (3 cases, 15%). There was evidence of correlation of loss at 5q with a positive node status. Cytogenetic studies on these same patients only detected clonal changes in 40% (8/20) specimens and did not detect the majority of abnormalities seen by the GM technique. We propose this technology for the evaluation of prostate and other heterogeneous cancers as a rapid and efficient way to detect genetic copy number changes.

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.

Background: Mammary gland branching morphogenesis is a highly regulated developmental process often disrupted in breast cancer. Results: A chemical genetic screen in primary three-dimensional culture revealed that activation of the aryl hydrocarbon receptor promotes desmosomes to block branching. Conclusion: Down-regulation of desmosomes is required for proper mammary branching morphogenesis. Significance: Desmosomes are a novel mechanism through which exposure to environmental pollutants may affect mammary development., During the process of branching morphogenesis, the mammary gland undergoes distinct phases of remodeling to form an elaborate ductal network that ultimately produces and delivers milk to newborn animals. These developmental events rely on tight regulation of critical cellular pathways, many of which are probably disrupted during initiation and progression of breast cancer. Transgenic mouse and in vitro organoid models previously identified growth factor signaling as a key regulator of mammary branching, but the functional downstream targets of these pathways remain unclear. Here, we used purified primary mammary epithelial cells stimulated with fibroblast growth factor-2 (FGF2) to model mammary branching morphogenesis in vitro. We employed a forward chemical genetic approach to identify modulators of this process and describe a potent compound, 1023, that blocks FGF2-induced branching. In primary mammary epithelial cells, we used lentivirus-mediated knockdown of the aryl hydrocarbon receptor (AHR) to demonstrate that 1023 acts through AHR to block branching. Using 1023 as a tool, we identified desmosomal adhesion as a novel target of AHR signaling and show that desmosomes are critical for AHR agonists to block branching. Our findings support a functional role for desmosomes during mammary morphogenesis and also in blocking FGF-induced invasion.

Background Genetic profiling by RNA microarray of normal and neoplastic colonic tissue is used to identify biomarkers for development of a diagnostic tool for the inherited colon cancer syndromes. The utility of this method is to 1) properly classify patients where genetic diagnosis is incomplete 2) suggest molecular pathways that are shared between the syndromes and 3) identify molecular targets for prevention and treatment.

Despite improvements in early detection and treatment, cancer remains a major cause of mortality. Death from cancer is largely due to metastasis, which results in spreading of tumor cells to other parts of the body. The metastatic process is poorly understood, is often unpredictable, and usually results in incurable disease. There are no therapies specifically designed to target metastases or to block the metastatic process. Development and pre-clinical testing of new cancer therapies is limited by the scarcity of in vivo models that authentically reproduce human tumor growth and metastatic progression. Here, we report development of novel models for breast tumor growth and metastasis, which exist in the form of transplantable tumors derived directly from patients. These tumor grafts not only represent the diversity of human breast cancer, but also maintain essential features of the original patients’ tumors, including histopathology, clinical markers, hormone responsiveness, and metastasis to specific sites. Genomic features, such as gene expression profiles and DNA copy number variants, are also well maintained between the original specimens and the tumor grafts. We found that co-engraftment of primary human mesenchymal stem cells with tumor grafts helps to maintain the phenotypic stability of the tumors, and increases tumor growth by promoting angiogenesis and reducing necrosis. Remarkably, tumor engraftment is also a prognostic indicator of disease outcome: newly diagnosed women whose primary breast tumor successfully engrafted in mouse mammary glands had significantly reduced survival compared to patients whose tumors did not engraft. Thus, orthotopic breast tumor grafting marks a first step toward personalized medicine by replicating the diversity of human breast cancer through patient-centric models for tumor growth, metastasis, drug efficacy, and prognosis.

Protein aggregation cardiomyopathy is a life-threatening manifestation of a multisystem disorder caused by the exchange mutation in the gene encoding the human small heat shock protein αB-crystallin (hR120GCryAB). Genetic studies in mice have established cardiac hR120GCryAB expression causes increased activity of glucose 6-phosphate dehydrogenase (G6PD) and “reductive stress” (Rajasekaran et al., Cell 130: 427–439, 2007). However, the initiating molecular events in the pathogenesis of this novel toxic gain-of-function mechanism remain poorly defined. In an integrated systems approach using gene expression profiling, we identified a “biosignature,” whose features can be validated to predict the onset, rate of progression, and clinical outcome of R120GCryAB cardiomyopathy. At the 3 mo disease-related but compensated stage, we demonstrate that transcripts were only upregulated in three distinct pathways: stress response (e.g., Hsp70, Hsp90), glutathione metabolism (Gpx1, Gpx3, glutathione S-transferase), and complement and coagulation cascades in hR120GCryAB transgenic mouse hearts compared with either hCryAB WT transgenic mice or nontransgenic controls. In 6 mo old myopathic hearts, ribosomal synthesis and cellular remodeling associated with increased cardiac hypertrophy were additional upregulated pathways. In contrast, the predominant downregulated pathways were for oxidative phosphorylation, fatty acid metabolism, intermediate metabolism, and energetic balance, supporting their primary pathogenic roles by which G6PD-dependent reductive stress causes cardiac decompensation and overt heart failure in hR120GCryAB cardiomyopathy. This study extends and confirms our previous findings that reductive stress is a causal mechanism for hR120G CryAB cardiomyopathy and demonstrates that alteration in glutathione pathway gene expression is an early biosignature with utility for presymptomatic detection.

Transcription factors with identical DNA‐binding specificity often activate different genes in vivo . Yeast Ace2 and Swi5 are such activators, with targets we classify as Swi5‐only, Ace2‐only, or both. We define two unique regulatory modes. Ace2 and Swi5 both bind in vitro to Swi5‐only genes such as HO , but only Swi5 binds and activates in vivo . In contrast, Ace2 and Swi5 both bind in vivo to Ace2‐only genes, such as CTS1 , but promoter‐bound Swi5 fails to activate. We show that activation by Swi5 is prevented by the binding of the Forkhead factors Fkh1 and Fkh2, which recruit the Rpd3(Large) histone deacetylase complex to the CTS1 promoter. Global analysis shows that all Ace2‐only genes are bound by both Ace2 and Swi5, and also by Fkh1/2. Genes normally activated by either Ace2 or Swi5 can be converted to Ace2‐only genes by the insertion of Fkh‐binding sites. Thus Fkh proteins, which function initially to activate SWI5 and ACE2 , subsequently function as Swi5‐specific antiactivators.

Purpose. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, has been found to radiosensitize various human cancer cells. However, its potential to act as an effective therapeutic agent is diminished by its toxicity levels. The purposes of this study were to determine the mechanism by which LY294002 radiosensitizes. Materials and methods. Cell growth curves and clonogenic assays were performed with increasing LY294002 exposure times proximate to the radiation dose. Protein levels of downstream PI3K effectors were analyzed. Detection of phosphorylated histone H2AX (γH2AX) was used to identify DNA double-strand breaks at various time points post-radiation. Results. LY294002 significantly radiosensitized HeLa cervical cancer cells when administered for just 12 h following radiation. Cell growth curves also decreased with brief LY294002 application. DNA double-strand breaks are typically repaired within 2–6 h following radiation. Interestingly, at 48, 72, and 96 h post-irradiation, γH2AX was still significantly elevated in cells radiated in combination with LY294002. Protein expressions of ATM and ATR downstream effectors showed no differences among the treated groups, however, DNA-PK activity was significantly inhibited by LY294002. Conclusions. These results lead us to conclude that the central mechanism by which LY294002 radiosensitizes is via DNA-PK inhibition which induces DNA double-strand break repair inhibition. We are currently investigating radiosensitization induced by DNA-PK-specific inhibition in efforts to find a less toxic, yet equally effective, chemotherapeutic agent than LY294002.

BACKGROUND The early detection and characterization of metastatic melanoma are important for prognosis and management of the disease. Molecular methods are more sensitive in detecting occult lymph node metastases compared with standard histopathology and are reported to have utility in clinical diagnostics. METHODS Using real-time quantitative reverse transcriptase-polymerase chain reaction ([q]RT-PCR), the authors examined 36 samples (30 melanomas, 4 benign nevi, and 2 reactive lymph nodes) for the expression of 20 melanoma-related genes that function in cell growth and differentiation (epidermal growth factor receptor [EGFR], WNT5A, BRAF, FOS, JUN, MATP, and TMP1), cell proliferation (KI-67, TOP2A, BUB1, BIRC5, and STK6), melanoma progression (CD63, MAGEA3, and GALGT), and melanin synthesis (TYR, MLANA, SILV, PAX3, and MITF). In addition, samples were tested for mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3). RESULTS Hierarchical clustering analysis of the expression data was able to distinguish between the melanoma and nonmelanoma samples and further stratified the melanoma samples into two groups differentiated by high expression of the genes involved in β-catenin activation (EGFR and WNT5A) and the MAPK/ERK pathway (BRAF, FOS, and JUN). Eighteen of the 28 patients (64%) were found to have mutations in either exon 15 of BRAF (V599 substitution) or codon 61 of NRAS. The mutations were mutually exclusive and did not appear to be associated with the different expression subtypes. CONCLUSIONS The results of the current study demonstrate that real-time qRT-PCR can be analyzed using hierarchical clustering to identify expression patterns that differentiate between melanomas and other tissue types. Using a supervised analysis of the data, the authors found that the best discriminators for molecularly distinguishing between melanoma, benign nevi, and lymph nodes were MLANA, CD63, and BUB1. These markers could have diagnostic utility for the detection of melanoma micrometastasis in sentinel lymph nodes. Cancer 2005. © 2005 American Cancer Society.

Objective: The detection and identification of chromosomal translocations has important implications in the diagnosis, prognosis, and treatment of patients with cancer. Current approaches to translocation detection often have significant shortcomings, including limited sensitivity and/or specificity, and can be difficult to apply to formalin-fixed paraffin-embedded (FFPE) clinical samples. We developed a new approach, antibody detection of translocations (ADOT), that avoids many of the shortcomings of current techniques. Methods: ADOT combines a transcriptional microarray-based approach with a novel antibody-based detection method. Microarrays were designed that probe for every possible fusion transcript in Ewing's sarcoma, both previously identified and theoretical. These include EWS/FLI, EWS/ERG, EWS/ETV1, EWS/ETV4, EWS/FEV and others. Total RNA, purified from Ewing's sarcoma cells or tumors (that express different types of translocations), is hybridized to the custom microarrays. A high-affinity antibody (S9.6) that binds to RNA-DNA duplexes formed during hybridization is used as a detection reagent. Results: We demonstrate the feasibility of ADOT by examples in which both known, and unknown, translocation fusion transcripts are identified from cell lines, tumor xenografts, and primary tumor samples. Translocation fusion points identified by ADOT were confirmed using traditional RT-PCR and sequencing. Furthermore, ADOT is able to detect translocations in FFPE tumor samples in which case RT-PCR fails. The sensitivity of ADOT to detection of translocations is also determined. Conclusion: ADOT allows for the accurate and sensitive identification of translocations and also provides exon-level information about the specific fusion transcript produced. Furthermore, the technique is readily generalizable to any potential fusion transcript, and can also detect previously undescribed fusions. This suggests that ADOT may be used as a diagnostic tool for pediatric sarcoma or other translocation-based cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4864. doi:10.1158/1538-7445.AM2011-4864

Background Familial adenomatous polyposis (FAP) is a colon cancer syndrome with a prevalence of 1:10,000. Patients have 100's to 1000's of precancerous colonic polyps and nearly 100% risk of developing colon cancer at an average age of 39 years in the absence of colon surveillance and surgery. Mutations in the APC gene lead to FAP as well as an attenuated form (AFAP) which presents with variable phenotypic expression, reduced polyp numbers and reduced cancer risk as compared to FAP. Current methods for the clinical diagnosis of genetic diseases most commonly involve analysis of germline DNA. Germline DNA-based diagnosis can be incomplete, for example no mutation is found in approximately 20% of FAP and 50% of AFAP patients. The objective of this study is to determine a molecular profile of the colonic epithelia from patients with APC mutations leading to FAP or AFAP then to use this information to establish a gene expression signature for diagnosis and for better understanding of the disease in the primary affected tissue.

Purpose To evaluate gene expression patterns in patients with advanced cervix cancer before and during chemoradiation in a multi-institutional cooperative group setting. Methods RTOG C0128 was designed as a Phase II trial of radiation therapy with concomitant chemotherapy and Celecoxib at 400 mg twice daily for one year. Tumor samples were obtained for microarray gene expression analysis before treatment and at the time of the first implant (paired sample). RNA was extracted, linearly amplified, and purity was assessed by gel electrophoresis. Each sample was hybridized against a universal RNA mixture on a customized spotted array consisting of >10,000 genes. Gene expression pre-treatment was compared with clinical characteristics. Changes in gene expression following radiation were assessed within the paired samples (same patient) and then compared across all paired samples. Data were normalized using the AROMA software, and clustering analysis was performed using Ward's method in Spotfire. Differences in paired samples were calculated with Significance Analysis of Microarrays (SAM). Results From August 2001 to March 2004, 84 patients were accrued to the trial. Tissue was obtained prior to initiation of therapy from 34 patients (40%). FIGO stages of the patients providing tissue were IB (23%), II (57%), and IIIA–IVA (20%). RNA quality was sufficient in 22 pre-treatment and 14 post-treatment samples. Among pre-treatment samples, no significant differences in gene expression were observed by FIGO stage, age, or race. However, between comparison of histologic subtypes (adenocarcinoma, n =5; squamous cell carcinoma, n =17) demonstrated 45 genes differentially expressed with a false discovery rate of 0.018. Cluster analysis segregated unpaired samples into 2 groups: 18/22 comprising pre-treatment samples and 10/14 in group 2 representing post-treatment samples. In all 13 paired samples, gene expression after chemoradiation was significantly upregulated in 91 genes and downregulated in 251 genes (false discovery rate of 0.0018). Genes significantly upregulated included bax, cdk inhibitor 1, MMP2, and adhesion molecules PECAM1, VCAM1, and ICAM2. Genes significantly downregulated included topoisomerase II alpha, myc, H2AX, MSH2, RAD51, RAD53, PCNA, and cell cycle-regulating molecules chk1, CDK2, cyclinB1, cyclin D3, cdc2, and cdc25. Conclusions Microarray analysis was successfully performed in a multi-institutional cooperative group trial. Gene expression significantly correlated with histology, but not stage, age or race. Cluster analysis identified two groups of gene expression profiles correlating with pre or post-treatment acquisition of tissue. Notably, paired samples showed significant changes in gene expression following chemoradiation, including several downregulated radiation response genes. Further analysis comparing gene expression to clinical outcomes, acute and late toxicities awaits maturation of clinical data. Hopefully, this data will lead to the development of molecularly based therapies.

Newts have the remarkable ability to regenerate lost appendages including their forelimbs, hindlimbs, and tails. Following amputation of an appendage, the wound is rapidly closed by the migration of epithelial cells from the proximal epidermis. Internal cells just proximal to the amputation plane begin to dedifferentiate to form a pool of proliferating progenitor cells known as the regeneration blastema. We show that dedifferentiation of internal appendage cells can be initiated in the absence of amputation by applying an electric field sufficient to induce cellular electroporation, but not necrosis or apoptosis. The time course for dedifferentiation following electroporation is similar to that observed following amputation with evidence of dedifferentiation beginning at about 5 days postelectroporation and continuing for 2 to 3 weeks. Microarray analyses, real-time RT-PCR, and in situ hybridization show that changes in early gene expression are similar following amputation or electroporation. We conclude that the application of an electric field sufficient to induce transient electroporation of cell membranes induces a dedifferentiation response that is virtually indistinguishable from the response that occurs following amputation of newt appendages. This discovery allows dedifferentiation to be studied in the absence of wound healing and may aid in identifying genes required for cellular plasticity.

Lens regeneration in adult newts is possible by transdifferentiation of the pigment epithelial cells (PECs) of the dorsal iris. The same cells in the ventral iris are not capable of such a process. To understand this difference in regenerative competency, we examined gene expression of 373 genes in the intact dorsal and ventral irises as well as in irises during the process of lens regeneration. We found similar signatures of gene expression in dorsal and ventral with several cases of even higher levels in the ventral iris. Such transcriptional activity in the regeneration-incompetent ventral iris was unexpected and calls for a revision of our views about mechanisms of lens regeneration induction.

Background Despite the fundamental biological importance and clinical relevance of characterizing the effects of chronic hypoxia exposure on central nervous system (CNS) development, the changes in gene expression from hypoxia are unknown. It is not known if there are unifying principles, properties, or logic in the response of the developing CNS to hypoxic exposure. Here, we use the small vertebrate zebrafish (Danio rerio) to study the effects of hypoxia on connectivity gene expression across development. We perform transcriptional profiling at high temporal resolution to systematically determine and then experimentally validate the response of CNS connectivity genes to hypoxia exposure. Results We characterized mRNA changes during development, comparing the effects of chronic hypoxia exposure at different time-points. We focused on changes in expression levels of a subset of 1270 genes selected for their roles in development of CNS connectivity, including axon pathfinding and synapse formation. We found that the majority of CNS connectivity genes were unaffected by hypoxia. However, for a small subset of genes hypoxia significantly affected their gene expression profiles. In particular, hypoxia appeared to affect both the timing and levels of expression, including altering expression of interacting gene pairs in a fashion that would potentially disrupt normal function. Conclusions Overall, our study identifies the response of CNS connectivity genes to hypoxia exposure during development. While for most genes hypoxia did not significantly affect expression, for a subset of genes hypoxia changed both levels and timing of expression. Importantly, we identified that some genes with interacting proteins, for example receptor/ligand pairs, had dissimilar responses to hypoxia that would be expected to interfere with their function. The observed dysynchrony of gene expression could impair the development of normal CNS connectivity maps. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2638-x) contains supplementary material, which is available to authorized users.

With the rapidly falling cost and availability of high throughput sequencing and microarray technologies, the bottleneck for effectively using genomic analysis in the laboratory and clinic is shifting to one of effectively managing, analyzing, and sharing genomic data. Here we present three open-source, platform independent, software tools for generating, analyzing, distributing, and visualizing genomic data. These include a next generation sequencing/microarray LIMS and analysis project center (GNomEx); an application for annotating and programmatically distributing genomic data using the community vetted DAS/2 data exchange protocol (GenoPub); and a standalone Java Swing application (GWrap) that makes cutting edge command line analysis tools available to those who prefer graphical user interfaces. Both GNomEx and GenoPub use the rich client Flex/Flash web browser interface to interact with Java classes and a relational database on a remote server. Both employ a public-private user-group security model enabling controlled distribution of patient and unpublished data alongside public resources. As such, they function as genomic data repositories that can be accessed manually or programmatically through DAS/2-enabled client applications such as the Integrated Genome Browser. These tools have gained wide use in our core facilities, research laboratories and clinics and are freely available for non-profit use. See http://sourceforge.net/projects/gnomex/ , http://sourceforge.net/projects/genoviz/ , and http://sourceforge.net/projects/useq .