Genomic Instability and TP53 Genomic Alterations Associate With Poor Antiproliferative Response and Intrinsic Resistance to Aromatase Inhibitor Treatment.

PURPOSE: Although aromatase inhibitor (AI) treatment is effective in estrogen receptor-positive postmenopausal breast cancer, resistance is common and incompletely explained. Genomic instability, as measured by somatic copy number alterations (SCNAs), is important in breast cancer development and prognosis. SCNAs to specific genes may drive intrinsic resistance, or high genomic instability may drive tumor heterogeneity, which allows differential response across tumors and surviving cells to evolve resistance to treatment rapidly. We therefore evaluated the relationship between SCNAs and intrinsic resistance to treatment as measured by a poor antiproliferative response. PATIENTS AND METHODS: SCNAs were determined by single nucleotide polymorphism array in baseline and surgery core-cuts from 73 postmenopausal patients randomly assigned to receive 2 weeks of preoperative AI or no AI in the Perioperative Endocrine Therapy-Individualizing Care (POETIC) trial. Fifty-six samples from the AI group included 28 poor responders (PrRs, less than 60% reduction in protein encoded by the MKI67 gene [Ki-67]) and 28 good responders (GdRs, greater than 75% reduction in Ki-67). Exome sequencing was available for 72 pairs of samples. RESULTS: Genomic instability correlated with Ki-67 expression at both baseline (P < .001) and surgery (P < .001) and was higher in PrRs (P = .048). The SCNA with the largest difference between GdRs and PrRs was loss of heterozygosity observed at 17p (false discovery rate, 0.08), which includes TP53. Nine of 28 PrRs had loss of wild-type TP53 as a result of mutations and loss of heterozygosity compared with three of 28 GdRs. In PrRs, somatic alterations of TP53 were associated with higher genomic instability, higher baseline Ki-67, and greater resistance to AI treatment compared with wild-type TP53. CONCLUSION: We observed that primary tumors with high genomic instability have an intrinsic resistance to AI treatment and do not require additional evolution to develop resistance to estrogen deprivation therapy.

Molecular characterisation of aromatase inhibitor-resistant advanced breast cancer: the phenotypic effect of ESR1 mutations.

BACKGROUND: Several thousand breast cancer patients develop resistance to aromatase inhibitors (AIs) each year in the UK. Rational treatment requires an improved molecular characterisation of resistant disease. MATERIALS AND METHODS: The mutational landscape of 198 regions in 16 key breast cancer genes and RNA expression of 209 genes covering key pathways was evaluated in paired biopsies before AI treatment and at progression on AI from 48 patients. Validity of findings was assessed in another five ESR1-mutated tumours progressing on AI. RESULTS: Eighty-nine mutations were identified in 41 matched pairs (PIK3CA in 27%; CDH1 in 20%). ESR1 (n = 5), ERBB2 (n = 1) and MAP2K4 (n = 1) had mutations in the secondary sample only. There was very high heterogeneity in gene expression between AI-resistant tumours with few patterns apparent. However, in the ESR1-mutated AI-resistant tumours, expression of four classical oestrogen-regulated genes (ERGs) was sevenfold higher than in ESR1 wild-type tumours, a finding confirmed in the second set of ESR1-mutated tumours. In ESR1 wild-type AI-resistant tumours ERG expression remained suppressed and was uncoupled from the recovery seen in proliferation. CONCLUSIONS: Major genotypic and phenotypic heterogeneity exists between AI-resistant disease. ESR1 mutations appear to drive oestrogen-regulated processes in resistant tumours.

A systems immunology approach identifies the collective impact of 5 miRs in Th2 inflammation.

Allergic asthma is a chronic inflammatory disease dominated by a CD4+ T helper 2 (Th2) cell signature. The immune response amplifies in self-enforcing loops, promoting Th2-driven cellular immunity and leaving the host unable to terminate inflammation. Posttranscriptional mechanisms, including microRNAs (miRs), are pivotal in maintaining immune homeostasis. Since an altered expression of various miRs has been associated with T cell-driven diseases, including asthma, we hypothesized that miRs control mechanisms ensuring Th2 stability and maintenance in the lung. We isolated murine CD4+ Th2 cells from allergic inflamed lungs and profiled gene and miR expression. Instead of focusing on the magnitude of miR differential expression, here we addressed the secondary consequences for the set of molecular interactions in the cell, the interactome. We developed the Impact of Differential Expression Across Layers, a network-based algorithm to prioritize disease-relevant miRs based on the central role of their targets in the molecular interactome. This method identified 5 Th2-related miRs (mir27b, mir206, mir106b, mir203, and mir23b) whose antagonization led to a sharp reduction of the Th2 phenotype. Overall, a systems biology tool was developed and validated, highlighting the role of miRs in Th2-driven immune response. This result offers potentially novel approaches for therapeutic interventions.

A novel long non-coding RNA Myolinc regulates myogenesis through TDP-43 and Filip1.

Myogenesis is a complex process required for skeletal muscle formation during embryonic development and for regeneration and growth of myofibers in adults. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) play key roles in regulating cell fate decision and function in various tissues. However, the role of lncRNAs in the regulation of myogenesis remains poorly understood. In this study, we identified a novel muscle-enriched lncRNA called 'Myolinc (AK142388)', which we functionally characterized in the C2C12 myoblast cell line. Myolinc is predominately localized in the nucleus, and its levels increase upon induction of the differentiation. Knockdown of Myolinc impairs the expression of myogenic regulatory factors and formation of multi-nucleated myotubes in cultured myoblasts. Myolinc also regulates the expression of Filip1 in a cis-manner. Similar to Myolinc, knockdown of Filip1 inhibits myogenic differentiation. Furthermore, Myolinc binds to TAR DNA-binding protein 43 (TDP-43), a DNA/RNA-binding protein that regulates the expression of muscle genes (e.g. Acta1 and MyoD). Knockdown of TDP-43 inhibits myogenic differentiation. We also show that Myolinc-TDP-43 interaction is essential for the binding of TDP-43 to the promoter regions of muscle marker genes. Finally, we show that silencing of Myolinc inhibits skeletal muscle regeneration in adult mice. Altogether, our study identifies a novel lncRNA that controls key regulatory networks of myogenesis.

Functionally Conserved Noncoding Regulators of Cardiomyocyte Proliferation and Regeneration in Mouse and Human.

BACKGROUND: The adult mammalian heart has little regenerative capacity after myocardial infarction (MI), whereas neonatal mouse heart regenerates without scarring or dysfunction. However, the underlying pathways are poorly defined. We sought to derive insights into the pathways regulating neonatal development of the mouse heart and cardiac regeneration post-MI. METHODS AND RESULTS: Total RNA-seq of mouse heart through the first 10 days of postnatal life (referred to as P3, P5, P10) revealed a previously unobserved transition in microRNA (miRNA) expression between P3 and P5 associated specifically with altered expression of protein-coding genes on the focal adhesion pathway and cessation of cardiomyocyte cell division. We found profound changes in the coding and noncoding transcriptome after neonatal MI, with evidence of essentially complete healing by P10. Over two-thirds of each of the messenger RNAs, long noncoding RNAs, and miRNAs that were differentially expressed in the post-MI heart were differentially expressed during normal postnatal development, suggesting a common regulatory pathway for normal cardiac development and post-MI cardiac regeneration. We selected exemplars of miRNAs implicated in our data set as regulators of cardiomyocyte proliferation. Several of these showed evidence of a functional influence on mouse cardiomyocyte cell division. In addition, a subset of these miRNAs, miR-144-3p, miR-195a-5p, miR-451a, and miR-6240 showed evidence of functional conservation in human cardiomyocytes. CONCLUSIONS: The sets of messenger RNAs, miRNAs, and long noncoding RNAs that we report here merit further investigation as gatekeepers of cell division in the postnatal heart and as targets for extension of the period of cardiac regeneration beyond the neonatal period.

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.

Impact of mutational profiles on response of primary oestrogen receptor-positive breast cancers to oestrogen deprivation.

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy.

Genetic analysis of the cardiac methylome at single nucleotide resolution in a model of human cardiovascular disease.

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.

Noncoder: a web interface for exon array-based detection of long non-coding RNAs.

Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called 'noncoder'. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de.

Hypoxia-induced alternative splicing in endothelial cells.

BACKGROUND: Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max). CONCLUSIONS/SIGNIFICANCE: For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.

Deeply dissecting stemness: making sense to non-coding RNAs in stem cells.

Adult stem cells are an important source for the regeneration of damaged body parts. Unlike fish and amphibians, the regeneration capacity of human tissues is rather limited. Therefore, one might ask for reasons that led to the loss of regenerative capacity during evolution. Although intensive efforts have been made, we still cannot answer this question definitively. Recent advances in so-called "-omics" (e.g. transcriptomics, proteomics) technologies allowed researchers to obtain detailed views of both mRNA and protein expression levels at different time points during regeneration and tissue repair. It is now possible to make a series of snap shots to characterize stem cell activities at various stages. Recent findings have revealed an enormous plasticity of different cell types reaffirming the landscape model of cell differentiation. Apparently, differentiation of stem cells into a certain lineage is not a fixed process but rather a delicate balance, in which different signaling pathways are involved. To understand this balance, it is utmost importance to profile and catalog changes that occur during the differentiation process of stem cells both at mRNA and protein levels. In this review, we survey the impact of expression profiling on stem cell research with a particular emphasis on non-coding RNAs.

Gene Array Analyzer: alternative usage of gene arrays to study alternative splicing events.

Exon arrays are regularly used to analyze differential splicing events. GeneChip Gene 1.0 ST Arrays (gene arrays) manufactured by Affymetrix, Inc. are primarily used to determine expression levels of transcripts, although their basic design is rather similar to GeneChip Exon 1.0 ST Arrays (exon arrays). Here, we show that the newly developed Gene Array Analyzer (GAA), which evolved from our previously published Exon Array Analyzer (EAA), enables economic and user-friendly analysis of alternative splicing events using gene arrays. To demonstrate the applicability of GAA, we profiled alternative splicing events during embryonic heart development. In addition, we found that numerous developmental splicing events are also activated under pathological conditions. We reason that the usage of GAA considerably expands the analysis of gene expression based on gene arrays and supplies an additional level of information without further costs and with only little effort.

Jumonji domain-containing protein 6 (Jmjd6) is required for angiogenic sprouting and regulates splicing of VEGF-receptor 1.

JmjC domain-containing proteins play a crucial role in the control of gene expression by acting as protein hydroxylases or demethylases, thereby controlling histone methylation or splicing. Here, we demonstrate that silencing of Jumonji domain-containing protein 6 (Jmjd6) impairs angiogenic functions of endothelial cells by changing the gene expression and modulating the splicing of the VEGF-receptor 1 (Flt1). Reduction of Jmjd6 expression altered splicing of Flt1 and increased the levels of the soluble form of Flt1, which binds to VEGF and placental growth factor (PlGF) and thereby inhibits angiogenesis. Saturating VEGF or PlGF or neutralizing antibodies directed against soluble Flt1 rescued the angiogenic defects induced by Jmjd6 silencing. Jmjd6 interacts with the splicing factors U2AF65 that binds to Flt1 mRNA. In conclusion, Jmjd6 regulates the splicing of Flt1, thereby controlling angiogenic sprouting.

Identification of right heart-enriched genes in a murine model of chronic outflow tract obstruction.

The right ventricle (RV) differs in several aspects from the left ventricle (LV) including its embryonic origin, physiological role and anatomical design. In contrast to LV hypertrophy, little is known about the molecular circuits, which are activated upon RV hypertrophy (RVH). We established a highly reproducible model of RVH in mice using pulmonary artery clipping (PAC), which avoids detrimental RV pressure overload and thus allows long-term survival of operated mice. Magnetic resonance imaging revealed pathognomonic changes with striking similarities to human congenital heart disease- or pulmonary arterial hypertension-patients. Comparative, microarray based transcriptome analysis of right- and left-ventricular remodeling identified distinct transcriptional responses to pressure-induced hypertrophy of either ventricle, which were mainly characterized by stronger transcriptional responses of the RV compared to the LV myocardium. Hierarchic cluster analysis revealed a RV- and LV-specific pattern of gene activity after induction of hypertrophy, however, we did not find evidence for qualitatively distinct regulatory pathways in RV compared to LV. Data mining of nearly three thousand RV-enriched genes under PAC disclosed novel potential (co)-regulators of long-term RV remodeling and hypertrophy. We reason that specific inhibitory mechanisms in RV restrict excessive myocardial hypertrophy and thereby contribute to its vulnerability to pressure overload.

C-It: a knowledge database for tissue-enriched genes.

MOTIVATION: Due to the development of high-throughput technologies such as microarrays, it has become possible to determine genome-wide expression changes in a single experiment. Although much attention has been paid to identify differentially expressed genes, the functions of tens of thousands of genes in different species still remain unknown. RESULTS: C-It is a knowledge database that has its focus on 'uncharacterized genes'. C-It contains expression profiles of various tissues from human, mouse, rat, chicken and zebrafish. By applying our previously introduced algorithm DGSA (Database-Dependent Gene Selection and Analysis), it is possible to screen for uncharacterized, tissue-enriched genes in the species mentioned above. C-It is designed to include further expression studies, which might provide more comprehensive coverage of gene expression patterns and tissue-enriched splicing isoforms. We propose that C-It will be an excellent starting point to study uncharacterized genes. AVAILABILITY: C-It is freely available online without registration at http://C-It.mpi-bn.mpg.dehttp://C-It.mpi-bn.mpg.de.

Exon Array Analyzer: a web interface for Affymetrix exon array analysis.

SUMMARY: The Exon Array Analyzer (EAA) is a web server, which provides a user-friendly interface to identify alternative splicing events analyzed with Affymetrix Exon Arrays. The EAA implements the Splice Index algorithm to identify differential expressed exons. The use of various filters allows reduction of the number of false positive hits. Results are presented with detailed annotation information and graphics to identify splice events and to facilitate biological validations. To demonstrate the versatility of the EAA, we analyzed exon arrays of 11 different murine tissues using sample data provided by Affymetrix (http://www.affymetrix.com). Data from the heart were compared with other tissues to identify exons that undergo heart-specific alternatively splicing, resulting in the identification of 885 differentially expressed probe sets in 649 genes. AVAILABILITY: The web interface is available at http://EAA.mpi-bn.mpg.de/. Detailed documentation is available on the EAA web site (http://EAA.mpi-bn.mpg.de/supp.php) including screen shots, example analyzes and step by step instructions. CONTACT: thomas.braun@mpi-bn.mpg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.