Exon Microarray Research Articles

The Impact of Cell Source, Culture Methodology, Culture Location, and Individual Donors on Gene Expression Profiles of Bone Marrow-Derived and Adipose-Derived Stromal Cells

Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells.

DEK and WT1 Affect Alternative Splicing of Genes Involved in Hematopoietic Cell Lineage and Resistance to Chemotherapy in Acute Myeloid Leukemia Cells.

AbstractAbstract 2392In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole-exome/genome technologies in myelodysplastic syndromes (MDS) and in acute myeloid leukemia (AML). In MDS the frequency of somatic spliceosomal mutations (SSM) range from 1–3% for U2AF1 in RARS/RCMD-RS to more than 70% for SF3B1 in ARSI. These values are significantly lower in AML whereas AML cells cumulate numerous splicing defects. Beside SSMs, one can propose that alternative splicing (AS) might be disturbed by other processes such as abnormal protein-protein interactions. DEK and WT1 are 2 oncogenes overexpressed in most patients with AML. They physiologically influence AS through physical interactions with the heterodimer U2AF1/U2AF2 involved in the recognition of splice acceptor site by the splicing machinery. It is therefore possible that the leukemogenic overexpression of DEK or WT1 might deregulate AS in AML cells, even in the absence of SSM. Here we show that DEK and WT1 affect AS in AML cells. Exon expression profiling was performed in triplicate with MOLM13, KASUMI and KG1 AML cells stably knocked down or not for DEK and WT1 through shRNA. The efficiency of shRNA-mediated silencing was confirmed by western blot and total RNA was analyzed using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). Microarray data were cross-compared between cell lines and only statistically significant modifications (p<0.05) shared by MOLM13, KASUMI and KG1 cells were selected. DEK and WT1 knock-down induced the transcriptional deregulation of 1613 (813 up) and 3280 (998 up) genes in AML cell lines, respectively. AS events were selected and annotated with fasterDB database (http://fasterdb.com/faster/home.pl) for genes displaying either no or low (<2) differential transcription. With this approach, differential expression of DEK coincided with changes in 1049 AS events over 934 genes. Those were distributed in 4 alternative acceptor sites (ACC), 222 first exons (AFE), 257 last exons (ALE), 539 spliced exon (ASE), 6 deletions (DEL), 21 donor sites (DON). Differential expression of WT1 led to modifying 1371 alternative splicing entities over 1198 genes. Those were distributed in 6 ACC, 385 AFE, 343 ALE, 590 ASE, 13 DEL, and 34 DON. Genes with AS events were then sorted based on gene function with DAVID bioinformatics resources version 6.7 (http://david.abcc.ncifcrf.gov/). The results indicated that, in both DEK- and WT1-dependent assays, a large subset of genes were related to hematopoietic cell lineage followed by other functional categories such as calcium signaling, ATP-binding cassette (ABC) transporters, and focal adhesion pathways that have been previously reported to be affected in AML cells and involved in resistance to chemotherapy. Differential expression of WT1 modified AS of CD3E, CD9, CSF1R, CSF3R, CR2, GP1BA, ITGA1, ITGA3, ITGB3, IL1A and IL6 while that of DEK led to modulate AS of CD19, CD1d, CD36, CD3G, CSF1R, CR2, ITGA1, ITGA2B, ITGA4, ITGA6, IL7, HLA-DRB5, and MME. Microarray data were validated by exon specific RT-PCR. Exon expression profiling of fresh AML bone marrow samples with or without U2AFs mutations and various levels of DEK or WT1 expression is currently in progress and will be presented. In conclusion in AML cells, DEK and WT1 overexpression affects AS of numerous key genes involved in hematologic differentiation, leukemogenesis and resistance to chemotherapy. These posttranscriptional effects occur in the absence of transcriptional change and therefore highlight hitherto unknown phenotypic alterations having putative diagnostic, prognostic and therapeutic interests. DEK and WT1 are overexpressed in more than 80% of AML and target AS via U2AFs that are mutated in only 0–8% of cases. Accordingly present results strongly suggest that other factors than SSM divert AS in AML. Disclosures:No relevant conflicts of interest to declare.

Abundance of Alternative Splicing Events and Differentiation Stage-Specific Changes in Splicing Suggest A Major Role in Regulation of Gene Expression During Late Erythropoiesis

Abstract 978Alternative pre-mRNA splicing plays a major role in development and differentiation by re-modeling the transcriptome to generate mRNAs that encode the biologically appropriate cell type-specific proteome. Earlier studies employing RT-PCR and exon microarrays demonstrated a small number of splicing changes during erythroid differentiation, one of which (in protein 4.1R) is critical for mechanical stability of the membrane skeleton. Here we report that the landscape of splicing changes executed during terminal erythropoiesis is far more extensive and highly dynamic, ultimately affecting the expression of many more proteins than previously recognized. Highly purified populations of FACS-sorted cells representing erythroblasts at distinct stages during terminal erythroid differentiation from proerythroblasts to orthochromatic erythroblasts of both human and mouse origin were used as the source of RNA for RNA-seq analysis. In total, hundreds of millions of sequence reads were obtained from three biological replicates for four (mouse) or five (human) cell populations, and reads were aligned to the Ensembl-annotated transcriptome using the Bowtie aligner. Transcript-level estimates were obtained using the streaming transcript abundance estimation tool, eXpress, expression of individual exons in “exon-inclusion” isoforms relative to total isoforms was represented as Ψ (psi), or percent spliced in, and statistical significance estimates adjusted for multiple comparisons by the Benjamini-Hochberg method. Thousands of alternative splicing events were predicted in genes with diverse functions in transcription, RNA processing, protein synthesis, membrane receptors, cytoskeletal structure, etc. Initial RT-PCR studies indicate that a high proportion of predicted alternative splicing events can be validated. Comparison of Ψ values across the differentiation series revealed that hundreds of alternative exons in erythroid transcripts exhibit substantial differences in splicing efficiency between proerythroblasts and orthrochromatic erythroblasts (ΔΨ>20%), suggesting that their splicing efficiency is regulated. Both increases and decreases in exon splicing efficiency were observed, indicating that multiple splicing regulatory pathways are active and that both splicing enhancer and splicing silencer factors are involved in the regulation. Interestingly, some of the splicing switches introduce premature translation termination codons, leading us to hypothesize that splicing-coupled nonsense mediated decay may down-regulate expression of a class of erythroid transcripts. To begin exploring mechanisms that regulate the late erythroid alternative splicing program, we used the RNA-seq data to derive differentiation stage-specific expression profiles of known splicing factors. Major changes in the expression profile of many splicing regulators were observed. hnRNPA1 was strongly down-regulated in late erythroblasts, in concert with up-regulation of the protein 4.1R splicing switch it has been shown to inhibit. Because many other exons are up-regulated with similar kinetics, hnRNPA1 may be a general inhibitor of alternative splicing in early erythroblasts. In contrast, RNA-seq data indicate that several other splicing factors including MBNL1, a known splicing factor in muscle and brain whose activity is disturbed in myotonic dystrophy, are substantially up-regulated in late erythropoiesis. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts, in conjunction with the better studied transcriptional program, plays a major role in regulating gene expression to insure synthesis of the appropriate constellation of proteins both quantitatively and qualitatively as the cells are remodeled in preparation for production of mature red cells. Disclosures:No relevant conflicts of interest to declare.

Genome-Wide Expression Analysis in Down Syndrome: Insight into Immunodeficiency

Down syndrome (DS) is caused by triplication of Human chromosome 21 (Hsa21) and associated with an array of deleterious phenotypes, including mental retardation, heart defects and immunodeficiency. Genome-wide expression patterns of uncultured peripheral blood cells are useful to understanding of DS-associated immune dysfunction. We used a Human Exon microarray to characterize gene expression in uncultured peripheral blood cells derived from DS individuals and age-matched controls from two age groups: neonate (N) and child (C). A total of 174 transcript clusters (gene-level) with eight located on Hsa21 in N group and 383 transcript clusters including 56 on Hsa21 in C group were significantly dysregulated in DS individuals. Microarray data were validated by quantitative polymerase chain reaction. Functional analysis revealed that the dysregulated genes in DS were significantly enriched in two and six KEGG pathways in N and C group, respectively. These pathways included leukocyte trans-endothelial migration, B cell receptor signaling pathway and primary immunodeficiency, etc., which causally implicated dysfunctional immunity in DS. Our results provided a comprehensive picture of gene expression patterns in DS at the two developmental stages and pointed towards candidate genes and molecular pathways potentially associated with the immune dysfunction in DS.

A model system for assessing and comparing the ability of exon microarray and tag sequencing to detect genes specific for malignant B-cells

BackgroundMalignant cells in tumours of B-cell origin account for 0.1% to 98% of the total cell content, depending on disease entity. Recently, gene expression profiles (GEPs) of B-cell lymphomas based on microarray technologies have contributed significantly to improved sub-classification and diagnostics. However, the varying degrees of malignant B-cell frequencies in analysed samples influence the interpretation of the GEPs. Based on emerging next-generation sequencing technologies (NGS) like tag sequencing (tag-seq) for GEP, it is expected that the detection of mRNA transcripts from malignant B-cells can be supplemented. This study provides a quantitative assessment and comparison of the ability of microarrays and tag-seq to detect mRNA transcripts from malignant B-cells. A model system was established by eight serial dilutions of the malignant B-cell lymphoma cell line, OCI-Ly8, into the embryonic kidney cell line, HEK293, prior to parallel analysis by exon microarrays and tag-seq.ResultsWe identified 123 and 117 differentially expressed genes between pure OCI-Ly8 and HEK293 cells by exon microarray and tag-seq, respectively. There were thirty genes in common, and of those, most were B-cell specific. Hierarchical clustering from all dilutions based on the differentially expressed genes showed that neither technology could distinguish between samples with less than 1% malignant B-cells from non-B-cells. A novel statistical concept was developed to assess the ability to detect single genes for both technologies, and used to demonstrate an inverse proportional relationship with the sample purity. Of the 30 common genes, the detection capability of a representative set of three B-cell specific genes - CD74, HLA-DRA, and BCL6 - was analysed. It was noticed that at least 5%, 13% and 22% sample purity respectively was required for detection of the three genes by exon microarray whereas at least 2%, 4% and 51% percent sample purity of malignant B-cells were required for tag-seq detection.ConclusionA sample purity-dependent loss of the ability to detect genes for both technologies was demonstrated. Taq-seq, in comparison to exon microarray, required slightly less malignant B-cells in the samples analysed in order to detect the two most abundantly expressed of the selected genes. The results show that malignant cell frequency is an important variable, with fundamental impact when interpreting GEPs from both technologies.

Theory on the Coupled Stochastic Dynamics of Transcription and Splice-Site Recognition

Eukaryotic genes are typically split into exons that need to be spliced together to form the mature mRNA. The splicing process depends on the dynamics and interactions among transcription by the RNA polymerase II complex (RNAPII) and the spliceosomal complex consisting of multiple small nuclear ribonucleo proteins (snRNPs). Here we propose a biophysically plausible initial theory of splicing that aims to explain the effects of the stochastic dynamics of snRNPs on the splicing patterns of eukaryotic genes. We consider two different ways to model the dynamics of snRNPs: pure three-dimensional diffusion and a combination of three- and one-dimensional diffusion along the emerging pre-mRNA. Our theoretical analysis shows that there exists an optimum position of the splice sites on the growing pre-mRNA at which the time required for snRNPs to find the 5′ donor site is minimized. The minimization of the overall search time is achieved mainly via the increase in non-specific interactions between the snRNPs and the growing pre-mRNA. The theory further predicts that there exists an optimum transcript length that maximizes the probabilities for exons to interact with the snRNPs. We evaluate these theoretical predictions by considering human and mouse exon microarray data as well as RNAseq data from multiple different tissues. We observe that there is a broad optimum position of splice sites on the growing pre-mRNA and an optimum transcript length, which are roughly consistent with the theoretical predictions. The theoretical and experimental analyses suggest that there is a strong interaction between the dynamics of RNAPII and the stochastic nature of snRNP search for 5′ donor splicing sites.

THE EFFECT OF HYPOXIA ON GLOBAL GENE EXPRESSION AND ALTERNATIVE SPLICING OF HUMAN MESENCHYMAL STEM CELLS

ObjectivesHypoxia preconditioning has become a novel strategy to improve the in vivo therapeutic potential of bone marrow-derived mesenchymal stem cells (MSCs). However, the underlying molecular mechanisms responsible for the hypoxia-induced...

Deregulation of Specific Sets of Genes Detected by Microarray Analysis of Marrow Stromal Fibroblast Cells Stimulated by IR and VR Light

Conflicting reports have questioned whether low level laser treatment can have an effect on bone. Purpose:The purpose of this study was to examine how light affects gene expression. Methods: Clones of marrow fibroblast stem cells were isolated from human bone marrow and used for primary cell cultures. The cells were grown without light as a control and with light under eight different conditions: two wavelengths of light(830-IR and 633nmVR) at four different energy levels(0.5.1.0,1.5 and 2.0 joules /cm2). Results: Light stimulated some cultures to proliferate up to 40% faster depending on the type of light stimulation using BrdU markers. For the light-stimulated cultures, western blot data for osteocalcin, alkaline phosphatase and RUNX 2, markers of osteoblast differentiation and bone formation, did not show a pattern of increased or early gene expression compared to control cultures. Affymetrix Human exon microarrays of 22,000 protein-encoding genes (40 markers per gene) showed distinct sets of genes being deregulated for each of the eight experimental conditions. A two-fold screen for gene expression differences between control and light-stimulated cultures revealed a complex network of gene expression interactions involving dose response and silencing of inhibitor genes for candidate genes such as RANKL, IL-1A and MMP10. Pathway analysis showed the top VR biological gene networks were skeletal and muscular system development and function, tissue development and amino acid metabolism, consistent with bone turnover. The top biological functions associated with IR stimulation are skin condition, genetic disorders, cancer, immune response. Conclusion: This study provides strong evidence for light stimulating different sets of genes according to wavelength and energy level that can alter bone turnover. The data provides a molecular explanation for the conflicting reports in the current literature.

HPMC supplementation reduces abdominal fat content, intestinal permeability, inflammation, and insulin resistance in diet‐induced obese mice

The effects of hydroxypropyl methylcellulose (HPMC), a highly viscous nonfermentable soluble dietary fiber, were evaluated on adipose tissue inflammation and insulin resistance in diet-induced obese (DIO) mice fed a high-fat (HF) diet supplemented with either HPMC or insoluble fiber. DIO C57BL/6J mice were fed a HF diet supplemented with 6% HPMC or 6% microcrystalline cellulose (MCC). Gene expression analyses of epididymal adipose tissue by exon microarray and real-time PCR along with glucose and insulin tolerance and intestinal permeability were assessed. HPMC-fed mice exhibited significantly reduced body weight gain and adipose tissue weight as well as reduced areas under the curve for 2-h insulin and glucose responses. HPMC significantly decreased HF diet-induced intestinal permeability. Overall, HPMC enhanced insulin sensitivity and glucose metabolism and downregulated genes related to inflammation and immune response, adipogenesis, and oxidative stress markers. Pathway analysis of microarray data identified lipid metabolism, inflammatory disease, and acute phase response pathways as being differentially regulated by HPMC. These results suggest HPMC consumption ameliorates HF diet effects on obesity-induced insulin resistance, adipose tissue inflammatory and immune responses, weight gain, as well as intestinal permeability.

508 Identification of Fusion Transcripts in Colorectal Cancer by Combined RNA-seq and Exon Microarray Analyses

508 Identification of Fusion Transcripts in Colorectal Cancer by Combined RNA-seq and Exon Microarray Analyses

A systematic comparison and evaluation of high density exon arrays and RNA-seq technology used to unravel the peripheral blood transcriptome of sickle cell disease

BackgroundTranscriptomic studies in clinical research are essential tools for deciphering the functional elements of the genome and unraveling underlying disease mechanisms. Various technologies have been developed to deduce and quantify the transcriptome including hybridization and sequencing-based approaches. Recently, high density exon microarrays have been successfully employed for detecting differentially expressed genes and alternative splicing events for biomarker discovery and disease diagnostics. The field of transcriptomics is currently being revolutionized by high throughput DNA sequencing methodologies to map, characterize, and quantify the transcriptome.MethodsIn an effort to understand the merits and limitations of each of these tools, we undertook a study of the transcriptome in sickle cell disease, a monogenic disease comparing the Affymetrix Human Exon 1.0 ST microarray (Exon array) and Illumina’s deep sequencing technology (RNA-seq) on whole blood clinical specimens.ResultsAnalysis indicated a strong concordance (R = 0.64) between Exon array and RNA-seq data at both gene level and exon level transcript expression. The magnitude of differential expression was found to be generally higher in RNA-seq than in the Exon microarrays. We also demonstrate for the first time the ability of RNA-seq technology to discover novel transcript variants and differential expression in previously unannotated genomic regions in sickle cell disease. In addition to detecting expression level changes, RNA-seq technology was also able to identify sequence variation in the expressed transcripts.ConclusionsOur findings suggest that microarrays remain useful and accurate for transcriptomic analysis of clinical samples with low input requirements, while RNA-seq technology complements and extends microarray measurements for novel discoveries.

Deep brain stimulation induces rapidly reversible transcript changes in Parkinson's leucocytes

Subthalamic deep brain stimulation (DBS) reversibly modulates Parkinson's disease (PD) motor symptoms, providing an unusual opportunity to compare leucocyte transcripts in the same individuals before and after neurosurgery and 1 hr after stimulus cessation (ON- and OFF-stimulus). Here, we report DBS-induced reversibility and OFF-stimulus restoration in 12 of 16 molecular functions and 3 of 4 biological processes shown in exon microarrays to be differentially expressed between PD patients and controls, post-DBS from pre-DBS and OFF from ON states. Intriguingly, 6 of 18 inflammation and immune-related functions exhibited reversibility, and the extent of stimulus-induced changes correlated with the neurological DBS efficacy, suggesting mechanistic implications. A minimal list of 29 transcripts that changed in all three comparisons between states discriminated pre-surgery and OFF states from post-surgery and controls. Six of these transcripts were found to be able to distinguish between PD patients and both healthy controls and patients with other neurological diseases in a previously published whole blood 3’ array data study of early PD patients. Our findings support the future use of this approach for identifying targets for therapeutic intervention and assessing the efficacy of current and new treatments in this and other neurological diseases.

Gene Expression Changes Associated With the Progression of Intraductal Papillary Mucinous Neoplasms

The diagnosis of high-grade intraductal papillary mucinous neoplasm (IPMN) is difficult to distinguish from low-grade IPMN. The aim of this study was to identify potential markers for the discrimination of high-grade and invasive (HgInv) IPMN from low- and moderate-grade dysplasia IPMN. Laser capture microdissection was used to isolate distinct foci of low-grade, moderate-grade, high-grade, and invasive IPMN from paraffin-embedded archival tissue from 14 patients who underwent resection for IPMN. Most samples included multiple grades in the same specimen. Affymetrix Human Exon microarrays were used to compare low- and moderate-grade dysplasia IPMN with HgInv IPMN. Sixty-two genes were identified as showing significant changes in expression (P ≤ 0.05 and a 2-fold cutoff), including up-regulation of 41 in HgInv IPMN. Changes in gene expression are associated with biological processes related to malignant behavior including cell motion, cell proliferation, response to hypoxia, and epithelial-to-mesenchymal transition. In addition, altered signaling in several transforming growth factor β-related pathways was exhibited in the progression of IPMN to malignancy. This study identifies a set of genes associated with the progression of IPMN to malignancy. These genes are potential markers that could be used to identify IPMN requiring surgical resection.

Abstract 3945: HIFs regulate alternative splicing of HIF target genes

Abstract Alternative splicing is the process in which identical pre-mRNA molecules are spliced in different ways, allowing individual gene to express multiple mRNAs or isoforms that encode proteins with diverse and even antagonistic functions. Alternative splicing occurs in over 70% of human genes, is important in normal development and many diseases including cancer. It is well known that most solid-tumors are hypoxic and the hypoxic microenvironment is a driving force for tumor progression and metastasis by hypoxia-inducible-factor (HIF)-mediated transcriptional programs that increase genes involved in tumor growth, angiogenesis, and metastasis. Here, we report a novel finding that transcriptional factor HIF not only activates gene expression, also regulates RNA splicing of its target genes, generating isoforms that favor tumor growth. Using an Exon Microarray we found that approximately 1000 genes are predicted to undergo hypoxia-mediated AS in Hep3B cells, 100 of those genes are hypoxia inducible genes. Using RT-PCR, RT-qPCR and WB we validated that the first 10 HIF target genes, CA9, ANGPTL4, WNK1, PLOD2, ADM, DUOX2, ENO2, P4HA2, EGLN3, and PDK1 undergo hypoxia-mediate AS, as predicted by exon array. Interestingly, AS of these genes is regulated by HIFs, not by hypoxia since: 1) siRNA knockdown of ARNT, HIF1, or HIF2 inhibits hypoxic induction of HIF target genes as well as inhibits hypoxia-mediated splicing changes; and 2) Normoxic HIF activity by deleting VHL, inhibiting PHD, or expressing normoxic-stable HIF protein led to hypoxic splicing patterns of HIF target genes, demonstrating HIF activity is sufficient to induce alternative splicing of HIF target genes. However, AS of HIF target genes cannot be explained by increased transcription of these genes since no splicing changes were observed when these genes were activated by hybrid protein with HIF transactivation domains replaced by VP16, E2F1, or SP1 transactivation domain. Further experiments indicated that the N-terminal transactivation domains (N-TAD) of HIF1α or HIF2α are responsible for splicing changes of these HIF-target genes. In summary, we have uncovered a novel function of HIFs in the regulation of target gene splicing. The discovery of HIF's role in mRNA splicing of its target genes will change the dogma concerning the role of HIF in cancer progression. We propose that HIFs promote cancer progression not only through transcription activation but also through alteration of mRNA splicing of HIF targets. RO1 CA134687 NIH/NCI (Hu) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3945. doi:1538-7445.AM2012-3945

Abstract 1166: PTEN loss and ERG over-expression as prognostic biomarkers in prostate cancer and identification of downstream biomarkers with potential therapeutic value

Abstract Genomic deletion of the PTEN tumor suppressor gene and formation of the TMPRSS2:ERG gene fusion are the two most recurrent genomic aberrations in prostate cancer. The lipid phosphatase activity of PTEN negatively regulates the PI3Kinase-AKT signaling pathway, which controls numerous downstream targets such as cell cycle checkpoints, DNA damage repair with maintenance of chromosomal stability and integrity. ERG is a member of the ETS transcription factor family, whose members are implicated in numerous cellular processes including membrane remodelling, angiogenesis, differentiation, proliferation, and tumourigenesis. Emerging evidence suggests that formation of the fusion gene may promote prostatic tumourigenesis, progression, and invasive disease by elevating motility and invasiveness. The simultaneous manifestation of both PTEN loss and TMPRSS2:ERG fusion is associated with poor prognosis. This study is addressing pathways downstream of PTEN and effectors of ERG over-expression to identify additional biomarkers of prognostic and therapeutic potential. In silico genomic copy number analyses demonstrated that patient samples harboring a genomic deletion of PTEN have a greater number of genomic aberrations, in keeping with the model that loss of PTEN leads to heightened genomic instability. Mining of publically available gene expression datasets have been performed to further examine signalling pathway aberrations specific to each rearrangement. PTEN loss and ERG over-expression are being modeled in the histologically normal prostate epithelial cell line RWPE-1 using shRNA knockdown or transgene directed ectopic over-expression, respectively. Biochemical, proliferation, migration and invasion assays will be performed to determine if knockdown of PTEN leads to the expected AKT activation and increased proliferation; whereas over-expression of ERG in RWPE-1 cells correlates with heightened motility and invasive potential. Gene expression microarray profiling will also be carried out for each derivative RWPE-1 cell line using the Agilent SurePrint G3 Human Exon microarrays. This cell model system will be used to validate transcriptional changes associated with PTEN loss and ERG over-expression in prostate cancer gene expression datasets and identify potential novel downstream therapeutic targets and predictive biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1166. doi:1538-7445.AM2012-1166

Hepatocyte maturation is arrested in mouse liver lacking the splicing factor SRSF3 (SRp20)

SRSF3 is the smallest member of the highly conserved SR protein family that has numerous roles in mRNA metabolism, from transcription to translation. To investigate the role of SRSF3 in postnatal hepatic function, we used Cre‐loxP‐mediated gene technology to delete SRSF3 specifically in hepatocytes in mice. Deletion of SRSF3 inhibits hepatocyte maturation as livers from mice lacking SRSF3 exhibit a fetal phenotype characterized by increased expression of the fetal isozyme of pyruvate kinase (M‐PK), persistent expression of alpha‐fetoprotein and H19 mRNA. The livers also show increased expression of the fetal isoform of the insulin receptor (IR‐A). The expression of several genes that regulate glucose and lipid metabolism in the mature liver is significantly reduced in SRSF3−/− liver, and glycogen levels are decreased. Global gene expression and exon microarray analyses identified multiple splicing and expression changes in the SRSF3−/− liver. Interestingly, some of these changes alter expression of major hepatic transcription factors that play crucial role in development and metabolism. Thus, SRSF3 directs a genetic program required for morphological and functional differentiation of hepatocytes and the hepatocyte SRSF3 knock‐out mice provide a new model to study metabolic dysregulation and liver disease.This work has been supported by VA Merit Grant Award.

HPMC supplementation reduces weight gain, intestinal permeability, inflammation, and insulin resistance in dietinduced obese mice

Consumption of diets high in soluble dietary fibers has been suggested to induce biomarkers for anti‐obesity and anti‐inflammation, and improve insulin resistance. To investigate the effects of hydroxypropyl methylcellulose (HPMC), a highly viscous soluble dietary fiber, on obesity‐induced metabolic complications, obese C57BL/6J mice were fed high fat diets with either 6 % HPMC or 6 % microcrystalline cellulose (MCC) for five weeks. Body weight gain and adipose tissue weight were significantly lower in HPMC fed mice compared with MCC. High‐fat diet induced intestinal permeability was lowered by HPMC. IPA analysis of exon microarray data from adipose tissue identified lipid metabolism and acute phase response pathways were regulated by HPMC. We confirmed that genes related to inflammation and immune response, and oxidative stress markers were down‐regulated by the HPMC diet. HPMC feeding markedly reduced area under the curve for 2‐hr insulin and glucose responses, indicating enhanced insulin sensitivity and glucose metabolism. These data suggest that consumption of HPMC ameliorates obesity‐induced insulin resistance via reduction of weight gain and anti‐inflammatory and immune response in adipose tissue as well as enhancing intestinal barrier function.

The Sam68 STAR RNA-Binding Protein Regulates mTOR Alternative Splicing during Adipogenesis

We report that mice ablated for the Sam68 RNA-binding protein exhibit a lean phenotype as a result of increased energy expenditure, decreased commitment to early adipocyte progenitors, and defects in adipogenic differentiation. The Sam68(-/-) mice were protected from obesity, insulin resistance, and glucose intolerance induced with a high-fat diet. To identify the alternative splice events regulated by Sam68, genome-wide exon usage profiling in white adipose tissue was performed. Adipocytes from Sam68(-/-) mice retained intron 5 within the mTOR transcript introducing a premature termination codon, leading to an unstable mRNA. Consequently, Sam68-depleted cells had reduced mTOR levels resulting in lower levels of insulin-stimulated S6 and Akt phosphorylation leading to defects in adipogenesis, and this defect was rescued by the exogenous expression of full-length mTOR. Sam68 bound intronic splice elements within mTOR intron 5 required for the usage of the 5' splice site. We propose that Sam68 regulates alternative splicing during adipogenesis.

Abstract A21: MALAT-1 is essential for lung cancer metastasis in a novel human knockout model

Abstract The highly conserved long non-coding RNA MALAT-1 (Metastasis-Associated in Lung Adenocarcinoma Transcript 1) had been discovered as a prognostic marker associated with poor survival and development of distant metastasis in lung adenocarcinoma. Since then, it has been found to be deregulated in numerous tumor entities and has been linked to splicing. However, its functional relevance in tumor cells remains to be elucidated. Knockdown models for MALAT1 have been described but suffer from insufficient silencing efficiency of the highly abundant, nuclear non-coding RNA (ncRNA). In this project, we have developed a novel strategy to create ncRNA knockouts in human cancer cell lines. We have successfully used a synthetic Zinc Finger Nuclease engineered to target the 5′-region of MALAT1 to stably and biallelically integrate RNA-destabilizing elements into the genome of human lung cancer cells (A549). This approach resulted in a specific and more than 1000-fold silencing of MALAT1 in individual clones compared to a less than 5-fold silencing using siRNAs. Thus, this approach can be used to create functional knockouts of coding as well as non-coding genes also in human tumor cell lines allowing loss-of-function studies also of non-conserved ncRNAs in the future. Phenotypically, the MALAT1-Knockout cells (KO) greatly differ from their parental cell line and wildtype clones (WT): Next to morphological changes, the migration of the KO cells is largely impaired as shown in scratch assays. In xenograft assays after i.v. injection, the KO cells form significantly fewer and smaller lung metastases than their WT counterparts. Since no large difference was observed after subcutaneous injection of the WT and the KO cells, this indicates a specific, active and essential function of MALAT1 in metastasis. Exon microarrays of the WT and KO cell lines have revealed multiple migration- or metastasis-associated transcripts deregulated by loss of MALAT1 and hence potential target genes. These analyses also uncovered, that splicing is likely not the only functional mechanism of MALAT1 in the nucleus. Taken together, we have developed a novel, highly effective approach for the knockout of genes that can be used for non-coding as well as coding RNAs in human tumor cells as well as cells from other species. Knockout of MALAT1 in human lung cancer cells revealed its essential function in metastasis as well as genes directly and indirectly targeted by MALAT1. Citation Format: Tony Gutschner, Moritz Eißmann, Monika Hämmerle, Marion Stentrup, Catherina Hildenbrandt, Matthias Groß, Martin Zörnig, Sven Diederichs. MALAT-1 is essential for lung cancer metastasis in a novel human knockout model [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A21.

A New Mouse Model in which the Proximal Tandem Ig Element of Titin is Truncated (IGKO) - Assessment of Diastolic Function

Titin is a giant protein that functions as a molecular spring and contributes to the passive diastolic stiffness of the left ventricle (LV). Titin's spring region consists of three sequence elements: the tandem Ig, the N2B and the PEVK elements. Mouse models exist in which either of the latter two elements (N2B KO, PEVK KO) has been deleted, but considering that these elements are also likely to play roles in signaling, we made a model in which we deleted 2/3 of the proximal tandem Ig element (IGKO), a region that is not known to contain signaling functions. Homozygous KO mice survive to adulthood and are fertile with no change in LV Weight to Body Weight (LVW/BW) ratio (3.67±0.07mg/g WT vs 3.51±0.04mg/g IgKO). A custom titin exon microarray confirmed the deletion of the expected exons with no other changes. Gel electrophoresis showed that the IGKO expresses an 88kDa smaller titin isoform but no other changes in isoform expression. Immuno-electron microscopy indicates that the remaining spring elements extend to a higher degree than in WT myocardium. Mechanical measurements in cardiomyocytes (see also poster by Methawasin) and myofibers show that the deletion increases passive stiffness in titin in a sarcomere length dependence manner, without adaptations in the passive stiffness of the extracellular matrix. Initial isolated intact heart data and in-vivo pressure volume measurement suggest that the IGKO has increased stiffness in-vivo. Further phenotypic characterization is currently underway. In summary, shortening the proximal tandem Ig element of titin produces a diastolic stiffness phenotype.