Affymetrix Exon Arrays Research Articles

Hemoglobin overexpression and splice signature as new features of inflammatory breast cancer?

IntroductionInflammatory Breast Cancer (IBC) is the most aggressive form of breast carcinoma characterized by rapid onset of inflammatory signs and its molecular fingerprint has not yet been elucidated. ObjectivesThe objective of this study was to detect both gene expression levels and alternate RNA splice variants specific for IBC. MethodsW e performed splice-sensitive array profiling using Affymetrix Exon Array and quantitative RT-PCR analyses in 177 IBC compared to 183 non-IBC. We also assessed the prognostic value of the identified candidate genes and splice variants. ResultsA 5-splice signature (HSPA8, RPL10, RPL4, DIDO1 and EVL) was able to distinguish IBC from non-IBC tumors (p<10-7). This splice signature was associated with poor metastasis-free survival in hormone receptor-negative non-IBC (p=0.02), but had no prognostic value in IBC. PAM analysis of dysregulated genes in IBC compared to non-IBC identified a 10-gene signature highly predictive of IBC phenotype and conferring a poor prognosis in non-IBC. The genes most commonly upregulated in IBC were 3 hemoglobin genes able to reliably discriminate IBC from non-IBC (p<10-4). Hb protein expression in epithelial breast tumor cells was confirmed by immunohistochemistry. ConclusionIBC has a specific spliced transcript profile and is characterized by hemoglobin gene overexpression that should be investigated in further functional studies.

Dietary Fat Quantity and Type Induce Transcriptome-Wide Effects on Alternative Splicing of Pre-mRNA in Rat Skeletal Muscle

Background: Fat-enriched diets produce metabolic changes in skeletal muscle, which in turn can mediate changes in gene regulation.Objective: We examined the high-fat-diet-induced changes in skeletal muscle gene expression by characterizing variations in pre-mRNA alternative splicing.Methods: Affymetrix Exon Array analysis was performed on the transcriptome of the gastrocnemius/plantaris complex of male obesity-prone Sprague-Dawley rats fed a 10% or 60% fat (lard) diet for 2 or 8 wk. The validation of exon array results was focused on troponin T (Tnnt3). Tnnt3 splice form analyses were extended in studies of rats fed 10% or 30% fat diets across 1- to 8-wk treatment periods and rats fed 10% or 45% fat diets with fat sources from lard or mono- or polyunsaturated fats for 2 wk. Nuclear magnetic resonance (NMR) was used to measure body composition.Results: Consumption of a 60% fat diet for 2 or 8 wk resulted in alternative splicing of 668 and 726 pre-mRNAs, respectively, compared with rats fed a 10% fat diet. Tnnt3 transcripts were alternatively spliced in rats fed a 60% fat diet for either 2 or 8 wk. The high-fat-diet-induced changes in Tnnt3 alternative splicing were observed in rats fed a 30% fat diet across 1- to 8-wk treatment periods. Moreover, this effect depended on fat type, because Tnnt3 alternative splicing occurred in response to 45% fat diets enriched with lard but not in response to diets enriched with mono- or polyunsaturated fatty acids. Fat mass (a proxy for obesity as measured by NMR) did not differ between groups in any study.Conclusions: Rat skeletal muscle responds to overconsumption of dietary fat by modifying gene expression through pre-mRNA alternative splicing. Variations in Tnnt3 alternative splicing occur independently of obesity and are dependent on dietary fat quantity and suggest a role for saturated fatty acids in the high-fat-diet-induced modifications in Tnnt3 alternative splicing.

Abstract 4908: Neuropilin-1 is up-regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality

Abstract Aims: Androgen-targeted therapies (ATTs) are the mainstay treatment for metastatic prostate cancer (PCa). However, ATTs promote adaptation of tumour cells and lead to castration resistant disease (CRPC). We have recently identified the cell surface receptor, Neuropilin-1 (NRP1) as increased during EMT and in CRPC. However, the role of NRP1 in the prostate epithelium is poorly understood. This study aims to determine whether the inhibition of NRP1 will be a feasible therapeutic strategy for blocking PCa metastasis and therapy resistance. Methods: qPCR and western blotting were used to assess NRP1 expression in PCa cell lines. NRP1 expression in CRPC was assessed using a murine LNCaP xenograft model of castration. NRP1 was knocked down with shRNA sequences from the pLKO.1 lentiviral construct. For metastasis assays, PC3 cells were microinjected into the zebrafish yolk sac and metastatic dissemination imaged 5 days later. NRP1 expression in radical prostatectomy (RP) samples from Mayo Clinic (545 patients) and Johns Hopkins Medical Institutions (JHMI; 188 patients) cohorts was quantified by Affymetrix exon arrays and multivariable analysis performed. Wound scratch migration and invasion assays were performed with the WoundMaker™ tool and IncuCyte™ FLR imaging systems. Results: NRP1 levels were elevated in humanCRPC xenografts, metastatic and castrate resistant clinical PCa samples (p &amp;lt;0.0001), and PCa cell lines. NRP1 suppression significantly reduced metastasis of human xenografts in zebrafish and the migratory and invasive behaviour of metastatic PCa cells (p=0.0002). Multivariable analysis identified NRP1 as a significant independent prognostic indicator of metastasis and prostate cancer specific mortality in two large clinical cohorts (Mayo Clinic and JHMI; p=0.008/0.048 and 0.013/0,034 respectively). We show that NRP1 knockdown promotes E-Cadherin expression and loss of vimentin in mesenchymal PCa cells. Conclusion: These results will provide the preclinical data necessary to rationalise the use of anti-NRP1 directed adjuvant therapies for clinical use in PCa patients receiving ATTs, and will pave the way for larger scale preclinical and clinical trials in the PCa setting. Citation Format: Marianna Volpert, Brian Tse, Ellca Ratther, Nataly Stylianou, Mannan Nouri, Melanie Lehman, Stephen McPherson, Mani Roshan-Moniri, Mandeep Takhar, Nicholas Erho, Mohamed Alshalafa, Elai Davicioni, Robert Jenkins, Ashley Ross, Jeffrey Karnes, Robert Den, Ladan Fazli, Martin Gleave, Elizabeth Williams, Paul Rennie, Ralph Buttyan, Pamela Russell, Colleen Nelson, Brett Hollier. Neuropilin-1 is up-regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4908. doi:10.1158/1538-7445.AM2017-4908

Splice variants as novel targets in pancreatic ductal adenocarcinoma

Despite a wealth of genomic information, a comprehensive alternative splicing (AS) analysis of pancreatic ductal adenocarcinoma (PDAC) has not been performed yet. In the present study, we assessed whole exome-based transcriptome and AS profiles of 43 pancreas tissues using Affymetrix exon array. The AS analysis of PDAC indicated on average two AS probe-sets (ranging from 1–28) in 1,354 significantly identified protein-coding genes, with skipped exon and alternative first exon being the most frequently utilised. In addition to overrepresented extracellular matrix (ECM)-receptor interaction and focal adhesion that were also seen in transcriptome differential expression (DE) analysis, Fc gamma receptor-mediated phagocytosis and axon guidance AS genes were also highly represented. Of note, the highest numbers of AS probe-sets were found in collagen genes, which encode the characteristically abundant stroma seen in PDAC. We also describe a set of 37 ‘hypersensitive’ genes which were frequently targeted by somatic mutations, copy number alterations, DE and AS, indicating their propensity for multidimensional regulation. We provide the most comprehensive overview of the AS landscape in PDAC with underlying changes in the spliceosomal machinery. We also collate a set of AS and DE genes encoding cell surface proteins, which present promising diagnostic and therapeutic targets in PDAC.

MP88-10 INHIBITION OF LIM AND SH3 DOMAIN PROTEIN 1 DELAYS CISPLATIN-RESISTANT BLADDER TUMOR PROGRESSION

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2017MP88-10 INHIBITION OF LIM AND SH3 DOMAIN PROTEIN 1 DELAYS CISPLATIN-RESISTANT BLADDER TUMOR PROGRESSION Takashi Dejima, Ario Takeuchi, Masaki Shiota, Masatoshi Eto, Martin Gleave, and Christopher Ong Takashi DejimaTakashi Dejima More articles by this author , Ario TakeuchiArio Takeuchi More articles by this author , Masaki ShiotaMasaki Shiota More articles by this author , Masatoshi EtoMasatoshi Eto More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Christopher OngChristopher Ong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2735AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer is the 6th most common cancer in the USA. The current standard therapy for the first line of metastatic or local advanced bladder cancer is combination therapy with cisplatin (CDDP) and gemcitabine (GEM). However 5-years survival is still below 50%; therefore additional therapy is needed. LIM-SH3 domain protein 1 (LASP1), identified from cDNA library of metastatic axillary lymph nodes of breast cancer, has been shown to promote cancer progression and invasion in several malignancies. In bladder cancer, LASP1 expression associates with cell invasion and can be used for detecting bladder cancer. However, the anti-tumor effects of LASP1 knockdown in vivo as well as combination therapy with chemotherapy remain unclear. In this study, we investigated the anti-cancer activity of LASP1 knockdown in bladder cancer. METHODS LASP1 gene expression of tumor samples is analyzed using the Affymetrix Exon Array. The LASP1 expression in several bladder cancer cell lines was assessed by Western blot analysis and quantitative reverse transcription-PCR. Silencing of LASP1 in vitro was achieved using siRNA. Cell growth was measured by crystal violet assay. Cell cycle distribution was analyzed by flow cytometry after double thymidine block. The In vivo effect of LASP1 antisense oligonucleotide (ASO) treatment was assessed in the T24 CDDP-R (cisplatin-resistant) orthotopic bladder cancer model. RESULTS High LASP1 expression correlated with metastatic recurrence rate between patients. The LASP1 expression is higher in UC1 and T24 cells than in UC13 and UC6 cells. Knockdown of LASP1 using siRNA inhibited cell growth, and was accompanied by an increase in p21 and p27, and a decreased of cyclin D1. Flow cytometry revealed that LASP1 knockdown induced G1 arrest. Conversely, stable LASP1 overexpression drove cell growth with an increase of cyclin D1 in UC6 and UC3 cells. The treatment of CDDP and GEM induced LASP1 expression in Western Blotting. Furthermore, compared with parental cell line, LASP1 is higher in T24 CDDP-R and RT112 CDDP-R cells than in parental cells. LASP1 ASO inhibited cell growth in RT112 CDDP-R and T24 CDDP-R cells. In the orthotopic bladder cancer model, systemic LASP1 ASO administration to athymic nude mice delayed tumor progression in T24 CDDP-R cells. CONCLUSIONS These data revealed that LASP1 inhibition might be as a promising novel therapeutics modality in the treatment of chemoresistant bladder cancer. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1177-e1178 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Takashi Dejima More articles by this author Ario Takeuchi More articles by this author Masaki Shiota More articles by this author Masatoshi Eto More articles by this author Martin Gleave More articles by this author Christopher Ong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Age-dependent increase in Kalirin-9 and Kalirin-12 transcripts in human orbitofrontal cortex.

KALRN (KAL) is a Rho GEF that is highly involved in regulation of the actin cytoskeleton within dendrites. There are several isoforms of the protein that arise from differential splicing of KALRN's 66 exons. KAL isoforms have different functions in development. For example, overexpression of the KAL9 and KAL12 isoforms induce dendritic elongation in early development. However, in mature neurons KAL9 overexpression reduces dendritic length, a phenotype also observed in normal human ageing. We therefore hypothesized that KAL9 would have increased expression with age, and undertook to evaluate the expression of individual KALRN exons throughout the adult lifespan. Postmortem human brain grey matter from Brodmann's area (BA) 11 and BA47 derived from a cohort of 209 individuals without psychiatric or neurodegenerative disease, ranging in age from 16 to 91 years, were analysed for KALRN expression by Affymetrix exon array. Analysis of the exon array data in an isoform-specific manner, as well as confirmatory isoform-specific qPCR studies, indicated that the longer KAL9 and KAL12 isoforms demonstrated a statistically significant, but modest, increase with age. The small magnitude of the age effect suggests that inter-individual factors other than age likely contribute to a higher degree to KAL9 and KAL12 expression. In contrast to KAL9 and KAL12, global KALRN expression did not increase with age. Our work suggests that global measures of KALRN gene expression may be misleading and future studies should focus on isoform-specific quantification.

Abstract 500: DNA Methylation Profiling Reveals an Epithelial/Endothelial-Mesenchymal Transition-like Signature of Intima-Media Cells in the Ascending Aorta of Bicuspid Aortic Valve Patients

Introduction: Individuals with bicuspid aortic valves (BAV) are at increased risk of ascending aortic aneurysm than individuals with tricuspid aortic valves (TAV), but the underlying mechanism is not fully understood. Aberrant DNA methylation has been described in various human diseases, and we have shown that key enzymes in the methylation machinery are differentially expressed in the aortic intima-media of BAV and TAV patients. In the present study, we assessed the hypothesis that DNA methylation may play an important role during aneurysm formation in BAV. We undertook a global methylation approach to delineate biological processes associated with BAV aortopathy, using TAV as a reference. Methods: Ascending aortic biopsies were collected from 21 BAV and 24 TAV patients, with either a non-dilated or a dilated aorta, at the time of surgery. Global DNA methylation was measured in the intima-media layer using Illumina 450k Array. Gene expression was analyzed in the same samples using Affymetrix Exon Array. Results: Compared with TAV, the BAV dilated aorta was hypomethylated (P=0.031), correlating with an up-regulation of global gene expression. A total of 4913 differentially methylated regions (DMRs) were identified and Hallmark analysis of the DMR-associated genes with a fold change of 10% (n=3147) showed a gene signature of Epithelial Mesenchymal Transition (EMT) (FDR q=2.91e-29). This was further confirmed by functional annotation analysis of hypomethylated DMRs using the Genomic Regions Enrichment of Annotations Tool (Stanford University), showing association to actin filament bundle (P=7.09e-12), stress fibers (P=1.72e-11) and adherence junctions (P=2.97e-8). Interestingly, analysis of non-dilated BAV and TAV aorta revealed that genes involved in EMT were the most differentially methylated genes prior to dilatation (FDR q=1.18e-6). We further confirmed the EMT-related molecular signature by immunostaining of some key players of EMT. In conclusion, epigenetic profiling clearly revealed differential methylation between BAV and TAV aorta, particularly in EMT-related genes. Aberrant EMT in the ascending aorta prior to dilatation may constitute the basis for the increased aneurysm susceptibility in BAV patients.

Gene Expression Signatures Predictive of Bevacizumab/Erlotinib Therapeutic Benefit in Advanced Nonsquamous Non-Small Cell Lung Cancer Patients (SAKK 19/05 trial).

We aimed to identify gene expression signatures associated with angiogenesis and hypoxia pathways with predictive value for treatment response to bevacizumab/erlotinib (BE) of nonsquamous advanced non-small cell lung cancer (NSCLC) patients. Whole-genome gene expression profiling was performed on 42 biopsy samples (from SAKK 19/05 trial) using Affymetrix exon arrays, and associations with the following endpoints: time-to-progression (TTP) under therapy, tumor-shrinkage (TS), and overall survival (OS) were investigated. Next, we performed gene set enrichment analyses using genes associated with the angiogenic process and hypoxia response to evaluate their predictive value for patients' outcome. Our analysis revealed that both the angiogenic and hypoxia response signatures were enriched within the genes predictive of BE response, TS, and OS. Higher gene expression levels (GEL) of the 10-gene angiogenesis-associated signature and lower levels of the 10-gene hypoxia response signature predicted improved TTP under BE, 7.1 months versus 2.1 months for low versus high-risk patients (P = 0.005), and median TTP 6.9 months versus 2.9 months (P = 0.016), respectively. The hypoxia response signature associated with higher TS at 12 weeks and improved OS (17.8 months vs. 9.9 months for low vs. high-risk patients, P = 0.001). We were able to identify gene expression signatures derived from the angiogenesis and hypoxia response pathways with predictive value for clinical outcome in advanced nonsquamous NSCLC patients. This could lead to the identification of clinically relevant biomarkers, which will allow for selecting the subset of patients who benefit from the treatment and predict drug response.

From transcriptomic to protein level changes in TDP-43 and FUS loss-of-function cell models

The full definition of the physiological RNA targets regulated by TDP-43 and FUS RNA-binding proteins (RBPs) represents an important issue in understanding the pathogenic mechanisms associated to these two proteins in amyotrophic lateral sclerosis and frontotemporal dementia. In the last few years several high-throughput screenings have generated a plethora of data, which are difficult to compare due to the different experimental designs and models explored. In this study by using the Affymetrix Exon Arrays, we were able to assess and compare the effects of both TDP-43 and FUS loss-of-function on the whole transcriptome using the same human neuronal SK-N-BE cell model. We showed that TDP-43 and FUS depletion induces splicing and gene expression changes mainly distinct for the two RBPs, although they may regulate common pathways, including neuron differentiation and cytoskeleton organization as evidenced by functional annotation analysis. In particular, TDP-43 and FUS were found to regulate splicing and expression of genes related to neuronal (SEPT6, SULT4A1, TNIK) and RNA metabolism (DICER, ELAVL3/HuC, POLDIP3). Our extended analysis at protein level revealed that these changes have also impact on the protein isoform ratio and content, not always in a direct correlation with transcriptomic data. Contrarily to a loss-of-function mechanism, we showed that mutant TDP-43 proteins maintained their splicing activity in human ALS fibroblasts and experimental cell lines. Our findings further contribute to define the biological functions of these two RBPs in physiological and disease state, strongly encouraging the evaluation of the identified transcriptomic changes at protein level in neuronal experimental models.

Abstract 1079: Transcriptional profiling reveals uterine leiomyoma subtypes with distinct pathways and biomarkers of tumorigenesis

Abstract Uterine leiomyomas, also known as fibroids, are benign smooth muscle tumors with an estimated prevalence of 77% among women of reproductive age. Although benign, they frequently cause a variety of health complications and are the leading cause of hysterectomy worldwide. Recent studies utilizing high-throughput sequencing have revealed recurrent and mutually exclusive mutations in a variety of genes, suggesting that distinct molecular pathways are involved in the initiation of leiomyoma tumorigenesis. The consequences of these mutations are poorly understood and expression signatures associated with these driver mutations may provide further insights. The aim of this study was to explore transcriptional differences between genetic subgroups of leiomyomas utilizing Affymetrix exon arrays and RNA-sequencing. We performed transcriptional profiling of ∼100 leiomyomas harboring different genetic drivers; these included leiomyomas harboring MED12 mutations, HMGA2/HMGA1 rearrangements, biallelic inactivation of FH and COL4A5-COL4A6 deletions. We identified a long non-coding transcript of RAD51B to be specifically upregulated in MED12 mutant leiomyomas. Genes involved in key metabolic pathways, including pentose phosphate pathway and glutathione biosynthesis, were specifically upregulated in FH-deficient leiomyomas. AKR1B10 was the most significantly upregulated gene in leiomyomas of the FH type. This gene was not expressed in other leiomyomas and may serve as a robust biomarker for FH-deficient leiomyomas. Overexpression of the proto-oncogene PLAG1 was specifically seen in leiomyomas harboring HMGA2 or HMGA1 aberrations, suggesting that these tumors exert their tumorigenic effect through dysregulation of PLAG1. This was supported by identification of one uterine leiomyoma harboring a translocation of PLAG1 resulting in a similar overexpression. While this study highlights the importance of stratifying each leiomyoma into a specific subgroup, it also shows that several genes are commonly dysregulated among all leiomyomas. Targeting these genes would be ideal in developing treatments for this disease. Citation Format: Miika Mehine, Eevi Kaasinen, Esa Pitkänen, Nanna Sarvilinna, Hanna-Riikka Heinonen, Netta Mäkinen, Jari Sjöberg, Lauri A. Aaltonen. Transcriptional profiling reveals uterine leiomyoma subtypes with distinct pathways and biomarkers of tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1079. doi:10.1158/1538-7445.AM2015-1079

Abstract P2-05-06: Identification of specific gene signatures and alternative splice variants using exon array in Inflammatory breast Cancer

Abstract Inflammatory Breast Cancer (IBC) is a rare form of breast cancer with a particular phenotype and aggressiveness. Although significant progress has been made in the management of IBC, the prognosis remains poor. Intensive research on IBC has allowed identification of several candidate genes and pathways, but molecular findings unique to IBC have yet to be identified to improve treatment and survival. Transcriptomic studies have revealed a marked heterogeneity of IBC with limited gene overlap between studies, preventing the identification of a sensitive and specific signature in IBC. In order to detect both gene expression levels and alternate RNA splice isoforms, we chose to perform splice-sensitive array profiling using Affymetrix Exon Array in a well-defined series of 33 IBC (core biopsies from previously untreated T4d carcinomas) compared with 28 stage I to non-inflammatory stage III breast cancers. Gene expression analysis allowed the identification of classical molecular subtypes in both IBC and non-IBC, with overrepresentation of basal-like (17/33 vs 9/28) but no luminal A in IBC (vs 11/28). Based on Fold-Change (FC) &amp;gt; 1.5 and p-value &amp;lt; 0.05, 495 genes were significantly dysregulated between IBC and non-IBC, including in particular up-regulated hemoglobin genes and down-regulated ER-related genes in IBC compared to non-IBC. Most activated pathways were hematopoeitic cell lineage, cytokine-cytokine receptor interaction, chemokine signalling pathway and complement and coagulation cascade. We defined a 21-gene signature discriminating IBC from non-IBC with 8% error rate. To get rid of genes associated with molecular subtypes like ER-related genes, an analysis restricted to basal-like BC (17 IBC compared to 9 non-IBC) was performed allowing the definition of a 29-gene signature discriminating the whole groups of IBC from non-IBC with 3.3% error rate. Validation of this signature in the gene expression datasets from the World IBC Consortium will be presented. Specific exon expression analysis revealed that when based on FC splicing-index and p-value, 266 exons representing 177 distinct genes were differentially regulated between IBC and non-IBC. After manually curation of results, 13 splice events representing 12 distinct genes were retained as good candidates for alternative splicing in IBC (EVL, RPL10, MYH10, HSPA8, DOCK7, DIDO1, RPL4, TRAK1, RGS1, LMO4, SMARCA4, ZNF337). To confirm gene-signatures specific to IBC, altered pathways and major splice variants, we are performing a validation study using quantitative RT-PCR in the screening set (n=33) and in an independent series of 140 IBC compared to 200 non-stage-matched non-IBC. Finally the most dysregulated genes will also be studied at the protein level using immunohistochemistry. Citation Format: Florence Lerebours, Sophie Vacher, Marie Le Cann, Sophie Rondeau, David Gentien, Steven Van Laere, François Bertucci, Pierre de la Grange, Ivan Bieche. Identification of specific gene signatures and alternative splice variants using exon array in Inflammatory breast Cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-06.

MP49-13 LIM-SH3 DOMAIN PROTEIN 1 KNOCKDOWN INHIBITS CELL GROWTH AND ENHANCES ACTIVITY OF CISPLATIN IN BLADDER CANCER.

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-13 LIM-SH3 DOMAIN PROTEIN 1 KNOCKDOWN INHIBITS CELL GROWTH AND ENHANCES ACTIVITY OF CISPLATIN IN BLADDER CANCER. Takashi Dejima, Ario Takeuchi, Tetsutaro Hayashi, Jeffrey Leong, Tabitha Tombe, Kevin Tam, Htoo Oo, Peter Black, Seiji Naito, Martin Gleave, and Christopher Ong Takashi DejimaTakashi Dejima More articles by this author , Ario TakeuchiArio Takeuchi More articles by this author , Tetsutaro HayashiTetsutaro Hayashi More articles by this author , Jeffrey LeongJeffrey Leong More articles by this author , Tabitha TombeTabitha Tombe More articles by this author , Kevin TamKevin Tam More articles by this author , Htoo OoHtoo Oo More articles by this author , Peter BlackPeter Black More articles by this author , Seiji NaitoSeiji Naito More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Christopher OngChristopher Ong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.517AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer is the 6th most common cancer in the USA. The current standard therapy for the first line of metastatic or local advanced bladder cancer is combination therapy with cisplatin (CDDP) and gemcitabine (GEM). However 5-years survival is still below 50%; therefore additional therapy is needed. LIM-SH3 domain protein 1 (LASP1), identified from cDNA library of metastatic axillary lymph nodes of breast cancer, has been shown to promote cancer progression and invasion in several malignancies. In bladder cancer, LASP1 expression associates with cell invasion and can be used for detecting bladder cancer. However, the anti-tumor effects of LASP1 knockdown in vivo as well as combination therapy with chemotherapy remain unclear. In this study, we investigated the anti-cancer activity of LASP1 knockdown in bladder cancer. METHODS LASP1 gene expression of tumor samples is analyzed using the Affymetrix Exon Array. The LASP1 expression in several bladder cancer cell lines was assessed by Western blot analysis and quantitative reverse transcription-PCR. Silencing of LASP1 in vitro was achieved using siRNA. The In vivo effect of LASP1 antisense oligonucleotide (ASO) treatment was assessed in the T24 CDDP-R (cisplatin-resistant) orthotopic bladder cancer model. RESULTS High LASP1 expression correlated with recurrence rate between patients receiving adjuvant chemotherapy or not. This difference is increased in patients after adjuvant chemotherapy. The LASP1 expression is higher in UC1 and UC15 cells than in UC13 and UC6 cells. Knockdown of LASP1 using siRNA inhibited cell growth, and was accompanied by an increase in p21 and p27. Conversely, stable LASP1 overexpression drove cell growth with increase of cyclinD1 in UC13 and UC6 cells. The treatment of CDDP and GEM induced LASP1 expression in vitro. Combination treatment with LASP1 knockdown using siRNA and CDDP inhibited cell growth in UC1 cells. Furthermore, compared with parental cell line, LASP1 is higher in T24 CDDP-R and RT112 CDDP-R cells than in parental cells. In the orthotopic bladder cancer model, systemic LASP1 ASO administration to athymic nude mice delayed tumor progression in T24 CDDP-R cells. CONCLUSIONS These data revealed that LASP1 inhibition might be as a promising novel therapeutics modality in the treatment of bladder cancer, as well as a possible synergy with chemotherapy. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e607-e608 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Dejima More articles by this author Ario Takeuchi More articles by this author Tetsutaro Hayashi More articles by this author Jeffrey Leong More articles by this author Tabitha Tombe More articles by this author Kevin Tam More articles by this author Htoo Oo More articles by this author Peter Black More articles by this author Seiji Naito More articles by this author Martin Gleave More articles by this author Christopher Ong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Genome wide transcriptome analysis reveals ABA mediated response in Arabidopsis during gold (AuCl(-) 4) treatment.

The unique physico-chemical properties of gold nanoparticles (AuNPs) find manifold applications in diagnostics, medicine and catalysis. Chemical synthesis produces reactive AuNPs and generates hazardous by-products. Alternatively, plants can be utilized to produce AuNPs in an eco-friendly manner. To better control the biosynthesis of AuNPs, we need to first understand the detailed molecular response induced by AuCl−4 In this study, we carried out global transcriptome analysis in root tissue of Arabidopsis grown for 12- h in presence of gold solution (HAuCl4) using the novel unbiased Affymetrix exon array. Transcriptomics analysis revealed differential regulation of a total of 704 genes and 4900 exons. Of these, 492 and 212 genes were up- and downregulated, respectively. The validation of the expressed key genes, such as glutathione-S-transferases, auxin responsive genes, cytochrome P450 82C2, methyl transferases, transducin (G protein beta subunit), ERF transcription factor, ABC, and MATE transporters, was carried out through quantitative RT-PCR. These key genes demonstrated specific induction under AuCl4− treatment relative to other heavy metals, suggesting a unique plant-gold interaction. GO enrichment analysis reveals the upregulation of processes like oxidative stress, glutathione binding, metal binding, transport, and plant hormonal responses. Changes predicted in biochemical pathways indicated major modulation in glutathione mediated detoxification, flavones and derivatives, and plant hormone biosynthesis. Motif search analysis identified a highly significant enriched motif, ACGT, which is an abscisic acid responsive core element (ABRE), suggesting the possibility of ABA- mediated signaling. Identification of abscisic acid response element (ABRE) points to the operation of a predominant signaling mechanism in response to AuCl−4 exposure. Overall, this study presents a useful picture of plant-gold interaction with an identification of candidate genes involved in nanogold synthesis.

Abstract 3413: A genetic analysis of splicing in neuroblastoma identifies opposing functions for FUBP1 splice variants in MYC regulation

Abstract Neuroblastoma, a tumor of the peripheral neural crest, is the most common cancer of infancy and the most common extracranial solid tumor of childhood. Large scale sequencing efforts have identified recurrent mutations in only a handful of genes in a minority of patients, but these efforts have so far focused on the exome, limiting identification of mutations leading to changes in alternative splicing. Regulation of alternative splicing is a critical and tightly regulated cellular function involving a complex interplay between cis-acting sequences on the pre-mRNA transcript and accessory trans-acting splicing factors. To elucidate genetic control of splicing, we used a splicing quantitative trait loci (sQTL) analysis in a backcrossed mouse model of neuroblastoma. We utilized Affymetrix Exon Arrays to profile mRNA expression at the exon level for superior cervical ganglia and cerebella (n=102) which we coupled with genotypes at 350 SNP and microsattlelite locations. With whole-genome sequencing data available for the inbred parental strains, our approach provided us with localized sequences for each individual mouse. In addition to identifying novel candidate trans-acting splicing factors, these sQTL identify novel candidate intronic regulatory sequences critical for splicing and reveal strain-specific splicing events leading to functional consequences in neuroblastoma. Among these, we show that a subtle splicing event within FUBP1 modulates levels of the MYC oncoprotein in human neuroblastoma-derived cell lines and correlates with outcome in children with neuroblastoma. Citation Format: Justin Chen, Christopher S. Hackett, Shile Zhang, Young K. Song, Annette Molinaro, David A. Quigley, Allan Balmain, William C. Gustafson, Terry A. Van Dyke, Pui-Yan Kwok, Javed Khan, William A. Weiss. A genetic analysis of splicing in neuroblastoma identifies opposing functions for FUBP1 splice variants in MYC regulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3413. doi:10.1158/1538-7445.AM2014-3413

Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones.

Loss of nuclear TDP-43 characterizes sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether (1) RNA splicing dysregulation is present in lower motor neurones in ALS and in a motor neurone-like cell model; and (2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurones obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. We found altered expression of spliceosome components in motor neurones and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43-depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, which were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurones, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism.

Global molecular changes in rat livers treated with RXR agonists: a comparison using transcriptomics and proteomics

AbstractThe ability of the retinoid X receptors (RXRs) specific agonists (targretin [TRG] and UAB30) to alter rat liver gene and protein expression was determined using Affymetrix Exon arrays and high‐performance liquid chromatography – tandem mass spectrometry (LC‐MS/MS). TRG profoundly increases triglycerides levels while UAB30 does not. The expression patterns of transcripts or proteins from rat liver treated with TRG or UAB‐30 were different from controls and each other. There were six times more gene transcripts identified than proteins. Differentially expressed RNAs or proteins were mapped into known gene ontology (GO) categories and GeneGo Metacore (KEGG) pathway maps. The GO categories which were highly overrepresented with differentially expressed RNAs (P &lt; 10−16) were also overrepresented at the protein level. This high concordance of GO Terms was achieved despite the fact that typically ≤1/3 of the elements identified by gene expression were identified by proteomics. Within these GO categories, the magnitude of alterations induced by RXR agonists at the transcript and protein levels were correlated. When GO categories with moderate overrepresentation (10−5 &lt; P &lt; 10−9) were examined, there was greater discordance between the transcript and protein data. Examination of KEGG pathway maps with highly significant changes at both the protein and the RNA levels showed that the individual proteins/genes altered were often the same and changes were of similar magnitude; while KEGG pathways showed limited statistical significance and exhibited minimal overlap. Finally, metabolomics analysis of liver and serum identified altered expression of metabolites related to fatty acid oxidation and bile acid metabolism that were consistent with transcript/protein changes. We observed significant concordance between genomics and proteomics implying either can identify pathways modulated and can indirectly predict resulting physiologic effects.

Genetic Association Of a MAPK8 Expression Quantitative Trait Locus With Pre-Capillary Pulmonary Hypertension In Sickle Cell Disease

Sickle cell disease (SCD) is associated with pleiotropic clinical outcomes, the severity of which exhibits remarkable inter-individual variability. Investigations of the pathophysiology of SCD have focused on the adverse effects of vaso-occlusion, chronic inflammation, and hemolysis. SCD is also characterized by up-regulation of the hypoxic response to chronic anemia. We postulated that the up-regulated hypoxic response in SCD contributes to altered gene expression that might impact pulmonary hypertension, a complication associated with early mortality.To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 sickle cell anemia untreated with hydroxyurea and 15 Chuvash polycythemia (CP) patients characterized by homozygous VHLR200W-induced constitutive up-regulation of hypoxia inducible factors in the absence of anemia or hypoxia. Gene expression of both cohorts was profiled on identical Affymetrix exon arrays. The degree and direction of differential gene expression were highly correlated between sickle cell anemia and CP (Spearman's ρ = 0.73 between regression coefficients of differential gene expression), suggesting that 53% of expression variation in sickle cell anemia is related to hypoxic transcriptional responses. At 5% false discovery rate (FDR), 1040 genes exhibited a >1.15 fold change in both sickle cell anemia and VHLR200W homozygotes, among which 297 were up-regulated and 743 down-regulated. Hypoxia strongly induced inflammatory response pathways but suppressed T-cell activation in sickle cell anemia. MAPK8, encoding a mitogen-activated protein kinase important for stress-induced apoptosis, T-cell differentiation and inflammatory responses, was a hypoxia down-regulated gene and played a central role in hypoxic gene regulation in sickle cell anemia according to gene network analysis.To assess the genetic contribution to hypoxic transcriptional variation among SCD patients, we mapped expression quantitative trait loci (eQTL) for the 1,040 hypoxia response genes. Association mapping with a focus on local regulatory polymorphisms in 61 SCD patients identified eQTL for 103 of the hypoxia response genes at 5% FDR. We further tested the hypothesis that these hypoxic eQTL potentially underlie heterogeneity in risk of pulmonary hypertension in an additional SCD cohort (University of Illinois cohort). In this cohort, the A allele of an eQTL of MAPK8, rs10857560, was associated with pre-capillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency=0.66; OR=4.4, P=0.00037, n=238). This association was confirmed in another independent cohort (Walk-PHaSST cohort) (allele frequency=0.65; OR=7.2, P=0.0025, n=519). The homozygous AA genotype of rs10857560, which was associated with decreased MAPK8 expression, was present in all 14 identified pre-capillary pulmonary hypertension cases among the combined 757 SCD patients (P = 6 x 10-6 by the Fisher exact test). Our study demonstrates a prominent hypoxic transcription component in SCD that in part contributes to pre-capillary pulmonary hypertension. Disclosures:No relevant conflicts of interest to declare.

Genome-Wide Analysis of Differentially Expressed Genes and Splicing Isoforms in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common malignant renal epithelial tumor and also the most deadly. To identify molecular changes occurring in ccRCC, in the present study we performed a genome wide analysis of its entire complement of mRNAs. Gene and exon-level analyses were carried out by means of the Affymetrix Exon Array platform. To achieve a reliable detection of differentially expressed cassette exons we implemented a novel methodology that considered contiguous combinations of exon triplets and candidate differentially expressed cassette exons were identified when the expression level was significantly different only in the central exon of the triplet. More detailed analyses were performed for selected genes using quantitative RT-PCR and confocal laser scanning microscopy. Our analysis detected over 2,000 differentially expressed genes, and about 250 genes alternatively spliced and showed differential inclusion of specific cassette exons comparing tumor and non-tumoral tissues. We demonstrated the presence in ccRCC of an altered expression of the PTP4A3, LAMA4, KCNJ1 and TCF21 genes (at both transcript and protein level). Furthermore, we confirmed, at the mRNA level, the involvement of CAV2 and SFRP genes that have previously been identified. At exon level, among potential candidates we validated a differentially included cassette exon in DAB2 gene with a significant increase of DAB2 p96 splice variant as compared to the p67 isoform. Based on the results obtained, and their robustness according to both statistical analysis and literature surveys, we believe that a combination of gene/isoform expression signature may remarkably contribute, after suitable validation, to a more effective and reliable definition of molecular biomarkers for ccRCC early diagnosis, prognosis and prediction of therapeutic response.

Abstract A117: Vitamin D, parathyroid hormone, Cyp2r1, and breast cancer susceptibility in mice

Abstract Vitamin D is a group of fat-soluble secosteroids. Apart from its main physiological role in calcium homeostasis, several studies have demonstrated a wide range of functions of vitamin D which can be associated with cancer. Even though the exact mechanism underlying the protective action of vitamin D against cancer is not clearly understood, there is strong experimental evidence showing that vitamin D participates in cell growth regulation, apoptosis and proliferation in normal and malignant breast cells. This evidence has lead to the hypothesis that high levels of vitamin D might reduce the risk of breast cancer. In order to be biologically active, vitamin D must be converted to its active form 1,25(OH)2D3. CYP2R1 is a major vitamin D hydroxylase that catalyzes the first step of this activation producing 25(OH)D3. Cyp2r1 is located within the SuprMam1 locus, a mammary tumour susceptibility locus identified in the BALB/c-Trp53+/- mouse model of spontaneous breast cancer (1). Affymetrix data comparing T cell gene expression have identified Cyp2r1 to be differentially expressed between BALB/c and C57BL/6 mouse strains (0.68 fold reduction in BALB/c, the mammary tumour susceptible strain). As lower CYP2R1 levels should result in lower vitamin D levels and may contribute to the higher cancer susceptibility of BALB/c mice, we studied the Cyp2r1 gene expression and plasma 25(OH)D3 levels in SM09 congenic-mice (BALB/c SuprMam loci in C57BL/6 background) in comparison to C57BL/6-controls. A real-time quantitative PCR of Cyp2r1 in different tissues from SM09 and control mice revealed that there is a significant difference in Cyp2r1 expression in mammary glands of the two strains (2.76 fold reduction in SM09, p=0.04). But this difference was not observed in liver or lymph node tissues of the same mice. Further investigation of the major regulators of vitamin D pathway found that differences in plasma 25(OH)D3 levels were not reliably detected, and there was no difference in plasma phosphate or calcium levels between the congenic and control mouse strains. However, 3-fold higher levels of parathyroid hormone (Pth) were measured in female mice carrying the BALB/c allele of the SuprMam1 locus. Further, expression profiling of the major genes involved in the vitamin D pathway by affymetrix exon array on mammary glands from SM09 and control mice indicated many were differentially expressed, indicating disruption of the vitamin D pathway. Further investigations are underway to determine if genetic differences in the Pth or Cyp2R1 gene are responsible for this disruption, and if this is of biological significance for tumour growth in vivo. 1. Blackburn AC, Hill LZ, Roberts AL, et al. Genetic mapping in mice identifies DMBT1 as a candidate modifier of mammary tumors and breast cancer risk. Am J Pathol. 2007; 170:2030–41. Citation Format: Madara Ratnadiwakara, Rohan B. Williams, Anneke C. Blackburn. Vitamin D, parathyroid hormone, Cyp2r1, and breast cancer susceptibility in mice. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A117.

Human Induced Pluripotent Stem Cells As a Tool to Model a Form of Leber Congenital Amaurosis

Our purpose was to investigate genes and molecular mechanisms involved in patients with Leber congenital amaurosis (LCA) and to model this type of LCA for drug screening. Fibroblasts from two unrelated clinically identified patients with a yet undetermined gene mutation were reprogrammed to pluripotency by retroviral transduction. These human induced pluripotent stem cells (hiPSCs) were differentiated into neural stem cells (NSCs) that mimicked the neural tube stage and retinal pigmented epithelial (RPE) cells that could be targeted by the disease. A genome-wide transcriptome analysis was performed with Affymetrix Exon Array GeneChip(®), comparing LCA-hiPSCs derivatives to controls. A genomic search for alteration in all genes known to be involved in LCA revealed a common polymorphism on the GUCY2D gene, referenced as the LCA type I (OMIM *600179 and #204000), but the causative gene remained unknown. The hiPSCs expressed the key pluripotency factors and formed embryoid bodies in vitro containing cells originating from all three germ layers. They were successfully differentiated into NSC and RPE cells. One gene, NNAT, was upregulated in LCA cell populations, and three genes were downregulated, GSTT1, TRIM61 and ZNF558, with potential correlates for molecular mechanisms of this type of LCA, in particular for protein degradation and oxidative stress. The two LCA patient-specific iPSC lines will contribute to modeling LCA phenotypes and screening candidate drugs.