Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids

Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transitamplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis. Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited because of the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles, we performed in vitro aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50 μmol/L aspirin or vehicle control for 72 hours and subjected to bulk RNA sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant (q < 0.10) DEGs prior to deconvolution. We provide replication of these findings in an independent population-based RNA-sequencing dataset of mucosal biopsies (BarcUVa-Seq), where a significant enrichment for overlap of DEGs was observed (P < 2.2E−16). Single-cell deconvolution revealed changes in cell composition, including a decrease in transit-amplifying cells following aspirin treatment (P = 0.01). Following deconvolution, DEGs included novel putative targets for aspirin such as TRABD2A (q = 0.055), a negative regulator of Wnt signaling. Weighted gene co-expression network analysis identified 12 significant modules, including two that contained hubs for EGFR and PTGES2, the latter being previously implicated in aspirin chemoprevention. In summary, aspirin treatment of patient-derived colon organoids using physiologically relevant doses resulted in transcriptome-wide changes that reveal altered cell composition and improved understanding of transcriptional pathways, providing novel insight into its chemopreventive properties. Prevention Relevance: Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transit-amplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis.

[1]  G. Casey,et al.  Controlling for cellular heterogeneity using single-cell deconvolution of gene expression reveals novel markers of colorectal tumors exhibiting microsatellite instability , 2021, Oncotarget.

[2]  Stephanie A. Bien,et al.  Genetic Effects on Transcriptome Profiles in Colon Epithelium Provide Functional Insights for Genetic Risk Loci , 2021, Cellular and molecular gastroenterology and hepatology.

[3]  G. Casey,et al.  Ethanol exposure drives colon location specific cell composition changes in a normal colon crypt 3D organoid model , 2021, Scientific reports.

[4]  David A. Drew,et al.  Aspirin in the Prevention of Colorectal Neoplasia. , 2020, Annual review of medicine.

[5]  O. Sansom,et al.  Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1 , 2020, Cellular and molecular gastroenterology and hepatology.

[6]  N. Nishioka,et al.  Effect of Low-dose and Standard-dose Aspirin on PGE2 Biosynthesis Among Individuals with Colorectal Adenomas: A Randomized Clinical Trial , 2020, Cancer Prevention Research.

[7]  R. Kalla,et al.  Faculty Opinions recommendation of Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis. , 2020 .

[8]  S. Gallus,et al.  TEMPORARY REMOVAL: ASPIRIN AND THE RISK OF COLORECTAL AND OTHER DIGESTIVE TRACT CANCERS: AN UPDATED META-ANALYSIS THROUGH 2019. , 2020, Annals of oncology : official journal of the European Society for Medical Oncology.

[9]  Stephanie A. Bien,et al.  Modeling the effect of prolonged ethanol exposure on global gene expression and chromatin accessibility in normal 3D colon organoids , 2020, PloS one.

[10]  H. Weiler,et al.  The Adult Murine Intestine is Dependent on Constitutive Laminin-γ1 Synthesis , 2019, Scientific Reports.

[11]  Aviv Regev,et al.  Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis , 2019, Cell.

[12]  G. Toffolo,et al.  Obesity is associated with impaired responsiveness to once‐daily low‐dose aspirin and in vivo platelet activation , 2019, Journal of thrombosis and haemostasis : JTH.

[13]  Ash A. Alizadeh,et al.  Determining cell-type abundance and expression from bulk tissues with digital cytometry , 2019, Nature Biotechnology.

[14]  I. Nicholl,et al.  A novel mechanism for the anticancer activity of aspirin and salicylates , 2019, International journal of oncology.

[15]  Damian Szklarczyk,et al.  STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets , 2018, Nucleic Acids Res..

[16]  Helen E. Parkinson,et al.  The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019 , 2018, Nucleic Acids Res..

[17]  R. Wolff,et al.  Power in pairs: assessing the statistical value of paired samples in tests for differential expression , 2018, BMC Genomics.

[18]  Christoph Hafemeister,et al.  Comprehensive integration of single cell data , 2018, bioRxiv.

[19]  Mathieu Lemire,et al.  Discovery of common and rare genetic risk variants for colorectal cancer , 2018, Nature Genetics.

[20]  N. Cook,et al.  Effects of aspirin on risks of vascular events and cancer according to bodyweight and dose: analysis of individual patient data from randomised trials , 2018, The Lancet.

[21]  Á. Lanas,et al.  New use of low-dose aspirin and risk of colorectal cancer by stage at diagnosis: a nested case–control study in UK general practice , 2017, BMC Cancer.

[22]  A. Kong,et al.  Mechanisms of colitis‐accelerated colon carcinogenesis and its prevention with the combination of aspirin and curcumin: Transcriptomic analysis using RNA‐seq , 2017, Biochemical pharmacology.

[23]  N. Gassler,et al.  Low acyl-CoA synthetase 5 expression in colorectal carcinomas is prognostic for early tumour recurrence. , 2017, Pathology, research and practice.

[24]  Emma M Schatoff,et al.  WNT Signaling and Colorectal Cancer , 2017, Current Colorectal Cancer Reports.

[25]  Jiang Chang,et al.  A functional polymorphism located at transcription factor binding sites, rs6695837 near LAMC1 gene, confers risk of colorectal cancer in Chinese populations , 2016, Carcinogenesis.

[26]  Tri M Phan,et al.  Unlocking Aspirin's Chemopreventive Activity: Role of Irreversibly Inhibiting Platelet Cyclooxygenase-1 , 2016, Cancer Prevention Research.

[27]  Lian-xin Wang,et al.  Significant Modules and Biological Processes between Active Components of Salvia miltiorrhiza Depside Salt and Aspirin , 2016, Evidence-based complementary and alternative medicine : eCAM.

[28]  Yin Cao,et al.  Aspirin and colorectal cancer: the promise of precision chemoprevention , 2016, Nature Reviews Cancer.

[29]  Zhongming Zhao,et al.  ccmGDB: a database for cancer cell metabolism genes , 2015, Nucleic Acids Res..

[30]  Tanya Barrett,et al.  The Gene Expression Omnibus Database , 2016, Statistical Genomics.

[31]  Jeffrey S. Morris,et al.  The Consensus Molecular Subtypes of Colorectal Cancer , 2015, Nature Medicine.

[32]  R. Wolff,et al.  Differential Gene Expression in Colon Tissue Associated With Diet, Lifestyle, and Related Oxidative Stress , 2015, PloS one.

[33]  J. Potter,et al.  Tissue-specific patterns of gene expression in the epithelium and stroma of normal colon in healthy individuals in an aspirin intervention trial , 2015, BMC Medical Genetics.

[34]  Matthew E. Ritchie,et al.  limma powers differential expression analyses for RNA-sequencing and microarray studies , 2015, Nucleic acids research.

[35]  M. Gala,et al.  Molecular Pathways: Aspirin and Wnt Signaling—A Molecularly Targeted Approach to Cancer Prevention and Treatment , 2014, Clinical Cancer Research.

[36]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[37]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[38]  Á. Lanas,et al.  Reappraisal of the clinical pharmacology of low‐dose aspirin by comparing novel direct and traditional indirect biomarkers of drug action , 2014, Journal of thrombosis and haemostasis : JTH.

[39]  Hiroyuki Miyoshi,et al.  In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture , 2013, Nature Protocols.

[40]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[41]  Alexander van Oudenaarden,et al.  The Lgr 5 intestinal stem cell signature : robust expression of proposed quiescent ‘ þ 4 ’ cell markers , 2012 .

[42]  A. Zauber,et al.  Colonoscopic polypectomy and long-term prevention of colorectal-cancer deaths. , 2012, The New England journal of medicine.

[43]  Hans Clevers,et al.  Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. , 2011, Gastroenterology.

[44]  J. Roh,et al.  Prolonged Use of Aspirin Alters Human and Rat Intestinal Cells and Thereby Limits the Absorption of Clopidogrel , 2011, Clinical pharmacology and therapeutics.

[45]  Takuji Tanaka,et al.  Selective PGE2 Suppression Inhibits Colon Carcinogenesis and Modifies Local Mucosal Immunity , 2011, Cancer Prevention Research.

[46]  H. Ishwaran,et al.  CEACAM-7: a predictive marker for rectal cancer recurrence. , 2010, Surgery.

[47]  E. Birney,et al.  Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt , 2009, Nature Protocols.

[48]  Jing Chen,et al.  ToppGene Suite for gene list enrichment analysis and candidate gene prioritization , 2009, Nucleic Acids Res..

[49]  Steve Horvath,et al.  WGCNA: an R package for weighted correlation network analysis , 2008, BMC Bioinformatics.

[50]  Shuji Ogino,et al.  Aspirin and the risk of colorectal cancer in relation to the expression of COX-2. , 2007, The New England journal of medicine.

[51]  J. Meyerhardt,et al.  Long-term use of aspirin and nonsteroidal anti-inflammatory drugs and risk of colorectal cancer. , 2005, JAMA.