Omics Analysis of Educated Platelets in Cancer and Benign Disease of the Pancreas

Simple Summary Tumor cells are known to produce and secrete pro-coagulants that recruit blood particles such as platelets, inducing hypercoagulability. However, platelets can also influence tumor carcinogenesis and metastasis, creating a reciprocal, vicious loop with the tumors. Confrontation of platelets with tumor cells via transfer of tumor-associated biomolecules or influencing platelets biology (“education”) is an emerging concept, that has been recently proposed to create innovative platforms for biomarkers within blood-based “liquid biopsies”. In this study, we explore the intrinsic regulation and the potential “education” of platelets using -omics profiling in pancreatic cancer patients. Our results showed: (i) a high activity on RNA splicing that can lead to subsequent platelets education; (ii) enrichment of specific modified forms (isomiRs) of canonical miRNAs; and (iii) inhibition of SPARC transcription by specific class of isomiRs. Moreover, we created an interactive tool to visualize expected correlations, to facilitate further investigations on additional potential biomarkers and therapeutic tools. Abstract Pancreatic ductal adenocarcinoma (PDAC) is traditionally associated with thrombocytosis/hypercoagulation and novel insights on platelet-PDAC “dangerous liaisons” are warranted. Here we performed an integrative omics study investigating the biological processes of mRNAs and expressed miRNAs, as well as proteins in PDAC blood platelets, using benign disease as a reference for inflammatory noise. Gene ontology mining revealed enrichment of RNA splicing, mRNA processing and translation initiation in miRNAs and proteins but depletion in RNA transcripts. Remarkably, correlation analyses revealed a negative regulation on SPARC transcription by isomiRs involved in cancer signaling, suggesting a specific ”education” in PDAC platelets. Platelets of benign patients were enriched for non-templated additions of G nucleotides (#ntaG) miRNAs, while PDAC presented length variation on 3′ (lv3p) as the most frequent modification on miRNAs. Additionally, we provided an actionable repertoire of PDAC and benign platelet-ome to be exploited for future studies. In conclusion, our data show that platelets change their biological repertoire in patients with PDAC, through dysregulation of miRNAs and splicing factors, supporting the presence of de novo protein machinery that can “educate” the platelet. These novel findings could be further exploited for innovative liquid biopsies platforms as well as possible therapeutic targets.

[1]  T. Wurdinger,et al.  Tumor-Educated Platelet RNA for the Detection and (Pseudo)progression Monitoring of Glioblastoma , 2020, Cell reports. Medicine.

[2]  E. Giovannetti,et al.  Co-expression analysis of pancreatic cancer proteome reveals biology and prognostic biomarkers , 2020, Cellular Oncology.

[3]  B. Tannous,et al.  Blood platelet RNA enables the detection of multiple sclerosis , 2020, Multiple sclerosis journal - experimental, translational and clinical.

[4]  Juan Peng,et al.  Role of PI3K/AKT pathway in cancer: the framework of malignant behavior , 2020, Molecular Biology Reports.

[5]  T. Wurdinger,et al.  RNA-Sequencing of Tumor-Educated Platelets, A Novel Biomarker for Blood Based Sarcoma Diagnostics. , 2020 .

[6]  Alex A Henneman,et al.  iq: an R package to estimate relative protein abundances from ion quantification in DIA-MS-based proteomics , 2020, Bioinform..

[7]  X. Liu,et al.  Early detection of pancreatic ductal adenocarcinoma using methylation signatures in circulating tumour DNA , 2019, Annals of Oncology.

[8]  S. Carr,et al.  Proteomic analyses of ECM during pancreatic ductal adenocarcinoma progression reveal different contributions by tumor and stromal cells , 2019, Proceedings of the National Academy of Sciences.

[9]  E. Giovannetti,et al.  Unravelling the Diagnostic Dilemma: A MicroRNA Panel of Circulating MiR-16 and MiR-877 as A Diagnostic Classifier for Distal Bile Duct Tumors , 2019, Cancers.

[10]  T. Wurdinger,et al.  Tumor-educated platelets. , 2019, Blood.

[11]  A. Saraya,et al.  Tu2033 – Quantitation of Methylation Load of Tumor Suppressor Gene Promoter Methylation in Pancreatic Cancer , 2019, Gastroenterology.

[12]  T. Wurdinger,et al.  RNA sequencing and swarm intelligence–enhanced classification algorithm development for blood-based disease diagnostics using spliced blood platelet RNA , 2019, Nature Protocols.

[13]  R. Turkington,et al.  Pancreatic cancer: A review of clinical diagnosis, epidemiology, treatment and outcomes , 2018, World journal of gastroenterology.

[14]  A. Dziembowski,et al.  5′ and 3′ modifications controlling RNA degradation: from safeguards to executioners , 2018, Philosophical Transactions of the Royal Society B: Biological Sciences.

[15]  E. Giovannetti,et al.  Abstract 2596: Spliced messenger RNA of tumor-educated platelets yields a new diagnostic prospective for pancreatic cancer , 2018, Clinical Research (Excluding Clinical Trials).

[16]  C. Dubois,et al.  Effects of platelets on cancer progression. , 2018, Thrombosis research.

[17]  G. Cimmino,et al.  Splicing of platelet resident pre-mRNAs upon activation by physiological stimuli results in functionally relevant proteome modifications , 2018, Scientific Reports.

[18]  Lei Li,et al.  Integrating multiple fitting regression and Bayes decision for cancer diagnosis with transcriptomic data from tumor-educated blood platelets. , 2017, The Analyst.

[19]  M. Makary,et al.  Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers , 2017, Proceedings of the National Academy of Sciences.

[20]  K. Koike,et al.  Thromboembolisms in Advanced Pancreatic Cancer: A Retrospective Analysis of 475 Patients. , 2017, Pancreas.

[21]  E. Giovannetti,et al.  Swarm Intelligence-Enhanced Detection of Non-Small-Cell Lung Cancer Using Tumor-Educated Platelets , 2017, Cancer cell.

[22]  T. Wurdinger,et al.  Platelet RNA as a circulating biomarker trove for cancer diagnostics , 2017, Journal of thrombosis and haemostasis : JTH.

[23]  E. Giovannetti,et al.  Never let it go: Stopping key mechanisms underlying metastasis to fight pancreatic cancer. , 2017, Seminars in cancer biology.

[24]  M. Mayr,et al.  MicroRNA Biomarkers and Platelet Reactivity: The Clot Thickens , 2017, Circulation research.

[25]  Hyunju Lee,et al.  Integration of MicroRNA, mRNA, and Protein Expression Data for the Identification of Cancer-Related MicroRNAs , 2017, PloS one.

[26]  R. Xu,et al.  The TGF-β/Smad4 Signaling Pathway in Pancreatic Carcinogenesis and Its Clinical Significance , 2017, Journal of clinical medicine.

[27]  Christina Backes,et al.  miEAA: microRNA enrichment analysis and annotation , 2016, Nucleic Acids Res..

[28]  T. Wurdinger,et al.  Non-invasive prostate cancer detection by measuring miRNA variants (isomiRs) in urine extracellular vesicles , 2016, Oncotarget.

[29]  Henrik Zetterberg,et al.  Identification of tissue-specific cell death using methylation patterns of circulating DNA , 2016, Proceedings of the National Academy of Sciences.

[30]  Jiuyong Li,et al.  Identification of miRNA-mRNA regulatory modules by exploring collective group relationships , 2016, BMC Genomics.

[31]  A. Andrén-sandberg,et al.  Pancreatic cancer and thromboembolic disease, 150 years after Trousseau. , 2015, Hepatobiliary surgery and nutrition.

[32]  M. O. oude Egbrink,et al.  Platelets: an unexploited data source in biomarker research. , 2015, The Lancet. Haematology.

[33]  Pieter Wesseling,et al.  RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics , 2015, Cancer cell.

[34]  E. Ma,et al.  Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments , 2015, Proceedings of the National Academy of Sciences.

[35]  A. Franco,et al.  Platelets at the interface of thrombosis, inflammation, and cancer. , 2015, Blood.

[36]  D. Bartel,et al.  Predicting effective microRNA target sites in mammalian mRNAs , 2015, eLife.

[37]  Michael Hackenberg,et al.  sRNAtoolbox: an integrated collection of small RNA research tools , 2015, Nucleic Acids Res..

[38]  G. Cimmino,et al.  Activating stimuli induce platelet microRNA modulation and proteome reorganisation , 2015, Thrombosis and Haemostasis.

[39]  B. Frey,et al.  Widespread intron retention in mammals functionally tunes transcriptomes , 2014, Genome research.

[40]  B. Ylstra,et al.  Nontemplated nucleotide additions distinguish the small RNA composition in cells from exosomes. , 2014, Cell reports.

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

[42]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..

[43]  S. Nie,et al.  Target proteomic profiling of frozen pancreatic CD24+ adenocarcinoma tissues by immuno-laser capture microdissection and nano-LC-MS/MS. , 2013, Journal of proteome research.

[44]  M. Periyasamy,et al.  miR-23b regulates cytoskeletal remodeling, motility and metastasis by directly targeting multiple transcripts , 2013, Nucleic acids research.

[45]  P. Gunaratne,et al.  The Repertoire and Features of Human Platelet microRNAs , 2012, PloS one.

[46]  Q. Ye,et al.  microRNA-29a suppresses cell proliferation by targeting SPARC in hepatocellular carcinoma. , 2012, International journal of molecular medicine.

[47]  Ugo Boggi,et al.  High-Throughput MicroRNA (miRNAs) Arrays Unravel the Prognostic Role of MiR-211 in Pancreatic Cancer , 2012, PloS one.

[48]  Chen Wang,et al.  A combined approach identifies three mRNAs that are down-regulated by microRNA-29b and promote invasion ability in the breast cancer cell line MCF-7 , 2012, Journal of Cancer Research and Clinical Oncology.

[49]  Min Pan,et al.  A miRNA-regulatory network explains how dysregulated miRNAs perturb oncogenic processes across diverse cancers , 2012, Genome research.

[50]  Nahum Sonenberg,et al.  The mechanics of miRNA-mediated gene silencing: a look under the hood of miRISC , 2012, Nature Structural &Molecular Biology.

[51]  I. Baek,et al.  Preliminary Study Analyzing the Methylated Genes in the Plasma of Patients with Pancreatic Cancer , 2012, Scandinavian journal of surgery : SJS : official organ for the Finnish Surgical Society and the Scandinavian Surgical Society.

[52]  Lars Bullinger,et al.  Integrative nucleophosmin mutation-associated microRNA and gene expression pattern analysis identifies novel microRNA - target gene interactions in acute myeloid leukemia , 2011, Haematologica.

[53]  T. Wurdinger,et al.  Blood platelets contain tumor-derived RNA biomarkers. , 2011, Blood.

[54]  Renato V Iozzo,et al.  Proteoglycans in cancer biology, tumour microenvironment and angiogenesis , 2011, Journal of cellular and molecular medicine.

[55]  M. O. oude Egbrink,et al.  The Role of Blood Platelets in Tumor Angiogenesis , 1985 .

[56]  Sonja Gillen,et al.  Reduced expression of the membrane skeleton protein beta1-spectrin (SPTBN1) is associated with worsened prognosis in pancreatic cancer. , 2010, Histology and histopathology.

[57]  Ugo Boggi,et al.  MicroRNA-21 in pancreatic cancer: correlation with clinical outcome and pharmacologic aspects underlying its role in the modulation of gemcitabine activity. , 2010, Cancer research.

[58]  Connie R. Jimenez,et al.  On the beta-binomial model for analysis of spectral count data in label-free tandem mass spectrometry-based proteomics , 2010, Bioinform..

[59]  D. Wagner,et al.  Platelets: guardians of tumor vasculature. , 2009, Cancer research.

[60]  D. Tollervey,et al.  The Many Pathways of RNA Degradation , 2009, Cell.

[61]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[62]  Paul Ahlquist,et al.  MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins , 2008, Proceedings of the National Academy of Sciences.

[63]  C. Brodie,et al.  SPARC-induced increase in glioma matrix and decrease in vascularity are associated with reduced VEGF expression and secretion , 2008, International journal of cancer.

[64]  E. Izaurralde,et al.  Getting to the Root of miRNA-Mediated Gene Silencing , 2008, Cell.

[65]  S. Barni,et al.  Venous thromboembolism predicts poor prognosis in irresectable pancreatic cancer patients. , 2007, Annals of oncology : official journal of the European Society for Medical Oncology.

[66]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[67]  K. Swoboda,et al.  Escaping the Nuclear Confines: Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets , 2005, Cell.

[68]  D. Dixon,et al.  Change in protein phenotype without a nucleus: translational control in platelets. , 2004, Seminars in thrombosis and hemostasis.

[69]  Enrique Casado,et al.  PI3K/Akt signalling pathway and cancer. , 2004, Cancer treatment reviews.

[70]  E Helene Sage,et al.  Enhanced expression of SPARC/osteonectin in the tumor-associated stroma of non-small cell lung cancer is correlated with markers of hypoxia/acidity and with poor prognosis of patients. , 2003, Cancer research.

[71]  R. Hruban,et al.  SPARC/osteonectin is a frequent target for aberrant methylation in pancreatic adenocarcinoma and a mediator of tumor–stromal interactions , 2003, Oncogene.

[72]  K Motamed,et al.  SPARC (osteonectin/BM-40). , 1999, The international journal of biochemistry & cell biology.

[73]  J. Gorski,et al.  Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction. , 1988, The Journal of clinical investigation.

[74]  Samuel C. Belete,et al.  Circulating Tumor Cells and Cell-Free DNA in Pancreatic Ductal Adenocarcinoma. , 2019, The American journal of pathology.

[75]  Evan S. Glazer,et al.  TGF-β Inhibitors in Metastatic Pancreatic Ductal Adenocarcinoma , 2019, Journal of Gastrointestinal Cancer.

[76]  L. Edelstein,et al.  Noncoding RNAs in Platelet Biology , 2017 .

[77]  E. Giovannetti,et al.  Role of CYB5A in pancreatic cancer prognosis and autophagy modulation. , 2014, Journal of the National Cancer Institute.

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

[79]  J. Dunn,et al.  Transcript profiling of human platelets using microarray and serial analysis of gene expression. , 2003, Blood.

[80]  A. Falanga,et al.  Activation of clotting factors in cancer. , 2009, Cancer treatment and research.

[81]  Wolfgang Schima,et al.  Pancreatic adenocarcinoma , 2006, European Radiology.

[82]  D. Botstein,et al.  Exploring the new world of the genome with DNA microarrays , 1999, Nature Genetics.