Transcriptomics and Genomics in Prostate Cancer Prof. Dr. Guido Jenster Experimental Urological Oncology Erasmus MC Rotterdam [email protected] Basic Research: The prostate cancer problem Transcriptomics and genomics of prostate cancer - What is the PCa problem and how to address it? - DNA sequencing - Common, rare and private mutations - Personalized Medicine - Metastasis - RNA sequencing - Small RNAs - Long RNAs - Liquid biopsies The prostate cancer problem How to address the prostate cancer problem: Early detection and cure via Hormone-, chemo-, immuno- Prevention surgery/radiation therapy therapy Bone metastasis Functional: Markers: Therapy targets: Understanding the -cancer risk? -which new medicines? components and -cancer present? -which order and combination? processes responsible -treatment needed? -therapy resistance? for cancer development -which treatment? and progression -treatment successful? Markers and therapy targets for prostate cancer Markers and therapy targets: An inventory of the differences between normal and cancer cells: Fusion genes DNA Gene mutations Novel RNAs RNA Small RNAs PCa nanobodies Protein Androgen receptor Metabolites Hormones Fatty acids Morphology Exosomes Cellular behavior Pathology / Imaging Genes and pathways responsible for prostate cancer initiation and progression TMPRSS2-ERG fusion PTEN inactivation Myc overexpression P53 AR inactivation amplification DNAseq Data Analysis Copy Number SNVs / InDels Abberations TF Binding B-Allele Frequency DNAseq data Chromatin Interactions Structural Variations Methylation Active Chromatin Identify Read Integration Sites Barcode PCa genomics: The messy genome FISH : chromosome painting In the messy cancer genomes, the secret of the tumor characteristics are hidden Van Bokhoven et al., The Prostate 57: 226-244 (2003) PCa genomics: The messy genome The Molecular Taxonomy of Primary Prostate Cancer How does this help an individual patient? TCGA, Cell 163, 4, 2015, Pages 1011–1025 PCa genomics: The messy genome TCGA, Cell 163, 4, 2015, Pages 1011–1025 Actionable targets PARP inhibitors Mateo et al., N Engl J Med. 2015 373(18):1697-708 PI3K and AKT inhibitors Kinase inhibitors Markers and therapy-targets for prostate cancer How many changes are identified in the VCaP cell line? On chromosome level: Breaks : 35 Base pair level: Breaks: 764 Small changes: 246,653 ~ 10 common ~ 20 rare ~ 500 proteins ~ 470 novel/private mutated Teles Alves et al., Human Genetics (2013) Markers and therapy-targets for prostate cancer The most common gene changes The most logical markers and therapy targets Majority and newly discovered Percentage tumors with gene change THADA MAP4K3 BRAF TMPRSS2-ERG MPP5-FAM71D ~50% ARHGEF3 LRBA RAF1 GPS2-MPP2 PRRT2 Common Rare Private The prostate cancer problem Personalized oncology / Precision medicine Search for changes in the tumor (such as DNA) that indicate: “Who should be treated when and with which medicines” Tumor type determines choice and order of therapy Type of DNA mutation and tumor type determine choice and order of therapy However, for many (rare/private) mutations we have no medicine PCa genomics: The messy genome The issue with heterogeneity and clonality Gundem et al., Nature 520: 353-357 (2015) PCa genomics: The messy genome Will the Personalized Medicine approach cure these patients? Gundem et al., Nature 520: 353-357 (2015) PCa genomics: Heterogeneity, clonal origin and re-population Cooper CS et al., Nat Genet. 2015 Apr;47(4):367-72. Su F et al., Eur Urol. 2018 Jun 22. pii: S0302-2838(18)30430-5 ‘All’ tumors represented: Biopsy Blood Urine Identify trunk mutations Primary tumor single cell sequencing Gundem et al., Nature 520: 353-357 (2015) PCa genomics: The messy genome The Molecular Taxonomy of Primary Prostate Cancer TCGA, Cell 163, 4, 2015, Pages 1011–1025 Basic Research: The prostate cancer problem Transcriptomics and genomics of prostate cancer - What is the PCa problem and how to address it? - DNA sequencing - Common, rare and private mutations - Personalized Medicine - Metastasis - RNA sequencing - Small RNAs - Long RNAs - Liquid biopsies Which type of RNA is most abundant? How many different genes do we have per type? Table 6-1 Molecular Biology of the Cell (© Garland Science 2008) Transcriptomics of PCa tissue: discovery of novel PCa-associated transcripts How many genes do humans have? 1) Up to 25,000 2) 25,000 – 60,000 3) 60,000 – 100,000 4) More than 100,000 RNAseq Data Analysis sdRNAs; tRFs PCA3 RNA modification Differential expression AR T877A ARv7 Alternative splicing & SNV / InDels RNAseq data Promoters Read-Through & Novel Transcripts Fusion Transcripts EPCAT176 (SChLAP1) TMPRSS2-ERG Small RNAs in prostate cancer progression Early detection and cure via Hormone-, chemo-, immuno- Prevention surgery/radiation therapy therapy Bone metastasis NAP: Normal Adjacent Prostate BPH: Benign Prostate Hyperplasia PCa: Organ-confined Prostate Cancer LN: PCa Lymph Node Metastases TURP-PCa: Castration Resistant PCa RNA pools of 4 fresh frozen patient samples for each group - size fractionation 18-35 nt - adaptor ligation, cDNA amplification and Solexa/Illumina sequencing (30-35 bp) Inventory of small RNAs in prostate and PCa samples Martens et al., Oncogene 2012;31(8):978-91. Families of small nucleolar RNA (snoRNA) snoRNA H/ACA-box snoRNA C/D-box scaRNAs PseudoUridylation of rRNA Methylation of rRNA Modification of RNA N= ±120 N= ±260 N= ±25 SNORA SNORD H/ACA and C/D-box snRNA Name Abbr. Function Size (nt) Post-transcriptional gene Micro RNA miRNA 21-24 regulation Small Nucleolar RNA snoRNA Modification of rRNAs 70-130 Small Cajal Body-specific RNAs scaRNA Modification of snRNAs 80-350 ? Hypothesis: Not only pre-miRNAs, but also other ncRNAs are specifically processed to smaller (functional ?) fragments snoRNAs from the GAS5 locus are up-regulated in prostate cancer GAS5 SNORDS NAP BPH PCa LN SNORD81 233 157 1,024 1,905 SNORD47 4 7 76 94 SNORD80 9 4 5 22 SNORD79 28 40 200 359 SNORD78 545 168 2,469 10,658 SNORD44 2,396 3,111 11,391 11,117 SNORD77 SNORD76 SNORD75 36 88 Prostate Cancer SNORD74 853 645 3,235 5,166 GAS5 mRNA 20 20 31 45 From Exon Arrays Normal adjacent prostate Martens-Uzunova ES et al., Oncotarget. 2015 Fragments generated from the GAS5 locus 05 32 07 300 08 404 11 117 11 846 800 325 800 405 Fragments generated from the GAS5 locus Basic Research: The prostate cancer problem Transcriptomics and genomics of prostate cancer - What is the PCa problem and how to address it? - DNA sequencing - Common, rare and private mutations - Personalized Medicine - Metastasis - RNA sequencing - Small RNAs - Long RNAs - Liquid biopsies A novel EMC Prostate Cancer-Associated Transcript (EPCAT) A novel EMC Prostate Cancer-Associated Transcript (EPCAT) Junction track Compilation of RNAseq reads 20 matched NAP 20 PCa Gene Left Gene Left EPCAT966 Gene Right Transcriptomics of PCa tissue: validation of novel PCa-associated transcripts Novel genes: Erasmus MC Prostate Cancer-Associated Transcripts EPCATs are long noncoding RNAs and not conserved Transcriptomics of PCa tissue: validation of novel PCa-associated transcripts No PCa: n=117 + 74 (cystoprostatectomy / pelvic exenteration / TURP BPH) Local PCa: n=481 Radical prostatectomy LN metastases: n=119 TURP-PCa: n=120 (65 CRPC; 55 HS) Böttcher R et al., Oncotarget. 2015 Feb 28;6(6):4036-50. Prostate Cancer Biomarkers: validation of novel PCa-associated transcripts PCa-associated transcripts (EPCATs) In situ hybridization EPCAT176 / SChLAP1 EPCAT176 overall survival Böttcher R. et al., Oncotarget. 2015 28;6(6):4036-50. Prostate Cancer Biomarkers: novel transcripts Prostate Cancer Biomarkers: novel transcripts Tissue-specific circRNAs PCa cells tissue Chen S, …Jenster G, ...Boutros P, He H, Cell in press Basic Research: The prostate cancer problem Transcriptomics and genomics of prostate cancer - What is the PCa problem and how to address it? - DNA sequencing - Common, rare and private mutations - Personalized Medicine - Metastasis - RNA sequencing - Small RNAs - Long RNAs - Liquid biopsies Prostate Cancer Research: Liquid biopsy CELL-FREE CIRCULATING TUMOR PROTEIN RNA CELLS METABOLITES CELL-FREE DNA EXTRACELLULAR VESICLES (EV-RNA; EV- PROTEINS; EV-DNA) PLATELETS VIRUSES MICRO- Urine ORGANISMS Serum Prostate Cancer Research: Liquid biopsy cell-free DNA Targeted cfDNA mutational analysis (72 mCRPC genes) WGS cfDNA mutational analysis (low pass) Wyatt AW et al., J Natl Cancer Inst. 2017 Dec 1;109(12) Viswanathan SR et al., Cell. 2018 Jul 12;174(2):433-447.e19 Diagnosis and prognosis of urogenital diseases The URINOME Project RNA and DNA from: Kidney Bladder Prostate Representing: Infection Cancer Stones Chronic disease Congenital diseases Transplantation Children, women and men ERG RNAseq of urine: TMPRSS2-ERG fusion transcript detected in a man with PCa Markers and therapy-targets for prostate cancer The most common gene changes The most logical markers and therapy targets Newly discovered Percentage tumors with gene change THADA MAP4K3 SKIL TMPRSS2-ERG PCATs/PCA3 MPP5-FAM71D ~50% sdRNAs ARHGEF3 LRBA HES6 circRNAs GPS2-MPP2 PRRT2 AR variants Common Biomarkers/Targets? Rare Private .