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 and Markers TMPRSS2
~50% Percentage tumors with gene change -ERG
Common
targets for for cancer -targets prostate targets therapy and markers logical most The ARHGEF3 THADA
The most common gene changesThegene most common Rare
Majority MAP4K3 LRBA
and RAF1 BRAF
newly
discovered GPS2
-MPP2 MPP5
Private
-FAM71D PRRT2
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 and Markers TMPRSS2
~50% Percentage tumors with gene change Common -ERG
targets for for cancer -targets prostate Biomarkers/ PCATs AR AR circRNAs sdRNAs variants /PCA3 Targets
?
targets therapy and markers logical most The ARHGEF3 THADA
The most common gene changesThegene most common Rare
MAP4K3 LRBA
Newly discovered Newly
HES6 SKIL
GPS2
-MPP2 MPP5 Private
-FAM71D PRRT2