Exploring drivers of gene expression in The Cancer Genome Atlas

Presentation at the MCW Physiology Department Seminar

Abstract: The Cancer Genome Atlas (TCGA) has greatly advanced cancer research by generating, curating, and publicly releasing deeply measured molecular data from thousands of tumor samples. In particular, gene expression measures, both within and across cancer types, have been used to determine the genes and proteins that are active in tumor cells. To more thoroughly investigate the behavior of gene expression in TCGA tumor samples, we introduce a statistical framework for partitioning the variation in gene expression due to a variety of molecular variables including somatic mutations, transcription factors (TFs), microRNAs, copy number alternations, methylation, and germ-line genetic variation. As proof-of-principle, we identify and validate specific TFs that influence the expression of PTPN14 in breast cancer cells. We provide a freely available, user-friendly, browseable interactive web-based application for exploring the results of our transcriptome-wide analyses across 17 different cancers in TCGA.

doi: https://doi.org/10.1101/227926

Transcript

1. Exploring Drivers of Gene Expression in The Cancer Genome Atlas ANDR EA R AU, PHD PHYSIOLOGY DEPARTM ENT SEM INAR M EDICAL COLLEGE OF W ISCONSIN M ARCH 28, 2018 1 http://www.andrea-rau.com, @andreamrau
2. The Cancer Genome Atlas (TCGA) - Collaboration between the National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI) to accelerate the understanding of the molecular basis of cancer - Comprehensive, multi-dimensional maps of key genomic changes in 33 cancer types - Publically available data (multi-tiered data depending on patient identifiability) - Widely used by the research community (1000+ studies of cancer in publications by TCGA research network and independent researchers) 2 Diagnose – Treat – Prevent 2005 2006 2011 2013 2016 NIH launch Pilot project 2008 Glioblastoma report Ovarian cancer report Pan-cancer analysis 2014 10k cases complete NCI Genomic Data Commons opens
3. 3 Source: https://cancergenome.nih.gov/abouttcga
4. 4 Source: https://cancergenome.nih.gov/abouttcga Ø Basal-like subtype of breast cancer is molecularly similar to the serous subype of ovarian cancer, suggesting a common path of development and similar response to therapeutic strategies (TCGA Network et al., 2012) Ø Stomach cancer is made up of four subtypes, including one characterized by infection with Epstein-Barr virus (TCGA Network et al., 2014) Ø Identification of targetable genomic alterations in lung squamous cell carcinoma led to NCI’s Lung-Map Trial (TCGA Network et al., 2012)
5. Gene expression in cancer • Cancer results from a gene not normally expressed in a cell being switched on and expressed at high levels due to mutations or alterations in gene regulation • Epigenetic, transcription, post-transcription, translation, post-translation, … • Tumor suppressor genes: active in normal cells to prevent uncontrolled cell growth (e.g. p53) • Oncogenes: overexpression can lead to uncontrolled cell growth (e.g. MYC) • Gene expression profiling often used to accurately classify tumors • Studying how to control transcriptional activation of gene expression in cancer can potentially lead to new therapeutic treatments for cancer 5
6. 6 Gene expression TTTGCA AAACGT TF Transcription factor expression Copy number alterations Transcriptional regulation in cancer genomes Promoter methylation microRNA expression …GCAGCGTTCGA… …GCAACGTTAGA… Somatic mutations within tumors, Germline genetic variation
7. Our big question How is gene expression influenced by other genomic & epigenomic mechanisms in cancer genomes? Does a TCGA pan-cancer analysis reveal patterns among subsets of cancer types? 7
8. Inferring global transcriptional regulation in cancers using TCGA • Jiang et al. (2015) used TCGA gene expression data and ChIP-Seq data from ENCODE on 150 transcription factor profiles to search for cancer-associated TFs • Analyzed data across genes within each sample to determine if TF targets were significantly up- or down-regulated (after adjusting for confounding factors) 8 TCGA-A3FO TCGA-A2MZ TCGA-A8JD gene 1 . . . . gene 20,000 Image source: Figure 2 from Jiang et al. (2015)
9. Inferring drivers of expression at the gene-level using TCGA • Here, rather than fixing each sample and analyzing across genes, we aimed to make inferences specific to each gene • Motivating question: for a specific gene in a specific cancer type, what are the relative molecular drivers of its expression? 9 TCGA-A3FO TCGA-A2MZ TCGA-A8JD gene 1 . . . . gene 20,000
10. 10 Gene expression TTTGCA AAACGT TF Transcription factor expression Copy number alterations TCGA Data Sources Promoter methylation microRNA expression …GCAGCGTTCGA… …GCAACGTTAGA… Somatic mutations within tumors, Germline genetic variation RNA-seq (tumor) miRNA-seq (tumor) RNA-seq as proxy (tumor) Exome sequencing (presence of nonsynonymous mutations, tumor) Illumina methylation arrays (tumor) Affymetrix 6.0 genotyping arrays (tumor vs normal) Affymetrix 6.0 genotyping arrays (normal)
11. Cancers in TCGA with all requisite data Cancer (sample size) Breast invasive carcinoma (506): BRCA Head and neck squamous cell carcinoma (245):HNSC Brain lower grade glioma (262): LGG Skin cutaneous melanoma (320): SKCM Thyroid carcinoma (265): THCA Sarcoma (210): SARC Pheochromoctyoma and paraganglioma (144): PCPG Lung adenocarcinoma (144): LUAD Esophageal carcinoma (113): ESCA Bladder urothelial cancer (109): BLCA Liver hepatocellular carcinoma (110): LIHC Kidney renal clear cell carcinoma (228): KIRC Pancreatic adenocarcinoma (131): PAAD Kidney renal papillary cell carcinoma (95): KIRP Stomach adenocarcinoma (138): STAD Prostate adenocarcinoma (132): PRAD Cervical squamous cell carcinoma (136): CESC 11 Note: analyses restricted to the largest population, individuals of self-reported European ancestry. Central nervous system Breast Endocrine system Gastro- intestinal Gynecologic Head and neck Skin Soft tissue Thoracic Urologic
12. Statistical model: linear mixed effects where g is an n x 1 vector of the total genetic effects of the individuals with ! ~ # 0, &'( ) and A is interpreted as the genetic relationship matrix (GRM) between individuals We fit1 this LMM for every gene in each cancer type, where: y = gene expression for a given gene A is estimated2 from the germline genetic data as a covariance matrix taken across SNPs, weighted by allele frequency X is a matrix of fixed effects representing non-genetic factors (methylation, somatic mutations, CNA, TF, miRNAs) 12 s: data management, estimation of the genetic relationships from SNPs, mixed linear Ps, estimation of the linkage disequilibrium structure, and GWAS simulation. We focus plained by all the SNPs on the X chromosome and testing the hypotheses of dosage e tool to estimate and partition complex trait variation with large GWAS data sets. wide association undreds of SNPs uman complex cture of human lained. For most y explain a small s not been any ing heritability.’’ mber of common ith large effects, ntly proposed a t of phenotypic y ¼ Xb þ g þ 3 with V ¼ As2 g þ Is2 3 ; (Equation 2) where g is an n 3 1 vector of the total genetic effects of the individuals with g $ Nð0; As2 g Þ, and A is interpreted as the genetic relationship matrix (GRM) between individuals. We can therefore estimate s2 g by the restricted maximum likelihood (REML) approach,10 relying on the GRM esti- mated from all the SNPs. Here we report a versatile tool called genome-wide complex trait analysis (GCTA), which implements the method of estimating variance explained by all SNPs, and extend the method to partition the genetic variance onto each of the chromosomes and also to esti- 1 Via REML estimation on standardized expression residuals. 2 Using the GCTA software (Yang et al., 2011)
13. TF and miRNA target genes - Expression measures available: ◦ ~850 TFs (combined list from IPA and TRRUST databases) ◦ ~800 miRNAs - TFs and miRNAs each potentially target multiple genes, each gene is potentially targeted by multiple TFs/miRNAs ◦Definitive mapping is unknown! - Many available methods / databases for predicted TF-target and miRNA-target pairs (via text-mining, bioinformatics approaches …) 13
14. Sparse representation of TF & miRNA effects • Primary goal: infer relative contribution of molecular drivers of gene expression by estimating the proportion of variance explained • Secondary goal: identify specific TFs / miRNAs influencing expression for a specific gene • Obstacles for our work: • Too many TFs and miRNAs to include all of them (p >> n problem) • Even if we could, a potential list of hundreds of TFs is not helpful… • TFs and miRNAs that target a specific gene are not definitely known • Solution: sparse Principal Component representation of TFs / miRNAs 14 Dimension reduction + enhanced interpretability!
15. Sparse Principal Component Analysis (sPCA) • Principal components = linear combinations of original variables accounting for the most possible variability: ! = #$ %$ + #' %' … + #) %) Large weights (loadings) = important contribution to the PC. When large number of (potentially irrelevant) variables, interpretation can be difficult… • Sparse PCA = variable weights set to 0 for irrelevant variables: ! = * ∗ %$ + #' %' … + 0 * %) 15 Image courtesy of Kim Anh Lê Cao (https://www.slideshare.net/AustralianBioinformatics/tuesday-session-8kimanhlecao1) mixOmics R package
16. Sparse Principal Component Analysis (sPCA) • TFs and miRNAs with non-zero sPCA loadings correspond to those that contribute most to variation in overall TF / miRNA expression • Number of non-zero loadings in each sPC must be chosen by user ↦ 10 • Select first 5 (uncorrelated) sPCs for both TF and miRNA data for inclusion in the X matrix of fixed effects 16
17. Back to the model: Quantities of interest • Heritability in gene expression: Price et al. (2011) and Gamazon et al. (2015) V = Var(y) = Var(genetic) + Var(residual) Heritability = Var(genetic)/Var(y) = !" # !$ # • Contribution to overall variance by the fixed effects: %&'()* +): essentially a corrected R2 metric for LMMs Nakagawa & Schlielzeth (2013) • This “partitioning” of variance provides us with estimates for the relative contribution of each component on gene expression 17 s: data management, estimation of the genetic relationships from SNPs, mixed linear Ps, estimation of the linkage disequilibrium structure, and GWAS simulation. We focus plained by all the SNPs on the X chromosome and testing the hypotheses of dosage e tool to estimate and partition complex trait variation with large GWAS data sets. wide association undreds of SNPs uman complex cture of human lained. For most y explain a small s not been any ing heritability.’’ mber of common ith large effects, ntly proposed a t of phenotypic y ¼ Xb þ g þ 3 with V ¼ As2 g þ Is2 3 ; (Equation 2) where g is an n 3 1 vector of the total genetic effects of the individuals with g $ Nð0; As2 g Þ, and A is interpreted as the genetic relationship matrix (GRM) between individuals. We can therefore estimate s2 g by the restricted maximum likelihood (REML) approach,10 relying on the GRM esti- mated from all the SNPs. Here we report a versatile tool called genome-wide complex trait analysis (GCTA), which implements the method of estimating variance explained by all SNPs, and extend the method to partition the genetic variance onto each of the chromosomes and also to esti-
18. Pan-cancer trends in expression drivers • CNAs represent most consistent driver of expression üCorresponds to previous reports of relative importance of aneuploidy versus somatic mutations or germline polymorphisms • PRAD and KIRP have highest number of genes with large germline genetic drivers of expression • LUAD and LIHC have largest number of genes affected by miRNA variation • Distinct clustering of cancers observed for the molecular drivers of some genes 18
19. 19 p53-DNA repair pathway: • Major oncogenic pathway, responsible for maintaining fidelity of DNA replication/cell division • BRCA1 and BRCA2 have large variance components for TF expression in {LGG, SARC, LUAD, SKCM}
20. 20 LGG SKCM Weighted sPC TF loadings (BRCA2) • Similar TF programs for BRCA2 in these two cancers, with some unique differences
21. 21 Pan-cancer trends: MYC expression
22. Efficiently exploring results • Interactive web-based R/Shiny Application called Exploring Drivers of Gene Expression (EDGE) in TCGA: http://ls-shiny-prod.uwm.edu/edge_in_tcga/ • Exploratory results can be queried and visualized by gene and cancer site (among other fun stuff) 22
23. PTPN14 locus background • Non-receptor protein tyrosine phosphatase that regulates many breast cancer pathways ◦ Positive regulator of Her2 ◦ Positive regulator of TGFB ◦ Negative regulator of HIPPO pathway (e.g., YAP) • Implicated in breast cancer growth and metastasis (suggest tumor suppressor role, but might also have some oncogenic functions). • A PTPN14 polymorphism is implicated with ER+ breast cancer risk in AA • However, somatic mutations or copy number variants of PTPN14 do not appear to be prevalent in breast cancer • The transcriptional regulators of PTPN14 appear to be unknown 23
24. Suggestive data from ENCODE → Largest variance components for PTPN14 in EDGE-in-TCGA app are CNAs and TFs 24 ENCODE CHIP-seq analysis of PTPN14 in T47D cells ChIP-seq data from ENCODE (breast cancer cell line T47D) suggest that FOXA1 and GATA3 bind to the PTPN14 promoter
25. PTPN14 TF results from EDGE in TCGA app 25
26. PTPN14 promoter assay validation • Flister lab (MCW) performed promoter reporter construct on GATA3 and FOXA1 for PTPN14 in breast cancer cell lines • Expression of GATA3 and FOXA1 down- regulated PTPN14 • Next step → investigate how GATA3 and FOXA1 influence breast cancer outcomes… 26
27. Wrapping up and future work • Layering on information from NHGRI-EBI GWAS catalogue and GTEx consortium • Expanding analyses to data beyond TCGA (e.g., BRIDGES EU Study) • Potential extensions to incorporate pertinent clinical information (e.g., disease progression-free survival) 27
28. Acknowledgements Paul L. Auer Hallgeir Rui Michael Flister • Anthony San Lucas (MD Anderson) • Paul Scheet (MD Anderson) 28
29. Appendix SOME SCREENSHOTS OF EDGE-IN-TCGA SHINY APP (JUST IN CASE!) 29