1. www.reactome.org Hinxton 30 March 2011 1 Reactome Functional Interaction (FI) Network Steve Jupe

2. www.reactome.org Hinxton 30 March 2011 2 Motivation for Pathway and Network Analysis in Cancer “Exclusivity principle” in cancer. once a gene is mutated in a pathway, the selective growth advantage incurred by a second mutated gene in the same pathway is minimal. rare for a single tumor to have multiple genes altered in the same pathway. Analyzing mutated genes in a pathway context. reveals relationships among these genes. facilitates hypothesis generation on roles of these genes in disease phenotype. identifies genes on the affected pathways as therapeutic targets.

3. www.reactome.org Hinxton 30 March 2011 3 Analysis Workflow – Cancer Microarray Experiment: Gene expression profiling of pancreatic tumour (vs normal tissue). Output: list of genes that show significant changes in expression. Downstream analysis: 1.Validation of the top genes sorted by fold-change (or p-value): artificial cut-off, information loss; 2.Manual reviewing and gene classification based on literature, databases and own prior experience: time-consuming and bias; 3.GO annotation and pathway analysis (e.g. Reactome): what biological terms or functional categories are represented in the gene list more often than expected by chance; 4.Network-based analysis. - identify markers not as individual genes but as sub-networks extracted from protein interaction databases and the resulting sub-networks provide novel hypotheses for pathways involved in cancer.

4. www.reactome.org Hinxton 30 March 2011 4 What is the Functional Interaction Network? A reliable interaction network based on manually curated pathways extended with verified interactions Covers almost 50% of human Uniprot proteins A resource for construction of FI sub-networks based on gene sets Tools that: Provide the underlying evidence for FIs Build and analyze network modules of highly-interacting groups Perform functional enrichment to annotate modules Expand the network by finding genes related to the experimental data display source pathway diagrams and overlay with a variety of information sources such as cancer gene index annotations. Method and practical application: A human functional protein interaction network and its application to cancer data analysis, Wu et al. 2010 Genome BiologyWu et al. 2010 Genome Biology

5. www.reactome.org Hinxton 30 March 2011 5 Pairwise Problem Input1-Input2, Input1-Catalyst, Input1-Activator, Input1- Inhibitor, Input2-Catalyst, Input2- Activator, Input2-Inhibtior, Catalyst-Activator, Catalyst- Inhibitor, Activator-Inhibitor Reaction Functional Interaction Convert reactions in pathways into pair-wise relationships Functional Interaction: an interaction in which two proteins are involved in the same reaction as input, catalyst, activator and/or inhibitor, or as components in a complex

6. www.reactome.org Hinxton 30 March 2011 6 Reactome Functional Interaction (FI) Network [15%] Wu et al. Genome Biology 2010 Construction of the Reactome FI Network

7. www.reactome.org Hinxton 30 March 2011 7 Coverage after Prediction 10,956 proteins (9,542 genes) 209,988 FIs 5% of network shown here

8. www.reactome.org Hinxton 30 March 2011 8 Reactome Pathway Diagram with FI Overlay

9. www.reactome.org Hinxton 30 March 2011 9 Limitations of web FI overlay Simple viewer – no extra tools Not easy to produce networks Not dynamic – you can’t move the pathway diagram objects

10. www.reactome.org Hinxton 30 March 2011 10 Projecting Experimental Data onto FI Network

11. www.reactome.org Hinxton 30 March 2011 11 A FI Sub-Network Generated from Significant Network Modules based on a TCGA OV Expression Data Set

12. www.reactome.org Hinxton 30 March 2011 12 Cytoscape Cytoscape - open source platform for visualizing complex- networks and integrating these with any type of attribute data. Java-based Plug-in development framework First public release: July 2002, v0.8 Current release: 2.8.1 Website: http://www.cytoscape.org

13. www.reactome.org Hinxton 30 March 2011 13

14. www.reactome.org Hinxton 30 March 2011 14 Reactome FI Cytoscape Plug-In Cytoscape v2.7.0 or later. Reactome Homepage (see URL below) Menu Bar -> Tools, FI Cytoscape Plugin Save jar file caBigR3.jar into Cytoscape plugins folder, restart.caBigR3.jar http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/plugins/caBigR3.jar Also available as Java Web Start: Cytoscape.jnlpCytoscape.jnlp http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/Cytoscape.jnlp

15. www.reactome.org Hinxton 30 March 2011 15 FI Network Analysis Pipeline Select gene list (mutated, over- expressed, down-regulated, amplified or deleted genes) Project genes of interest onto Reactome F.I. Network Identify Disease/Cancer Subnetwork Apply Clustering Algorithms Apply Pathway/GO Annotation to each cluster Generate Biological Hypothesis! Predict Disease Gene Function Classify Patients & Samples T2D OvCa GBM Breast Prostate TCGA ICGC

16. www.reactome.org Hinxton 30 March 2011 16 Using the Reactome FIs Plug-in Choose Plugins, Reactome FIs. FI plug-in supports three file formats: Simple gene set: one line per gene. Gene/sample number pair. Contains two required columns, gene and number of samples having gene mutated, and an optional third column listing sample names (delimited by semi-colon ;). NCI MAF (mutation annotation file).

17. www.reactome.org Hinxton 30 March 2011 17 File Formats MSI2 PTPRT PELO SLC18A1 TACC2 FAM148B PRC1 MSTN ATP6V1G2 APOE IMPA2 AGER XPO5 MEST RREB1 BAT1 WIPI1 CATSPERB SSR1 VEGFA EGFL8 Simple Gene ListGene/Sample Number PairsNCI MAF (mutation annotation file)

18. www.reactome.org Hinxton 30 March 2011 18 Loading a file Choose file Choose file format and parameters Click OK

19. www.reactome.org Hinxton 30 March 2011 19 Results Display Constructed network is displayed in the Network View panel using an FI specific visual style

20. www.reactome.org Hinxton 30 March 2011 20 Pop-up menu Right-click an empty space in the network view panel.

21. www.reactome.org Hinxton 30 March 2011 21 Fetch FI annotations Details information on selected FIs. Three edge attributes are created: FI Annotation, FI Direction, and FI Score. Edges display direction attribute values. --> for activating/catalyzing --| for inhibition solid line for complexes or inputs --- for predicted FIs. VizMapper tab, Edge Source Arrow Shape and Edge Target Arrow Shape values give details.

22. www.reactome.org Hinxton 30 March 2011 22 Analyze network functions Pathway or GO term enrichment analysis for the displayed network. Use Data Panel to: Filter enrichment results by FDR value. Check “Hide nodes in not selected rows” to display only nodes in the selected row or rows To analyze an unlinked set of genes choose the "Show genes not linked to others" option when importing data

23. www.reactome.org Hinxton 30 March 2011 23 Cluster FI network Runs spectral partition based network clustering (Newman, 2006) on the displayed FI network.Newman, 2006 Nodes in different network modules will be shown in different colours (max 15 colours).

24. www.reactome.org Hinxton 30 March 2011 24 Analyze module functions Pathway or GO term enrichment analysis on individual network modules. Use filter to remove small network modules Filter by FDR Button selects nodes in the network corresponding to selected row or rows in the Data Panel.

25. www.reactome.org Hinxton 30 March 2011 25 Other features - Query FI source Right-click edge, select "Reactome FI/Query FI Source". If the FI was extracted from a curated pathway the original data source(s) is displayed Double click a row in the table to show details If the FI is predicted the evidence is displayed.

26. www.reactome.org Hinxton 30 March 2011 26 Other features - Fetch FIs for node Select node in the network panel, right click for pop-up menu, select "Reactome FI, Fetch FIs" FI partners for the selected node are displayed in two sections: Partners that are displayed and partners that are not displayed. Select undisplayed partners and click Add

27. www.reactome.org Hinxton 30 March 2011 27 Other features - Show pathway diagram (KEGG) Select a pathway in "Pathways in Network/Modules" tabs, right click, select "Show Pathway Diagram” KEGG pathways - diagram page is opened. Nodes listed in "Nodes" column are highlighted in red.

28. www.reactome.org Hinxton 30 March 2011 28 Other features - Show pathway diagram (Reactome) Reactome pathway diagrams open in a separate window. For human data the manually drawn pathway diagram is displayed. For other species an automated layout is generated. Nodes in the Cytoscape network are highlighted in blue on the diagram Detailed information is viewed using a pop-up menu item "View Instance". Zoom slider bottom of the window. Overview window top-right corner

29. www.reactome.org Hinxton 30 March 2011 29 Cancer Gene Index annotations Cancer Gene Index annotations can be added in two ways: popup menu "Load Cancer Gene Index" when no object is selected (left) popup menu "Fetch Cancer Gene Index" for a selected node (right)

30. www.reactome.org Hinxton 30 March 2011 30 Load Cancer Gene Index This method loads the tree of NCI disease terms in the left panel. Select a disease term in the tree to select all diagram objects that have this annotation or one of its sub-terms.

31. www.reactome.org Hinxton 30 March 2011 31 Fetch Cancer Gene Index This method is for viewing detailed annotations for the selected gene or protein. Annotations are sortable by PubMed ID, Cancer type, status, and several other criteria.

32. www.reactome.org Hinxton 30 March 2011 32 Cancer Gene Survival Analysis Based on a server-side R script that runs either coxph or Kaplan-Meyer survival analysis. Requires appropriate data file (see online). Popup menu “Analyze Module Functions, Survival Analysis...” Results are displayed with a tab "Survival Analysis" (right). You can display multiple analyses. Only Kaplan-Meyer analysis will show a plot file. You can click the file to view the actual plot (far right).

33. www.reactome.org Hinxton 30 March 2011 33 Summary Reactome is a highly reliable, human-curated database of biological pathways and reactions. Expanded the human curated Reactome pathways by importing pathways from other databases and integrating predicted FIs: ~50% coverage. Functional interaction network provides a powerful way to visualize cancer and other disease genes to reveal functional relationships among these genes.

34. www.reactome.org Hinxton 30 March 2011 34 Future Implement multiple network clustering algorithms Currently spectral partition only Future: edge-betweenness, MCL, and hierarchical clustering Integrate with other data types: gene expressions, CNVs… Seamless integration with Reactome pathway diagrams Expand known cancer pathways by using FIs Integrate miRNA/target interactions to study miRNA activities on cancers

35. www.reactome.org Hinxton 30 March 2011 35 Software Demo