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

www.reactome.org Hinxton 30 March 20111

Reactome Functional Interaction (FI) Network

Steve Jupe


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.


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.


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 Biology

http://genomebiology.com/content/11/5/R53


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


Reactome Functional Interaction (FI) Network [15%]

Wu et al. Genome Biology 2010

Construction of the Reactome FI Network


Coverage after Prediction

• 10,956 proteins (9,542 genes)

• 209,988 FIs

• 5% of network shown here


Reactome Pathway Diagram with FI Overlay


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


Projecting Experimental Data onto FI Network


A FI Sub-Network Generated from Significant Network Modules based on a TCGA OV Expression Data Set


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



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.

• http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/plugins/caBigR3.jar

• Also available as Java Web Start: Cytoscape.jnlp

• http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/Cytoscape.jnlp

http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/plugins/caBigR3.jar

http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/plugins/caBigR3.jar

http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/Cytoscape.jnlp

http://reactomedev.oicr.on.ca:8080/caBigR3WebApp/Cytoscape/Cytoscape.jnlp


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

T2DOvCaGBM

BreastProstate

TCGAICGC


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).


File Formats

MSI2PTPRTPELOSLC18A1TACC2FAM148BPRC1MSTNATP6V1G2APOEIMPA2AGERXPO5MESTRREB1BAT1WIPI1CATSPERBSSR1VEGFAEGFL8

Simple Gene ListGene/Sample Number PairsNCI MAF (mutation annotation file)


Loading a file

• Choose file

• Choose file format and parameters

• Click OK


Results Display

• Constructed network is displayed in the Network View panel using an FI specific visual style


Pop-up menu

• Right-click an empty space in the network view panel.


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.


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


Cluster FI network

• Runs spectral partition based network clustering (Newman, 2006) on the displayed FI network.

• Nodes in different network modules will be shown in different colours (max 15 colours).

http://www.ncbi.nlm.nih.gov/pubmed/16723398


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.


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.


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


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.


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


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)


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.


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.


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).


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.


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


Software Demo

Documents

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