Fig 1.
Overview of DSPathNet, a novel computational framework to construct a drug-specific signaling pathway network (SPNetwork): metformin as a case.
Step 1: we collected the metformin upstream genes from multiple sources and inferred metformin downstream genes from metformin-induced gene expression data. We also compiled one human SPNetwork. Step 2: we utilized the metformin upstream and downstream genes as seeds to generate a metformin-specific SPNetwork from the human SPNetwork. The process involved longitudinal and lateral movements. Step 3: we utilized disease genes and genome-wide association studies (GWAS) data to evaluate if the metformin-specific SPNetwork was enriched with disease genes for type 2 diabetes (T2D) and cancer, genes associated with metformin action. Furthermore, we derived a crosstalk network of metformin action for T2D and cancer in order to identify key components in the metformin signal transduction via network topological and functional analysis. The nodes in orange correspond to the drug-related upstream genes, the nodes in green to the drug-related downstream genes, and the nodes in red to the nodes common to the upstream and downstream gene networks.
Table 1.
Comparison of genes in metformin-specific signaling pathway network with T2D and cancer genes and genes with smallest P-value less than 0.05 in five GWAS data sets.
Fig 2.
Gene Set Enrichment Analysis (GSEA) enrichment score curves of metformin-induced probes in three treatments vs. treatment 1.
The four sets of probes of metformin treatments were obtained from the gene expression profiles from Connectivity Map. The three treatment instance IDs are 2, 3, and 4. The graphs on the top panels represent the ranked, non-redundant, and up-regulated probes in the second, third, and fourth treatment groups compared with probes in the first treatment group. The graphs on the bottom panels represent the ranked, non-redundant, and down-regulated probes in second, third, and fourth treatment groups compared with probes in the first treatment group. In each graph, probes on the far left (red) correlated with the most up-regulated probes in the treatment 1 and probes on the far right (blue) correlated with the most down-regulated probes in treatment 1. In each graph, the vertical black lines indicate the position of each of the probes of the studied probe set in the ordered, non-redundant data set. The green curve denotes the ES (enrichment score) curve, the running sum of the weighted enrichment score in GSEA.
Fig 3.
Metformin-specific signaling pathway network (SPNetwork).
A) A four-way Venn diagram summarizes the number of shared genes among metformin upstream genes represented by ‘Upstream genes’, metformin downstream genes (‘TF genes’), metformin upstream extended genes in the metformin upstream network (‘Upstream extended genes’), and metformin downstream extended genes in the metformin downstream network (‘Downstream extended genes’). B) Metformin-specific SPNetwork with 477 nodes and 1366 edges. The nodes and edges in orange correspond to nodes and edges only in the metformin upstream network. The nodes and edges in green correspond to the nodes and edges only in the metformin downstream network. And the nodes and edges in red correspond to the nodes and edges common to the metformin upstream network and the metformin downstream network. C) Degree distributions and average degrees (vertical lines) of the four gene sets in the metformin-specific SPNetwork. The four gene sets are 41 common nodes, 174 nodes only in the metformin upstream network (SPNetwork_up), 262 nodes only in the metformin downstream network, all 477 nodes in the metformin-specific SPNetwork (SPNetwork_down). The Y-axis represents the proportion of proteins having a specific degree. D) The subnetwork of the 38 hub nodes extracted from metformin-specific SPNetwork. The legends for orange nodes and edges, red nodes and edges, and green nodes and edges are same as those in the subFig B. The nodes in yellow correspond to the genes that exist in the pathway ‘MAPK signaling pathway’ according to KEGG annotation.
Table 2.
Summary of genes and hypergeometric tests at each step in the process of metformin-specific SPNetwork construction.
Fig 4.
Functional comparison of the common genes, upstream network genes, and downstream network genes.
The common genes were those found in both metformin upstream network and downstream network. The upstream network genes were those only belonging to the metformin upstream network. The downstream network genes were those only belonging to the metformin downstream network. A) Proportion of genes of interest in Gene Ontology (GO) molecular function domain. B) Comparison of proportion of enriched pathway in the three gene sets at the first-level category of KEGG annotation. C) The clustering of enriched pathways for the three gene sets at second-level category of KEGG annotation.
Fig 5.
(A) Comparison of gene-level P-value distribution in T2D GWAS among three gene sets from the metformin-specific SPNetwork, human SPNetwork, and genes covered by T2D GWAS. (B) Interactions were extracted from metformin-specific SPNetwork. These interactions occur between T2D-related genes, namely, their smallest P-value less than 0.05 in T2D GWAS. The legends for orange nodes, red nodes, and green nodes are same as in Fig 3.
Fig 6.
Common genes and a crosstalk subnetwork between T2D and cancer.
(A) The four-way Venn diagram summarizes the number of shared genes among the four gene sets with smallest P-value less than 0.05 in the T2D GWAS and the three types of cancer GWAS data sets (breast, pancreatic, prostate) in metformin-specific SPNetwork. (B) Degree comparison of common genes among the four gene sets in A, common genes’ direct interactors, and all genes in metformin-specific SPNetwork. (C) A crosstalk subnetwork of metformin action for T2D and cancer with three modules and enriched pathways. The legends for orange nodes, red nodes, and green nodes are same as in Fig 3. The nodes with underlines are key components in the metformin signal transduction process.
Table 3.
KEGG pathways overrepresented in genes in three modules of the reduced common network between T2D and cancer for metformin.
Fig 7.
A novel metformin action pathway.
Solid lines indicate the proposed mechanisms as supported by experimental evidence from literature. The two black dashed lines indicate the drug effects. The red dashed line indicates the relationship is existed but the direction is unknown. The arrows beside the gene names or biological processes indicate the metformin effects. Up-arrows indicate the corresponding genes or processes are up-regulated while the down-arrows indicate the corresponding genes or process are down-regulated.