Identification of a Ser585/PI3-kinase transcriptional network. (A) Gene ontology (GO) (www.geneontology.org/) classifications are shown for the 138 Ser585-regulated genes identified by microarray screening. (B) Connectivity mapping19 of the 138 Ser585-regulated genes indicated negative connectivity with the expression change induced by the PI3-kinase inhibitor, LY294002. A negative connectivity (red) represents genes that are either induced by Ser585 and repressed by LY294002 or repressed by Ser585 and induced by LY294002. All 453 experiments in the Connectivity Map database were ranked by their connectivity score with the 138 Ser585-regulated genes identified in our studies. Each of the LY294002 comparisons (n = 17) is highlighted as a black bar on the heat map, and colors represent a negative (red), no connection (gray), or positive connection (green) between the 138 Ser585-regulated genes identified in our studies and individual LY294002-induced gene expression changes for each array in the Connectivity Map database. A cluster of LY294002 experiments (13 of 17) lie within the red region, indicating a statistically significant enrichment of our 138 Ser585-regulated genes and the LY294002 differentially expressed genes (P = .009). (C) Analysis of the overlap between the 138 Ser585-regulated gene set identified in our studies and the LY294002-sensitive genes in the Connectivity database. We pooled the data from the 6 top-ranked MCF7 cell LY294002 microarrays that showed significant overlap with our 138 gene set (bracketed asterisks) and performed a Wilcoxon rank sum test to compare a ranked list of LY294002 sensitive genes (Lod-ranking of LY294002-regulated genes) with our 138 gene set. Each blue line corresponds to an individual probe identified from our screen. Ser585-regulated genes (blue lines) cluster with top-ranked LY294002-sensitive genes (red region). Plotting this overlap shows a significant enrichment above a random distribution (dotted line) in Ser585-regulated genes (probe frequency) as the Lod-ranking of LY294002-regulated genes increases (P < .001). Of the 138 genes identified in our screen, 53 (38%) correspond to LY294002-sensitive genes encompassed by the gray portion of the graph (LY294002 cutoff; P = .01). (D) The 53 Ser585/PI3-kinase–regulated genes identified in panel C together with their fold change were uploaded into the IPA and overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Networks of genes were then algorithmically generated based on their connectivity to known functional, biochemical, and disease interactions. A significant overlap with genes involved in cancer and cell death was observed (P < .001, right-tailed Fisher exact test). (E) The 53 Ser585/PI3-kinase–regulated genes identified in panel C were subjected to IPA mapping of network interactions. This analysis showed that 27 of 53 genes constitute a gene network with red denoting Ser585-induced genes and green denoting Ser585-repressed genes. Node shape denotes the indicated gene function or biologic process.
