IRS isoforms mediate distinct gene expression profiles, functional pathways, and breast cancer subtype association.

<p>(A) Venn diagrams depicting four distinct IRS isoform gene signatures were derived from overlapping and differential global gene expression patterns in response to IGF-I. (B) Target gene validation confirms both distinct and overlapping patterns of IRS-regulated gene expression. Gene expression was normalized to RPLP0 and is presented as fold-change of treatment (black bars) vs. serum-free (white bars) conditions. Error bars represent standard deviation and all results are representative of at least three independent replicates. (C) IRS gene signature enrichment in breast tumor subtypes in the UNC337 cohort. Median expression values are represented here in graphical format with p-values included for each of the IRS gene signatures.</p

Multivariate Cox regression analysis of RFS & OS in Luminal B breast cancer tumors.

<p>Multivariate Cox regression analysis of RFS & OS in Luminal B breast cancer tumors.</p

IRS adaptor protein isoforms define tumor cell biology and regulate global gene expression profiles.

<p>(A) Monolayer growth and motility of T47D-YA (YA), T47D-YA-IRS-1 (#10 and #20) and T47D-YA-IRS-2 (#1 and #6) were measured by MTT assay and (B) scratch-wound healing assay in response to IGF-I treatment. The graphs are presented as fold-change response vs. non-treated control and error bars represent standard deviation. (C) IGF-induced gene expression is IRS-dependent. cDNA microarray analysis was performed on IRS-null YA, IRS-1, and IRS-2 clones. The graph represents IGF-regulated probes in comparison to untreated samples that met both fold (1.5) and p-value (0.05) cutoff values. Hierarchical clustering was carried out on log2-transformed using Gene Cluster 3.0 and visualized in Java TreeView.</p

IRS proteins regulate TGFβ2 mRNA expression and breast cancer cell motility.

<p>(A) Expression of TGFβ1 and TGFβ2 by qPCR in T47D-YA-IRS-1 (#10 and #20) and T47D-YA-IRS-2 (#1 and #6). (B) IGF-induced TGFβ2 expression in MCF10A, MCF-7L, MCF-7 ATCC, MDA-231 and F11 cells. For A & B, all cells were exposed to 5nm IGF-I for 4 hours prior to harvesting mRNA. Gene expression was normalized to RPLP0 and is presented as fold-change of treatment (black bars) vs. serum-free (white bars) conditions. (C) TGFβ2 expression was assessed by qPCR in an IRS-gene deletion mouse models (left) and IRS-overexpressing SH-EP neuroblastoma cells (right). (D) IRS-1, IRS-2 and TGFβ2 expression in a panel of patient breast tumors. Arrows indicate invasive breast carcinoma. Yellow bars signify high gene expression, blue bars signify low gene expression. E) pSMAD2 was examined by immunoblot at the indicated time points in MCF-7 cells. (F) Cell motility was examined by modified Boyden chamber assay. MCF-7 cells were incubated in the presence of neutralizing antibodies to either TGFβ1 or TGFβ2 and IGF-induced motility assessed. Error bars represent standard deviation and all results are representative of at least three independent replicates.</p

Gene set enrichment analysis for each IRS gene signature was performed using DAVID (Database of Annotation, Visualization and Integrated Discovery, v6.7).

<p>P-value indicates modified Fisher’s exact Probability Value and a high E-Scores (Enrichment Scores) indicates significant gene enrichment in the annotation cluster.</p