Estrogen and IGF-I Independently Down-Regulate Critical Repressors of Breast Cancer Growth.

Abstract

Abstract Background: Estrogen receptor and insulin-like growth factor (IGF) signaling pathways are important for both normal mammary gland development and breast cancer pathogenesis. Despite this evidence, cross-talk between these two critical mitogenic and survival pathways remains poorly understood, particularly at the level of downstream gene transcriptional networks.Methods: We performed microarray analysis on ER-positive MCF-7 breast cancer cells following stimulation with either estradiol (10nM) or IGF-I (10nM) for 3hr or 24hr. We defined an E2-IGF gene expression signature and examined the effect of the gene signature on time to recurrence in ER+ tumors in the publicly available NKI dataset. Q-RT-PCR was used to validate the effects of IGF-I and E2 on mRNA of repressed genes. To examine gene regulation further, MCF-7 cells were incubated with various signaling inhibitors and gene expression measured.Results: We found 183 probesets to be co-regulated by E2 and IGF-I after 3hr of stimulation and 454 probesets to be co-regulated at the 24hr time point. Patients with tumors that had the same set of genes up- and down-regulated had a poor outcome (p=7.04E-07). E2-IGF co-regulated genes showed a significant enrichment for down-regulated genes (p<0.001). The top 10% of down-regulated genes contained four (BLNK, SOCS2, CCNG2, and ING4) that have previously been shown to have tumor suppressor function and/or show loss in human breast tumors. We confirmed that E2 and IGF-I indeed repressed levels of these candidate genes. Blockade of ER by ICI 182780 (ICI) completely reversed E2-mediated repression but had little or no effect on IGF-mediated repression of these genes. Similarly, blockade of IGF-IR was able to completely reverse IGF-I-mediated repression of all four genes, but had essentially no effect upon E2-mediated repression. Inhibition of the PI3K pathway with LY294002 (20μM) affected short-term (3hr) down-regulation of BLNK and SOCS2 by both E2 and IGF-I, and CCNG2 by IGF-I. By 24 hours, LY completely reversed both E2 and IGF-I mediated repression of CCNG2, and significantly reversed both E2 and IGF-I repression of BLNK and SOCS2. Blockade of MEK1/2 with U0126 (10μM) had no effect on short-term repression by E2 or IGF-I, though after 24hr it affected both E2 and IGF-I repression of BLNK and SOCS2.Conclusions: E2 and IGF-I co-regulate a set of genes that affect breast cancer outcome. E2 and IGF-I down-regulate several critical tumor suppressor genes. While the down-regulation is independent of each other at the level of ER and IGF-IR, for some genes, there is convergence on the PI3K pathway for repression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1126.