The transactivation of enhanced growth factor receptor (EGFR) by G protein–coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase sphingosine kinase-1 (SphK1). We show that E2 stimulates SphK1 activation and the release of sphingosine 1-phosphate (S1P), by which E2 is capable of activating the S1P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease–dependent manner. Thus, these findings reveal a key role for SphK1 in the coupling of the signals between three membrane-spanning events induced by E2, S1P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., “criss-cross” transactivation.

We previously demonstrated that sphingosine kinase-1 (SphK1) is an important mediator in the cytoplasmic signaling of estrogens, including Ca(2+) mobilization, ERK1/2 activation, and the epidermal growth factor receptor transactivation. Here we report for the first time that SphK1 activity is causally associated with endocrine resistance in MCF-7 human breast cancer cells. Enforced overexpression of human SphK1 in MCF-7 cells resulted in enhanced cell proliferation and resistance to tamoxifen-induced cell growth arrest and apoptosis. Tamoxifen-resistant (TamR) MCF-7 cells selected by prolonged exposure to 4-hydroxytamoxifen, exhibited higher levels in SphK1 expression and activity, compared with the control cells. Inhibition of SphK1 activity by either specific pharmaceutical inhibitors or the dominant-negative mutant SphK1(G82D) restored the antiproliferative and proapoptotic effects of tamoxifen in the TamR cells. Furthermore, silencing of SphK1, but not SphK2, expression by the specific small interference RNA also restored the tamoxifen responsiveness in the TamR cells. Thus, blockade of the SphK1 signaling pathway may reprogram cellular responsiveness to tamoxifen and abrogate antiestrogen resistance in human breast cancer cells.

Subject-Peroxisome proliferator-activated receptor (PPAR)-␥ agonists are emerging as potential protectors against inflammatory cardiovascular diseases including atherosclerosis and diabetic complications. However, their molecular mechanism of action within vasculature remains unclear. We report here that PPAR␥ agonists, thiazolidinedione class drugs (TZDs), or 15-deoxy-⌬12,14 -prostaglandin J 2 (15d-PGJ2) were capable of activating diacylglycerol (DAG) kinase (DGK), resulting in attenuation of DAG levels and inhibition of protein kinase C (PKC) activation. The PPAR␥ agonist-induced DGK was completely blocked by a dominant-negative mutant of PPAR␥, indicating an essential receptor-dependent action. Importantly, the suppression of DAG-PKC signaling pathway was functional linkage to the anti-inflammatory properties of PPAR␥ agonists in endothelial cells (EC), characterized by the inhibition of proinflammatory adhesion molecule expression and adherence of monocytes to the activated EC induced by high glucose. These findings thus demonstrate a novel molecular action of PPAR␥ agonists to suppress the DAG-PKC signaling pathway via upregulation of an endogenous attenuator, DGK.

The MYB oncogene encodes a transcription factor, Myb, which is essential for normal haemopoiesis and also for the proliferation of most acute leukaemias (reviewed in ref. 1). While a number of Myb target genes have been reported previously, these do not completely account for key elements of MYB’s activity, including its pro-leukaemic and differentiation-suppressing functions. We hypothesised that this reflects the fact that previous screens may have not been sufficiently comprehensive and/or employed the most appropriate cell systems. Thus we have embarked upon a program to identify and validate Myb targets critical for these functions. Here we report results from extensive expression profiling studies using a conditionally myb-transformed myeloid cell line, ERMYB (2). We have used ~44,000-element Illumina Beadchips in conjunction with a kinetic profiling strategy that selects candidates based on rapid, statistically-significant and consistent responses to both activation and inactivation of Myb. This has resulted in the identification of a substantial number of candidate Myb-activated and -repressed genes (381 and 502, respectively). In addition, we have used this cell system to identify candidate Myb-regulated microRNAs. Inspection of the list of candidate Myb-activated genes revealed several previously-described Myb targets including myc, bcl2, gstm1 and mpo, providing additional confidence in our approach. Our focus to date has been on novel candidates that may mediate myb’s ability to enhance proliferation, suppress differentiation and possibly suppress apoptosis. Q-RT-PCR was used as an initial validation step for a number of such targets; to date 13/14 genes identified by array screening have been confirmed by this method in ERMYB cells. A second approach to validation is to confirm correlation with Myb over-expression in a second cell system (FDB-1) (3). As in primary cells, enforced Myb expression can suppress differentiation and promote proliferation of these cells in the presence of GM-CSF (4). Amongst the Myb-activated genes are gfi1 and nucleostemin/gnl3, which are involved in stem-cell functions, cellular proliferation and in the case of gfi1, lineage-specific functions. Strikingly, candidate Myb-repressed genes include several important positive regulators of haemopoietic differentiation and/or negative regulators of proliferation, namely gata3, sfpi1/pu.1, cebpb, junb, klf’s-3,-6 -13 and btg1. Most of these genes have evolutionarily conserved internal or proximal candidate Myb binding sites. Our progress in validating these by chromatin immunoprecipitation will be presented. Finally, we have identified a number of microRNAs that are potentially regulated by Myb. These include members of the miR-17–92 cluster and mir-146b, which appear to be activated and repressed by Myb, respectively. These have been validated by Q-PCR for both the mature miR and the precursor pri-miR transcript. Interestingly, the mir-17–92 cluster has been strongly implicated in oncogenesis and cell cycle regulation (5), while miR-146a/b may have tumour suppressor activity.