Epidermal Growth Factor Induces G Protein-Coupled Receptor 30 Expression in Estrogen Receptor-Negative Breast Cancer Cells

Abstract

Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.

GPER1 is regulated by insulin in cancer cells and cancer-associated fibroblasts

“…Short hairpin constructs against human GPER1 (shGPER1) and CTGF (shCTGF) were generated and used as described previously (Albanito et al 2008). In brief, they were generated in the lentiviral expression vector pLKO.1 purchased from Euroclone, Milan, Italy.…”

“…As regards the regulation of GPER1, our previous studies have shown that some important growth-factormediated transduction pathways such as EGFR (Albanito et al 2008, Vivacqua et al 2009) and IGF1 (Bartella et al 2012 are involved in the expression and function of GPER1 in cancer cells. Interestingly, high levels of expression of GPER1 in breast, endometrial, and ovarian tumors have been associated with a higher risk of developing metastatic disease and poor survival (Prossnitz & Barton 2011).…”

GPCRs and cancer

“…Moreover, it has been shown that the activation of the Gα s protein by GPER is responsible for the estrogen, phyto-and xenoestrogens stimulation of adenylate cyclase and the ensuing increase in cAMP in breast cancer cells [140,141] . The signaling events upon GPER activation by both estrogens and notably ER antagonists can lead to gene transcription as well as to the growth and migration in diverse hormone-sensitive tumors like breast, endometrial and ovarian cancer [142][143][144][145][146][147][148][149] . Notably, GPER was also involved in the stimulatory effects elicited by estrogens and ER antagonists in cancer-associated fibroblasts [147,150] .…”

In Vivo Functions of GPR30/GPER-1, a Membrane Receptor for Estrogen: From Discovery to Functions In Vivo

“…it has been reported that the aP-1 site that exists in the second intron is a transcriptional regulatory region of GPR30 activated in response to EGF [16]. There is a possibility that some of the transcript products for GPR30 are expressed in response to stimulation, because the aP-1 site in intron 2 cannot transcribe Exons1 and 2 of GPR30.…”