The assembly and maturation of invadopodia can be initiated
The assembly and maturation of invadopodia can be initiated by chemical stimuli such as the epidermal growth factor (EGF), as well as the mechanical signals from extracellular matrix (ECM). Upon binding, EGF activates the EGF receptor (EGFR), which further activates several intracellular signaling pathways (Cortesio et al., 2008), including the Src kinase which mediates the assembly of the invadopodium precursor and the phosphoinositide-3 kinase (PI-3K), necessary for anchoring the precursor to the cell membrane (Beaty and Condeelis, 2014). In addition to EGF, several other growth factors have been shown to initiate invadopodia formation. For example, transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and heparin binding EGF (HB-EGF) can induce invadopodia formation in several types of cancer ixazomib receptor (Díaz et al., 2013, Eckert et al., 2011, Pignatelli et al., 2012, Rajadurai et al., 2012, Yamaguchi et al., 2005). Moreover, autocrine signals can lead to the induction of invadopodium formation (Patsialou et al., 2009). In addition to growth factors, a number of signals present in the tumor microenvironment, such as hypoxia, pH or direct interactions with stromal cells were shown to be associated with invasion and metastasis of cancer cells (Gould and Courtneidge, 2014). The production of the pro-inflammatory lipid Prostaglandin E2 (PGE2) is elevated in breast cancer due to high expression of the prostaglandin-endoperoxidase synthase 2 (COX2), which is a critical determinant for invasiveness of human breast cancer cells (Bocca et al., 2014, Miglietta et al., 2010, Park et al., 2012). However, the direct link between elevated PGE2 and increased invasiveness of human breast cancer cells remains elusive. PGE2 activity is mediated by four G-protein coupled receptor isotypes called EP1, −2, −3 and −4 (Narumiya et al., 1999, Pierce and Regan, 1998). The activation of the PGE2 receptor EP4 subtype (EP4) by its ligand PGE2 is known as a mechanism that increases the invasive capability of cancer cells (Konya et al., 2013). Hence, it is likely that EP4 is involved in the PGE2-mediated increase of cancer invasiveness. EP4 is capable of activating distinct intracellular signaling pathways. Its signaling is associated with the stimulating G alpha protein (Gαs), which leads to an increase of the intracellular secondary messenger cyclic adenosine monophosphate (cAMP) (Regan, 2003). Furthermore, EP4 is also capable of inhibiting the formation of cAMP by activating the inhibiting G alpha protein (Gαi) (Fujino and Regan, 2006). The activated EP4 can be phosphorylated by a G protein-coupled receptor kinase (GRK), which enables the recruitment of β-arrestin to EP4 (Konya et al., 2013, Yokoyama et al., 2013). The EP4/β-arrestin complex then activates the membrane-bound Src, which is able to transactivate the EGFR receptor, thus showing that the EP4/β-arrestin/Src complex facilitates cell migration and metastasis (Buchanan et al., 2006, Kim et al., 2010). Moreover, it has been demonstrated that Src, activated by the EP4 signaling pathway, can also activate PI-3K (Yokoyama et al., 2013). This signaling cascade was demonstrated previously to be active in the migration and metastasis processes in colorectal carcinomas (Buchanan et al., 2006, Konya et al., 2013). Taken collectively, these data demonstrate that stimulation of EP4 increases invasive behavior of cancer cells and that the EP4 and EGFR pathways cross-talk. However, the direct relationship between EP4 stimulation by PGE2 and invadopodia formation has not been studied yet. Here we focus on testing if EP4 stimulation influences invadopodia assembly and ECM degradation. Using in vitro invadopodia and spheroid assays as well as intravital imaging, our results provide novel insights into the effects of EP4 activation on breast cancer invasion. Understanding the interplay between different signaling receptors promoting cancer cell invasive behavior may provide novel targets to prevent or reduce cancer dissemination and metastasis.