Regulation of c-Jun multisite phosphorylation
c-Jun is a major component of the transcription factor AP-1, a paradigm for transcriptional response to extracellular signals. c-Jun activation by N-terminal multisite phosphorylation mediates the nuclear response of MAPK signaling in many different organisms, giving rise to very different cellular responses, including cell cycle progression, differentiation and apoptosis. In turn, deregulation of c-Jun activation may give rise to pathological conditions, including tumor progression, neuroinflammation and degenerative loss of neurons. The challenging question is how can the same signalingpathway regulate the expression of genes involved in quite different cellular programs. In the last years, our research interest has focused on the regulation of c-Jun multisite phosphorylation and its possible role in the diverse outputs of c-Jun biological activity.
Our studies in cerebellar granule cells, Bergmann glial cells and breast cancer cells have shown that the extent of c-Jun N-terminal multisite phosphorylationby JNK reflects the degree of extracellular signals and plays an important role in determining the type of c-Jun biological output (Madeo et al. 2010, Albanito et al. 2011, Reddy et al. 2013). Importantly, we have elucidated that T91/T93 phosphorylation requires a priming event at theadjacent T95 site (Vinciguerra et al 2008). Also, we have demonstrated that abrogation of T95 phosphorylation protects cerebellar granule neurons (CGs) from cell death, whereas it has no effect on Jun-dependent neurite outgrowth innaïve PC12 cells (Reddy et al 2013). Moreover, we have shown that DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) phosphorylates c-Jun at T95 in fibroblasts from systemic sclerosis (SSc) patients and leads to activation of Wnt signaling by repressing the expression of Wnt inhibitor factor 1 (WIF-1) (Svegliati et al. 2014). Altogether our studies indicate that site-specific configurations of c-Jun phosphorylation may reflect either the grade or the type(s) of extracellular signals, hence allowing c-Jun to act downstream of either synergistic or antagonistic signaling pathways.
Currently, we are interested to assess how cellular signaling pathways regulates c-Jun/Ap-1 activation in the tumor microenvironments, particularly in tumor-infiltrating T cells, where c-Jun/AP1 cooperates with the NFAT transcription factor to induce a gene expression profile associated with cytotoxic T cell activation.
Signaling pathways downstreamthe alternate estrogen receptor GPER in breast cancer cells and cancer-associated fibroblasts.
Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a large number of evidence correlates the sustained exposure to exogenous estrogen with the high risk of the onset of various cancers. Mainly, these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors and upon activation translocate into the nucleus and regulate the expression of different target genes by binding directly specific DNA sequences. Beyond ERα and ERβ, estrogens signal through the G-protein coupled estrogen receptor GPER, a member belonging to the rhodopsine-like family of G-protein coupled receptors (GPCRs).GPER has been shown to unlock access to estrogens in breast cancer cells devoid of the classic Estrogen Receptor (ER), thus allowing the stimulatory actions of these steroids. In estrogen-responsive tumors, GPER expression correlates with negative clinical-pathological features such as larger tumor size, distant metastasis, and worse prognosis. In accordance with these observations, GPER signaling has been linked to ERα loss, which occurs in breast cancer cells undergoing tamoxifen resistance. GPER mediates rapid estrogen action via heterotrimeric G proteins, hence activating multiple intracellular pathways regulating crucial mechanisms involved in breast cancer growth, invasion, and metastasis. Moreover, GPER also signals in cancer-associated fibroblasts (CAFs), therefore contributing to the expression of pro-tumorigenic factors within the tumor microenvironment, as inflammatory cytokines and angiogenic factors.
Early studies have shown that GPER binds estrogens and in turn functionally cross-reacts with diverse cell signaling systems, including epidermal growth factor receptor (EGFR) pathway and the mitogen-activated protein kinases (MAPK) pathway. Our previous studies have demonstrated for the first time that E2 induce rapid c-fos up-regulation through GPR30-mediated mechanism in various cancer types, including breast cancer cells, endometrial cancer cells, thyroid cancer cells and ovarian cancer cells (Maggiolini et al. 2004, Vivacqua et al. 2006a, Vivacqua et al. 2004b, Albanitoat al. 2007). On the other hand, we have shown that in breast cancer cells GPER responds to tamoxifen by inducing c-fos expression, which in turn contribute to JNK/cJun-dependent cell death by forming the AP-1 transcription factor (Madeo et al. 2010). All together these studies suggested that depending on the contextual tumor microenvironment, the functional output of the GPER/cFos axis may depend on whether or not there is a concomitant activation of c-Jun pro-apoptotic N-terminal phosphorylation.
More recently, we have discovered a functional cross-talk between GPER and Notch signaling both in breast cancer cells and CAFs. In particular, we demonstrated that E2/GPER signaling induced ligand-independent activation of Notch1 and Notch target genes, including the EMT-transcription factor Snail, whose activation required the tripartite NICD-CSL-MALM-1 transcriptional complex. Most importantly, breast cancer cells acquired invasion properties and lost Cadherin expression in a GPER/Notch dependent fashion(Pupo et al. 2014, De Francesco et al. 2018).