RNA interference-directed silencing of VPAC1 receptor inhibits VIP effects on both EGFR and HER2 transactivation and VEGF secretion in human breast cancer cells
Highlights
► Proliferation was impaired in VPAC1-receptor siRNA transfected breast cancer cells. ► VPAC1-receptor silencing abolished VIP stimulation of VEGF165 secretion. ► VPAC1-receptor silencing abolished VIP transactivation of EGFR and HER2. ► EGFR and HER2 transactivation are likely involved in VIP-stimulated VEGF secretion.
Introduction
Breast cancer is the most frequent carcinoma and the second leading cause of cancer mortality among women in the Western world (Jemal et al., 2010). It poses a major public health problem that has led to increasing efforts in classification, prognostication, prediction, and treatment (Weigel et al., 2010). In its early stages, breast cancer growth is estrogen-dependent so that estrogen-deprivation therapies cause repression of breast tumors and improve the cure rate (Popat and Smith, 2006, Bombonati and Sgroi, 2011). Unfortunately, more than 60% of breast cancers are advanced, estrogen-independent tumors (hormone refractory), making estrogen-deprivation therapies ineffective. In these cases, the primary treatment approach is cytoreductive surgery followed by adjuvant chemotherapy and/or radiotherapy (Popat and Smith, 2006); however, the prognosis is poor and new therapies are needed for this numerous group of patients.
The study of breast tumor growth and metastasis development has shown the primary role of angiogenesis, i.e. the formation of new blood vessels from the existing vascular network (Boudreau and Myers, 2003). The main positive regulator of this important process is vascular endothelial growth factor (VEGF), a protein involved in endothelial cell activation, proliferation, migration, invasion, and survival (Donovan and Kummar, 2006, Roskoski, 2007). VEGF represents a family of homodimeric glycoproteins of which VEGF-A isoforms have been thoroughly studied and shown to be involved in tumor-induced angiogenesis; among them, VEGF165 and VEGF121 are considered the main soluble and biologically active members (De Castro et al., 2006, Roskoski, 2007). VEGF initiates intracellular signaling cascades by binding to a family of membrane receptors provided with tyrosine-kinase activity in their intracellular segment; VEGFR-2 regulates vascular endothelial function, i.e. the angiogenic response, and its therapeutic inhibition is now having an impact in the clinic for cancer therapies (De Castro et al., 2006, Holmes et al., 2007). VEGF has been shown to act in an autocrine manner as a survival factor in breast carcinoma cells (Bachelder et al., 2001). Furthermore, increased VEGF levels correlate with poor prognosis and decreased overall survival in both node-positive and negative breast cancer women (Linderholm et al., 1998, Linderholm et al., 2000).
The human epidermal growth factor family of receptors (EGFR/HER) mediates cell proliferation and differentiation mechanisms in many neoplasms including breast cancer (Mass, 2004, Sebastian et al., 2006). These receptors have tyrosine kinase activity, and binding of ligands results in hetero- or homo-dimerization and phosphorylation of the intracellular domain. Overexpression of EGFR and HER2 (which are preferred interacting partners for dimerization) in breast cancer and other epithelial cancers is associated with a poorer clinical prognosis and predicts a bad response to endocrine therapy (Atalay et al., 2003, Lin and Winer, 2004). However, several strategies targeting these receptors, including monoclonal antibodies directed towards the extracellular domain and small molecule tyrosine kinase inhibitors have demonstrated therapeutical benefits (Popat and Smith, 2006, Singer et al., 2008, Ross et al., 2009). EGFR/HER signaling pathways have been shown to contribute to the regulation of both angiogenesis and metastasis. In this context, tumor progression has been associated with EGFR-mediated angiogenesis via upregulation of VEGF and metalloproteinases (MMPs) (Ongusaha et al., 2004). Moreover, dual silencing of HER2 and VEGF results in inhibition of breast cancer cell growth and invasiveness (Tai et al., 2010). There is increasing evidence on that HER proteins can induce intracellular signaling not only in response to cognate ligands but also following transactivation by G-protein coupled receptors (GPCRs) and other cross-talking receptors (Bhola and Grandis, 2008, Higashiyama et al., 2008).
Vasoactive intestinal peptide (VIP) belongs to glucagon-secretin superfamily and broadly distributes into the organism (Fahrenkrug and Said, 2000). This neuroendocrine peptide exerts pleiotropic physiological effects including the regulation of the immune response, exocrine and endocrine secretions, muscle relaxation, and cell proliferation and differentiation (Moody et al., 2003, Moody et al., 2011). VIP and the related molecule pituitary adenylate cyclase-activating peptide (PACAP) bind to VPAC1 and VPAC2 receptors with high affinity whereas VIP and PACAP bind to PAC1 receptor with low and high affinity, respectively (Laburthe and Couvineau, 2002, Couvineau et al., 2010). These receptors are coupled to G proteins and their occupation activates adenylate cyclase and, less commonly, inositol triphosphate (IP3)–Ca2+ and other signaling pathways (Chorny et al., 2006, Dickson and Finlayson, 2009). VIP is involved in growth, differentiation and function in normal and neoplastic breast (García-Fernández et al., 2005, Moody and Jensen, 2006), and VPAC1 receptor is overexpressed in human breast carcinoma (Moody and Gozes, 2007). We have shown that VIP induces the expression and secretion of VEGF (Valdehita et al., 2007) and the transactivation of EGFR and HER2 (Valdehita et al., 2009) in human breast cancer cells. In the present study, using human estrogen-dependent (T47D) and estrogen-independent (MDA-MB-4687) breast cancer cells, we show that the involvement of VIP in angiogenesis and EGFR/HER pathways is mediated by VPAC1 receptor. We also show that VIP stimulation of VEGF secretion is dependent of VIP transactivation of EGFR/HER members. These observations were made by means of in vitro silencing of VPAC1 receptor with small interfering RNA (siRNA) which is a powerful tool for controlling cellular processes of gene silencing at post-transcriptional level (Lochmatter and Mullis, 2011). Results give value to the consideration of VIP antagonists and VPAC1-receptor silencing by siRNA transfection as potential molecular therapies in human breast cancer.
Section snippets
Cell culture
Estrogen-dependent (T47D) and estrogen-independent (MDA-MB-468) breast carcinoma cell lines were supplied by the American Type Culture Collection (Manassas, VA, USA). T47D cells were cultured in RPMI-1640 (Life Technologies, Barcelona, Spain) supplemented with 1% antibiotic/antimycotic, 10% fetal bovine serum (FBS), and insulin (8 μg/ml). MDA-MB-468 cells were grown in the same medium in the absence of insulin.
Transfecting VPAC1-receptor siRNA into human breast cancer cells
T47D and MDA-MB-468 breast cancer cells were placed for 24 h in 6-well plates at a
Validation of VPAC1-receptor siRNA transfection procedure
In order to find the best experimental conditions, three different VPAC1-receptor siRNA oligomers were tested at three concentrations (20, 50 and 100 nM) and three time periods (24, 48 and 72 h) followed by addition of RPMI medium supplemented with 5% FBS, The corresponding siRNA sequences (5′–3′) were: S1 sense GGAGGAGUGUAGCUAUGUG and S1 antisense CACAUAGUCACACUCCUCC, S2 sense GGCUUGUGCAACAAUAAAU and S2 antisense AUUUAUUGUUGCACAAGCC, and S3 sense GGAGUGUGACUAUGUGCAG and S3antisense
Discussion
We report in the present study the leading role of VPAC1 receptor in VEGF secretion and EGFR/HER2 transactivation in human breast cancer cell lines, as shown by means of VPAC1-receptor silencing with siRNA. Furthermore, we show that VIP transactivation of EGFR/HER2 can cause VEGF secretion, thus linking VIP effects on EGFR/HER and angiogenesis pathways.
We used two human breast cancer cell lines that represent two stages in the progression of breast carcinoma. Estrogen-dependent T47D cells
Acknowledgements
This study was supported by a Grant from the Ministerio de Educación y Ciencia (SAF2007-63794). A.V. was a fellow from the University of Alcalá.
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