**3.4 Estrogen receptor and co-regulators**

Since ER action is mainly controlled through transcriptional factors and co-regulator molecules, it seems likely some of these may be implicated in endocrine resistance. For example, increased AP1 and NFKB transcriptional activity has been associated with endocrine resistance (Johnston et al., 1999; Zhou et al., 2007). And similarly when ER coactivators are over-expressed or phosphorylated. For example, NCOA3 (A1B1 or SRC3) over-expression leads to constitutive ER-mediated transcription, which confers resistance both *in vitro* and in xenograft models and is associated with reduced responsiveness to tamoxifen in patients (Ali & Coombes, 2002; Osborne et al., 2003; Ring & Dowsett, 2004). Another ER co-activator associated with resistance is PELP1 (Fig 2) which is a cytoplasmic scaffold protein that modulates ER interaction with SRC, leading to activation of SRC and the ERK family kinases and also promotes oestrogen activation of PI3K (Gururaj et al., 2006). Interestingly, ER cytoplasmic complex composed of ERα, PI3K, SRC and focal adhesion kinase (FAK; also known as pTK2) is formed as a result of the transient methylation of ER at R260 by protein arginine N-methyltransferase 1 (pRMT1). This complex activates AKT and could confer resistance to endocrine therapy but this methylation event which is frequent in breast cancer has yet to be linked to resistance (Le Romancer et al., 2008).

### **3.5 Growth factor receptor pathways**

Perhaps the most important factors that affect the response to endocrine therapy are those that can modulate alternative proliferation and survival in the tumours in which the ER signalling pathway is effectively inhibited. These alternative growth pathways can do so by the establishment of a bidirectional cross talk with ER signalling. These pathways will act as ER-independent drivers of cancer proliferation and survival and are involved in both *de novo* and *acquired* resistance (Normanno et al., 2005). Increased expression of EGFR, ERBB2 and IGF1R along with their downstream components such as ERK and PI3K can modulate tamoxifen resistance (Faridi et al., 2003; Hutcheson et al., 2003; McClelland et al., 2001). ERBB2 has been reported to be over-expressed in association with down regulation of the Xlinked tumour suppressor forkhead box p3 (FOXP3) and the zinc finger transcription factor GATA4 (Hua et al., 2009; Zuo et al., 2007). Other factors that might affect ERBB2 expression are the presence of the paired-domain transcription factor PAX2 and the ER co-activator NCOA3 which compete for binding and regulating ERBB2 transcription and, in turn, responsiveness to endocrine therapy. However, like GATA4 and FOXP3, PAX2 was also shown to be down-regulated in tamoxifen resistant breast cancers in the presence of NCOA3 and an over-expressed ERBB2 (Hurtado et al., 2008). The SRC substrates BCAR1 and BCAR3 have both been reported to elicit endocrine resistance *in vitro* (Dorssers et al., 1993). BCAR1 binds and activates SRC leading to phosphorylation of EGFR and the signal transducer and activator of transcription 5B (STAT5B) (Riggins et al., 2007). On the other hand, BCAR3 is believed to activate RAC and p21-activated kinase 1 (pAK1), which is a mediator of endocrine resistance itself through ER phosphorylation, and through the activation of SRC in association with BCAR1 (Cai et al., 2003; Rayala et al., 2006; Riggins et al., 2003; van Agthoven et al., 1998).

The de-regulation of several growth pathways including EGFR, ERBB2 and IGF1R are implicated in endocrine resistance (Faridi. et al., 2003; Miller et al., 2009). Many events might trigger this de-regulation such as activating mutations in PIK3CA and loss of heterozygosity or methylation of PTEN, activation of AKT, over-expression of ERBB2 and activation of IGF1R and ERBB3 following the loss of PTEN (Arpino et al., 2008; Miller et al., 2009; Riggins et al., 2007). However, following de-regulation of these pathways acquisition of endocrine resistance might be effected by a number of possible activities as summarised by Musgrove & Sutherland, (2009): "decreased ER expression mediated by ERK activation; loss of ERmediated repression of EGFR and ERBB2 and consequent activation of mitogenic signalling cascades; ligand-independent activation of ER or its co-activators through phosphorylation; up-regulation of key cell cycle regulators, for example MYC and the D and E-type cyclins, through constitutive activation of mitogenic signalling pathways; and the inhibition of apoptosis through constitutive activation of survival signalling".
