**3.6 Cell cycle signalling molecules**

458 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

MAPK or EGFR/ERBB2 blockade but not by blocking MEK/MAPK or PI3K/AKT signalling, indicating that additional kinases might be involved in this hypersensitive state. Nicholson et al., (2004) also developed an MCF7 cell line (MCF-7X cells) that is resistant to estrogen withdrawal but not hypersensitive to it. These cells could be growth inhibited by fulvestrant, implying that the ER pathway is still involved in their proliferation. However, the PI3K/AKT pathway was demonstrated to be the main factor promoting their growth without the involvement of EGFR/ERBB2 or IGF-1Rsignalling, suggesting that the adaptation to estrogen withdrawal can occur in the absence of increased sensitivity to

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

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

estrogen and does not require activation of classical growth factor receptors.

breast cancer has yet to be linked to resistance (Le Romancer et al., 2008).

**3.4 Estrogen receptor and co-regulators** 

**3.5 Growth factor receptor pathways** 

In order for cancer cells to bypass the inhibition of cell proliferation elicited by endocrine agents, one would expect down-regulation of effector molecules involved in the induction of apoptosis while those involved in proliferation, especially during G1 phase, are up regulated. Over-expressed cell cycle regulators include MYC, cyclin E1, cyclin D1, cyclin D1b, as well as p21 and p27, and a de-activated RB gene (Prall et al., 1998; Wang et al., 2008). Over-expression of MYC and cyclin D1 leads to an abundance of CDK complexes that are directly associated with increased cellular proliferation and/or relief of the inhibitory effects of the negative cell cycle regulators p21 and p27, a phenomenon that is also achieved through activation of ERBB2, AKT and SRC (Caldon et al., 2009; Chu et al., 2008; Hui et al., 2002; Perez-Tenorio et al., 2006). Cyclin D1 can also interact with several transcription factors including ER and STAT3 (Coqueret et al., 2002). Tamoxifen actually enhances the binding of cyclin D1 to ER at the expense of STAT3, hence activating both transcription factors and consequently establishing endocrine resistance (Ishii et al., 2008). Other important molecules are those involved in apoptosis. In particular, the pro-apoptotic molecules such as BIK (BCL2-interacting killer) and caspase 9 are down regulated in endocrine resistant cancers while those which are considered as anti-apoptotic molecules such as BCL-XL and its second messenger ceramide, are up regulated (Mandlekar et al., 2001; Riggins et al., 2005) . The expression of these molecules is also affected by signalling through PI3K/AKT, TNF, IFN and NFKB.
