**3.3 Adaptation to estrogen withdrawal**

Breast cancer cells can acquire a state of hypersensitivity to estrogen that renders them resistant to endocrine therapy. MCF7 cells cultured in estrogen-free medium to produce long-term estrogen deprived cells (LTED) mimics the effects of ablative endocrine therapy (Santen et al., 2003) and produces cells that are highly sensitised to substantially lower concentrations of estrogen as compared with wild-type MCF-7 cells (Masamura et al., 1995). Growth factor signalling and ER expression was significantly higher in these cells. Treatment with estrogen resulted in rapid association of ER and phosphorylation of SHC, an adaptor protein involved in tyrosine kinase receptor signalling, and increased activation of both SRC and the RAS/RAF/MEK/MAPK signalling pathways (Song et al., 2002a,b; Song et al., 2004). Exposure of these cells to fulvestrant blocked MAPK activation indicating that this pathway may be a downstream effector of the ER non-genomic pathway (Santen et al., 2003; Song et al., 2002a,). However, a high AKT and MAPK level in LTED cells was associated with increased resistance to endocrine therapy and a worse outcome.

In another version of MCF7 LTED cells, enhanced transcriptional activity of ER was associated with increased activation of growth factor pathways that in turn trans-activate ER (Johnston & Dowsett, 2003). After prolonged culture in the absence of estradiol, the ER in these cells functions independently from exogenous estradiol, which was suggested to be due to a super-sensitivity of LTED to residual estrogen present in the medium (Chan et al., 2002; Martin et al., 2003). These cells also showed increasedlevels of phosphorylation of ER at serine 118, a known target for several intracellular kinases. Furthermore, IGF-1R and ERBB2 signalling was significantly increased in these cells concurrently with increased MAPK activation. Interestingly, the phosphorylation of ER at serine 118 was blocked by

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 459

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

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

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

The phenomenon of epithelial cells undergoing a transition towards a mesenchymal phenotype was first identified as programmed events occurring during embryonic developmental processes (Greenberg & Hay, 1982). Since then EMT has since been described in various pathological conditions. During the process of cancer metastasis, a minority of

apoptosis through constitutive activation of survival signalling".

**3.6 Cell cycle signalling molecules** 

through PI3K/AKT, TNF, IFN and NFKB.

**4. Epithelial mesenchymal transition** 

Agthoven et al., 1998).

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