**2. Mechanisms of estrogen receptor induced cell proliferation**

ERα and ERβ are transcribed from distinct genes located on separate chromosomes (6 and 14, respectively) (Green et al., 1986; Kuiper et al., 1996). These receptors differ in their tissue distribution, with ERα being highly expressed in the pituitary gland, ovaries (thecal and interstitial cells), uterus, liver, kidneys, adrenals and the mammary glands while ERβ is found mainly in the prostate, bone, ovaries (granulosa cells), lungs and in various parts of the central and peripheral nervous system (Emmen et al., 2005; Kuiper et al., 1997). Nevertheless, ERα and ERβ do overlap in their expression in some tissues (Zilli et al., 2009). More importantly, the two receptors have different roles in breast development. Only ERα appears to be essential for ductal growth although both receptors are present in the breast. ERα-knockout mice show very little growth of mammary ducts, while ERβ-knockout mice develop a normal mammary gland with regular ductal branching (Förster et al., 2002; Lubahn et al., 1993). This suggests that ERβ might be exerting pro-differentiative and antiproliferative functions. In addition, increased ERα/ERβ ratio in breast cancer as compared with benign tumours and normal tissues suggest that ERα is most closely associated with breast cancer pathogenesis, while ERβ can protect against the mitogenic activity of estrogens in pre-malignant lesions (Roger et al., 2001; Shaw et al., 2002). It has even been suggested that the estrogen-induced proliferation of ER+ breast cancer cells can be inhibited by ERβ over-expression (Ström et al., 2004; Williams et al., 2008). Thus ERα remains the main focus of attention in studies on breast cancer. Unless otherwise specified, 'ER' in this review will refer to ERα.

In what is now referred to as the nuclear or genomic action of ER, binding of estrogen induces activation of the receptor by initiating its dissociation from cognate heat shock proteins, and leads to conformational changes, dimerisation and autophosphorylation (Osborne & Schiff, 2005). The activated ER binds to estrogen response elements (EREs) located in the promoter regions upstream of estrogen-regulated genes. Frasor et al., (2003) observed from microarray analysis of gene expression in MCF-7 cells that about 70% of such estrogen-regulated genes were actually down-regulated following treatment with estradiol. Many of these genes are transcriptional repressors, or genes with anti-proliferative or proapoptotic function. On the other hand, there is increased expression of genes inducing cell proliferation and survival. Up-regulation of gene expression is mediated through two domains; activating function-1 (AF-1) and activating function-2 (AF-2). AF-1 is a hormone

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 455

pathways by these receptors down-regulates the expression of ER and PR causing reduction in estrogen dependency while activating the transcriptional function of ER, which suggests a contribution of this cross talk to the relative resistance to endocrine therapies in tumours with amplified ERBB2 expression (Bayliss et al., 2007; Brinkman and El-Ashry, 2009;

The two types of ER actions, genomic and non-genomic, are not mutually exclusive and do overlap. For example, ER induces the expression of transcripts for both TGFα and amphiregulin (Normanno et al., 1993; Saeki et al., 1991) which can both bind and activate EGFR resulting in activation of MAPK and AKT signaling which are also activated by direct interaction with ER (Salomon et al., 1995). ER binding to membrane caveolin-1 leads to the activation of specific G proteins resulting in the activation of SRC and in turn of matrix metalloproteinases that cleave transmembrane precursors of the EGFR ligand, heparin binding-EGF (HB-EGF) (Levin, 2003; Razandi et al., 2003). Fig 2 illustrates the major

It should be noted that most tumours are heterogeneously composed and a biphasic response to treatment could reflect the survival and eventual clonal outgrowth of an

Since all endocrine therapies target ER, the expression of the latter is the main predictor of the outcome of such therapies. The *de novo* resistance is clearly caused by the lack of ER expression which can be due to histone deacetylation (Parl, 2003) or associated with aberrant methylation of ER CpG islands that deactivates chromatin (Ottaviano et al., 1994; Weigel & deConinck, 1993 ). Interestingly, ER expression can be restored in ER-ve breast cancer following co-treatment with DNMT1 and HDAC inhibitors (Robertson et

It was initially thought that acquired resistance might be due to missing or non-functional variants of ER. However, only 17–28% of patients with acquired endocrine resistance lack ER expression (Gutierrez et al., 2005; Johnston et al., 1995 ). Furthermore, approximately 20% of tamoxifen-resistant patients will eventually respond to second-line treatment with aromatase inhibitors or fulvestrant (Howell et al., 2005; Osborne et al., 2002). Although a number of exon-deleted receptor isoforms have been described, their frequency is

Furthermore, these mutations have been detected in ER-ve tumours (Herynk & Fuqua 2004). A mutation that results in a hypersensitive receptor that shows enhanced binding of coactivators in the presence of low estrogen levels (a single amino acid substitution changing lysine 303 to arginine) was found in 20 of 59 hyperplastic breast lesions (Fuqua et al., 2000). However, the role and frequency of such mutations in primary breast carcinomas and their

Patients carrying inactive alleles of cytochrome P450 2D6 (CYP2D6) (approximately 8% of Caucasian women) fail to convert tamoxifen to its active metabolite, endoxifen (4-hydroxy-N-desmethyl-tamoxifen), and are consequently less responsive to tamoxifen, which is considered to be a significant factor in resistance to therapy (Hoskins et al, 2009). The baseline levels of endoxifen are elevated in patients carrying the wild-type CYP2D6 and who

relation to endocrine resistance needs to be exploredin a larger number of patients.

Creighton et al., 2010; Lopez-Tarruella and Schiff, 2007).

identified downstream events involving ER activation.

**3. Mechanisms of endocrine resistance** 

intrinsically resistant minor sub-population.

**3.1 Alterations in ER expression or function** 

al., 2000; Rountree et al., 2000;Yang et al., 2001).

insufficient to account for resistance.

independent domain located at the N-terminus of the receptor with its function regulated by phosphorylation. AF-2 is the site where ligand-binding actually occurs and is therefore hormone dependant. Almost all gene promoters are activated through both AF-1 and AF-2, though some are activated independently by AF-1 or AF-2 (Gronemeyer 1991; Osborne et al., 2001). Subsequent to formation of the ER-ligand complex, binding of co-regulatory molecules such as nuclear-receptor co-activator 1 (NCOA1 or SRC1), NCOA2 (TIF2) and NCOA3 (AIB1, TRAM1, RAC3 or ACTR) (Leo & Chen 2000; McKenna et al., 1999) enhance the transcriptional activity of ER accompanied by increased activity of histoneacetyltransferase (HAT) at the promoter site. Other co-regulatory molecules can also partly suppress the transcriptional activity of ER by recruitment of histone-deacetylase complexes such as nuclear-receptor co-repressor 1 (NCOR1) and NCOR2 that influence ER-induced transcription (Chen & Evans, 1995; Horlein et al., 1995). Several of these groups of molecules have been reported to have prominent roles in cancer. AIB1 (SRC-3) is over-expressed in almost two thirds of all breast cancers and associated with a shorter disease-free survival in patients receiving tamoxifen as adjuvant treatment (Osborne et al., 2003). In untreated patients, high levels of AIB1 were associated with improved outcome, consistent with studies that suggest the possibility of an association between an enhanced agonistic effect of tamoxifen and the high levels of co-activators. However, ER can also co-operate with FOS/JUN and bind with other transcription factors such as AP-1 (activator protein-1) and SP-1 (specificity protein-1) at their specific sites on DNA (Kushner et al., 2000; Ray et al., 1997; Safe 2001) commonly designated as serum response elements (SRE).

In addition to its classical mode of action through a nuclear-located receptor, estrogen has also been reported to interact with membrane associated receptors, leading to a more rapid reaction than would be expected from a transcriptionally mediated response, such as initiation of cAMP production (Rosner et al., 1999; Zivadinovic et al., 2005) and activation of intrinsic kinases present in other plasma membrane receptors such as insulin-like growth factor-1 receptor (IGF-1R), epidermal growth factor receptor (EGFR) and ERBB2 (Bunone et al., 1996; Campbell et al., 2001; Font de Mora & Brown 2000) as well as receptors for fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). It has been suggested that interaction of SERMs including tamoxifen with such membrane associated receptors may be responsible for their agonist behaviour. There is however much controversy over this issue, with other studies discounting the involvement of such postulated receptors as G proteincoupled receptor as targets of estrogen action (Otto et al., 2008). However this may be, any non-genomic interactions of estrogen would depend on the levels of the above-mentioned kinases, and they will likely be modest in ER+ breast cancer cells that express low levels of tyrosine kinase receptors such as EGFR and ERBB2 (Normanno et al., 2005).

Ligand independent activation of ER can occur via the downstream signaling cascades transmitted through membrane receptor tyrosine kinases such as EGFR, ERBB2, and IGF1R In particular. MAPK/ERK, PI3K/AKT, p90RSK and p38 MAPK pathways can specifically activate ER at key positions (serine 118 and 167 and threonine 311) in the AF-1 domain and in other domains (Bunone et al., 1996; Campbell et al., 2001; Joel et al., 1998; Kato et al., 1995). Expression of ligands and receptors such as transforming growth factor-α (TGFα), IGF1 and IGF1R can be increased by estrogen and those can then initiate signalling while expression of other receptors such as EGFR and ERBB2 is decreased by estrogen signaling (Kushner et al., 2000; Massarweh et al., 2008; Umayahara et al., 1994; Vyhlidal et al., 2000; Yarden et al., 2001). In addition, activation of the PI3K/AKT and the p42/44 MAPK pathways by these receptors down-regulates the expression of ER and PR causing reduction in estrogen dependency while activating the transcriptional function of ER, which suggests a contribution of this cross talk to the relative resistance to endocrine therapies in tumours with amplified ERBB2 expression (Bayliss et al., 2007; Brinkman and El-Ashry, 2009; Creighton et al., 2010; Lopez-Tarruella and Schiff, 2007).

The two types of ER actions, genomic and non-genomic, are not mutually exclusive and do overlap. For example, ER induces the expression of transcripts for both TGFα and amphiregulin (Normanno et al., 1993; Saeki et al., 1991) which can both bind and activate EGFR resulting in activation of MAPK and AKT signaling which are also activated by direct interaction with ER (Salomon et al., 1995). ER binding to membrane caveolin-1 leads to the activation of specific G proteins resulting in the activation of SRC and in turn of matrix metalloproteinases that cleave transmembrane precursors of the EGFR ligand, heparin binding-EGF (HB-EGF) (Levin, 2003; Razandi et al., 2003). Fig 2 illustrates the major identified downstream events involving ER activation.
