**3.1 Alterations in ER expression or function**

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

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 insufficient to account for resistance.

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

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

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 457

had low levels of the metabolite when co-treated with paroxetine, a selective serotonin reuptake inhibitor (prescribed to alleviate tamoxifen-associated hot flashes) that can inhibit CYP enzymes. Heterozygous patients showed a better outcome when treated with tamoxifen, as compared with untreated patients suggesting a role for cytochrome

The presence of ER variants was also hypothesized to have a role in endocrine resistance. A reduced response to endocrine therapy has been associated with the presence of a new

It has been reported that ERß transcript levels were about 2-fold higher than those of ERα in tamoxifen-resistant as compared with tamoxifen-sensitive patients (Speirs et al., 1999) and that ERß bound to tamoxifen,raloxifen or the anti-estrogen ICI 164 384, increased transcription of AP-1-dependent genes (Paech et al., 1997). Other studies show that ERβ has a negative effect on ERα-promoted transcription (Hall & McDonnell 1999; Pettersson et al., 2000) or no correlation with response or resistance to endocrine treatment (Cappelletti et al., 2004). Development of antibodies distinguishing between the ER types and their variants has led to identification of responses in ERβ+ve but ERα-ve cancers and a potential role for the carboxy-terminally truncated variants of ERβ (ERβ2 and ERβ5) in tamoxifen responsiveness (Honma et al., 2008; Murphy and Watson, 2006). In addition to ERβ, the oestrogen-related receptor ERRγ was found to be over-expressed and mediated tamoxifen

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

P450 enzyme variants in regulating the response to tamoxifen (Wegman et al., 2005).

truncated variant of ER, ER36, in addition to the full-length receptor (Shi et al., 2009).

resistance in lobular invasive breast cancer models (Riggins et al., 2008).

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

**3.2 Estrogen receptor β**

**3.3 Adaptation to estrogen withdrawal** 

Fig. 2. Proposed cellular mechanisms mediated through the estrogen receptor. 1. Classical genomic mode of action, in which estrogen binds to an inactive ER complex, causing dissociation from heat shock and other cognate proteins, receptor dimerisation and phosphorylation (P). This can then interact directly with estrogen response elements (ERE) on target genes in concert with histone acetyl transferase (HAT) and several other coactivators (coA) or by association with the ubiquitous transcriptional factors FOS/JUN and with NCOA1 and AIB1 co-activators at API/SPI sites termed serum response element (SRE). 2. Cytoplasmically located ER can be phosphorylated by the action of AKT, SRC and ERK/MAPK serine/threonine kinases, downstream of signalling events initiated by various growth factors' interaction with their respective tyrosine kinase containing receptors and mediated through RAS or PI3K. This ligand- independent activated receptor can initiate transcription through the SRE. 3. Binding of estrogen to membrane–associated ER may induce assembly of complexes with either PI3K/FAK/SRC leading to activation through ERK of the transcriptional activator NCOA3 or with PELP1/SRC resulting in up-regulation of mTOR and NFKB through AKT. These mediate an action through other transcriptional response elements (TRE) on a variety of target genes without involving direct interaction of ER with chromatin. The latter mechanisms are referred to as the non-genomic pathways, that are postulated to explain those observed effects of estrogen which are too rapid to be accounted for by mechanism 1. Further 'crosstalk' between ER and RTKs may involve participation of PELP1.

had low levels of the metabolite when co-treated with paroxetine, a selective serotonin reuptake inhibitor (prescribed to alleviate tamoxifen-associated hot flashes) that can inhibit CYP enzymes. Heterozygous patients showed a better outcome when treated with tamoxifen, as compared with untreated patients suggesting a role for cytochrome P450 enzyme variants in regulating the response to tamoxifen (Wegman et al., 2005).

The presence of ER variants was also hypothesized to have a role in endocrine resistance. A reduced response to endocrine therapy has been associated with the presence of a new truncated variant of ER, ER36, in addition to the full-length receptor (Shi et al., 2009).
