1.5.3.1 Ligand independent ER activation
Most resistant tumours continue to express ER (Johnston et al., 1995; Guiterrez et
al., 2001). Although this does not dictate continued dependence on ER signalling,
nevertheless a cohort of resistant patients do respond to second-line endocrine treatment (Howell et al., 1996; Buzdar et al., 2001) indicating maintained reliance on ER signalling for resistant growth and disease progression.
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It has been suggested that ligand-independent activation of ER (independent or downstream of any non-genomic ER input) is linked to endocrine resistance, with ER becoming activated despite E2 deprivation or presence of tamoxifen. Such events can promote hypersensitivity to any residual oestrogens (Chan et al., 2002) or tamoxifen agonism (Shou et al., 2004). Phosphorylation of 2 key AF-1 residues in ER have been associated with resistance, serine 118 (S118) and serine 167 (S167). Both are reported to be important for ER activity (Arnold et al., 1994; Thrane et al., 2013). Phosphorylation of S118 has been shown to be mediated by not only oestrogen but also by growth factor ligands such as EGF and IGF-1 (Joel et al., 1998; Lannigan et al., 2003) as well as downstream growth factor signalling molecules ERK1/2 and p38 MAPK (Kato et al., 1998; Joel et al., 1998; Gutierrez et al., 2005; Britton et al., 2006). Further the PI3K/AKT signalling pathway has also been implicated in phosphorylation of S167 (Campbell et al., 2001). Subsequently, up- regulation of these signalling pathways has been associated with endocrine resistance (Shim et al., 2000; Campbell et al., 2001; Gutierrez et al., 2005). There is also cell model evidence that some of the target growth factor receptors in such pathways (e.g. erbB family members HER2 and EGFR) are oestrogen-repressed and antihormone-induced elements, and so increases in such receptors have also been implicated in the development of resistance, cross-talking with ER through their downstream kinase signalling to drive acquired resistant cell growth (Gee et al., 2003; Nicholson et al., 2005; Britton et al., 2006). HER2 induction in resistance is believed to be a consequence of loss of the transcription factor PAX2, which is involved in its oestrogen/ER-mediated repression (Hurtado et al., 2008)
Most of the above research was carried out in relation to tamoxifen treatment and in light of the mechanism of action of Faslodex, ligand independent activation of ER should be reduced as the ER protein is degraded and activity of both AF-2 and AF-1 are blocked. Nevertheless, as explained previously 250mg Faslodex failed to sufficiently suppress ER level leading to the approval of 500mg Faslodex. While this dosage was found to be biologically and clinically superior, clinical trials also indicate that this higher dose fails to completely suppress ER expression and
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function (Kuter et al., 2012; Di Leo et al., 2010; Robertson et al., 2009) and thus it remains feasible that ligand independent ER activation may contribute to Faslodex resistance. Indeed, the responses observed to further endocrine agents following Faslodex failure at least in a proportion of patients implies some retained importance for this receptor (Vergote et al., 2003), and trials are ongoing to determine if higher doses, or combination with alternative endocrine agents, can promote superior ER blockade (Robertson et al., 2009; Di Leo et al., 2010; Kuter et
al., 2012; Mehta et al., 2012).
1.5.3.2 Loss of reliance on ER signalling
A proportion of resistant tumours appear to be ER independent: they are no longer reliant on ER signalling for resistant cell growth and survival and thus are not growth inhibited by further antihormone treatments. It seems that such tumours have acquired the ability to employ alternative signalling pathways which ultimately leads to the emergence of a resistant cancer cell population. This has been observed in a significant number of patients who fail to respond to second-line endocrine therapy (Buzdar et al., 2001). The hyperactivation of growth factor signalling pathways specifically has again been heavily implicated in ER- independent tumour growth and survival (Normanno et al., 2005).
In MCF-7-derived acquired tamoxifen-resistant and Faslodex-resistant cells, HER2 and/or EGFR expression is elevated in comparison to wild-type cells. These antioestrogen-resistant cells can also show increased EGFR/HER2 heterodimerisation and activity. Such partners activate MAPK and PI3K/AKT signalling pathways driving antioestrogen-resistant cell growth (McClelland et al., 2001; Knowlden et al., 2003; Jordan et al., 2004). While in the tamoxifen resistant cells hyperactivated EGFR/HER2 signalling activates ER in a ligand independent manner (Britton et al., 2006), such growth factor receptor signalling must function independently of ER where acquired Faslodex resistant cells lose this receptor. Faslodex has also been shown to differentially regulate EGFR ligands leading to an increase in EGFR signalling reducing Faslodex response in MCF-7 cells (Zhang et al., 2013). Nf-κB may also be involved, as its p65 subunit has been shown to increase in Faslodex resistant MCF-7 cells (Gu et al., 2002; Riggins et al., 2005)
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1.5.3.3 Impact of targeting growth factor pathways in resistance
The potential importance of growth factor signalling pathways in driving resistant cell growth, either through their cross-talk with ER or in an ER-independent manner, has been demonstrated by using agents that specifically target these growth factor receptor pathways. EGFR or HER2 blockade in either acquired tamoxifen-resistant or Faslodex-resistant MCF-7 cells using gefitinib or trastuzumab respectively results in potent growth inhibition, whereas in antihormonal untreated cells inhibition is small due to the low basal levels of such growth factor receptors in the presence of functional E2/ER signalling which represses these receptors (McClelland et al., 2001; Knowlden et al., 2003). Co-treatment with such targeted agents alongside antihormones such as tamoxifen can also subvert emergence of resistance in MCF-7 cells (Gee et al., 2003; Leary et
al., 2010). Targeted therapy of the downstream kinases such as MAPK or PI3K/AKT
has also been shown to inhibit growth of such antiestrogen-resistant cells (McClelland et al., 2001; Knowlden et al., 2003; Jordan et al., 2004). Immunohistochemical analysis of clinical breast cancer tissue has also shown increased expression and/or activity of EGFR, HER2 and MAPK members in samples from de novo ER+ (and ER-) tamoxifen resistant patients as well as in some patients who acquired resistance (Gutierrez et al., 2005) including samples obtained from tamoxifen-treated ER+ primary elderly patients (Gee et al., 2005).
In total, such findings have provided the rationale for the introduction of trials looking at EGFR/HER2 targeted therapies alongside antihormones to control resistance in the clinic (Christofanilli et al., 2010; Osborne et al., 2011; Carlson et al., 2012). Emerging data indicates that some tamoxifen or AI treated breast cancer patients (notably those who are also HER2+) can respond to such targeted agents (Gutteridge et al., 2010; Osborne et al., 2011) but responses can be short-lived, indicating further contributory resistance mechanisms. The impact of growth factor–targeted treatments in Faslodex resistant disease remains to be established in the clinic. However, it is already clear that up-regulation of EGFR, HER2 along with MAPK activity is not observed in all Faslodex-resistant cell models where, for example, other members of the erbB family have been reported to contribute to
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reduced efficacy of Faslodex (Sonne-Hansen et al., 2010; Thrane et al., 2013). Further, ER has been observed to be lost in some cell models of Faslodex resistance while retained in others (Liu et al., 2004; Nicholson et al., 2007). Importantly, the long-term biological effects of Faslodex in the clinic are not well reported and thus the prevalence of growth factor pathways and relation to any retained ER following the development of Faslodex resistance are unknown. Further studies are clearly required to understand both response and resistance mechanisms to Faslodex, within and beyond growth factor signalling, particularly given the limited success of antihormone (tamoxifen and AI’s) alongside further targeted therapies in the clinic.