The Dark Side of Antihormonal Action in Breast Cancer

Julia M.W. Gee, Andrew Stone, Richard A. McClelland, Stephen Hiscox, Iain R. Hutcheson, Nicola J. Jordan, Heidi M. Fiegl, Martin Widschwendter, Victoria E. Shaw, Denise Barrow and Robert I. Nicholson


4.1 Introduction 64

4.2 Compensatory Pathways Are Induced by Antihormonal Treatment 67

4.2.1 Oestrogen Repression and the Antihormone-Induced Compensatory Genes

EGFR and HER2 67

4.2.2 Microarrays Reveal the Considerable Diversity of Antihormone-Induced Compensatory Genes 68

4.3 Long-Term Use of Antihormones Also Suppresses Expression of Tumour Suppressor/Pro-Apoptotic Genes 73

4.4 Therapeutic Implications 75

References 77

Abstract Antihormones are of substantial benefit in treating oestrogen receptora positive (ER+) breast cancer. However, their anti-tumour effect is limited by emergence of resistance. Our in vitro studies are highlighting a new underlying concept: that antihormones are not passive bystanders but alongside growth inhibitory effects promote adverse compensatory mechanisms within tumour cells. These mechanisms involve drug-induction of signalling elements normally suppressed by oestrogen (E2)-occupied ER While best exemplified by the tyrosine kinases epidermal growth factor receptor and HER2, microarrays reveal the true diversity of the induced signalling kinases, where their potential to promote resistance is exacerbated under paracrine conditions. Such drug-induced events permit initial ER+ breast cancer cell survival, allow development and maintenance of resistance, and also promote gain of invasiveness under conditions of poor intercellular contact. In addition, prolonged antihormonal exposure is associated with epigenetic

Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University Cardiff CF10 3NB, UK e-mail: [email protected]

S. Hiscox et al. (eds.), Therapeutic Resistance to Anti-Hormonal Drugs in Breast Cancer, DOI 10.1007/978-1-4020-8526-0.4, © Springer Science+Business Media B.V. 2009

silencing of classical E2-induced tumour suppressors, an event which further contributes to resistance. Based on proof of principle experiments targeting induced signalling events alongside antihormones or restoring E2-induced suppressor genes through DNA methylation inhibitor-containing strategies, it is our belief that continued deciphering of these mechanisms will reveal improved treatments for breast cancer.

Keywords Antihormone ■ Resistance ■ Microarray ■ Compensatory signalling ■ Tumour suppressor

4.1 Introduction

Antihormones that deplete oestrogen (E2)/oestrogen receptor a (ER) signalling promote worthwhile tumour remissions and significant survival benefits in many ER positive (ER+) patients in the advanced and adjuvant setting. Some of these agents act by competing with E2 for binding to its target receptor in breast cancer cells, resulting in an ER conformational change that blocks one of the two receptor Activation Functions, AF-2, as exemplified by "partial" antioestrogens such as tamoxifen. Others, notably aromatase inhibitors, severely deplete the oe-strogenic environment which theoretically should drive the ER into a fully inactive state, potentially equating with improved responses seen with such agents versus tamoxifen. However, the efficacy of all current antihormones is incomplete since there are a proportion of ER+ patients who exhibit an apparent intrinsic resistance, while despite differences in response duration according to antihormone type, acquisition of resistance also remain inevitable for 40% initial responders in the adjuvant setting and almost all patients with advanced metastatic disease (Nicholson and Johnston 2005; Normanno et al. 2005). Unfortunately, resistance can also associate with increased metastatic capacity of breast cancer cells (Hiscox et al. 2004, 2006a) and invariably ultimately equates with poorer outlook clinically.

Many mechanisms have been associated with antihormone resistance in ER+ breast cancer. Of note, based on pre-clinical studies including our own primarily with tamoxifen, changes in the dominant growth factor receptors (e.g. the receptor tyrosine kinases HER2 and the additional erbB family member epidermal growth factor receptor [EGFR]) and their downstream kinases (notably Ras/Raf/mitogen-activated protein kinases [MAPKs] and phosphoinositide 3-kinase [PI3K]/protein kinase B [AKT] signalling) have been substantially implicated in driving resistance (Gee et al. 2005a; Nicholson et al. 2007). Such signalling can be apparent de novo in the tamoxifen refractory state (e.g. associated with HER2 amplification), or can be a feature gained in the relapsed tumour cells (Knowlden et al. 2003). In some instances, these growth factor signalling pathways can harness the ER to promote growth despite presence of antihormones. Indeed, growth factor pathway/ER cross-talk at the nuclear level (including growth factor-driven ER phosphorylation and coactivator recruitment; Britton et al. 2006; Arpino et al. 2008), as well as non-genomic events at the plasma membrane (Fan et al. 2007), have been cumulatively implicated in promoting agonistic behaviour of tamoxifen in resistance and E2 hypersensitivity in the steroid-deprived environment (Nicholson et al. 2004). In addition, using classical signalling techniques such as Western blotting and immunocytochemistry as applied to our acquired tamoxifen resistant in vitro breast cancer cell model (TAMR), we have been able to identify that a further important growth factor receptor input in driving acquired tamoxifen resistance is the insulin-like growth factor receptor (IGF1R) that acts to facilitate EGFR signalling via activation of the tyrosine kinase (TK) Src kinase (Knowlden et al. 2005). Of note, when extreme, growth factor pathways can also work in an ER independent manner and can even promote ER loss in vitro and clinically in a small proportion of patients (Holloway et al. 2004; Munzone et al. 2006).

Such growth factor-driven mechanisms and their cross-talk with ER are being exploited therapeutically. Targeting of functional ER in recurrent disease through use of further antihormonal measures, such as the "pure" antioestrogen fulvestrant (faslodex ®) which reduces ER level, can be beneficial in breast cancer (Howell 2006; Chia and Gradishar 2008). However, intrinsic and acquired resistance again remain a significant hurdle. Based on promising model data, diverse clinical studies are also exploring the value of anti-erbB pharmacological inhibitors/antibodies and also agents blocking candidate downstream signalling (including MAPK inhibitors, farnesyltransferase inhibitors and mammalian target of rapamycin inhibitors), where the aim is again to treat resistance, and (alongside antihormones) delay this state and restore response (Johnston et al. 2008). However, to date many of these studies have proved relatively disappointing, with therapeutic resistance again a pervading problem (see Chapter 10). For example, in breast cancer while clinical benefit was commonly seen in our acquired resistance study with the anti-EGFR agent gefitinib, this largely comprised disease stabilisation that invariably culminated in disease relapse (Agrawal et al. 2005). Based on model systems, these hurdles were perhaps predictable since, for example, our EGFR+ TAMR cells showed initial growth inhibitory responses to the anti-EGFR agent gefitinib but were ultimately subject to resistance, an event also associated with a further gain in aggressive behaviour (Jones et al. 2004), while one of our EGFR+ fulvestrant resistant models (FASRLT) proved largely de novo resistant to this agent. In addition, EGFR blockade exerted only partial inhibitory effects on invasion of such acquired resistant models (Hiscox et al. 2004). Moreover, breast cancer xenograft studies (Shou et al. 2004) indicated that EGFR targeting alongside antihormones delayed (rather than prevented) development of resistance, although pan-erbB approaches can be more successful in such models (Arpino et al. 2007). Problems of resistance appear to extend to the anti-HER2 agent herceptin. While of improved value in treating breast cancer patients when combined with chemotherapy, responses to herceptin as a single agent only occur in ~ 30% of HER2+ patients (McKeage and Perry 2002) and again relapse is invariably the ultimate outcome following initial response to various herceptin treatment strategies (Nahta et al. 2006). While there are many reasons likely to contribute to the relatively disappointing performance of these types of agents clinically, it is likely that there are additional important determinants of endocrine resistance and associated progression in breast cancer that remain to be defined. Indeed, there is emerging data that clinical antihormone resistance is associated with heterogeneous gene expression profiles (Miller et al. 2008), suggesting that resistance involves multiple underlying pathways.

Use of high through-put microarray strategies has potential to be particularly enlightening in determining the breadth of drivers for resistant growth and progression. Our group has embraced Affymetrix (HG-U133A GeneChip) microarray technology as applied to in vitro breast cancer models to focus on identifying deregulated members of the "kinome" and their key regulators in this context. Particular emphasis has initially been placed on the TK category of the kinome (as defined by Manning et al. 2002 and the "KinWeb" resource, since these have commonly been implicated in neoplastic development and progression and moreover are being intensively studied as targets for anti-cancer drug

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