A molecular model for the mechanism of acquired tamoxifen resistance in breast cancer

Abstract

Purpose

Oestrogen (E₂)-stimulated growth re-emerges after a c-Src inhibitor blocking E₂-induced apoptosis. A resulting cell line, MCF-7:PF, is selected with features of functional oestrogen receptor (ER) and over-expression of insulin-like growth factor-1 receptor beta (IGF-1Rβ). We addressed the question of whether the selective ER modulator (SERM), 4-hydroxytamoxifen (4-OHT) or other SERMs could target ER to prevent E₂-stimulated growth in MCF-7:PF cells.

Methods

Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was measured through real-time RT-PCR. Recruitment of ER or nuclear receptor coactivator 3 (SRC3) to the promoter of ER-target gene was detected by chromatin-immunoprecipitation (ChIP).

Results

4-OHT and other SERMs stimulated cell growth in an ER-dependent manner. However, unlike E₂, 4-OHT suppressed classical ER-target genes as does the pure antioestrogen ICI 182,780 (ICI). ChIP assay indicated that 4-OHT did not recruit ER or SRC3 to the promoter of ER-target gene, pS2. Paradoxically, 4-OHT reduced total IGF-1Rβ but increased phosphorylation of IGF-1Rβ. Mechanistic studies revealed that 4-OHT functioned as an agonist to enhance the non-genomic activity of ER and activate focal adhesion molecules to further increase phosphorylation of IGF-1Rβ. Disruption of membrane-associated signalling, IGF-1R and focal adhesion kinase (FAK), completely abolished 4-OHT-stimulated cell growth.

Conclusions

This study is the first to recapitulate a cellular model in vitro of acquired tamoxifen resistance developed in athymic mice in vivo. Importantly, it provides a rationale that membrane-associated pathways may be valuable therapeutic targets for tamoxifen resistant patients in clinic.

Introduction

The clinical application of the laboratory strategy of long-term adjuvant antihormone therapy for the treatment of breast cancer [1] has significantly improved breast cancer survival. Selection of patients whose tumours express the oestrogen receptor (ER) are more likely to respond to long-term adjuvant tamoxifen (TAM) [2], [3] or aromatase inhibitors (AIs) [4] than those without ER. However, acquired resistance to antihormone therapy remains a challenge as adjuvant therapy is extended [3], [5].

The evolution of acquired resistance to TAM treatment was discovered using MCF-7 tumours transplanted in athymic mice to mimic years of adjuvant treatment in patients [6], [7], [8]. Long-term therapy generates selection pressure for cell populations that evolve from acquired TAM resistance, ubiquitously observed in metastatic breast cancer, to eventually expose a vulnerability that is expressed as oestrogen (E₂)-induced apoptosis [8], [9], [10]. Acquired resistance to TAM or other selective ER modulators (SERMs) is unique in that the growth of resistant tumours is dependent on SERMs [6], [7], [8]. Acquired TAM resistance during the treatment of metastatic breast cancer occurs within one or two years [11], consistent with the model of SERM resistance in athymic mice [6], [8]. An AI (depleting E₂) or fulvestrant (ICI 182,780; a pure antioestrogen that destroys the ER) is effective as second-line therapy after TAM failure [12], [13]. Thus, it appears that acquired resistance to SERMs is initially able to utilise either E₂ or a SERM as the growth stimulus in ER-positive TAM-resistant breast tumours. However, no mechanism has been established to explain this paradox.

We describe a new model of antihormone-resistant breast cancer in vitro that exhibits the characteristics of acquired TAM resistance in vivo. The MCF-7:5C cell line emerged unexpectedly from an established MCF-7 cell line after long-term E₂ deprivation, i.e. simulated AI resistance [14]. The E₂-deprived cell lines [14], [15] created from MCF-7 cells have the unique ability to undergo E₂-induced apoptosis that has clinical significance for the treatment [16] and prevention of breast cancer [17]. We discovered that if a c-Src inhibitor is applied, E₂-induced apoptosis is initially blocked in MCF-7:5C cells [18], but with extended treatment, E₂-stimulated growth re-emerges [19]. A stable cell line, MCF-7:PF is established [19], [20]. Unexpectedly, the derived cell line MCF-7:PF was found to mimic the SERM/E₂-stimulated models in vivo [6], thereby providing the opportunity to decipher the mechanism of SERM-stimulated growth. Here, we provide evidence that 4-hydroxytamoxifen (4-OHT)-stimulated growth of MCF-7:PF is ER-dependent despite suppression of classical ER-target genes. However, 4-OHT functions as an agonist to enhance the non-genomic activity of ER and activates focal adhesion molecules to further increase phosphorylation of insulin-like growth factor-1 receptor beta (IGF-1Rβ). All of these events promote 4-OHT-stimulated cell growth. Overall, the sustained inhibition of nuclear ER-signalling causes broad activation of membrane-associated signalling to aid breast cancer cell survival during the selection process required for acquired TAM resistance.