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Genetic events during the transformation of a tamoxifen-sensitive human breast cancer cell line into a drug-resistant clone

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Abstract

Tamoxifen resistance is a serious clinical problem commonly encountered in the management of patients with breast cancer. The mechanisms leading to its development are unclear. Tamoxifen acts via multiple pathways and has diverse effects. Hence transformation from a tamoxifen-sensitive to a resistant phenotype could involve multiple genetic events. Knowledge of the genetic pathways leading to resistance may facilitate the development of novel therapeutic strategies. In this study, a variation of conventional comparative genomic hybridization (CGH) has been employed to detect genetic alterations associated with tamoxifen resistance. MCF-7, a tamoxifen-sensitive human breast cancer cells line, and its tamoxifen-resistant clone, CL-9 were used. Both cell lines showed extensive areas of concordance but consistent differences were seen with the acquisition of tamoxifen resistance. These differences included the amplification of 2p16.3∼p23.2, 2q21∼q34, 3p12.3∼p14.1, 3p22∼p26, 3q, 12q13.2∼q22, 13q12∼q14, 17q21.3∼q23, 20q11.2∼q13.1 and 21q11.2∼q21 as well as the deletion of 6p21.1, 6p23∼p25, 7q11.1∼q31, 7q35∼q36, 11p15, 11q24, 13q33, 17p, 18q12∼q21.1, 19p, 19q13.3, 22q13.1∼q13.2. These findings were supported by conventional cytogenetics and chromosome painting. The regions identified by CGH potentially harbor genes that could be important in the development of tamoxifen resistance.

Introduction

Tamoxifen is a non-steroidal anti-oestrogen with partial agonistic activity, extensively used in the treatment of breast cancer. Response to tamoxifen is frequently of limited duration due to the development of resistance and the disease ultimately progresses [1]. Resistance to tamoxifen is clinically important as it influences disease outcome for a large number of women. However, acquisition of resistance is not completely understood. An increased understanding of the mechanisms underlying resistance is crucial to improving breast cancer survival.

The activity of tamoxifen in oestrogen receptor (ER) positive breast cancer is thought to be due to its competition with oestrogen for binding to the ER. However, this cannot be the only mode of action, because the ER can frequently be normal in cases with endocrine resistance [2]. Tamoxifen has been shown to have a number of ER-independent effects including stimulation of transforming growth factor-betal (TGF-β1) 3, 4 and inhibition of insulin-like growth factor 1 (IGF-1) stimulated growth of breast cancer cells [5]. Control of endocrine responsive breast cancer is thus thought to involve complex interactions between steroid hormones and growth factor pathways: it is not merely an ER function [6]. It is likely that anti-oestrogen resistance involves changes in growth factor driven proliferation and in cell survival signaling networks. These probably involve genetic events. Therefore in this study we used CGH to assess the genomic alterations involved in the development of tamoxifen resistance.

Section snippets

Cell lines

MCF-7 a tamoxifen-sensitive breast cancer cell line, and CL-9, its tamoxifen resistant clone (a gift from Professor AL Harris, ICRF, Oxford [7]) were analyzed. CL-9 was isolated from wild type MCF-7 after transfection with mixed cDNA libraries, followed by selection of resistant clones in the presence of tamoxifen. CL-9 is several-fold more resistant to tamoxifen than MCF-7 both in vitro and in vivo but retains functional oestrogen receptors. Both cell lines were maintained in 25 cm2 tissue

Results

The CGH analysis detected many similarities between the two cell lines as might be expected (Fig. 2). Interestingly, consistent areas of differences between the two cell lines were identified involving amplification of chromosomes 2p16.3∼p23.2, 2q21∼q34, 3p12.3∼p14.1, 3p22∼p26, 3q, 12q13.2∼q22, 13q12∼q14, 17q21.3∼q23, 20q11.2∼q23, and 21q11.2∼q21 as well as the deletion of chromosomes 6p21.1, 6p23∼p25, 7q11.1∼q31, 7q35∼q36, 11p15, 11q24, 13q33, 17p, 18q12∼q21.1, 19p, 19q13.3, and 22q13.1∼ 13.2

Discussion

Tamoxifen activity in breast cancer is usually described in terms of its competitive interference with the binding of oestrogen to the oestrogen receptors α and β. The mechanisms underlying the development of tamoxifen resistance remain elusive. In this study, CGH was used to try to identify genetic events associated with tamoxifen resistance.

It is now recognized that tamoxifen resistance is generally not associated with the loss or abnormal function of the ER as only around 10% of

Acknowledgements

Work in the authors laboratory is supported by Yorkshire Cancer Research, the Medical Research Council and the Imperial Cancer Research Fund. R.A. is a clinical research training fellow funded by The British Cancer Research Action Group.

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