Tamoxifen resistance and epigenetic modifications in breast cancer cell lines

Abstract

Epigenetic mechanisms play crucial roles in many processes, including neoplasia, genomic imprinting, gene silencing, differentiation, embryogenesis and X chromosome inactivation. Their relevance in human disease and therapy has grown rapidly with the recent emergence of drugs that target for example DNA methylation or histone acetylation. Epigenetic effects were also recently highlighted by the deciphering of the mechanism of action of steroid hormones and anti-hormones acting through nuclear receptors. In this review, we focus on the epigenetic effects associated with long-term treatment of breast cancer cells with the antiestrogen (AE) tamoxifen, in the context of resistance appearance. We summarize the data obtained with a model cell line developed in our laboratory supporting a role for HP1 proteins in the irreversible inactivation of gene expression by long-term treatment with AE.

Figure 1. Overview of the working hypotheses concerning tamoxifen resistance

Estrogen receptor (ER) modifications may involve: mutations, alternative splicing or post-translational modifications. Coregulator dysfunction may involve variations of their cellular content as well as post-translational modifications. Interference with growth factor pathways may involve phosphorylation of the ER by growth factor-activated kinases [mitogen-activated protein kinase (MAPK), p90 ribosomal S6 kinase (RSK), serine threonine protein kinase B (AKT)], or the non-genomic action of ER on growth factor receptors. Intra-tumoral estrogens (synthesized either by breast, ovary or peripheral tissues) may compete with the binding of tamoxifen at the level of the ER. Tamoxifen metabolism and biodisponibility, involving liver metabolism and intracellular sequestration, were also investigated but with no clear indication that they could be involved in tamoxifen resistance.

Figure 2. Recruitment of HP1 protein to histones

A. Initial methylation of lysine 9 (K9) of histone H3 creates a high affinity binding site for HP1. Bound HP1 next recruits (protein-protein interaction) the histone methyltransferase (HMT) SU(VAR)3–9 that methylates adjacent nucleosomes. This in turn leads to the spread of HP1 along the chromatin fiber, which is supposed to adopt a more condensed conformation. B. Hypothetical model of recruitment of HP1 by tamoxifen-liganded estrogen receptor alpha bound on an estrogen response element (ERE). Corepressors NCoR and KAP-1 would be recruited next. The subsequent binding of HP1 to KAP-1 would allow the initiation of a series of cycles (as described in A) leading to the spreading of HP1 along the chromatin fiber and the inhibition of associated gene expression.

Figure 3. Irreversible transgene inactivation by 4-hydroxytamoxifen treatment in MVLN cells

Adapted from [28]. (A) MVLN cells were cultured for various times in DCC medium containing 200 nM OHTam. After OHTam treatment, they were stimulated for 48h with 1nM estradiol, and luminescence (per mg protein) was then recorded. (B) MVLN cells were cultured in either 200 nM OHTam, FCS medium or DCC medium for 30 days. They were then dispersed in FCS medium to obtain, 1 month later, separate clones whose luciferase activity was analyzed with a camera, and the percentage of luminous clone was determined.

Figure 4. Effect of HDAC-ER-GR chimeric receptor

A. Schematic representation of the chimeric construct: DNA-binding domain (DBD), ligand-binding domain (DBD), glucocorticoid receptor (GR), estrogen receptor (ER), histone deacetylase 1 (HDAC1). B. Adapted from [30]. As indicated in the figure, cell lines, as well as MVLN(−) [i.e., containing the tet-on system but devoid of chimeric construct], were treated for various times with 200 nM OHTam or 200 nM OHTam + 100 nM Bim. Recovered luciferase expression was induced in the presence of 1 nM E2 for 48 h. The results are expressed as the mean ± SD of triplicate values (RLU/mg protein) and as the percentage of luciferase activity under control condition at day 0.

Figure 5. Irreversible inactivation kinetics of luciferase transgene in MVLN, H-MVLN and K-MVLN

A. Schematic representation of the chimeric construct: androgen receptor (AR), heterochromatin protein 1 α (HP1α), and Krupple-associated box (KRAB) domain of the KOX-1 protein. B. Adapted from [31]. As indicated in the figure, cell lines, as well as MVLN(−) [i.e., containing the tet-on system but devoid of chimeric construct], were treated for various times with OHTam 100 nM or R1881 10 nM. Recovered luciferase expression was obtained and expressed as described in Figure 3.

Figure 6. Hypothetical models of irreversible inactivation of the luciferase transgene in MVLN cells

Recruitment of silencing complexes by: A. The chimeric receptor HP1α-ER(DBD)-AR(LBD), which drives the HP1 module near an estrogen responsive element (ERE). This in turn leads to the recruitment of the methyltransferase (HMT) SU(VAR)3–9 and the subsequent spreading of HP1 (as depicted in Figure 2A). B. The chimeric receptor KRAB module-ER(DBD)-AR(LBD), which drives the KRAB module near an ERE. This in turn would lead to the recruitment of the corepressor KAP-1 and then the protein HP1, followed by a sequence similar to the one described in Figure 2A [HDAC1 (histone deacetylase 1) could be involved in inhibitory protein complexes depicted in the figure]. C. The wild type estrogen receptor, which would operate through the mechanism depicted in Figure 2B.