Fulvestrant induces resistance by modulating GPER and CDK6 expression: implication of methyltransferases, deacetylases and the hSWI/SNF chromatin remodelling complex

Abstract

Background: Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance.

Methods: Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated.

Results: Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations.

Conclusion: The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.

Figure 1

Sensitivity of MCF-7 and FR.MCF-7 cells to FUL, TAM and E2. (A) MCF-7 and FUL-resistant (FR) MCF-7 cells were exposed to increasing concentrations of FUL or (C) TAM and cell proliferation was measured after 48 h. (B) Fulvestrant-resistant.MCF-7 cells were grown in hormone-deprived (DCC) medium±E2 for 96 h when cells were counted. Experiments were carried out in triplicate and error bars indicate s.e.m. and asterisks denote significance. (A, C) One-way ANOVA and Dunnett's post-test and (B) t-test.

Figure 2

Gene expression upon treatment with AZA and TSA. Fulvestrant-resistant.MCF-7 cells were pretreated for 24 h with 100 ng ml⁻¹ TSA and for 72 h with 2.5 μM AZA. Then, cells were lysed, RNA was extracted, mRNA reverse transcribed to cDNA and Q-PCR performed. The expression levels of GREB1, BCAR1, BCAR3, GPER, CDK6, ABCG2 and ERBB2 were standardised to GAPDH mRNA expression in FR.MCF-7 cells and naive MCF-7 cells. Experiments were carried out in duplicate and error bars indicate s.e.m.

Figure 3

Testing of specific gene products required for the cellular response to FUL. (A) MCF-7 cells were transfected with the indicated small interfering RNAs (siRNAs) or with control RNA in which no complementary cellular RNA exists, were treated with 500 nM FUL or solvent (Co) for 48 h and then cells were counted. (B, C) Fulvestrant-resistant.MCF-7 cells were exposed to 500 nM FUL alone or (B) in combination of 2.5 μM FTC or (C) in combination with 1 μg ml⁻¹ TRA, and the cell number was measured after 48 h. Experiments were carried out in triplicate, error bars indicate s.e.m. and asterisks denote significance (t-test).

Figure 4

Effects of FUL and PD0332991 on cell cycle regulators. (A) MCF-7 and FR.MCF-7 cells were grown to 70% confluence, or (B) treated with 500 nM FUL or solvent (Co) for the indicated times when they were lysed and similar amounts of protein subjected to electrophoretic separation and western blot analysis with the indicated antibodies. Equal sample loading was confirmed by Ponceau S staining and α-tubulin analysis. (C) MCF-7 and FR.MCF-7 cells were grown for 24 and 48 h when cells were counted and the duplication time calculated. Experiments were carried out in triplicate, error bars indicate s.e.m. and asterisk denotes significance (t-test). (D) MCF-7 and FR.MCF-7 cells were exposed to 25 and 50 nM PD0332991 and cell proliferation was measured after 48 h. Experiments were carried out in triplicate, error bars indicate s.e.m. and asterisks denote significance (t-test). (E) MCF-7 and FR.MCF-7 cells were grown to 70% confluence and treated with 50 nM PD0332911 or solvent (Co) for the indicated times when they were lysed and similar amounts of protein subjected to electrophoretic separation and western blot analysis with the indicated antibodies. Equal sample loading was confirmed by Ponceau S staining and α-tubulin analysis.

Figure 5

Effects of fulvestrant on SWI/SNF subunits and targets. MCF-7 and FR.MCF-7 cells were incubated with 500 nM FUL and harvested after 2, 8, 24 and 48 h of treatment. Cells were lysed, protein samples subjected to electrophoretic separation and to western blot analysis with the indicated antibodies. Equal sample loading was confirmed by Ponceau S staining and β-tubulin analysis.