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. 2017 Apr 20;36(16):2286-2296.
doi: 10.1038/onc.2016.382. Epub 2016 Oct 17.

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

A Harrod  1 , J Fulton  1 , V T M Nguyen  1 , M Periyasamy  1 , L Ramos-Garcia  1 , C-F Lai  1 , G Metodieva  2 , A de Giorgio  1 , R L Williams  1 , D B Santos  1 , P J Gomez  1 , M-L Lin  1 , M V Metodiev  2 , J Stebbing  1 , L Castellano  1 , L Magnani  1 , R C Coombes  1 , L Buluwela  1 , S Ali  1
Affiliations

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

A Harrod et al. Oncogene. .

Abstract

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CRISPR-Cas9-directed generation of the Y537S mutation in the ESR1 gene in MCF7 breast cancer cells. (a) Schematic representation of the ESR1 gene, exons 5-8, annotated for the positions of PCR primers used for RT-PCR analysis. (b) RT-PCR of MCF7 (WT) and MCF7-Y537S cell lines using primers in a. Expression of wild-type and mutant ER alleles was confirmed by RT-PCR, using primers in ESR1 Exon 5 (Primer 1, 5′-CCAGGGAAGCTACTGTTTGC-3′) and Exon 8 (Primer 3, 5′-GATGCATGCCGGAGTGTATG-3′), which generate a 700 bp product. The ESR1 transcript arising from the Y537S mutant allele was amplified as a 466 bp product using the exon 5 primer and the knock-in-specific primer. (Primer 2, 5′-TAGTGGGCGCGTGAAGTCTA-3′). (c) Sequencing chromatogram of the RT-PCR products for the exon 8 coding region for MCF7-Y537 S cells, showing expression of both mutant and wild-type alleles. (d) Frequency of RNA-seq reads for the Y537 and 537S codons in MCF7 and MCF7-Y537S cells. (e) Normalized intensity of ER tryptic peptide containing amino acid 537 from liquid chromatography mass spectrometry. Results are shown for three independent immunoprecipitated ER samples for MCF7 and MCF7-Y537S lines. No signal was obtained for the mutant peptide in MCF7 cells. For the MCF7-Y537S samples, the normalized intensity for each replicate sample is shown by circles of the same colour, demonstrating similar amounts of the two peptides in MCF7-Y537S cells.
Figure 2
Figure 2
MCF7-Y537S cells grow in an estrogen-independent manner. (a) MCF7 (WT) and Y537S cells were grown in the absence or presence of 10 nM estrogen over 12 days. (b, c) The cells were grown in the absence of estrogen (vehicle) or in the presence of 10 nM estrogen, together with 0, 1, 10, 100 or 1000 nM 4-hydroxytamoxifen (OHT; b) or faslodex (FAS; c). Mean growth at day 12 ±s.e.m. is shown relative to day 0 (n=6). (d) MCF7 and MCF7-Y537S cells, cultured in DMEM+10%FCS, were treated with OHT or FAS at the concentrations shown (nM). The graph shows mean growth at day 12±s.e.m. (n=6).
Figure 3
Figure 3
ER-Y537S mutant is globally recruited to ER binding regions in the absence of estrogen. (a) Overlap of significant ER binding sites as called by MACS analysis (P<1 × 10−5). (b) Heat map for ER binding events in MCF7 and MCF7-Y537S cells under vehicle (Veh) and estrogen (E2) treatment conditions. (c) Genome-wide profile of ER binding. (d) ChIP-seq tracks showing ER binding at two ER target genes. (e) Shown are the top enriched transcription factor binding motifs obtained using HOMER. See Supplementary Information for a complete list of motifs enriched in MCF7-WT and mutant cells. (f–k) ChIP was performed following treatment of MCF7 and Y537S cells cultured in hormone-depleted medium and treated with 10 nM estrogen for 45 min. Real-time PCR was used to determine enrichment at ER binding regions. PCR for two control regions near the TFF1 (control 1) and the PGR gene (control 2) is also shown. Bar charts represent the mean, ±s.e.m. (n=3). Asterisks represent statistically significant differences (unpaired t-test, P<0.05) for vehicle-treated MCF7-Y537S cells compared with MCF7 cells.
Figure 4
Figure 4
The Y537S mutation promotes estrogen-independent expression of ER target genes in breast cancer cells. (a) RNA-seq was performed using three replicate samples of hormone-depleted MCF7 and MCF7-Y537S cells, following addition of vehicle or estrogen for 8 h. Shown is a Venn diagram comparing differentially regulated genes (padj<0.05) identified from RNA-seq data. (b) The bar chart shows the normalized enrichment scores (NES) from Gene set enrichment analysis (GSEA) for ‘Hallmark' signalling pathways that are significantly up- or downregulated (q<0.00001) in pair-wise comparisons of the differentially regulated gene sets. (c) Hierarchical cluster analysis of genes that are differentially regulated in MCF7 cells ±E2 (padj<0.05) and for which an ER binding site is observed within 100 kb of the transcription start site. Bar charts show average normalized counts for all genes in the clusters. (d) E2 (1 nM), OHT (10 nM) or FAS (10 nM) were added to hormone-depleted MCF7 and MCF7-Y537S cells. Shown is RT-qPCR analysis of gene expression using RNA prepared 16 h following addition of ligands, relative to the vehicle-treated MCF7 cells (n=3). (e) Immunoblotting was performed using cell lysates prepared 24 h following addition of ligands.
Figure 5
Figure 5
Inhibition of MCF7-Y537S growth by the CDK7 inhibitor THZ1 in combination with anti-estrogens. (a) TFIIH interacts in an estrogen-independent manner with ER in MCF7-Y537S cells. Protein lysates from hormone-depleted MCF7 and MCF7-Y537S cells treated with estrogen (10 nM) for 3 h were immunoprecipitated with an antibody for ER, or with mouse immunoglobulins (IgG; control). Immunoprecipitates were immunoblotted with antibodies for the XPD subunit of TFIIH, the ER co-activator AIB1 and a control protein (Lamin A/C). (b) Cells were treated with increasing concentrations of THZ1 for 48 h. Mean growth is shown relative to that for vehicle (dimethylsulphoxide-treated cells) (n=6). GI50=concentration of THZ1 at which cell growth is inhibited by 50%. (c) Protein lysates prepared from cells treated with increasing concentrations of THZ1 for 4 h were immunoblotted for PolII and for phosphorylation of Ser2, Ser5 and Ser7 in the PolII C-terminal domain heptapeptide repeat. (d) Cells were grown in DMEM containing 10% FCS over a 12-day period in the presence of 10 or 100 nM FAS, together with 20, 50, 75 or 100 nM THZ1. Growth was assessed using the SRB assay and is shown relative to SRB values at day 0. The bars represent mean values ±s.e.m. for six replicates. (e–g) Immunoblotting was performed with protein lysates prepared 24 h after addition of FAS±75 nM THZ1.
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References

    1. Cuzick J, Sestak I, Baum M, Buzdar A, Howell A, Dowsett M et al. Effect of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: 10-year analysis of the ATAC trial. Lancet Oncol 2010; 11: 1135–1141. - PubMed
    1. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998; 339: 1609–1618. - PubMed
    1. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2002; 2: 101–112. - PubMed
    1. Ali S, Buluwela L, Coombes RC. Antiestrogens and their therapeutic applications in breast cancer and other diseases. Annu Rev Med 2011; 62: 217–232. - PubMed
    1. Johnston SR, Dowsett M. Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat Rev Cancer 2003; 3: 821–831. - PubMed

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