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.
Highlights
As the major driver of breast cancer development and progression, estrogen receptor-α (ER) is the pre-eminent target in 80% of breast cancers
This screening approach provides a rapid method for evaluating CRISPR activity and on this basis CRISPR058819 was used for generating the Y537S mutation in MCF7 cells
Mutations in the ER gene have recently been found to be common in advanced breast cancer and are likely to represent an important mechanism of endocrine resistance
Summary
As the major driver of breast cancer development and progression, estrogen receptor-α (ER) is the pre-eminent target in 80% of breast cancers. Functional studies following ectopic expression of ER in which L536 or Y537 were substituted by other amino acids showed ligand-independent activation of estrogen-responsive reporter genes and interaction with coactivator proteins.[13,20,21,22,23,24,25] ER mutated at these residues is inhibited by anti-estrogens, there is evidence for an attenuated response to anti-estrogens, at least for some substitutions.[10,14,26] it is possible that the observed resistance is reflective of the fact that studies to date have employed ectopic overexpression of the mutant proteins. CRISPR-Cas9-mediated genome editing provides a highly specific method for gene deletion and knock-in mutagenesis in mammalian cells,[27] potentially allowing more faithful evaluation of the functional importance of gene mutations in model systems
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