Investigating the functional importance of estrogen receptor alpha mutations in endocrine- resistant, metastatic breast cancer

Abstract

The growth of between 70% and 80% of breast cancers is driven by the expression of estrogen receptor-α(ER). This understanding has led to the development of endocrine therapies which inhibit ER activity. While these have vastly improved patient survival, around 30% of patients will relapse, invariably developing metastatic, incurable disease. Recurring mutations in the ER gene, ESR1, are detected in more than 20% of endocrinerefractory, metastatic tumours, implicating these as an important mechanism of resistance. However, the molecular consequences of many of these mutations are, as yet, poorly-defined. Here, I have explored the transcriptomic actions of ESR1 mutations using CRISPR-cas9 knock-in mutant models in the MCF-7 breast cancer cell line. I show that MCF7 cells expressing the L536R, Y537C, Y537N, Y537S or the D538G ESR1 mutations express a novel isoform of the transcription factor GATA4. Elevated expression of this isoform is seen in a broad range of cell line and patient-derived ER mutant models. Furthermore, I have generated knock-in models of the ESR1 exon 5 mutation E380Q. I show that, unlike wild-type (WT) parental cells, cells expressing the E380Q mutant are estrogen-independent for their growth. Moreover, I have shown that functionally homozygous, but not heterozygous E380Q mutant cells exhibit decreased sensitivity to selective estrogen receptor modulators and degraders (SERM/SERDs). Surprisingly, transcriptomic analysis by RNA-sequencing revealed that, unlike L536/Y537/D538 mutations, cells expressing E380Q have markedly reduced ER signalling relative to WT cells. Mutant clones were found to express an E380Q-specific gene signature that was associated with poor relapse-free survival in ER-positive breast cancer patients treated with endocrine therapy. Taken together, the results presented in this thesis highlight a role for the E380Q mutation in conferring resistance to estrogen-deprivation therapy. The significance of this mutation in mediating resistance to SERMs and SERDs is less clear, and may be influenced by continued expression of a WT allele. Crucially, work in this thesis provides evidence that the E380Q mechanism of action is distinct from that of mutations in the L536/Y537/D538 ER mutational hotspot.