Understanding the mechanisms of action of estrogen receptor mutations in metastatic breast cancer
File(s)
Author(s)
Harrod, Alison Elizabeth
Type
Thesis or dissertation
Abstract
Estrogen receptor (ER) is a key driver of breast cancer development and progression and is the target for endocrine therapies. Use of endocrine agents has contributed greatly to reductions in breast cancer mortality over the last three decades. However, many patients develop resistance to these therapies, for reasons that remain unclear. Recent studies have identified ESR1 (ER) mutations in metastatic breast cancer that are predicted to encode ER proteins that are constitutively active and resistant to anti-estrogens. However, functional studies to determine the action of these mutations is impeded by the lack of breast cancer cell lines encoding ESR1 mutations. Here, I have developed the engineered CRISPR-Cas9 genome editing system to incorporate ESR1 mutations in the genomically encoded gene in the well-studied estrogen responsive and ER-positive MCF7 breast cancer cell line. I show that MCF7 cells with genomically encoded tyrosine 537 to serine (Y537S) ER mutation are estrogen independent for their growth and show global ligand-independent ER recruitment to regulatory regions and frequently elevated expression of estrogen-responsive genes. Ligand-independent expression and growth was also found for fourteen further CRISPR knockin MCF7 cells encoding additional ESR1 mutations, with between two and four individual clones derived for each mutation. Partial resistance to anti-estrogens is also seen with the ESR1 mutations, as higher concentrations of 4-hydroxytamoxifen and Faslodex are required to achieve the same growth inhibition as are required for the parental (wild-type) cells. All ESR1 mutation knockin lines demonstrated sensitivity to CDK7 inhibitors alone and in combination with Faslodex, highlighting the utility of these lines for the assessment of potential therapeutic agents. Finally, the use of CRISPR to revert an ESR1 mutation back to wild-type in a long-term estrogen-deprived MCF7 cell line, led to the restoration of an estrogen-dependent phenotype, confirming the importance of these mutations for ligand-independence.
Version
Open Access
Date Issued
2018-09
Online Publication Date
2021-02-28T00:01:24Z
2021-03-30T14:06:14Z
Date Awarded
2019-03
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Ali, Simak
Buluwela, Laki
Sponsor
Cancer Research UK
Grant Number
P47093_WSCC
Publisher Department
Department of Surgery & Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)