ESR1 mutation profiling identifies potential drivers of metastatic breast cancer
Lead Research Organisation:
Imperial College London
Department Name: Surgery and Cancer
Abstract
Breast cancer is the most common cancer in the world, being responsible for 1 in 4 cancer diagnoses and 1 in 6 cancer deaths in women every year. The female hormone estrogen promotes both breast cancer development and its progression. The estrogen receptor (ER) protein mediates the cellular actions of estrogen. Estrogen binding to ER is necessary for activation of the ER. The activated estrogen-bound ER protein stimulates breast cancer development by inducing expression of the genes that work together to drive breast cancer growth. The majority (>70%) of breast cancers are ER-positive and standard-of-care drug treatments for ER-positive breast cancer work by blocking ER activity. These "endocrine therapies" are proven, effective treatments for reducing illness and death from breast cancer. However, many patients relapse. Rates of recurrence, inevitably of endocrine therapy refractory metastatic disease, remain constant even several decades after original diagnosis and surgical removal of the tumour from the breast, necessitating the development of new treatments.
Recent advances in DNA sequencing have revealed that the ER gene is mutated in up to 40% of patients whose tumours recur. These mutated ER proteins are constitutively active such that their activity is not blocked by endocrine therapies. Consequently, breast cancer patients with mutant ER cannot be controlled with endocrine therapies.
Established breast cancer cell lines originally isolated from patient tumours provide an invaluable resource for studying mechanisms of cancer cell growth and for identifying approaches for killing cancer cells and for developing and testing new cancer drugs. We have used state-of-the-art CRISPR technology to engineer all the common ER mutations in breast cancer cell lines. These cell lines recapitulate the resistance to endocrine therapies that is observed in patients. By comparing gene expression profiles in ER mutant cells with those in endocrine therapy responsive, ER wild-type breast cancer cells, we have been able to identify a very small number of genes that are specifically activated in breast cancer cells that are making mutant ER. We have found that these mutant ER-specific genes are strongly predictive of poor likelihood of patient survival, evidencing their potential importance for endocrine therapy resistant breast cancer. The most prominent of these genes is itself a regulator of gene expression and has a well-known function in regulating cancer cell survival in other tumour types.
We hypothesise that activation of this gene plays important roles in the enhanced survival and invasive properties of ER-mutant breast cancer. By using molecular approaches to manipulate the levels of this gene in ER wild-type and ER mutant breast cancer cells, either by over-expressing it or by "knocking-out" the gene, we can investigate the mechanisms by which this gene drives breast cancer cell survival, metastasis and to progress to identifying new treatment opportunities in this large group of very difficult to treat breast cancer patients.
Recent advances in DNA sequencing have revealed that the ER gene is mutated in up to 40% of patients whose tumours recur. These mutated ER proteins are constitutively active such that their activity is not blocked by endocrine therapies. Consequently, breast cancer patients with mutant ER cannot be controlled with endocrine therapies.
Established breast cancer cell lines originally isolated from patient tumours provide an invaluable resource for studying mechanisms of cancer cell growth and for identifying approaches for killing cancer cells and for developing and testing new cancer drugs. We have used state-of-the-art CRISPR technology to engineer all the common ER mutations in breast cancer cell lines. These cell lines recapitulate the resistance to endocrine therapies that is observed in patients. By comparing gene expression profiles in ER mutant cells with those in endocrine therapy responsive, ER wild-type breast cancer cells, we have been able to identify a very small number of genes that are specifically activated in breast cancer cells that are making mutant ER. We have found that these mutant ER-specific genes are strongly predictive of poor likelihood of patient survival, evidencing their potential importance for endocrine therapy resistant breast cancer. The most prominent of these genes is itself a regulator of gene expression and has a well-known function in regulating cancer cell survival in other tumour types.
We hypothesise that activation of this gene plays important roles in the enhanced survival and invasive properties of ER-mutant breast cancer. By using molecular approaches to manipulate the levels of this gene in ER wild-type and ER mutant breast cancer cells, either by over-expressing it or by "knocking-out" the gene, we can investigate the mechanisms by which this gene drives breast cancer cell survival, metastasis and to progress to identifying new treatment opportunities in this large group of very difficult to treat breast cancer patients.
Technical Summary
The estrogen receptor (ER), a member of the nuclear receptor superfamily of ligand-activated transcription factors, is the key driver of breast cancer (BC) in most patients. Endocrine therapies (ET) work by blocking estrogen biosynthesis (aromatase inhibitors (AI)), by binding to ER to inhibit its activity, or by promoting its degradation. ER mutations are present in 20-40% of metastatic, ET-resistant BC patients and are associated with poor survival.
The mutant ERs are constitutively active, so are insensitive to AI and have reduced sensitivity to anti-estrogens. Expression of ER mutations in BC cell lines results in elevated metastatic potential consistent with their presence in metastatic BCs. RNA-seq of CRISPR-Cas9 generated ER mutant BC cell lines has allowed us to identify altered signalling pathways and to reveal a small number of genes that are commonly over-expressed in ER mutant cells. Elevated expression of these genes is associated with poor survival of ER-mutant metastatic BC patients.
One of these genes is a previously unidentified isoform of GATA4, a member of the GATA transcription factor family. GATA3 already has an established pro-tumorigenic role in ER+ BC. Its prior presence at gene regulatory regions licenses the chromatin to facilitate recruitment of liganded ER. A similar "pioneer factor" role has also been described for GATA4 in osteoblasts. We hypothesize that expression of the novel GATA4 isoform lacking transcription activation domains but encoding the DNA binding domain and C-terminal regions, modifies the ER transcriptome towards a more metastatic state. Transcriptomic and epigenomic profiling of ER mutant cells will be used to determine the molecular actions of the variant GATA4, while its role in growth, invasion and metastasis will be established in vitro and in vivo. The overarching aim is to identify the variant GATA4-directed signalling pathways that can be targeted for treatment of therapy-refractory ER-mutant BC patients.
The mutant ERs are constitutively active, so are insensitive to AI and have reduced sensitivity to anti-estrogens. Expression of ER mutations in BC cell lines results in elevated metastatic potential consistent with their presence in metastatic BCs. RNA-seq of CRISPR-Cas9 generated ER mutant BC cell lines has allowed us to identify altered signalling pathways and to reveal a small number of genes that are commonly over-expressed in ER mutant cells. Elevated expression of these genes is associated with poor survival of ER-mutant metastatic BC patients.
One of these genes is a previously unidentified isoform of GATA4, a member of the GATA transcription factor family. GATA3 already has an established pro-tumorigenic role in ER+ BC. Its prior presence at gene regulatory regions licenses the chromatin to facilitate recruitment of liganded ER. A similar "pioneer factor" role has also been described for GATA4 in osteoblasts. We hypothesize that expression of the novel GATA4 isoform lacking transcription activation domains but encoding the DNA binding domain and C-terminal regions, modifies the ER transcriptome towards a more metastatic state. Transcriptomic and epigenomic profiling of ER mutant cells will be used to determine the molecular actions of the variant GATA4, while its role in growth, invasion and metastasis will be established in vitro and in vivo. The overarching aim is to identify the variant GATA4-directed signalling pathways that can be targeted for treatment of therapy-refractory ER-mutant BC patients.
