Investigating glucocorticoid receptor modulators as new therapies for breast cancer
Lead Research Organisation:
University of Leeds
Department Name: School of Medicine
Abstract
Breast cancer is the most common cancer diagnosed in women worldwide and between 10-20% of cases are of the highly aggressive and metastatic triple-negative breast cancer (TNBC) subtype. TNBCs lack expression of receptors for the hormones estrogen and progesterone and for the protein Her2. It is these receptors that are the target for many breast cancer therapies and so hormone therapy and the Her2 targeted drug trastuzumab (Herceptin) are ineffective in treating patients with TNBC. For patients with TNBC the only current therapeutic option is chemotherapy.
There is emerging evidence that TNBC has increased expression of the glucocorticoid receptor, a steroid hormone receptor which has roles in controlling cell fate, immunity and metabolism. The GR is a major target for treating inflammatory disease and many drugs that target GR are already in clinical use. Preliminary evidence suggests that repurposing existing drugs that modulate GR function may represent a new treatment option for TNBC.
The GR is a ligand activated transcription factor, which once activated by binding glucocorticoids (Gc) translocates into the nucleus to regulate gene transcription. Sites that GR can bind to across the genome are determined by the availability of pre-existing binding sites, meaning that changes to the tissue microenvironment that alter the binding of other transcription factors modify GR responses. In most tissues GR acts as a tumour suppressor. Currently the detrimental effect of increased GR activity in metastatic TNBC is unknown, but suggests a functional switch, possibly through crosstalk with other transcription factor pathways operating in TNBC.
This project aims to:
1. Define the function of glucocorticoid receptors in TNBC
2. Determine the therapeutic potential of glucocorticoid receptor modulators in TNBC
3. Remodel glucocorticoid receptor responses by the local environment and within the tumour
TNBC cell lines and primary cells (Breast Cancer Now tissue bank) grown as 3D mammosphere cultures will be treated with a range of GR modulators (from GlaxoSmithKline), alone and in combination with chemotherapeutic drugs. Cellular effects will be characterised using relevant endpoint assays including mammosphere number, size, proliferation, apoptosis, cell cycle progression and DNA damage.
Changes to the tissue microenvironment remodel GR responses through infiltration of immune cells and the subsequent activation of the proinflammatory transcription factor NFkB by inflammatory cytokines. The importance of TNBC-immune cell crosstalk in remodelling GR responses will therefore be determined using TNBC mammosphere/immune co-cultures or inflammatory cytokine cocktails and measuring GR effects on cell function, GR regulation of known target genes using PCR and genome binding using GR ChIP-PCR, and any regulatory role of NFkB confirmed by CRISPR/Cas9 knockdown.
Two other transcription factor pathways operating within the tumour have been shown to modify GR responses - HIF1A (hypoxic core at tumour centre) and YAP/TEAD (cell-cell contacts at tumour surface). Stable dual reporter TNBC cells expressing CRISPR/Cas9 fluorescent reporter genes induced by HIF1A (red) and TEAD transcription factors (green) will be grown as mammospheres and 'functional' layers separated by flow cytometry. Intratumour heterogeneity in GR responses will be determined by PCR and GR ChIP-PCR comparing different layers and regulatory roles of HIF1A/YAP/TEAD confirmed by CRISPR/Cas9 knockdown.
Through these approaches we will better understand crosstalk between major transcription factor pathways operating in tumours and determine the therapeutic value of GR modulators as new treatments for TNBC.
There is emerging evidence that TNBC has increased expression of the glucocorticoid receptor, a steroid hormone receptor which has roles in controlling cell fate, immunity and metabolism. The GR is a major target for treating inflammatory disease and many drugs that target GR are already in clinical use. Preliminary evidence suggests that repurposing existing drugs that modulate GR function may represent a new treatment option for TNBC.
The GR is a ligand activated transcription factor, which once activated by binding glucocorticoids (Gc) translocates into the nucleus to regulate gene transcription. Sites that GR can bind to across the genome are determined by the availability of pre-existing binding sites, meaning that changes to the tissue microenvironment that alter the binding of other transcription factors modify GR responses. In most tissues GR acts as a tumour suppressor. Currently the detrimental effect of increased GR activity in metastatic TNBC is unknown, but suggests a functional switch, possibly through crosstalk with other transcription factor pathways operating in TNBC.
This project aims to:
1. Define the function of glucocorticoid receptors in TNBC
2. Determine the therapeutic potential of glucocorticoid receptor modulators in TNBC
3. Remodel glucocorticoid receptor responses by the local environment and within the tumour
TNBC cell lines and primary cells (Breast Cancer Now tissue bank) grown as 3D mammosphere cultures will be treated with a range of GR modulators (from GlaxoSmithKline), alone and in combination with chemotherapeutic drugs. Cellular effects will be characterised using relevant endpoint assays including mammosphere number, size, proliferation, apoptosis, cell cycle progression and DNA damage.
Changes to the tissue microenvironment remodel GR responses through infiltration of immune cells and the subsequent activation of the proinflammatory transcription factor NFkB by inflammatory cytokines. The importance of TNBC-immune cell crosstalk in remodelling GR responses will therefore be determined using TNBC mammosphere/immune co-cultures or inflammatory cytokine cocktails and measuring GR effects on cell function, GR regulation of known target genes using PCR and genome binding using GR ChIP-PCR, and any regulatory role of NFkB confirmed by CRISPR/Cas9 knockdown.
Two other transcription factor pathways operating within the tumour have been shown to modify GR responses - HIF1A (hypoxic core at tumour centre) and YAP/TEAD (cell-cell contacts at tumour surface). Stable dual reporter TNBC cells expressing CRISPR/Cas9 fluorescent reporter genes induced by HIF1A (red) and TEAD transcription factors (green) will be grown as mammospheres and 'functional' layers separated by flow cytometry. Intratumour heterogeneity in GR responses will be determined by PCR and GR ChIP-PCR comparing different layers and regulatory roles of HIF1A/YAP/TEAD confirmed by CRISPR/Cas9 knockdown.
Through these approaches we will better understand crosstalk between major transcription factor pathways operating in tumours and determine the therapeutic value of GR modulators as new treatments for TNBC.