DiMeN Doctoral Training Partnership: Investigating glucocorticoid receptor modulators as new therapies for treating breast cancer

Triple negative breast cancer (TNBC) represents 10-20% of breast cancers diagnosed. TNBCs lack expression of the receptors normally used to stratify breast cancers - estrogen, progesterone and Her-2, which means current therapies that target these receptors are ineffective for TNBC. The only current therapeutic option for patients with TNBC, which is highly metastatic, is chemotherapy. Evidence is now emerging that TNBC have increased expression of a related steroid receptor, the glucocorticoid receptor (GR), which controls cell fate, immunity and metabolism. GR is a major drug target for treating inflammatory disease and a range of drugs that target GR are already in wide clinical use. Preliminary evidence suggests that repurposing existing drugs that modulate GR function may represent a new treatment option for TNBC.

GR is a ligand activated transcription factor. Once activated by binding glucocorticoids (Gc), GR translocates into the nucleus to regulate gene transcription. The sites GR can bind across the genome are determined by the availability of pre-existing binding sites, which means 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. 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.

The project will (i) define GR function in TNBC, (ii) therapeutic potential of GR modulators and (iii) remodeling GR 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 GR modulators of different pharmacologies (from GlaxoSmithKline), alone and in combination with chemotherapy and cellular effects characterized using relevant endpoint assays including mammosphere number, size, proliferation, apoptosis, cell cycle progression and DNA damage. We have shown that changes to the tissue microenvironment remodels GR responses through infiltration of immune cells and subsequent activation of the proinflammatory transcription factor NFkB by inflammatory cytokines. The importance of TNBC-immune cell crosstalk in remodeling 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.

We have recently shown that two other transcription factor pathways operating within the tumour, HIF1A (hypoxic core, tumour centre) and YAP/TEAD (cell-cell contacts, tumour surface) also modify GR responses. 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 determine by PCR and GR ChIP-PCR as above, comparing different layers and regulatory roles of HIF1A/ YAP/TEAD confirmed by CRISPR/Cas9 knockdown. Through innovative apprach, 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.

This project brings together a supervisory team with a track record of successful PhD student supervision, and significant expertise in breast cancer research (Prof Val Speirs, http://medhealth.leeds.ac.uk/profile/900/73/professor_valerie_speirs, University of Leeds and Prof Carlo Palmieri, https://www.liverpool.ac.uk/translational-medicine/staff/carlo-palmieri, University of Liverpool) and glucocorticoid receptor biology (Dr Laura Matthews, http://medhealth.leeds.ac.uk/profile/900/1658/laura_matthews, University of Leeds). The successful candidate will be based in Wellcome Trust Brenner building at St James’s University Hospital Campus, University of Leeds, the largest teaching hospital in Europe.

Training in all aspects of the project will be provided including 3D breast cancer cell culture; advanced bioinformatics of next generation sequencing data; CRISPR/Cas9; immunofluorescent microscopy; flow cytometry; PCR; ChIP-PCR and immunoblotting. These broad skillsets provide an excellent training opportunity for individuals hoping to pursue a research career in fields related to cell biology, molecular biology, cancer biology or pharmacology.