AbstractTriple negative breast cancer (TNBC) is a highly heterogeneous disease that accounts for around 15% of all breast cancers but is responsible for a disproportionately high rate of mortality. Characterised by lack of expression of the hormone receptors estrogen receptor α (ERα), progesterone receptor (PR) and devoid of amplification of the human epidermal growth factor receptor (HER2), these cancers cannot be treated with targeted therapies such as Tamoxifen and Herceptin which are often used in breast cancer. Thus, the current standard of care (SoC) for these patients is cytotoxic chemotherapy (often DNA damaging agents +/- anti-microtubule agents) with some patients responding well while others derive no clinical benefit. However, all patients experience unwanted and unpleasant side effects regardless of response. There are currently no biomarkers to predict which patients will respond to SoC and no targeted treatment approaches for those who do not respond. Subsequently, less than 30% of these patients will survive beyond 5 years.
An in-house, microarray-based gene expression analysis has identified a gene signature that has been shown to predict outcome in TNBC following SoC treatment. This project aims to build on findings from this analysis by identifying novel biomarkers and therapeutic targets that can be exploited to allow a personalised approach to the treatment of TNBC. Firstly, a hypothesis-led approach will characterise the role of NR3C1, which encodes the glucocorticoid receptor (GR) in TNBC as a biomarker and a therapeutic target. Secondly, a discovery-led approach will be taken by conducting a high throughput loss-of-function screen in a panel of TNBC cell lines to identify functional genes identified by the in-house analysis.
We have shown that high expression of GR at a gene and protein level is associated with an improved response to anthracycline-based chemotherapy. This relationship is unique to TNBC, with no association seen in other breast cancer subtypes. Additionally, high GR expression is indicative of patients who will respond poorly to taxane-based treatments, in accordance with previous publications. These results highlight how GR could be used as a predictive biomarker to discern the most effective chemotherapy option for TNBC patients at an early stage, forgoing the need for ineffective cytotoxic treatments. This will have the consequence of improving survival and reducing treatment-associated side effects.
The functional role of GR has also been interrogated using in vitro models of TNBC. We have shown that GR may be targeted to improve the response to anthracycline- and taxane-based chemotherapies in the luminal androgen receptor (LAR) subtype of TNBC. LAR TNBC accounts for 12% of all TNBCs and is characterised by androgen receptor (AR) driven growth and highlevels of chemoresistance. GR silencing using siRNA led to increased resistance to FEM (5- fluorouracil, epirubicin, mitomycin C) and docetaxel treatment in the LAR TNBC cell line, MDA-MB-453. Moreover, stimulation of GR using a synthetic agonist, dexamethasone, increased the sensitivity of this cell line to FEM chemotherapy. The GR-regulated effects on response to chemotherapy could be due to regulation of chemosensitivity mediators such as NF-κB or by the production of ROS in response to GR signalling. Given that these results were confined to the AR-expressing, LAR subtype of TNBC, it is likely that interactions between GR and AR are implicated in this process.
An siRNA screen guided by genes identified from the in-house bioinformatic analysis was conducted to identify genes that regulate viability, chemosensitivity and migration of TNBC cell lines. These genes could be exploited for use as targeted treatments for TNBC. Several genes were identified which regulated the survival and response to chemotherapy of cell lines spanning multiple molecular subtypes of TNBC. One gene of interest, COPZ1 was taken forward for further characterisation.
COPZ1 encodes a subunit of the coatomer complex which forms vesicles responsible for retrograde trafficking of cellular proteins from the Golgi to the endoplasmic reticulum (ER) as well as regulating autophagy and endosome maturation. COPZ1 silencing induces ER stress and cell death in multiple TNBC cell lines while having no effect on the normal breast cell line, 184A1. These effects can be attributed to cancer-specific silencing of the COPZ1 isoform, COPZ2 and can be counteracted by overexpression of COPZ2. Overexpression of COPZ1 leads to increased migration of the TNBC cell line, MDA-MB-468 highlighting its potential role in metastasis. The small molecule inhibitor of retrograde trafficking, brefeldin A led to dose dependent cytotoxicity and inhibition of migration in TNBC cells. These results highlight the potential for COPZ1 and/or retrograde trafficking to be targeted in the treatment of TNBC.
|Date of Award||Jul 2021|
|Sponsors||Northern Ireland Department for the Economy|
|Supervisor||Niamh Buckley (Supervisor) & Helen McCarthy (Supervisor)|
- Triple negative breast cancer
- personalised medicine
- glucocorticoid receptor
- retrograde trafficking
- siRNA screen