Current research

Multifactorial impacts on early breast carcinogenesis – assessing the combined effects of preventable factors on breast cancer using a novel Organ-On-a-Chip platform.

Breast Cancer UK has awarded a grant of £43,360 to Dr Elisabete Silva and colleagues, Drs Ruth MacKay, Sibylle Ermler and Emmanouil Karteris, Brunel University London, to fund research that examines the role of EDCs and saturated fatty acids on early stages of breast cancer. The project began in February 2020 and will finish February 2022.

Background and details of the research 

Lifestyle factors and environmental contaminants, including endocrine disrupting chemicals (EDCs), play a role in breast cancer development but human studies linking EDCs to cancer are often inconclusive. This is mainly because EDCs are studied individually and at concentrations that don’t match our day to day exposure. We are exposed to a cocktail of chemicals, which in combination, may have enhanced harmful effects.

Several studies support a link between a high fat diet and breast cancer, although this link is still debated, as what lies behind this effect is unclear. A high fat diet may not on its own be a serious contributor to breast cancer but its effect may be enhanced by the presence of EDCs in our bodies. The current project aims to address this knowledge gap. It will investigate the impact of complex mixtures of EDCs and saturated fatty acids (as found in a high fat diet) on breast tissue development and growth and initiation of breast cancer.

Dr Silva’s research will use an innovative “Breast-on-Chip” methodology developed at Brunel University. Different breast cell types are grown together in 3D cell culture and are fed with a constant flow of media and nutrients. This system is designed to mimic the physical environment of breast tissue. The approach provides a more robust and representative alternative to current laboratory and animal-based methods.

The EDCs to be tested include bisphenol A (plastic component), PBDEs (flame retardants), parabens (preservatives), benzophenones (UV filters), DDT (pesticide), polychlorinated biphenyls (used in hydraulic fluids, now banned) and galaxolide (perfume). The three most common fatty acids (lauric, palmitic and steric acids) and EDCs will be tested at concentrations that have been measured previously in human breast tissue.

The research aims to understand the impact of combinations of chemical exposures and lifestyle factors on early stages of breast carcinogenesis. This will provide a framework for further public information and practical advice on ways to reduce exposure to factors that contribute to breast cancer risk.

Previous funding: This is Dr Silva’s second BCUK grant. Her previous work investigated effects of low-dose mixtures of EDCs on breast cancer initiation using 3D breast cell culture. She found that low concentrations of 4 EDCs (propylparaben, BPA, DDT and benzophenone-3) added to breast cells grown in 3D cell culture caused changes resembling early stage breast cancer. When EDC mixtures were used, changes were more significant. Her new project uses a more sophisticated 3D model to examine the effects of EDCs in combination with saturated fatty acids

Evaluating the cancer-causing potential of bisphenol mixtures in primary mammary epithelial cells

Dr Michael Antoniou and Dr Robin Mesnage, King’s college London, were awarded a grant of £45,000 to fund research that examines the cancer-causing potential of bisphenols. Work began in April 2018 and is expected to last two years. They are collaborating with Dr Elisabete Silva, from Brunel University, who will assist with development of 3-dimensional breast cell culture.

Project details

The research will assess the breast cancer-causing and growth stimulating properties of a mixture of bisphenol substitutes, using cultures of human breast epithelial cells, which can form 3-dimensional breast-like cellular structures known as “mammospheres”. These are more representative of breast architecture than standard breast cell culture systems, as they can reproduce features of malignant changes which can be observed microscopically and monitored using molecular biology methods including transcriptomics (gene expression analysis) (1). This work is a continuation of a previously funded project which found bisphenols used as alternatives to BPA were oestrogenic and so able to stimulate growth of breast cancer cells in vitro (2).

Mixtures of endocrine disrupting chemicals, including those that can mimic oestrogen can have potent biological effects at concentrations at which they are inactive when tested individually (3). Different concentrations of bisphenol mixtures, including bisphenol A, bisphenol S, bisphenol F, bisphenol AP, bisphenol AF, bisphenol Z and bisphenol B will be tested for oestrogenic effects using human breast cells grown in cell culture and 3D cell culture systems. Changes will be monitored using microscopy and transcriptomics.

References

1. Marchese, S. and Silva, E. (2012). Disruption of 3D MCF-12A breast cell cultures by estrogens–an in vitro model for ER-mediated changes indicative of hormonal carcinogenesis. PLoS One 7(10): e45767.https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045767
2. Mesnage, R. et al. (2017). Editor’s Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells. Toxicological Sciences 158(2): 431-443.https://www.ncbi.nlm.nih.gov/pubmed/28591870
3. Silva, E. et al. (2002). Something from “nothing” – eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environmental Science and Technology 36: 1751–1756.https://pubs.acs.org/doi/abs/10.1021/es0101227

For more information about the research project see Science News and an interview with lead researcher Dr Michael Antoniou.

Examining how endocrine disrupting agents may modulate activity of fibroblasts generated from breast tissue of high and low mammographic density

Breast Cancer UK in collaboration with Animal Free Research UK have awarded a grant of £90,000 to Professor Valerie Speirs, University of Aberdeen, to fund a PhD studentship, which was awarded to Ms Kerri Palmer. The research project began October 2018 and will finish October 2021.

Project details

Breast density or mammographic density refers to how breasts appear on a mammogram. Breasts are made up of glandular tissue and fat tissue, held together by connective tissue (which contains cells known as fibroblasts). High breast density means there is a greater amount of connective and glandular tissue, compared to fat tissue. Women are 4-5 times more likely to get breast cancer if they have high breast density compared to those with low breast density (1, 2).

Breast density is partly inherited but is also influenced by the environment (3). It increases in response to hormones such as oestrogen, although how this occurs at the cellular level is unclear. Environmental factors that increase breast density include diet, alcohol (4) and synthetic hormones (3) such as hormone replacement therapy. Currently, it is unknown whether endocrine disrupting chemicals which mimic oestrogen contribute to changes in breast density. It is also unclear why high breast density increases breast cancer risk however it is believed to be associated with the activity of fibroblasts, (5). These cells are known to respond to hormones, but details are not fully understood.

This project is investigating the effects of oestrogen mimics on fibroblasts generated from human breast tissue, using a novel 3D in vitro human mammary gland model. This model uses different breast cell types grown together and represents the human breast more closely than epithelial breast cells grown in standard (2D) cell culture or animal models. The research will examine the effects of four oestrogen mimics on human fibroblasts derived from breast tissue of different breast density, in order to understand how these might affect breast density and in turn drive breast cancer development. The oestrogen mimics that will be used include the plastics component bisphenol A, plant-derived compounds genistein and resveratrol and the anti-miscarriage pill, diethylstilboestrol, which is no longer in use. RNA sequencing will be conducted on selected fibroblasts to identify which genes are affected by exposure to oestrogen mimics. Molecular biology techniques will be used to switch off or “silence” these genes. These fibroblasts will be incorporated into 3D breast cell models to establish if this influences the development of pre-invasive breast cancer.

The long-term objectives of the study are to identify biochemical pathways which mediate breast density, which could be modified to reduce the risk of breast cancer, and ultimately, identify a strategy which will help prevent breast cancer.

The research aims to replace two animal models of breast cancer, which are used currently by scientists, by using fully humanised animal free models.

For more details about breast density and Professor Speirs’ research project please see here

 

References

1. Boyd et al. (2011). Mammographic density and breast cancer risk: current understanding and future prospects. Breast Cancer Research 13(6): 223. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326547/
2. Sherratt, M. J. et al (2016). Raised mammographic density: causative mechanisms and biological consequences. Breast Cancer Research 18: 45. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855337/pdf/13058_2016_Article_701.pdf
3. Huo, C. W. et al. (2014). Mammographic density a review on the current understanding of its association with breast cancer. Breast Cancer Research and Treatment 144: 479–502. https://www.ncbi.nlm.nih.gov/pubmed/24615497
4. Voevodina, O. et al. (2013). Association of Mediterranean diet, dietary supplements and alcohol consumption with breast density among women in South Germany: a cross-sectional study. BMC Public Health 13: 203 https://www.ncbi.nlm.nih.gov/pubmed/23497280
5. Luo H. et al. (2015). Cancer-associated fibroblasts: a multifaceted driver of breast cancer progression. Cancer Letters 361(2):155-163. https://www.ncbi.nlm.nih.gov/pubmed/25700776

Oxysterols as functional biomarkers of Triple Negative Breast Cancer relapse

A grant of £45,000 was awarded to Dr James Thorne, University of Leeds, and Dr Hanne Røberg-Larsen (from the University of Oslo) to co-fund a 3 year PhD studentship which was awarded to Alex Websdale. The University of Leeds is co-funding the studentship. The project began in October 2018 and will continue until October 2021.

Details of the research

Many breast cancers are thought to be preventable through diet and lifestyle choices. Obesity and elevated cholesterol are risk factors for breast cancer initiation and progression, but exact mechanisms are not understood fully. Cholesterol levels are influenced by a variety of dietary and lifestyle factors, and high LDL-cholesterol has been linked to failure of cancer therapy.

The cholesterol metabolic pathway is altered in breast cancer, leading to increased products, known as oxysterols, some of which may promote tumour progression. Non-cancer “host” cells such as fat cells (adipocytes), support cells (fibroblasts) and immune cells (macrophages) convert cholesterol into oxysterols which are released into the surrounding tumour micro-environment. Oxysterols are then taken up by adjacent breast cancer cells and may trigger biochemical pathways which lead to chemotherapy resistance and breast cancer spread.

Host cells produce oxysterols under normal conditions, but it is unclear to what extent production is altered when these cells are in a cancerous environment. Each of the cell types of the tumour micro-environment exhibit their own potential for oxysterol production by making different amounts of oxysterol-producing and catabolising enzymes.

This project explores the hypothesis that oxysterols, released by non-cancer host cells into the tumour micro-environment, are taken up by adjacent breast cancer cells, eventually leading to chemotherapy resistance and tumour spread. As oxysterols may promote tumour progression, interventions which interfere with their production may prevent breast cancer relapse. This work is also investigating whether a dietary intervention can alter oxysterols produced by host cells, thereby preventing breast cancer recurrence.

Identifying nuclear receptor disruptors as risk factors in breast cancer

A grant of £15,000 was awarded to Dr Laura Matthews, University of Leeds, to identify which nuclear receptors play a role in breast cancer and identify chemicals which disrupt these receptors. The work began in September 2017 and is expected to be completed by December 2019.

Project details

Nuclear receptors are proteins that when activated move into the cell nucleus, bind DNA and regulate gene expression (switch genes on or off). Activation is by binding to a specific “ligand” – a hormone or cell by-product, depending on the receptor in question.

Two nuclear receptors – oestrogen and progesterone receptors – are known to be important in diagnosing and treating breast cancer. The amount of these proteins in tumours is used to assign patients to clinical groups which can help doctors decide the best therapy.  Evidence is now emerging that in some breast tumours, the level of other nuclear receptors, e.g. the androgen receptor, is altered. Understanding what causes these changes and how they might contribute to the development of breast cancer is important for identifying risk factors for breast cancer.

The research is using pre-existing RNA sequencing datasets to measure the levels of all 48 nuclear receptors in normal breast tissue and breast tumour tissue samples, to help identify common nuclear receptor gene signatures. Using these results, predictions can be made to identify environmental chemicals that regulate nuclear receptor expression and which metabolic pathways are changed following chemical exposure. Chemicals predicted to interfere with nuclear receptors and pathways are being experimentally tested using cell proliferation and migration assays, and a range of human breast cancer cell lines representing the main breast cancer sub-types. By uncovering novel chemical disruptors of nuclear receptor expression risk factors for breast cancer and novel therapeutic targets may be identified.