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Discovery of novel breast cancer targets using light-activatable chemical probes


   MRC DiMeN Doctoral Training Partnership

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  Dr Lianne Willems, Dr AN Holding  No more applications being accepted  Competition Funded PhD Project (Students Worldwide)

About the Project

Background

Breast cancer is the most common form of cancer diagnosed worldwide. In the majority of breast cancer cases (>70%) the main therapeutic target is the estrogen receptor (ER), a nuclear transcription factor that is activated by the hormone estrogen and regulates the expression of numerous target genes. The activity of the ER is controlled through protein-protein interactions (PPIs).1 Some of the ER’s interaction partners are known, but it is likely that many more remain undetected. We hypothesise that identifying and targeting these interactions offers an exciting new avenue for therapeutic intervention.

Project aims

While endocrine therapies for ER-positive breast cancers are currently available, there is an urgent need to develop new therapeutic strategies because of significant issues with drug resistance and incurable metastatic recurrence. The aims of this project are therefore to identify novel ER-dependent PPIs and explore alternative pathways to block ER activity, thereby contributing to the discovery of new breast cancer targets. 

Central to our approach is the finding that the ER is glycosylated with N-acetylglucosamine (GlcNAc) sugars.2 This form of glycosylation, termed O-GlcNAc modification, has been demonstrated to be capable of stabilising specific PPIs. It is currently not known, however, what effect(s) glycosylation has on the ER and if it plays a role in any of its interactions. The main focus of this project will therefore be on characterising ER-dependent PPIs that involve O-GlcNAc and studying the consequences of disrupting these interactions.

Experimental approach

In order to comprehensively identify the proteins that are binding to the ER inside the cell at any given moment, you will make use of photo-crosslinking probes.3 These probes are designed such that they bind to the ER and carry a latent reactive group, which induces covalent crosslinking to interacting proteins when it is activated through photoirradiation. Your project will consist of the following objectives:

1.   Synthesis of two series of new photo-crosslinker probes, one derived from the known ER binder Fulvestrant and the other from the sugar GlcNAc.

2.   Application of these chemical probes for the crosslinking of proteins in cell culture, enrichment of ER interaction partners, and identification of enriched proteins by mass spectrometry-based proteomics.

3.   Validation of identified proteins as true ER interaction partners in cell culture and characterisation of the consequences of disrupting these PPIs using target knockdown or knockout.

Training

You will build a highly valuable, multidisciplinary skillset in the latest chemical biology techniques. You will receive training in chemical synthesis (including carbohydrate chemistry and compound characterisation), molecular and cell biology (e.g. Western blotting, co-immunoprecipitation, Proximity Ligation Assay, luciferase-based activity assays, quantitative PCR, CRISPR/Cas9 knockout), and proteomics. You will also be supported in the quantitative analysis of ‘omics datasets. 

You will join a vibrant and supportive community of multidisciplinary researchers working across the departments of Chemistry and Biology. Lianne Willems is a carbohydrate chemist with specific interest in studying protein glycosylation in relation to human disease,4 and Andrew Holding is an experimental and computational biologist with expertise in cancer biology and the study of ER regulation.5

Benefits of being in the DiMeN DTP:

This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle, York and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.

We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.

Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: https://www.dimen.org.uk/blog 

Further information on the programme and how to apply can be found on our website:

https://www.dimen.org.uk/how-to-apply 


Funding Notes

Studentships are fully funded by the Medical Research Council (MRC) for 4yrs. Funding will cover tuition fees, stipend and project costs. We also aim to support the most outstanding applicants from outside the UK and are able to offer a limited number of full studentships to international applicants. Please read additional guidance here: https://www.dimen.org.uk/eligibility-criteria
Studentships commence: 1st October 2023
Good luck!

References

Miki Y, Iwabuchi E, Ono K, Sasano H, Ito K. Exploring Protein-Protein Interaction in the Study of Hormone-Dependent Cancers. Int J Mol Sci. 2018, 19(10), 3173. doi:10.3390/ijms19103173
https://www.mdpi.com/1422-0067/19/10/3173/htm
Jiang MS, Hart GW. A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J Biol Chem. 1997, 272(4), 2421-8. doi: 10.1074/jbc.272.4.2421.
https://www.jbc.org/article/S0021-9258(19)78521-4/fulltext
Yu SH, Boyce M, Wands AM, Bond MR, Bertozzi CR, Kohler JJ. Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners. Proc Natl Acad Sci U S A. 2012, 109(13), 4834-9. doi: 10.1073/pnas.1114356109. https://www.pnas.org/doi/10.1073/pnas.1114356109?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
Huxley KE, Willems LI. Chemical reporters to study mammalian O-glycosylation. Biochem Soc Trans. 2021, 49(2), 903-913. doi: 10.1042/BST20200839. https://portlandpress.com/biochemsoctrans/article/49/2/903/228375/Chemical-reporters-to-study-mammalian-O
Holding AN, Giorgi FM, Donnelly A, et al. VULCAN integrates ChIP-seq with patient-derived co-expression networks to identify GRHL2 as a key co-regulator of ERa at enhancers in breast cancer [published correction appears in Genome Biol. 2019 Jun 14;20(1):122]. Genome Biol. 2019, 20(1):91. doi:10.1186/s13059-019-1698-z
https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1698-z
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