Unravelling c-Met endosomal signalling to improve breast cancer therapy

Primary supervisor: Stephanie Kermorgant, Queen Mary University of London

Secondary supervisor: Louise Jones, Queen Mary University of London

Project

Background

Around 11,500 breast cancer patients die in the UK yearly, only 1 in 4 late-stage breast cancer patients survive for five years. The receptor tyrosine kinase (RTK) c-Met, overexpressed in 20-30 % of breast cancer across all subtypes, correlating with metastasis and poor prognosis, has been implicated in the growth and metastasis of breast cancer [1]. Considered a major target, several c-Met inhibitors have been approved. Unfortunately, treatment is active only in a subset of patients. Moreover, resistance to c-Met inhibition can occur. The mechanisms underlying these different therapeutic outcomes are unclear.

A growing body of literature reveals that RTKs do not solely signal from the plasma membrane as they rapidly internalise upon ligand binding. Endosomes are thought to play the role of localised signalling platforms, maintaining activated receptors and signals in close juxtaposition, thereby controlling signal specificity or longevity and fine tuning cell responses. We have established that c-Met endocytosis and trafficking through specific endosomes is required to assemble precise signalling cascades, which are necessary to trigger in vivo tumorigenesis and metastasis [2] and invasive breast cancer cell invasion [3]. However, c-Met signals and precise molecule relays that assemble on different endosomes are largely unknown.

We hypothesise that uncovering endosomal c-Met signalling pathways in invasive breast cancer will provide:

1. A new set of targets that may lead to improved therapeutic strategies.
2. Biomarkers to select patients that are likely to respond better to c-Met inhibition treatment.

Project

Aim 1- The student will determine the c-Met proximal interactome in specific endosomes in invasive breast cancer cell lines using state-of-the-art APEX2 proximity proteomics, established in our lab (with Prof. Cutillas) [4]. Following pathways enrichment bioinformatics and public databases interrogation (e.g. TCGA) to establish the clinical value of the hits, up to five hits will be validated. The expression or activity of the selected hits (using CRISPR, si/shRNA, pharmacological inhibitors) will be inhibited or their mutated form (which will be engineered to prevent their association with c-Met on endosome) expressed post-CRISPR KO. The influence on the cancer cells’ growth and invasion in a 3D spheroid assay (co-culture of cancer cells and patient-derived cancer associated fibroblasts in collagen: matrigel hydrogels) [5].

Aim 2- The student will investigate the prognostic value of c-Met endocytosis in human breast patient tissues using immunohistochemistry, co-immunofluorescence, VisioPharm and confocal microscopy (facilities onsite). Statistical analyses will determine whether c-Met endosomal localisation correlates with a reduced time to local or distant recurrence and poorest survival.

All techniques and equipments are set up and available on site. Training and technical assistance will be provided.

Many patients do not respond well to c-Met targeted therapies, despite the wealth of data showing how it promotes tumour progression. By elucidating the molecular mechanisms of c-Met signalling on endosomes we hope, in the future, to develop biomarker(s) that determine c-Met activity so as to improve the selection of patients to receive c-Met targeted therapy, but also to identify novel therapeutic targets. In the future, this work could be relevant to other cancers and extended to other membrane receptors.

References

1. Ho-Yen CM, Green AR, Rakha EA, Brentnall AR, Ellis IO, Kermorgant S, Jones JL. C-Met in invasive breast cancer: Is there a relationship with the basal-like subtype? Cancer 2014, 120:163-71.
2. Joffre C, Barrow R, Ménard L, Calleja V, Hart IR and Kermorgant S. A direct role for Met endocytosis in tumorigenesis. Nature Cell Biology 2011, 13:827-37.
3. Ménard L, Parker PJ, Kermorgant S. Receptor Tyrosine Kinase c-Met controls the cytoskeleton from different endosomes via different pathways. Nature Communications 2014, 5:3907.
4. Nollet M, Michailidis F, Dokal AD, Rajeeve V, Burden J, Godinho S, Nightingale TD, Cutillas P, Ketteler R, Kermorgant S. (2021) Proximity interactome of LC3B in normal growth conditions. https://biorxiv.org/cgi/content/short/2021.10.08.463639v1. Revised version in preparation for Nature Communications.
5. Fernandes M*, Hoggard B*, Jamme P*, Paget S, Truong MJ, Grégoire V, Vinchent A, Descarpentries C, Morabito A, Stanislovas J, Farage E, Meneboo JP, Sebda S, Bouchekioua-Bouzaghou K, Nollet M, Humez S, Perera T, Fromme P, Grumolato L, Figeac M, Copin MC, Tulasne D, Cortot AB, Kermorgant S# and Kherrouche Z#. MET exon 14 skipping mutation is an HGF-dependent oncogenic driver in vitro and in humanized HGF knock in mice. Molecular Oncology 2023, 17:2257-2274. *, co-first authors, # co-last and co-corresponding authors.