Investigating a novel cytoskeletal gene signature in neuroblastoma

Primary supervisor: Claire Wells, King’s College London

Secondary supervisor: Luigi Ombrato, Queen Mary University of London

Tertiary supervisor: Victoria Sanz-Moreno, Institute of Cancer Research

Project

Background: Neuroblastoma (NB) is a rare cancer that affects children, mostly under 5 years old. NB arises from rapidly proliferating neuroblasts in the tissues of the sympathetic nervous system, usually in the paraspinal ganglia or adrenal medulla, and metastasis is the primary cause of death. Neuroblastoma cells navigate through neuronal tissue and predominately metastasise to bone/bone marrow.

Project Description: Our collaborator Dr Vega, Seville University has recently carried out an in depth analysis of gene expression data from several cohorts of neuroblastoma patient tumour samples across disease severity and has identified a minimal gene signature which has the potential to predict disease progression. Within that signature PAK1, PAK2 and IQGAP3 protein expression was significantly modulated with increased disease severity. The Wells laboratory has a long-standing interest in PAK, identifying an interaction between IQGAP and PAK signalling. This project seeks to understand how changes in PAK1/PAK2/IQGAP3 expression are contributing to neuroblastoma progression. We will develop novel 3D models of neuroblastoma metastasis that more accurately reflect the tumour microenvironment (TME) and colonisation site.

Year 1: Explore gene signature in NB cell lines established to model disease progression via exploration of their differentiation potential using all-trans retinoic acid and other supplemented media/culture conditions. Test for an interaction between IQGAP3 and PAK1/PAK2 in NB cells using immunoprecipitation and proximity ligation assays. Localise PAK1/PAK2/IQGAP3 in the NB cells using advanced microscopy. Generate CRISPR KO PAK1/PAK2/IQGAP3 NB cells alongside NB cells overexpressing PAK1/PAK2/IQGAP3. Define the expression of PAK1/PAK2/IQGAP3 in NB tumour cell subpopulations with different metastatic potential through single cell expression data from patient samples and cell lines.

Year 2: Test the CRISPR modulated NB cell lines in morphology/proliferation/migration assays (2D/3D). Build a novel model of neuroblastoma migration towards a neuronal cell spheroid (with Dr Danovi, KCL, an expert in this area). The Ombrato lab has extensive expertise in studying metastatic colonisation and has developed 3D models of breast TME incorporating immune and stromal cells. Dr Ombrato will lead development of a similar model with neuroblastoma and development of a bone endothelial specific invasion assay to model colonisation. This work will also be supported by Prof Sanz-Moreno (ICR) an expert in studying 3D cancer cell morphology.

Year 3: Use the advanced invasion, TME and bone extravasation models to test the proliferative/invasive behaviour of CRISPR cells. Test the impact of PAK inhibitors on NB proliferation/invasion. Perform phosphoproteome screen in CRISPR and overexpressing cells across differentiation states. Perform a cytoskeletal phospho array to identify cytoskeletal signalling changes in CRISPR cells. Results will be cross referenced to the Vega Lab patient dataset.

Outcome: This project will deliver a step change in our understanding of the functional role of PAK1/PAK2/IQGAP3 in NB evolution, whilst generating novel experimental models. In tandem validation of the gene signature has prognostic potential and may lead to better stratification of patient treatment regimens and the opportunity to capitalise on targeted therapies.

Candidate background

This project would suit candidates with a background in biomedical sciences with an interest in biochemistry, advanced imaging techniques and bioinformatics.

Potential Research Placements

1. Aurora Negro, Cancer Research Technology
2. Simon Poland, Comprehensive Cancer Centre, King’s College London
3. Chris Tape, Cancer Institute, UCL

References

1. Samain et al., (2023) CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells SCIENCE ADVANCES Vol 9, Issue 42 DOI: 10.1126/sciadv.adi0244
2. Pipili et al., (2024) PAK6 acts downstream of IQGAP3 to promote contractility in triple negative breast cancer cells Cell Signal Doi 10.1016/j.cellsig.2024.111233. PMID: 38763182
3. Lizhou et al., (2020) Exosome-mediated RNAi of PAK4 prolongs survival of pancreatic cancer mouse model after loco-regional treatment. Biomaterials 264: 120369 doi.org/10.1016/j.biomaterials.2020.120369
4. Orgaz et al., (2020) Myosin II Reactivation and Cytoskeletal Remodeling as a Hallmark and a Vulnerability in Melanoma Therapy Resistance. Cancer Cell 37:85-103.e9. doi: 10.1016/j.ccell.2019.12.003
5. Ombrato et al., (2019) Metastatic-niche labelling reveals parenchymal cells with stem features. Nature 572:603-608.  doi: 10.1038/s41586-019-1487-6