Project ID CM-HD2024_10


Co Supervisor 1A Faculty of Life Sciences & Medicine, School of Cancer & Pharmaceutical Sciences, Comprehensive Cancer CentreWebsite

Co Supervisor 1B Faculty of Life Sciences & Medicine, School of Basic & Medical Biosciences, Randall Centre for Cell & Molecular BiophysicsWebsite

Investigating the role of PAK4 in the nucleus of breast cancer cells

Background: PAK4 kinase is overexpressed in breast cancer (BC) and has been associated with proliferation and matrix invasion. PAK4 has both cytoplasmic and nuclear localisations. However, very little is known about PAK4 nuclear function, whether it requires kinase activity and if it is related to cell invasion. Moreover, PAK4 transcriptional activity has not been fully explored. We have preliminary data suggesting PAK4 regulates its own expression. We work with CRUK to develop PAK kinase inhibitors (PAKi). It will be important to understand how these inhibitors might/might not interfere with PAK4 nuclear functionality. There is also evidence that PAK4i leads to double stranded DNA breaks (DSBs) in myeloma. This suggests that PAK4i could synergize with PARP inhibition.

We aim to investigate four key questions:
1. Is PAK4 regulating gene expression?
2. Is PAK4 associated with the repair of DSBs?
3. Does PAK4 nuclear function require kinase activity?
4. Does PAK4 nuclear function influence matrix invasion?

Rotation project: test re-expression of PAK4 in driving endogenous expression in PAK4-depleted cells (PAK4KO). Build PAK4 nuclear targeting constructs.

Year 1: Test appearance DSBs in PAK4KO/PAKi BC cells using immunofluorescence and investigate kinase dependence. Use CUT&RUN to identify PAK4 binding sites in genomic sequence. Optimise PAK4 nuclear localisation for RNA-Seq analysis.

Year 2&3 RNA-Seq of PAK4 driven gene expression using nuclear targeting /PAKi with a focus on DNA repair and cell invasion networks. Validate DNA interactions (Year 1) to identify gene regulation mechanisms. Identify DNA repair proteins that interact with PAK4 in BC. Test synergising PARPi with PAKi in BC proliferation. Test the ability of PAK4KO BC cells to invade 3D collagen when re-expressing kinase/nuclear localisation mutants.

Training: Genomics techniques including CUT&RUN, RNA-seq and library preparation, bioinformatic analysis of genomics datasets, high resolution live/fixed cell imaging, biochemistry, cell biology.

Representative Publications

Manuelli V, Cahill F, Wylie H, Gillett C, Correa I, Heck S, Rimmer A, Haire A, Van Hemelrijck M, Rudman S, Wells CM. (2022) Invadopodia play a role in prostate cancer progression. BMC Cancer. 22:386. doi: 10.1186/s12885-022-09424-4 Lizhou Xu, Farid N. Faruqu, Yau M. Lim, Kee Y. Lim, Revadee Liam-Or, Adam A Walters, Paul Lavender, David Fear, Claire M. Wells, Julie Tzu-Wen Wang, and Khuloud T. Al-Jamal (2020) Exosome-mediated RNAi of PAK4 prolongs survival of pancreatic cancer mouse model after loco-regional treatment. Biomaterials 264: 120369 Anna Dart, Gary Box , William Court , Madeline Gale, John Brown, Sarah Pinder, Sue Eccles and Claire M Wells* (2015) PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion. J Cell Biol. 211:863-79. doi: 10.1083/jcb.201501072.
Talbot CD, Walsh MD, Cutty SJ, Elsayed R, Vlachaki E, Bruce AEE, Wardle FC, Nelson AC. (2022). Eomes function is conserved between zebrafish and mouse and controls left-right organiser progenitor gene expression via interlocking feedforward loops. Front Cell Dev Biol. 10:982477. doi: 10.3389/fcell.2022.982477 Baranasic D, Hörtenhuber M, Balwierz PJ, Zehnder T, Mukarram AK, Nepal C, Várnai C, Hadzhiev Y, Jimenez-Gonzalez A, Li N, Wragg J, D’Orazio FM, Relic D, Pachkov M, Díaz N, Hernández-Rodríguez B, Chen Z, Stoiber M, Dong M, Stevens I, Ross SE, Eagle A, Martin R, Obasaju O, Rastegar S, McGarvey AC, Kopp W, Chambers E, Wang D, Kim HR, Acemel RD, Naranjo S, Łapiński M, Chong V, Mathavan S, Peers B, Sauka-Spengler T, Vingron M, Carninci P, Ohler U, Lacadie SA, Burgess SM, Winata C, van Eeden F, Vaquerizas JM, Gómez-Skarmeta JL, Onichtchouk D, Brown BJ, Bogdanovic O, van Nimwegen E, Westerfield M, Wardle FC, Daub CO, Lenhard B, Müller F. (2022). Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements. Nat Genet. 2022 Jul;54(7):1037-1050. doi: 10.1038/s41588-022-01089-w. Nelson, A.C., Cutty, S.J., Gasiunas, S.N., Deplae, I., Stemple, D.L., Wardle, F.C. (2017). In vivo regulation of the zebrafish endoderm progenitor niche by T-box transcription factors. Cell Reports, 19: 2782-95. doi: 10.1016/j.celrep.2017.06.011