EML4 is a poorly characterised microtubule-associated protein . The purpose of this cell biology-focused PhD project is to explore the molecular mechanisms through which EML4 regulates microtubule dynamics and cell migration. This research is important as it will not only provide new understanding of how the microtubule cytoskeleton is regulated in human cells, but also provide insights into how EML4-ALK oncogenic fusions promote metastasis in lung cancers .
EML4 protein is normally expressed in neuronal cells and we have discovered that EML4 promotes microtubule stability. Hence, we propose that the natural function of this protein is to stabilize microtubules in axons and dendrites of neuronal cells. However, in lung cancer, fusion of the EML4 gene with the gene encoding the ALK tyrosine kinase, leads to an oncogenic fusion protein that promotes proliferation and metastasis. We hypothesise that constitutive activation of ALK drives uncontrolled proliferation while the EML4 part of the fusion protein alters microtubule properties to promote cell migration and invasion. In support of this hypothesis, we have demonstrated that expression of recombinant EML4 or EML4-ALK causes a microtubule-dependent change in cell morphology and accelerated migration [3, 4].
Unexpectedly, we have also discovered that the wild-type EML4 protein interacts with the NEK9 kinase through the EML4 N-terminal domain that is present in the fusion proteins. Furthermore, overexpression of EML4 or an activated version of the NEK9 kinase alters cell morphology and migration, while depletion of NEK9 blocks the cell migration induced by EML4 and EML4-ALK. NEK9 is therefore an essential component through which EML4 regulates microtubule dynamics and cell migration.
Project Aim and Objectives
The aim of this project is to explore the underlying mechanisms through which EML4 and NEK9 cooperate to regulate microtubule-dependent cell migration. We will test the hypothesis that this is mediated by the NEK7 kinase that is a direct downstream substrate of NEK9, that together these alter the activation state of members of the Rho GTPase family that connects the microtubule and actin networks, and that the role of EML4 is to recruit these regulators to microtubules.
Figure 1. A model for the role of EML4 and EML4-ALK fusions in cell migration. EML4 interacts with Nek9 to cause localised activation of Nek7 on microtubules (MTs). This alters MT dynamics via phosphorylation of MT-associated proteins (MAPs) or MTs themselves. Stimulation of this pathway through expression of active Nek9 (RCC1) or Nek7 (Y97A), or the oncogenic EML4-ALK v3/5, alters cell morphology and increases migration in a manner that could enhance tumour metastasis. Patients with EML4-ALK variants (v3/5) that localise to MTs may benefit from treatment with ALK or NEK inhibitors, while patients with variants (v1/2) that do not localise to MTs will not.
The specific objectives of this project are:
(i) Cell-based reporter assays will be used to measure the activity of different Rho GTPases in cells in response to expression of EML4-ALK or NEK9 in isogenic U2OS and Beas-2B bronchial epithelial cells with inducible expression of these proteins, as well as lung cancer patient-derived cell lines.
(ii) We will test the consequences on cell morphology and migration in 2D and 3D cultures of expression of dominant-negative and activated mutants of these Rho GTPases, and use of selective chemical inhibitors of the Rho GTPases and downstream kinases, such as ROCK, MLCK or PAK1. Depending upon which Rho GTPase is implicated by these experiments, we will test the role of specific GEFs and GAPs for that particular G-protein by RNAi-mediated depletion. As Rho GTPase activation can be directly regulated by the microtubule network, these experiments will be done in untreated cells as well as cells in which the microtubule network is depolymerised with nocodazole and then allowed to regrow.
(iii) We will use gene-editing to add an auxin-inducible degron (AID) to the NEK9 gene in cells expressing EML4-ALK and test the consequences of inducible degradation of the endogenous kinase on cell morphology and migration in 2D and 3D, as well as on Rho GTPase activation.
Techniques that will be undertaken during the project
-Use of cell reporter assays to measure RhoGTPase activity.
-High-resolution fluorescence imaging by laser scanning confocal microscopy of fixed and live cells in 2D and 3D cultures.
-Quantitative analysis of imaging data using ImageJ, Fiji and Matlab codes.
-Use of CRISPR/Cas9-mediated gene editing to introduce an auxin-inducible degron tag to the endogenous NEK9 gene.
Available to UK/EU applicants only
Application information https://www2.le.ac.uk/research-degrees/doctoral-training-partnerships/bbsrc