Crosstalk between PAK1 signalling and intracellular trafficking

Epithelial cells are an important component of many different tissues and organs in the body. They form a sheet of highly adherent cells that line all inside our internal organs and externally to form our skin. Epithelial cells are an essential cell type for life, and alterations in their structure and function have important clinical implications. As epithelial tissues face many chemicals, biological and environmental challenges, a high proportion of cancers (about 85%) originates in epithelial cells. Dissecting the oncogenic drivers that perturb epithelial cells homeostasis and function will highlight potential therapeutic steps to ameliorate tumour progression.

Extensive research has focused on understanding how epithelial cells assemble themselves as tissues and how their function is regulated. A key process that determines tissue structure is the ability of epithelial cells to stick together and strongly. Such orderly organization of epithelial cell-cell adhesion is severely perturbed during tumorigenesis, in inflammatory, chronic and hyper-proliferative diseases. Thus, maintaining cell architecture is important for the function of epithelial tissues.

Here we aim to investigate the molecular events that subvert the normal epithelial architecture and disrupt contacts between neighbouring cells. Rac1 and Rabs are two classes of signalling molecules that control distinct cellular processes that are seemingly independent: cell adhesion and intracellular transport. We will study how Rac1 and Rab proteins combine their efforts during tumour progression to promote the removal of adhesion receptors (that glue cells together) from sites where cells contact each other.

We find that a Rac1 partner, the kinase PAK1, mediates a seamless integration between these two signalling molecules to integrate their function in space and time to precisely destroy cell-cell contacts. PAK1 gene is amplified or mutated in various cancers, where it is associated with more aggressive tumour phenotypes and poor outcome for patients. It is also activated by different oncogenes such as Rac1 itself or H-Ras, thereby increasing its significance and impact as therapeutics for cancer patients. We find that PAK1 directly modulate the function of a Rab regulator, which in turn can redirect trafficking of adhesion receptors to destroy tissue cohesion and architecture during transformation. Such molecular relationships and outcome will be validated in patient-derived tumour cells that represent the primary tumour status and the corresponding lymph node metastasis in each patient. By understanding the mechanisms via which Rac1 and Rab cross-talk is regulated by PAK1, our work will highlight potential approaches to interfere with tumour progression, by delaying the transition from benign to metastatic tumours.

In carcinomas with elevated Rac1 or PAK1 signalling, we hypothesise that a direct phosphorylation of RabGDIb by PAK1 promotes faster Rab retrieval and cycling leading to higher cadherin turnover and junction perturbation. Such defect leads to loss of tumour architecture and metastasis. We will explore these relationships in different model systems, including patient-derived tumour cells (primary and metastatic sites). We use a variety of approaches to investigate protein interactions, cellular and dynamic events and pathway finding. Two independent strategies will identify Rabs that are important for E-cadherin disruption: (i) differential interaction with phosphorylated RabGDIb and (ii) functional screen to select Rabs important for E-cadherin trafficking. We will reveal Rab subsets that cooperate in junction destabilization by cross-referencing the interaction and functional datasets. At least two new Rabs will be further investigated for function integration with Rac1 and PAK1 signalling. We will map Rab localization with E-cadherin cargo (i.e., photoactivatable proteins) and live imaging.

In parallel, we identify the intracellular routes modulated by PAK1>RabGDIb and their molecular mechanisms: the balance between cadherin fluid uptake (micropinocytosis, flotillin role) and delivery to junctions via recycling or exocytic post-Golgi transport. Following Rac1 activation, the dynamics and localization of RabGDIb (WT and mutants) and relevant Rabs will be done in selected intracellular compartments. The broad implications of PAK1>RabGDIb signalling will be investigated during cadherin turnover in cells stimulated with growth factors (EMT) or collective migration, which may engage distinct Rab subsets. Quantitative image analyses of cadherin dynamics will be performed in a microfluidic device with channels of distinct diameters and mechanical properties to modulate collective motility and migration mode of patient-derived tumour cells.