支架蛋白CNK3介导胶质母细胞瘤中的促侵袭信号转导

Glioblastoma (GBM) is the deadliest brain cancer with a five-year survival rate of 7% and a lack of targeted therapies for its treatment. One of the main factors limiting better treatment outcomes is the invasiveness of GBM cells, a property which reduces the efficacy of surgical resection and makes recurrence almost inevitable. The development of therapeutics for restraining cell invasion is therefore a crucial goal in the biomedical community. The invasive capacity of GBM cells is largely driven by a rewiring of signal transduction networks which results in abnormal activity of the motility-regulating RHO family GTPases, and activation of pro-invasive gene expression programs. However, the molecular mechanisms controlling this signalling switch are not fully understood, and their elucidation represents a significant challenge in biomedical research. Our goal is to improve the understanding of rewired signalling in GBM cells by studying the functions of the CNK scaffold protein family. Through genetic knockdown/knockout and in vitro cell migration and invasion assays with GBM cell lines we identified the CNK3 member as a new pro-invasive factor. Crucially, this is also the case in vivo, as loss of CNK3 expression greatly reduced tumour volume in an orthotopic mouse xenograft model. Using GTPase pulldown assays, we then determined that CNK3 supports cell motility by promoting the activation of the RHO GTPases RAC1 and CDC42, while inhibiting the activity of RHOA. Consistent with these findings, CNK3 is required for normal actin-based protrusion formation at the leading-edge of migrating cells, and limits cell contractility and cell-substrate adhesion. To determine the molecular mechanism of action by which CNK3 functions as a scaffold protein, we then identified its proximal interactome by TurboID and validated its functional binding partners through co-immunoprecipitation and functional rescue experiments. These include the RAC1/CDC42 GTPase-activating protein ARHGAP39, and the MAP4K kinases MAP4K4, TNIK, and MINK1. Finally, we identified upstream growth factors and receptor tyrosine kinases (RTKs) that regulate CNK3-dependent motility and RHO GTPase activity. Thus, CNK3 functions by linking signalling by upstream RTKs to downstream regulation of RHO GTPases. Current efforts focus on the identification of pro-motility transcription programs regulated by this signalling axis. Our research identifies the CNK3 scaffold protein as a new driver of motility in GBM cells and advances our understanding of the molecular mechanisms underlying their invasive behaviour. Ultimately, CNK3 and its binding partners could be new therapeutic targets for limiting the spread of GBM and improving patient survival.