Novel target identification & characterisation of key cell motility regulators in lung cancer metastasis

Lung cancer is the most common cancer killer worldwide. One of the major reasons for failing to cure this malignancy is the high rate of metastasis. Hence, understanding the mechanisms by which these cells metastasise is required to improve patients’ survival. Using the non-small cell lung cancer cell line A549 we optimised a 3D Invasion assay to enable a robotised high-throughput siRNA screen of large gene libraries. Despite thorough optimisation a pilot screen for the Phosphatome highlighted multiple issues with our set-up. Further optimisation work was conducted to improve the resolving power of the assay. However, as no clear explanation for the poor reproducibility could be uncovered, work began on a potential modulator of cell motility identified during a previous motility screen of the kinome. Microtubule affinity regulating kinases 4 (MARK4) is one of four members of the MARK family. A previously performed kinome siRNA screen for modulators of cell migration revealed that depletion of MARK4 reduced A549 cell migration. We validated this observation and additionally found that MARK4 silencing inhibited cell invasion through 3D collagen matrices. MARK4 depletion markedly changed cell structure, as exemplified by an increase tubulin network area. Follow-on experiments revealed an altered speed of microtubule polymerisation in MARK4-silenced cells. We observed that MARK4 downregulation promoted resistance to the chemotherapeutic agents Paciltaxel and Cisplatin, an effect potentially linked to a reduced proliferation in MARK4-silenced cells. MARK4 overexpression in NSCLC cells increased cell motility but did not impact the cell area or resistance to chemotherapy. A targeted siRNA cell motility screen of a selection of proposed MARK4 interacting proteins enabled us to connect MARK4 with Protein Phosphatase 2A and GSK3. Although further investigation is required into the signalling pathways upstream and downstream of MARK4, this work identified novel functions for this kinase and highlighted potential mechanisms underlying its effects.Open Acces