Harnessing soluble LRIG1 for pan-RTK targeting in glioblastoma

INTRODUCTION: The role of receptor tyrosine kinases (RTKs) in glio- blastoma is widely acknowledged. However, therapies based on RTK tar- geting have been continuously unsuccessful in GBM patients, highlighting the complexity of RTK signaling and biology. LRIG1 (Leucine-rich Repeats and ImmunoGlobulin domains protein 1) was identified as an endogenous inhibitor of epidermal growth factor receptor (EGFR) and other RTKs, and was confirmed as a tumor suppressor in various cancer types. We previ- ously identified the soluble form of LRIG1 as a potent inhibitor of GBM growth in vivo, irrespective of EGFR status. Here, we aim to shed light on the molecular mechanisms underlying its anti-cancer activity. MATERIAL AND METHODS: We used GBM cells overexpressing EGFRvIII, with or without soluble LRIG1 overexpression. In parallel, we generated a recom- binant human soluble LRIG1 protein (rh-sLRIG1) by expressing LRIG1 ectodomain in insect cells via baculovirus infection and subsequent His- tag purification. rh-sLRIG1 was applied in the medium of classical GBM cell lines and patient-derived GBM stem-like cells. Applying a variety of cell-based assays, cell proliferation, migration, cell morphology, as well as protein expression and protein-protein interactions were investigated. RESULTS: We confirmed that sLRIG1 efficiently reduced proliferation and invasion capacities of GBM cells, and modulated cytoskeleton proteins and cell shape. Inhibition of cell proliferation by sLRIG1 was independent of EGFR expression levels in GBM cells and interestingly, rh-sLRIG1 treatment was associated with downregulation of AXL, which constitutes a newly- identified regulatory function of LRIG1. We are currently addressing the impact of the LRIG1-AXL signaling axis on GBM invasion and resistance to EGFR inhibition. CONCLUSION: We identified AXL as a novel LRIG1 tar- get and provide evidence for the potential therapeutic application of recom- binant sLRIG1 in the inhibition of growth factor signaling in GBM.