The soluble form of pan-RTK inhibitor and tumor suppressor LRIG1 mediates downregulation of AXL through direct protein–protein interaction in glioblastoma
Neirinckx, Virginie; Hau, Ann-Christin; Schuster, Anneet al.
[en] Targeted approaches for inhibiting epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs) in glioblastoma (GBM) have led to therapeutic resistance and little clinical benefit, raising the need for the development of alternative strategies. Endogenous LRIG1 (Leucine-rich Repeats and ImmunoGlobulin-like domains protein 1) is an RTK inhibitory protein required for stem cell maintenance, and we previously demonstrated the soluble ectodomain of LRIG1 (sLRIG1) to potently inhibit GBM growth in vitro and in vivo.Here, we generated a recombinant protein of the ectodomain of LRIG1 (sLRIG1) and determined its activity in various cellular GBM models including patient-derived stem-like cells and patient organoids. We used proliferation, adhesion, and invasion assays, and performed gene and protein expression studies. Proximity ligation assay and NanoBiT complementation technology were applied to assess protein–protein interactions.We show that recombinant sLRIG1 downregulates EGFRvIII but not EGFR, and reduces proliferation in GBM cells, irrespective of their EGFR expression status. We find that sLRIG1 targets and downregulates a wide range of RTKs, including AXL, and alters GBM cell adhesion. Mechanistically, we demonstrate that LRIG1 interferes with AXL but not with EGFR dimerization.These results identify AXL as a novel sLRIG1 target and show that LRIG1-mediated RTK downregulation depends on direct protein interaction. The pan-RTK inhibitory activity of sLRIG1 warrants further investigation for new GBM treatment approaches.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Neirinckx, Virginie ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Département des sciences biomédicales et précliniques
Hau, Ann-Christin
Schuster, Anne
Fritah, Sabrina
Tiemann, Katja
Klein, Eliane
Nazarov, Petr V.
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
The soluble form of pan-RTK inhibitor and tumor suppressor LRIG1 mediates downregulation of AXL through direct protein–protein interaction in glioblastoma
Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2010;141(7):1117-1134.
Cancer Genome Atlas Research N. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061-1068.
Ekstrand AJ, Sugawa N, James CD, Collins VP. Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N-and/or C-terminal tails. Proc Natl Acad Sci U S A. 1992;89(10):4309-4313.
Keller S, Schmidt MHH. EGFR and EGFRvIII promote angiogenesis and cell invasion in glioblastoma: combination therapies for an effective treatment. Int J Mol Sci. 2017;18(6)..
Fan QW, Cheng CK, Gustafson WC, et al. EGFR phosphorylates tumorderived EGFRvIII driving STAT3/5 and progression in glioblastoma. Cancer Cell. 2013;24(4):438-449.
Greenall SA, Donoghue JF, Van Sinderen M, et al. EGFRvIII-mediated transactivation of receptor tyrosine kinases in glioma: mechanism and therapeutic implications. Oncogene. 2015;34(41):5277-5287.
Li L, Puliyappadamba VT, Chakraborty S, et al. EGFR wild type antagonizes EGFRvIII-mediated activation of Met in glioblastoma. Oncogene. 2015;34(1):129-134.
Brown PD, Krishnan S, Sarkaria JN, et al.; North Central Cancer Treatment Group Study N0177. Phase I/II trial of erlotinib and temozolomide with radiation therapy in the treatment of newly diagnosed glioblastoma multiforme: North Central Cancer Treatment Group Study N0177. J Clin Oncol. 2008;26(34):5603-5609.
Weller M, Butowski N, Tran DD, et al.; ACT IV trial investigators. Rindopepimut with temozolomide for patients with newly diagnosed, EGFRvIII-expressing glioblastoma (ACT IV): a randomised, double-blind, international phase 3 trial. Lancet Oncol. 2017;18(10):1373-1385.
Neyns B, Sadones J, Joosens E, et al. Stratified phase II trial of cetuximab in patients with recurrent high-grade glioma. Ann Oncol. 2009;20(9):1596-1603.
Schulte A, Liffers K, Kathagen A, et al. Erlotinib resistance in EGFRamplified glioblastoma cells is associated with upregulation of EGFRvIII and PI3Kp110?. Neuro Oncol. 2013;15(10):1289-1301.
Ma Y, Tang N, Thompson RC, et al. InsR/IGF1R pathway mediates resistance to EGFR inhibitors in glioblastoma. Clin Cancer Res. 2016;22(7):1767-1776.
Akhavan D, Pourzia AL, Nourian AA, et al. De-repression of PDGFR? transcription promotes acquired resistance to EGFR tyrosine kinase inhibitors in glioblastoma patients. Cancer Discov. 2013;3(5):534-547.
Neirinckx V, Hedman H, Niclou SP. Harnessing LRIG1-mediated inhibition of receptor tyrosine kinases for cancer therapy. Biochim Biophys Acta Rev Cancer. 2017;1868(1):109-116.
Hedman H, Nilsson J, Guo D, Henriksson R. Is LRIG1 a tumour suppressor gene at chromosome 3p14.3? Acta Oncol. 2002;41(4):352-354.
Mao F, Holmlund C, Faraz M, et al. Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma. Oncogenesis. 2018;7(2):13.
Wong VW, Stange DE, Page ME, et al. Lrig1 controls intestinal stemcell homeostasis by negative regulation of ErbB signalling. Nat Cell Biol. 2012;14(4):401-408.
Powell AE, Wang Y, Li Y, et al. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell. 2012;149(1):146-158.
Lindquist D, Kvarnbrink S, Henriksson R, Hedman H. LRIG and cancer prognosis. Acta Oncol. 2014;53(9):1135-1142.
Yi W, Holmlund C, Nilsson J, et al. Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1. Exp Cell Res. 2011;317(4):504-512.
Goldoni S, Iozzo RA, Kay P, et al. A soluble ectodomain of LRIG1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity. Oncogene. 2007;26(3):368-381.
Johansson M, Oudin A, Tiemann K, et al. The soluble form of the tumor suppressor Lrig1 potently inhibits in vivo glioma growth irrespective of EGF receptor status. Neuro Oncol. 2013;15(9):1200-1211.
Bougnaud S, Golebiewska A, Oudin A, et al. Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma. Oncotarget. 2016;7(22):31955-31971.
Dixon AS, Schwinn MK, Hall MP, et al. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem Biol. 2016;11(2):400-408.
Szpakowska M, Meyrath M, Reynders N, et al. Mutational analysis of the extracellular disulphide bridges of the atypical chemokine receptor ACKR3/CXCR7 uncovers multiple binding and activation modes for its chemokine and endogenous non-chemokine agonists. Biochem Pharmacol. 2018;153:299-309.
Szpakowska M, Nevins AM, Meyrath M, et al. Different contributions of chemokine N-terminal features attest to a different ligand binding mode and a bias towards activation of ACKR3/CXCR7 compared with CXCR4 and CXCR3. Br J Pharmacol. 2018;175(9):1419-1438.
Xu Y, Soo P, Walker F, et al. LRIG1 extracellular domain: structure and function analysis. J Mol Biol. 2015;427(10):1934-1948.
Wu G, Ma Z, Hu W, et al. Molecular insights of Gas6/TAM in cancer development and therapy. Cell Death Dis. 2017;8(3):e2700.
Antony J, Huang RY. AXL-Driven EMT state as a targetable conduit in cancer. Cancer Res. 2017;77(14):3725-3732.
Vouri M, An Q, Birt M, Pilkington GJ, Hafizi S. Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells. Oncotarget. 2015;6(18): 16183-16197.
Vajkoczy P, Knyazev P, Kunkel A, et al. Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival. Proc Natl Acad Sci U S A. 2006;103(15):5799-5804.
Bowman RL, Wang Q, Carro A, Verhaak RG, Squatrito M. GlioVis data portal for visualization and analysis of brain tumor expression datasets. Neuro Oncol. 2017;19(1):139-141.
Yu M, Li W, Wang Q, Wang Y, Lu F. Circadian regulator NR1D2 regulates glioblastoma cell proliferation and motility. Oncogene. 2018..
Ammoun S, Provenzano L, Zhou L, et al. Axl/Gas6/NFkappaB signalling in schwannoma pathological proliferation, adhesion and survival. Oncogene. 2014; 33(3):336-346.
Burchert A, Attar EC, McCloskey P, Fridell YW, Liu ET. Determinants for transformation induced by the Axl receptor tyrosine kinase. Oncogene. 1998; 16(24):3177-3187.
Yu X, Sharma KD, Takahashi T, Iwamoto R, Mekada E. Ligandindependent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling. Mol Biol Cell. 2002; 13(7):2547-2557.
Torigoe H, Yamamoto H, Sakaguchi M, et al. Tumor-suppressive effect of LRIG1, a negative regulator of ErbB, in non-small cell lung cancer harboring mutant EGFR. Carcinogenesis. 2018;39(5):719-727.
Gumienny TL, Macneil L, Zimmerman CM, et al. Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling. PLoS Genet. 2010;6(5): e1000963.
Learn CA, Hartzell TL, Wikstrand CJ, et al. Resistance to tyrosine kinase inhibition by mutant epidermal growth factor receptor variant III contributes to the neoplastic phenotype of glioblastoma multiforme. Clin Cancer Res. 2004;10(9):3216-3224.
Johnson H, Del Rosario AM, Bryson BD, Schroeder MA, Sarkaria JN, White FM. Molecular characterization of EGFR and EGFRvIII signaling networks in human glioblastoma tumor xenografts. Mol Cell Proteomics. 2012;11(12):1724-1740.
Hall A. The cytoskeleton and cancer. Cancer Metastasis Rev. 2009;28(1-2):5-14.
Iida K, Sakai R, Yokoyama S, et al. Cell softening in malignant progression of human lung cancer cells by activation of receptor tyrosine kinase AXL. Scientific Reports. 2017;7(1):17770.
Vouri M, Croucher DR, Kennedy SP, An Q, Pilkington GJ, Hafizi S. Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells. Oncogenesis. 2016;5(10):e266.
Gao CF, Xie Q, Su YL, et al. Proliferation and invasion: plasticity in tumor cells. Proc Natl Acad Sci U S A. 2005;102(30):10528-10533.
Zhang Z, Lee JC, Lin L, et al. Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat Genet. 2012;44(8):852-860.
Guo G, Gong K, Ali S, et al. A TNF-JNK-Axl-ERK signaling axis mediates primary resistance to EGFR inhibition in glioblastoma. Nat Neurosci. 2017;20(8):1074-1084.
Meyer AS, Miller MA, Gertler FB, Lauffenburger DA. The receptor AXL diversifies EGFR signaling and limits the response to EGFRtargeted inhibitors in triple-negative breast cancer cells. Sci Signal. 2013;6(287):ra66.
Huang PH, Mukasa A, Bonavia R, et al. Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci U S A. 2007; 104(31):12867-12872.
Moll HP, Pranz K, Musteanu M, et al. Afatinib restrains K-RAS-driven lung tumorigenesis. Sci Transl Med. 2018;10(446).
Jacobsen HJ, Poulsen TT, Dahlman A, et al. Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2, and HER3, effectively overcomes tumor heterogeneity and plasticity. Clin Cancer Res. 2015;21(18):4110-4122.