Four individually druggable MET hotspots mediate HGF-driven tumor progression
Cristina Basilico, … , Hans de Haard, Paolo Michieli
Cristina Basilico, … , Hans de Haard, Paolo Michieli
Published May 27, 2014
Citation Information: J Clin Invest. 2014;124(7):3172-3186. https://doi.org/10.1172/JCI72316.
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Four individually druggable MET hotspots mediate HGF-driven tumor progression

Abstract

Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF β chain binding site on blades 2–3 of the SEMA domain β-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2–3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2–3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.

Authors

Cristina Basilico, Anna Hultberg, Christophe Blanchetot, Natalie de Jonge, Els Festjens, Valérie Hanssens, Sjudry-Ilona Osepa, Gitte De Boeck, Alessia Mira, Manuela Cazzanti, Virginia Morello, Torsten Dreier, Michael Saunders, Hans de Haard, Paolo Michieli

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Figure 8

WT52 and WT46 cooperate in inhibiting HGF-mediated invasive growth.

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WT52 and WT46 cooperate in inhibiting HGF-mediated invasive growth. (A) ...
(A) Inhibition of HGF-induced MET autophosphorylation. A549 cells were stimulated with 1 nM recombinant HGF in the presence of increasing concentrations (0–200 nM) of WT52, WT46, or a 1:1 mixture of WT52 and WT46 (i.e., 200 nM of either WT52 or WT46 is compared with 100 nM of WT52 + 100 nM of WT46). An irrelevant IgG1 was used as negative control. MET autophosphorylation was determined by ELISA and is expressed as percent relative to control. (B) Inhibition of HGF-induced cell scattering. HPAF-II cells were stimulated with 0.14 nM HGF in the presence of increasing concentrations (0–200 nM) of antibodies as in A. Scatter activity was determined by microscopy and is expressed using the scoring system described in the text (0, total absence of cell scattering; 5, maximal cell scattering). (C) Inhibition of HGF-induced cell motility. HPAF-II cells were preincubated with 200 nM WT52, 200 nM WT46, or 100 nM WT52 + 100 nM WT46 and then stimulated with 0.14 nM HGF. Cell motility was monitored in real time using an xCELLigence RTCA device and is expressed as normalized cell index. (D) Inhibition of HGF-induced anchorage-independent cell growth. A549 cells were grown in soft agar in the presence of 0.34 nM HGF plus 200 nM antibodies as in C. Colony growth was determined after 2 weeks as described in the text. Statistical significance was determined by a Student’s t test (n = 4).