MET∆14 promotes a ligand-dependent, AKT-driven invasive growth

Abstract

MET is an oncogene encoding the tyrosine kinase receptor for hepatocyte growth factor (HGF). Upon ligand binding, MET activates multiple signal transducers, including PI3K/AKT, STAT3, and MAPK. When mutated or amplified, MET becomes a “driver” for the onset and progression of cancer. The most frequent mutations in the MET gene affect the splicing sites of exon 14, leading to the deletion of the receptor’s juxtamembrane domain (MET∆14). It is currently believed that, as in gene amplification, MET∆14 kinase is constitutively active. Our analysis of MET in carcinoma cell lines showed that MET∆14 strictly depends on HGF for kinase activation. Compared with wt MET, ∆14 is sensitive to lower HGF concentrations, with more sustained kinase response. Using three different models, we have demonstrated that MET∆14 activation leads to robust phosphorylation of AKT, leading to a distinctive transcriptomic signature. Functional studies revealed that ∆14 activation is predominantly responsible for enhanced protection from apoptosis and cellular migration. Thus, the unique HGF-dependent ∆14 oncogenic activity suggests consideration of HGF in the tumour microenvironment to select patients for clinical trials.

An mTOR feedback loop mediates the ‘flare’ (‘rebound’) response to MET tyrosine kinase inhibition

“…To enlighten what is behind the flare effect, we used two cell lines derived from two cancer cell types (EBC1 isolated from lung squamous carcinoma and HS746T from gastric carcinoma). These cell lines were carefully chosen to recapitulate the most common alterations of MET in cancer leading to MET ‘addiction’: gene amplification (EBC1 cells) and exon14 skipping (HS746T cells) 31 . Overnight JNJ-605 treatment abolished MET phosphorylation (Fig.…”

“…Immunoblotting. Cells were lysed in Laemmli Lysis Buffer as previously described 31 . Cell lysates were quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific™, Catalogue number 23225), and 15 µg of total proteins were resolved in polyacrylamide gels under denaturing conditions.…”

An mTOR feedback loop mediates the ‘flare’ (‘rebound’) response to MET tyrosine kinase inhibition

“…These cell lines were carefully chosen to recapitulate the most common alterations of MET in cancer leading to MET 'addiction': gene ampli cation (EBC1 cells) and exon14 skipping (HS746T cells). 31 Overnight JNJ-605 treatment abolished MET phosphorylation (Fig. 1A and 1B).…”

“…Immunoblotting: Cells were lysed in Laemmli Lysis Buffer as previously described. 31 Cell lysates were quanti ed using Pierce BCA Protein Assay Kit (ThermoFisher Scienti c ™ , Catalogue number 23225), and 15µg of total proteins were resolved in polyacrylamide gels under denaturing conditions. After transfer, membranes (ThermoFisher Scienti c ™ , Invitrolon ™ PVDF, Catalogue number LC2005) were blotted against antibodies listed in Supplementary Table 3.…”

The PSI Domain of the MET Oncogene Encodes a Functional Disulfide Isomerase Essential for the Maturation of the Receptor Precursor

“…Cells were lysed in Laemmli buffer as previously described [ 34 ]. Proteins were separated in denaturing polyacrylamide gels and blotted against antibodies listed in Supplementary Table S1 .…”