Transglutaminase 2 enhances hepatocyte growth factor signaling to drive the mesothelioma cancer cell phenotype

Abstract

Transglutaminase 2 (TG2) is an important mesothelioma cancer cell survival protein. However, the mechanism whereby TG2 maintains mesothelioma cell survival is not well understood. We present studies showing that TG2 drives hepatocyte growth factor (HGF)-dependent MET receptor signaling to maintain the aggressive mesothelioma cancer phenotype. TG2 increases HGF and MET messenger RNA and protein levels to enhance MET signaling. TG2 inactivation reduces MET tyrosine kinase activity to reduce cancer cell spheroid formation, invasion and migration. We also confirm that HGF/MET signaling is a biologically important mediator of TG2 action. Reducing MET level using genetic methods or treatment with MET inhibitors reduces spheroid formation, invasion and migration and this is associated with reduced MEK1/2 and ERK1/2. In addition, MEK1/2 and ERK1/2 inhibitors suppress the cancer phenotype. Moreover, MET knockout mesothelioma cells form 10-fold smaller tumors compared to wild-type cells and these tumors display reduced MET, MEK1/2, and ERK1/2 activity. These findings suggest that TG2 maintains HGF and MET levels in cultured mesothelioma cells and tumors to drive HGF/MET, MEK1/2, and ERK1/2 signaling to maintain the aggressive mesothelioma cancer phenotype.

Keywords: ERK1/2 signaling; MAPK signaling; hepatocyte growth factor (HGF); mesothelioma; transglutaminase 2 (TGM2).

Fig. 1

TG2 maintains the cancer phenotype via HGF/MET receptor. A TG2 knockout reduces HGF mRNA transcript levels. B/C/D TG2 knockout attenuates MET and ERK1/2 signaling, and spheroid formation and invasion. HGF treatment partially restores MET and ERK1/2 signaling and biological responses. E/F TG2 knockdown reduces NCI-Meso-17 MET and ERK1/2 signaling and invasion, and these responses are partially reversed by HGF treatment. G/H/I TG2 knockout reduces tumor formation, HGF and MET mRNA, MET protein level and ERK1/2 activity. Single asterisks indicate a significant decrease (n = 3, p ≤ 0.001) and double asterisks indicate a significant increase (n = 3, p ≤ 0.001).

Fig. 2

HGF/MET enhances the Meso-1 cell cancer phenotype. A/B HGF knockdown reduces Meso-1 cell invasion and migration. C/D/E Meso-1 cell MET knockdown reduces MET and ERK1/2 activity which is associated with reduced invasion and migration. F/G/H/I MET-inhibitor SGX-523 reduces Meso-1 ERK1/2 activity and spheroid formation, invasion, and migration. J/K/L HGF treatment activates MET, MEK1/2 and ERK1/2 signaling and increases invasion and migration. Single asterisks indicate a significant decrease (n = 3, p ≤ 0.001).

Fig. 3

HGF/MET maintains the NCI-Meso-17 cell cancer phenotype. A/B HGF-knockdown reduces NCI-Meso-17 cell invasion and migration. C/D/E MET-knockdown reduces ERK1/2 activity and cell invasion and migration. F/G/H/I Treatment with the SGX-523 MET inhibitor reduces spheroid formation, invasion and migration. J/K/L HGF treatment increases MET signaling and cell invasion and migration. Single asterisks indicate a significant decrease (n = 3, p ≤ 0.001) and double asterisks indicate a significant increase (n = 3, p ≤ 0.001).

Fig. 4

HGF activation of MET enhances the cancer phenotype. A/B HGF stimulates Meso-1 cell invasion and migration and this response requires MET receptor. C/D HGF stimulates NCI-Meso-17 cell MET, MEK1/2 and ERK1/2 activity and increases invasion, and these responses require MET receptor. E/F/G/H MET knockout cell lines display reduced cell proliferation, invasion and migration. I/J HGF treatment does not increase MEK1/2 or ERK1/2 activity in Meso1-MET-KOc1-4 cells. Single asterisks indicate a significant decrease (n = 3, p ≤ 0.001) and double asterisks indicate a significant increase (n = 3, p ≤ 0.001).

Fig. 5

ERK1/2 and MEK1/2 signaling is critical for MET-dependent activation of the cancer phenotype. A/B/C/D Treatment with MEK1/2 (U0126) or ERK1/2 (LY3214996) inhibitor reduces Meso-1 cell MEK1/2 or ERK1/2 activity leading to reduced spheroid formation, invasion and migration. E/F/G MET knockout cells display reduced tumor formation and these tumors display reduced MET, MEK1/2 and ERK1/2 activity and reduced levels of CD31. Bars = 50 microns. H These studies show that TG2 maintains HGF/MET mRNA and protein levels to facilitate HGF/MET signaling which increases MEK1/2 and ERK1/2 activity to drive the aggressive cancer phenotype. Single asterisks indicate a significant decrease (n = 3, p ≤ 0.001).