A systems view of epithelial-mesenchymal transition signaling states

Abstract

Epithelial-mesenchymal transition (EMT) is an important contributor to the invasion and metastasis of epithelial-derived cancers. While considerable effort has focused in the regulators involved in the transition process, we have focused on consequences of EMT to prosurvival signaling. Changes in distinct metastable and 'epigentically-fixed' EMT states were measured by correlation of protein, phosphoprotein, phosphopeptide and RNA transcript abundance. The assembly of 1167 modulated components into functional systems or machines simplified biological understanding and increased prediction confidence highlighting four functional groups: cell adhesion and migration, metabolism, transcription nodes and proliferation/survival networks. A coordinate metabolic reduction in a cluster of 17 free-radical stress pathway components was observed and correlated with reduced glycolytic and increased oxidative phosphorylation enzyme capacity, consistent with reduced cell cycling and reduced need for macromolecular biosynthesis in the mesenchymal state. An attenuation of EGFR autophosphorylation and a switch from autocrine to paracrine-competent EGFR signaling was implicated in the enablement of tumor cell chemotaxis. A similar attenuation of IGF1R, MET and RON signaling with EMT was observed. In contrast, EMT increased prosurvival autocrine IL11/IL6-JAK2-STAT signaling, autocrine fibronectin-integrin α5β1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFβR signaling. A relatively uniform loss of polarity and cell-cell junction linkages to actin cytoskeleton and intermediate filaments was measured at a systems level. A more heterogeneous gain of ECM remodeling and associated with invasion and migration was observed. Correlation to stem cell, EMT, invasion and metastasis datasets revealed the greatest similarity with normal and cancerous breast stem cell populations, CD49f(hi)/EpCAM(-/lo) and CD44(hi)/CD24(lo), respectively.

Fig. 1

a NSCLC model phenotypes for H292, H358, H358/TGFβ, H358/doxSnail, H358/doxZeb1, Calu6 and H1703 showing phase contrast and immunofluorescence images using anti-E-cadherin, anti-vimentin and DAPI co-staining. E-cadherin containing cell junctions (green) are visible in the epithelial lines H292 and H358. In contrast the metastable H358/TGFβ, H358/doxSnail and H358/doxZeb1 cells show a loss of E-cadherin expression, a gain of vimentin expression (red) and a characteristic scattered phenotype. Similarly the epigentically fixed NSCLC lines Calu6 and H1703 lack E-cadherin containing cell–cell junctions and express perinuclear vimentin, an intermediate filament component. b A scheme depicting EMT states within the primary tumor mass, in the tumor periphery and in disseminated tumor cells (DTCs). c Experimental schema for cell fractionation, protein, phosphoprotein and RNA measurements and network enrichment

Fig. 2

a Enriched functional annotations from combined proteome and transcript profiles. b Heterogeneity of EMT states is in part dependent on the EMT inducer, where Zeb1- and Snail-induced EMT were more closely related than TGFβ-induced EMT. c EMT dataset comparison to 18 datasets related stem cell, invasion/metastasis and EMT characteristics [–63]. The NSCLC EMT dataset was most closely related to normal and cancer-derived mammary stem cells, either by number of overlapping markers or when adjusted for dataset size (composite score)

Fig. 3

EMT associated cell–cell junction and cell-adhesion/ECM changes, grouped by system and assembled by protein–protein contacts. Systems decreased or lost with EMT included tight junctions (TJ), adherens junctions (AJ), desmosomal junctions (DJ) and adhesion to basement membrane (BM). Systems increased with EMT included myosin motor coupling with intermediate filament and actin cytoskeletal assemblies, proteases responsible for BM degradation and extracellular matrix remodeling, and integrin/cadherin switching allowing new adhesion signals. Components in green were attenuated during EMT while those in light brown/red were increased with EMT

Fig. 4

a Loss of autocrine RTK signaling networks through EGFR/ErbB2/ErbB3, Met/Ron and IGF1R signaling networks. Attenuated IL4R, Muc1 and Muc4 and integrin α6β4 components also were observed. b Attenuated RTK tyrosine phosphorylation in H292 and H358, (epithelial phenotype) Calu6 and H1703 (mesenchymal phenotype) cell models were confirmed by quantitation of RTK arrays. c EGFR is functional and responds to exogenous ligand in both epithelial and mesenchymal states. H292 and H358, (epithelial) Calu6 and H1703 (mesenchymal) cells were exposed to EGFR kinase inhibitor (3 μM erlotinib, 2 h) or DMSO control prior to stimulation with exogenous EGF (10 ng/ml, 10 min). The EGFR-dependent inhibition of substrates CBL and SHC were similar in epithelial and mesenchymal-like cells, relative for control HSPD1

Fig. 5

a Gain of new survival and cytokine networks. Increased signaling through IL-11/IL-6/gp130, TGFβ/BMP2, Axl/Tyro3, integrin α5β1, PDGFR and FGFR were observed. Downstream activation of BRAF/MAPK1 and PTK2/paxillin signaling were observed. Components in green were attenuated during EMT while those in light brown/red were increased with EMT. b Activating MAPK1/Erk phosphorylation (T185/Y187), associated with TGFβ-induced EMT, was confirmed by immunoblot

Fig. 6

a The transcription node regulating interferon response was modulated during EMT. a Reduced free radical scavenging capacity was correlated the mesenchymal state, associated with the NFE2L2 transcription node. Components in green were attenuated during EMT while those in light brown/red were increased with EMT. b Networks regulated during EMT associated with FOXA, Snail, Zeb, ATF, NFKB/Rel, MEF2and Myc transcription nodes