Cooperative Signaling between Oncostatin M, Hepatocyte Growth Factor and Transforming Growth Factor-β Enhances Epithelial to Mesenchymal Transition in Lung and Pancreatic Tumor Models

Abstract

Epithelial to mesenchymal transition (EMT) plays a dual role in tumor progression. It enhances metastasis of tumor cells by increasing invasive capacity and promoting survival, and it decreases tumor cell sensitivity to epithelial cell-targeting agents such as epithelial growth factor receptor kinase inhibitors. In order to study EMT in tumor cells, we have characterized 3 new models of ligand-driven EMT: the CFPAC1 pancreatic tumor model and the H358 and H1650 lung tumor models. We identified a diverse set of ligands that drives EMT in these models. Hepatocyte growth factor and oncostatin M induced EMT in all models, while transforming growth factor-β induced EMT in both lung models. We observed morphologic, marker and phenotypic changes in response to chronic ligand treatment. Interestingly, stimulation with 2 ligands resulted in more pronounced EMT compared with single-ligand treatment, demonstrating a spectrum of EMT states induced by parallel signaling, such as the JAK and PI3K pathways. The EMT changes observed in response to the ligand were reversed upon ligand withdrawal, demonstrating the ‘metastable’ nature of these models. To study the impact of EMT on cell morphology and invasion in a 3D setting, we cultured cells in a semisolid basement membrane extract. Upon stimulation with EMT ligands, the colonies exhibited changes to EMT markers and showed phenotypes ranging from modest differences in colony architecture (CFPAC1) to complex branching structures (H358, H1650). Collectively, these 3 models offer robust cell systems with which to study the roles that EMT plays in cancer progression.

Inducible expression of TGFβ, Snail and Zeb1 recapitulates EMT in vitro and in vivo in a NSCLC model

“…These were gradual changes that took a full 7 days to occur and are consistent with our previous work in this cell line [34,35]. Each model had a distinct endpoint, with aTGFb achieving the most pronounced EMT phenotype.…”

“…Cells were imaged by phase contrast prior to fixation. Immunofluorescence on 3D cultures was performed as previously described [35]. Antibodies used were Phalloidin Rhodamine (Invitrogen #R415) at 1:40 and Active Caspase 3 (BD Biosciences # 559565) at 1:100.…”

“…We chose H358 cells as the parental cell line for the engineered models of EMT, as we have shown previously that this cell line will undergo EMT in vitro [35,36]. We selected TGFb as the ligand for one of the models due to its well described role in EMT.…”

EGF augments TGFβ-induced epithelial–mesenchymal transition by promoting SHP2 binding to GAB1

“…Of course, the ability to increase SHP2 activity sufficiently to augment TGFβ-mediated EMT may not be unique to EGF. Growth factors including hepatocyte growth factor (HGF) and oncostatin-M (Argast et al, 2011), platelet-derived growth factor (PDGF) (Gotzmann et al, 2006) and IL-6 (Yao et al, 2010) have been reported to enhance TGFβ-mediated EMT, and signaling downstream of many of these ligands involves SHP2. It is unclear, however, whether SHP2 participates in the augmented EMT that accompanies the combination of these ligands with TGFβ.…”

Out of the Desert: The 4th TEMTIA Meeting on New Advances in Development, Fibrosis and Cancer

“…This is followed by a comprehensive review of the growth factor pathways regulating either EMT or MET with known relevance to metastasis, again with emphasis on their interdependence and interaction [Said and Williams, 2011]. Developmental scenarios/regulators are represented by work on dystrogylcan in chick gastrulation [Nakaya et al, 2011] and periostin in palate fusion [Kitase et al, 2011]; fibrotic kidney disease with angiotensin II downstream of the intrarenal renin-angiotensin system, which operates both through and independent of TGF-␤ 1 [Burns and Thomas, 2011], and carcinoma progression with articles on the E-cadherin repressor interactome in breast cancer systems [Hugo et al, 2011] and cooperation in EMT-regulating signals between oncostatin M, hepatocyte growth factor and TGF-␤ in carcinoma EMT, arising from a survey of some 60 growth factors/cytokines in 30 cell lines [Argast et al, 2011]. These articles capture the current flavor of complexity surrounding EMT regulation, with a large number of regulatory pathways across the different models.…”