TGFβ loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion

Abstract

The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotype in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83–01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83–01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion.

A Human Organotypic Microfluidic Tumor Model Permits Investigation of the Interplay between Patient-Derived Fibroblasts and Breast Cancer Cells

“…In a similar study to ours, Le bras et al . established a 3D organotypic co-culture model using a collagen matrix, human esophageal epithelial cells, and human skin fibroblasts and demonstrated enhanced tumor proliferation and growth[43]. Concordant with our work, gene expression analysis revealed more than a 1.5 fold-increase in GPNMB compared to non-invasive cultures.…”

MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma

“…80 On the other hand, ADAMTS-1 also facilitates cell invasion by cleaving heparin-binding epithelial growth factor–like growth factor and amphiregulin. 81,82 Interestingly, in our data from the protease arrays of the UMSCC1 cells, the secreted levels of ADAMTS-1 was identified as being significantly diminished in conditioned cell culture medium from UMSCC1 cells with increased miR-375 expression compared to its control line. The interactions of the theoretical miR-375 targets mentioned above with other invadopodia-associated proteins are shown in Supplemental Figure 1 (see supplemental digital content).…”

Intertwining of Activin A and TGFβ Signaling: Dual Roles in Cancer Progression and Cancer Cell Invasion

“…TGFβ has been shown to stimulate collective migration primarily through extracellular-regulated kinase 1/2 (ERK1/2) activation [ 79 ]. On the other hand, using a three-dimensional organotypic culture system, we have described that inhibition of TGFβ signaling increases collection into the underlying extracellular matrix in a fibroblast- and MMP-dependent manner [ 80 ]. Additional research has demonstrated that tumor cell knockout of TGFβ signaling, through deletion of the type II receptor, drives fibroblast-stimulated collective migration and metastasis [ 81 ].…”

“…However, it has been consistently shown that fibroblasts that lose responsiveness to TGFβ promote collective cell invasion [ 79 , 129 , 130 ]. These results suggest that, in contrast to activin A, TGFβ signaling in the tumor stroma is necessary to maintain an intact microenvironment and prevent tumor cell invasion, though it has been suggested that TGFβ may act through the reactive stroma, not the epithelial cell compartment, to promote tumor angiogenesis [ 2 , 4 , 6 , 80 , 134 ]. This is a significant point, where the functional consequences of TGFβ and activin A diverge.…”