Abstract 2389: Eps8: a negative regulator of myofibroblast differentiation and function
Authors: Frampton Steven J. , Jenei Veronika , Mellone Massimiliano , Hanley Christopher J. , Rucka Marta M. , Tod Joanne , Moutasim Karwan A. , King Emma V. , Thomas Gareth 
Published: August 2015

In this study we investigate the mechanisms regulating myofibroblast differentiation and function, and identify the adapter protein Epidermal growth factor receptor kinase substrate 8 (Eps8) as a negative regulator of the cancer-associated myofibroblast phenotype.

Cancer-associated fibroblasts (CAFs) are a heterogenous, poorly defined cell population. Most commonly CAFs are identified by de novo expression of α-smooth muscle actin (αSMA) incorporated into stress fibres generating enhanced contractility, which is associated with an enhanced secretory profile. Myofibroblasts promote a number of the hallmarks of malignancy, and clinical studies have shown that the presence of an αSMA-rich stroma is associated with poor outcome in several cancers, suggesting that stromal targeting may be a useful therapeutic adjunct. The aim of this study was to investigate the mechanisms regulating differentiation of the myofibroblastic CAF phenotype.

Fibroblasts (HFFF2 and primary fibroblasts extracted and cultured from normal skin, esophageal and oral mucosa) were treated with TGF-β1 to induce myofibroblast transdifferentiation. Target genes were suppressed using siRNA. Gene expression was examined using RNA-seq and qRT-PCR, and protein expression by Western blotting and immunofluorescence microscopy. Myofibroblast function was examined using gel contraction assays while the effect of the myofibroblast secretion profile on cancer cell migration was assessed using Transwell® migration assays.

RNA-seq confirmed the upregulation of myofibroblast-associated genes (FN1, COL1A1, CTGF, ACTA2) in TGF-β1-treated fibroblasts. Conversely, we found that the adaptor protein Eps8 was significantly down-regulated, and this occurred early in the transdifferentiation process. Eps8 sits at the heart of a complex system regulating reorganization of the actin cytoskeleton, and interacts with numerous intracellular proteins including palladin, F-actin and integrins. We found that siRNA knockdown of Eps8 in fibroblasts promoted myofibroblast differentiation, resulting in increased expression of αSMA, stress fibre formation and cell contractility and promotion of cancer cell motility. Suppression of Eps8 expression sensitized fibroblasts to TGF-β1 treatment and potentiated TGF-β1 signalling, in part through transcriptional upregulation and increased phosphorylation of SMAD2.

These data suggest that Eps8 expression provides tonic inhibition of myofibroblast transdifferentiation, at least in part by desensitization of cells to TGF-β1 mediated by the regulation of SMAD2 expression. Loss of Eps8 expression may play an important role in the generation of an αSMA-positive myofibroblastic CAF population.

Citation Format: Steven J. Frampton, Veronika Jenei, Massimiliano Mellone, Christopher J. Hanley, Marta M. Rucka, Joanne Tod, Karwan A. Moutasim, Emma V. King, Gareth Thomas. Eps8: a negative regulator of myofibroblast differentiation and function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2389. doi:10.1158/1538-7445.AM2015-2389