The role of Eps8 in myofibroblast differentiation

Abstract

Background
Myofibroblasts are central to the pathogenesis of fibrotic disease, but their presence in a range of solid tumours is also an important prognostic factor predicting poorer patient outcome. This specialised, highly contractile cell develops as a result of increases in intracellular and extracellular matrix tension and the activity of the cytokine TGFβ1. It is characterised in fibroblasts by the de novo expression of alpha smooth muscle actin (αSMA) stress fibres. Myofibroblasts have been shown to promote many of the ‘hallmarks of malignancy’ in neighbouring cancer cells including dysregulated growth, resistance to apoptosis, tumour cell invasion and metastasis.

Eps8 is a widely expressed adapter protein that has been identified as a critical regulator of actin organisation and cell motility in a range of cell types. In this study we have assessed the role of Eps8 in fibroblast-to-myofibroblast transdifferentiation.

Methods
Western blotting, quantitative real-time PCR, and RNA-seq techniques were used to assess alterations in expression profiles in cultured human fibroblasts as a result of TGFb1 treatment or Eps8 downregulation. Immunocytochemistry was used to assess the formation of stress fibres, while gel contraction; Transwell migration; xCELLigence proliferation and TGFb activation assays were used to assess effects on cell function. Tissue microarrays of a range of fibrotic human organs or oral cancer specimens were used to validate the in vitro findings and a mouse xenograft model was used to assess the effect of fibroblast Eps8 expression on human tumour development in vivo.

Results
We identified a novel role of Eps8 as a regulator of fibroblast-to-myofibroblast transdifferentiation and demonstrated that Eps8 is downregulated during myofibroblast transdifferentiation induced by TGFβ1 treatment or senescence-inducing stimuli. Suppression of Eps8 by the use of siRNA augments TGFβ1-induced myofibroblast transdifferentiation, and augments cancer cell migration in vitro and tumour growth in vivo. Eps8 knockdown upregulated SMAD2 expression and augmented TGFβ1-dependent SMAD2 phosphorylation and Nox4 induction. Although Eps8 has several known functions our investigations indicate that the formation of a tricomplex with its binding partners Abi1 and SOS1 is necessary for the regulation of myofibroblast transdifferentiation.

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