Overexpression of GRB2 Enhances Epithelial to Mesenchymal Transition of A549 Cells by Upregulating SNAIL Expression.

Payal Mitra,Pazhanichamy Kalailingam,Hui Tan,Thirumaran Thanabalu

doi:10.3390/cells7080097

Abstract

GRB2 is an adaptor protein which interacts with phosphorylated TGF-β receptor and is critical for mammary tumour growth. We found that TGF-β1-induced EMT increased GRB2 expression in A549 cells (non-small cell lung cancer). Overexpression of GRB2 (A549GRB2) enhanced cell invasion while knocking down GRB2 (A549GRB2KD) reduced cell migration and invasion, probably due to increased vinculin and reduced Paxillin patches in A549GRB2KD cell. TGF-β1-induced EMT was more pronounced in A549GRB2 cells and attenuated in A549GRB2KD cells. This could be due to the reduced expression of E-cadherin in A549GRB2 and increased expression of E-cadherin in A549GRB2KD cells, even before TGF-β1 stimulation. Expression of SNAIL was elevated in A549GRB2 cells and was further enhanced by TGF-β1 stimulation, suggesting that GRB2 down-regulates E-cadherin by enhancing the expression of SNAIL. The N-SH3 domain of GRB2 was critical for suppressing E-cadherin expression, while the C-SH3 domain of GRB2 mediating interaction with proteins such as N-WASP was critical for promoting invasion, and the SH2 domain was critical for suppressing E-cadherin expression and invasion. Thus, our data suggests that GRB2 enhances EMT by suppressing E-cadherin expression and promoting invasion probably through N-WASP to promote metastasis.

Highlights

Metastasis is responsible for most of cancer-related deaths [1]
We found that the expression of GRB2 increased in TGF-β1 treated cells compared to the control (Figure 1B), and this was not due to increased transcription, as determined by qPCR (Figure S1), suggesting that GRB2 is stabilised by TGF-β1 treatment
The results suggest that GRB2 may play a positive role in signalling pathways mediated by TGF-β1 in A549 cells

Summary

Introduction

Metastasis is responsible for most of cancer-related deaths [1]. Metastasis is a complex multistep process in which tumour cells migrate from the primary tumour site through the extracellular matrix, enter the blood circulation by forming new blood vessels (tumour angiogenesis) and migrate to distant organs and tissues (extravasation) and proliferate to form secondary tumours [2]. Epithelial cancer cells in primary tumours are strongly associated with each other via different types of cellular junctions such as tight junctions, adherens junctions and desmosomes [3]. Tumour cells have to break these tight cellular junctions before invading the surrounding tissues as single cells [1]. A variety of environmental stimuli such as extracellular matrix (collagen), and growth factors and cytokines such as transforming growth factor-β (TGF-β), Epidermal Growth Factor (EGF), Hepatocyte Epithelial-to-Mesenchymal Transition (EMT) facilitates invasion by attenuating cell–cell adhesion, reorganising the actin cytoskeleton and up-regulating the expression of mesenchymal markers [4,5,6].

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Results

Conclusion