Abstract
BackgroundThe mechanisms involved in lung cancer (LC) progression are poorly understood making discovery of successful therapies difficult. Adaptor proteins play a crucial role in cancer as they link cell surface receptors to specific intracellular pathways. Intersectin-1s (ITSN-1s) is an important multidomain adaptor protein implicated in the pathophysiology of numerous pulmonary diseases. To date, the role of ITSN-1s in LC has not been studied.MethodsHuman LC cells, human LC tissue and A549 LC cells stable transfected with myc-ITSN-1s construct (A549 + ITSN-1s) were used in correlation with biochemical, molecular biology and morphological studies. In addition scratch assay with time lapse microscopy and in vivo xenograft tumor and mouse metastasis assays were performed.ResultsITSN-1s, a prevalent protein of lung tissue, is significantly downregulated in human LC cells and LC tissue. Restoring ITSN-1s protein level decreases LC cell proliferation and clonogenic potential. In vivo studies indicate that immunodeficient mice injected with A549 + ITSN-1s cells develop less and smaller metastatic tumors compared to mice injected with A549 cells. Our studies also show that restoring ITSN-1s protein level increases the interaction between Cbl E3 ubiquitin ligase and Eps8 resulting in enhanced ubiquitination of the Eps8 oncoprotein. Subsequently, downstream unproductive assembly of the Eps8-mSos1 complex leads to impaired activation of the small GTPase Rac1. Impaired Rac1 activation mediated by ITSN-1s reorganizes the cytoskeleton (increased thick actin bundles and focal adhesion (FA) complexes as well as collapse of the vimentin filament network) in favor of decreased LC cell migration and metastasis.ConclusionITSN-1s induced Eps8 ubiquitination and impaired Eps8-mSos1 complex formation, leading to impaired activation of Rac1, is a novel signaling mechanism crucial for abolishing the progression and metastatic potential of LC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0543-1) contains supplementary material, which is available to authorized users.
Highlights
The mechanisms involved in lung cancer (LC) progression are poorly understood making discovery of successful therapies difficult
ITSN-1s protein and mRNA levels are downregulated in LC cells and tissues To address whether ITSN plays a role in LC, we examined ITSN-1s protein level in human LC cells by Western blot (WB) with ITSN-1 Ab compared to human bronchial cells
ITSN-1s mRNA levels were assessed in A549 cells compared to bronchial cells, and in adenocarcinoma tissue (Table 1), compared to non-LC tissue (Fig. 1b)
Summary
The mechanisms involved in lung cancer (LC) progression are poorly understood making discovery of successful therapies difficult. Adaptor proteins play a crucial role in cancer as they link cell surface receptors to specific intracellular pathways. The signal transduction initiated by receptor tyrosine kinases (RTK) plays a pivotal role in the regulation of proliferation and migration of cancer [4]. In most signaling pathways the adaptor proteins link cell surface receptors, including RTK, to specific intracellular pathways regulating the activation status of kinases and controlling the cross-talk between signaling cascades [5]. ITSN-1s is one such protein that plays a crucial role in intracellular signal transduction, linking activated receptors to downstream targets by binding to specific phospho-tyrosinecontaining sequences and proline-rich motifs [6,7,8]. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3Erk1/2 signaling balance downstream of transforming growth factor receptor type I leading to proliferation and neo-vascularization of lung endothelial cells [9]. Due to its multimodular structure, ITSN-1s is involved in multiple protein-protein interactions with a pivotal role in cell growth, apoptosis, cell cycle regulation, DNA damage repair and innate immune response [10, 11]
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