Integrin-Linked Kinase Links Integrin Activation to Invadopodia Function and Invasion via the p(T567)-Ezrin/NHERF1/NHE1 Pathway.

Maria Raffaella Greco,Khalid Alfarouk,Stefania Forciniti,Stephan Joel Reshkin,Rosa Angela Cardone,Loredana Moro,Stefania Cannone

doi:10.3390/ijms22042162

Abstract

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that β1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between β1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates β1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with β1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of β1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates β1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.

Highlights

Dissemination of metastatic cells to distant sites is the leading cause of cancer fatality, underlying the need for new therapeutic approaches focusing on invasive tumor cell spreading [1]
We started by analyzing the direct associations between integrin-linked kinase (ILK), β1-integrin p-ezrin, Na+/H+ exchanger type 1 (NHE1) and NHERF1 at proteolytically active invadopodia, utilizing in situ proximity ligation assay, which can measure endogenous protein–protein interactions occurring within 40 nm
Invadopodia extracellular matrix (ECM) proteolysis was visualized using a protocol based on the degradationdependent release of fluorescence of a quenched fluorophore (DQ Green-BSA) dissolved in Matrigel, where proteolysis of the ECM was measured as the amount of focal fluorescence unquenched by proteolysis [29]

Summary

Introduction

Dissemination of metastatic cells to distant sites is the leading cause of cancer fatality, underlying the need for new therapeutic approaches focusing on invasive tumor cell spreading [1]. Successful invasion processes require changes in tumor cell adhesion properties, cell motility and proteolytic remodeling of the extracellular matrix (ECM). It is well-established that metastatic cells have plasma membrane structures dedicated to driving their increased invasion, called invadopodia. Β1-integrin adhesion to the ECM promotes active, invadopodia focal ECM proteolysis through the phosphorylation of ezrin at T567 [25] This results in the formation of a “protein–protein” signal complex dedicated to the regulation of invadopodial proteolytic function and subsequent invasive and metastatic potential: the β1-integrin/p-ezrin/NHE1/p-NHERF1 “invadosome” localized in invadopodia that regulates their Na+/H+ exchanger type 1 (NHE1)-driven proteolytic activity [25,27,28]

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