Abstract
BackgroundThe mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and has been correlated with increased metastasis. We were the first to report binding between MUC1 and Intercellular adhesion molecule-1 (ICAM-1), which is expressed on stromal and endothelial cells throughout the migratory tract of a metastasizing breast cancer cell. Subsequently, we found that MUC1/ICAM-1 binding results in pro-migratory calcium oscillations, cytoskeletal reorganization, and simulated transendothelial migration. These events were found to involve Src kinase, a non-receptor tyrosine kinase also implicated in breast cancer initiation and progression. Here, we further investigated the mechanism of MUC1/ICAM-1 signalling, focusing on the role of MUC1 dimerization in Src recruitment and pro-metastatic signalling.MethodsTo assay MUC1 dimerization, we used a chemical crosslinker which allowed for the detection of dimers on SDS-PAGE. We then generated MUC1 constructs containing an engineered domain which allowed for manipulation of dimerization status through the addition of ligands to the engineered domain. Following manipulation of dimerization, we immunoprecipitated MUC1 to investigate recruitment of Src, or assayed for our previously observed ICAM-1 binding induced events. To investigate the nature of MUC1 dimers, we used both non-reducing SDS-PAGE and generated a mutant construct lacking cysteine residues.ResultsWe first demonstrate that the previously observed MUC1/ICAM-1signalling events are dependent on the activity of Src kinase. We then report that MUC1 forms constitutive cytoplasmic domain dimers which are necessary for Src recruitment, ICAM-1 induced calcium oscillations and simulated transendothelial migration. The dimers are not covalently linked constitutively or following ICAM-1 binding. In contrast to previously published reports, we found that membrane proximal cysteine residues were not involved in dimerization or ICAM-1 induced signalling.ConclusionsOur data implicates non-cysteine linked MUC1 dimerization in cell signalling pathways required for cancer cell migration.
Highlights
The mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and has been correlated with increased metastasis
MUC1/Intercellular adhesion molecule-1 (ICAM-1) binding induced signalling is mediated by Src kinase We first confirmed that Src kinase is a critical component of the MUC1/ICAM-1 signalling axis by Small interfering ribonucleic acid (siRNA) knockdown of Src in MUC1-CFP transfected HEK 293T cells
We assayed for calcium oscillations (Figure 2b) and migration (Figure 2c), and found that MUC1-CFP cells treated with Lipofectamine-only respond to ICAM-1 stimulation by generating calcium oscillations (Figure 2b) and cell migration (Figure 2c), indicating that the presence of the CFP tail does not interfere with this response
Summary
The mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and has been correlated with increased metastasis. We were the first to report binding between MUC1 and Intercellular adhesion molecule-1 (ICAM-1), which is expressed on stromal and endothelial cells throughout the migratory tract of a metastasizing breast cancer cell. We found that MUC1/ICAM-1 binding results in pro-migratory calcium oscillations, cytoskeletal reorganization, and simulated transendothelial migration. These events were found to involve Src kinase, a non-receptor tyrosine kinase implicated in breast cancer initiation and progression. We further reported that after interaction with ICAM-1, transendothelial migration invasion in MUC1 expressing cells is associated with increased MUC1-Src association, MUC1-cytoplasmic domain (MUC1-CD) phosphorylation, CrkL recruitment, and Rho-GTPase mediated cytoskeletal rearrangement [5,6,7]. The cytoplasmic portion consists of a 58-aa extracellular stub, a 28-aa transmembrane domain, and a 72-aa cytoplasmic domain, which contains seven conserved tyrosine residues, and has been shown to interact with diverse effectors [Reviewed in [12]] which is important since MUC1CD itself lacks tyrosine kinase activity
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