Investigating MUC1/ICAM-1 Binding Induced Signalling in Breast Cancer Metastasis

Background: The mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and hasbeen correlated with increased metastasis. We were the first to report binding between MUC1 and Intercellularadhesion molecule-1 (ICAM-1), which is expressed on stromal and endothelial cells throughout the migratory tractof a metastasizing breast cancer cell. Subsequently, we found that MUC1/ICAM-1 binding results in pro-migratorycalcium oscillations, cytoskeletal reorganization, and simulated transendothelial migration. These events were foundto 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 MUC1dimerization in Src recruitment and pro-metastatic signalling.Methods: To assay MUC1 dimerization, we used a chemical crosslinker which allowed for the detection of dimerson SDS-PAGE. We then generated MUC1 constructs containing an engineered domain which allowed formanipulation of dimerization status through the addition of ligands to the engineered domain. Followingmanipulation of dimerization, we immunoprecipitated MUC1 to investigate recruitment of Src, or assayed for ourpreviously observed ICAM-1 binding induced events. To investigate the nature of MUC1 dimers, we used bothnon-reducing SDS-PAGE and generated a mutant construct lacking cysteine residues.Results: We first demonstrate that the previously observed MUC1/ICAM-1signalling events are dependent on theactivity of Src kinase. We then report that MUC1 forms constitutive cytoplasmic domain dimers which arenecessary for Src recruitment, ICAM-1 induced calcium oscillations and simulated transendothelial migration. Thedimers are not covalently linked constitutively or following ICAM-1 binding. In contrast to previously publishedreports, we found that membrane proximal cysteine residues were not involved in dimerization or ICAM-1 inducedsignalling.Conclusions: Our data implicates non-cysteine linked MUC1 dimerization in cell signalling pathways required forcancer cell migration. BackgroundThe ability of malignant cells to escape from a primarytumour mass and migrate to distal sites to form meta-static tumors is the cause of mortality in the majority ofcarcinomas, including breast carcinoma. Approximately20% of breast cancers belong to the Luminal B geneticsubtype, typified by estrogen receptor positivity and aslow, steady rate of recurrence over time despite anti-estrogen therapy [1]. Estrogen is known to increase theexpression of MUC1 [2], a well-characterized memberof the mucin family of glycoproteins, and a correlationhas been demonstrated between MUC1 expression,resistance to anti-estrogen therapy and metastatic beha-viour [3]. We have been investigating the mechanism ofcell migration in the Luminal B breast cancer cell linesMCF7 and T47D, and were the first to demonstrate thatMUC1 mediates heterotypic cell-cell adhesion by bind-ing ICAM-1 [4], which is expressed on peritumoral stro-mal and endothelial cells. Subsequently, wedemonstrated that ICAM-1 binding triggers calcium* Correspondence: [email protected] of Laboratory Medicine and Pathology, 3-70 Heritage MedicalResearch Centre, University of Alberta, Edmonton, AB, T6G 2S2, CanadaFull list of author information is available at the end of the articleBernier et al. Molecular Cancer 2011, 10:93http://www.molecular-cancer.com/content/10/1/93 © 2011 Bernier et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited. oscillations which may activate proteins involved in focaladhesion disassembly and cell contraction. In keepingwith this, we further reported that after interaction withICAM-1, transendothelial migration invasion in MUC1expressing cells is associated with increased MUC1-Srcassociation, MUC1-cytoplasmic domain (MUC1-CD)phosphorylation, CrkL recruitment, and Rho-GTPasemediated cytoskeletal rearrangement [5-7].MUC1 (also known as DF3, CA15-3, or episialin) isexpressed apically on normal breast epithelia, but oftenloses this polarization and becomes underglycosylated inbreast cancer [8,9]. MUC1 is translated as a single poly-peptide, followed by conformational stress-induced clea-vage resulting in a heterodimer of non-covalentlyassociated extracellular and cytoplasmic portions [10,11](Figure 1). The extracellular portion consists of a vari-able number of 20-amino acid (aa) tandem repeats con-taining multiple sites for O-glycosylation, which imparta negative charge and result in a structure that canextend up to 500 nm from the cell surface. The cyto-plasmic portion consists of a 58-aa extracellular stub, a28-aa transmembrane domain, and a 72-aa cytoplasmicdomain, which contains seven conserved tyrosine resi-dues, and has been shown to interact with diverse effec-tors [Reviewed in [12]] which is important since MUC1-CD itself lacks tyrosine kinase activity.The signalling capacity of transmembrane proteinslacking kinase activity is often mediated by associatednon-receptor tyrosine kinases. In some instances, thesekinases are bound to pre-formed dimers of the receptor[[13], Reviewed in [14]]. Upon ligand binding, structuralchanges such as cysteine linkage, association with deter-gent resistant membrane fractions, and changes in clea-vage result in signal initiation [15-17]. Previous work byothers has demonstrated that constructs of the MUC1-CD form oligomers in vitro which are disulfide-linked,and in vivo which are dependent on the membrane-proximal cytoplasmicCQC motif [18,19] (Figure 1).Here, we investigated dimer formation in wild-typeMUC1 and the relationship between dimerization, Srcrecruitment and ICAM-1 induced signalling events. Wealso examined the role of membrane-proximal cytoplas-mic domain cysteine residues in these phenomena. Weconfirm that Src is an essential mediator of the pre-viously observed ICAM-1 binding pro-motility eventsand show that MUC1 forms constitutive cytoplasmicdomain dimers which are required for constitutive Srcrecruitment and ICAM-1 binding induced signalling.Contrary to previous reports, we found that dimers arenot disulfide linked constitutively or following ICAM-1ligation, and that membrane-proximal cysteine residuesare not required for dimerization or ICAM-1 inducedevents. Materials and methodsAntibodies and ReagentsCT2 Armenian Hamster monoclonal antibody (mAb)[20], directed against the last 17 C-terminal amino acidsof MUC1-CD, was generously provided by Dr. SandraGendler (Mayo Clinic, Scottsdale, AZ). Rabbit anti-SrcmAb, anti-Src polyclonal Abs, and anti-rabbit perox-idase conjugated secondary antibody were purchasedfrom Cell Signalling. Goat anti-mouse and anti-Arme-nian hamster peroxidase-conjugated secondary antibo-dies were purchased from Jackson ImmunoResearchLaboratories, Inc. Mouse anti-tubulin antibody was fromSigma-Aldrich. Disuccinimidyl suberate (DSS) was fromPierceNet. Protein G-Agarose was purchased fromRoche Diagnostics. ECL Plus Western Blotting detectionreagent was purchased from GE Healthcare (AmershamBiosciences). Gelatin Type A and phosphatase inhibitorcocktail were from Sigma-Aldrich. Protease inhibitor Figure 1 Schematic of constructs used in this study. “SS” indicates signal sequence, “ECD” indicates extracellular domain, “TMD” indicatestransmembrane domain and “CD” indicates cytoplasmic domain. On SDS-PAGE, full-length MUC1 dissociates at “cleavage site” and runs as twoseparate entities.Bernier et al. Molecular Cancer 2011, 10:93http://www.molecular-cancer.com/content/10/1/93Page 2 of 13