c-Src-dependent cross-talk between CEACAM6 and αvβ3 integrin enhances pancreatic adenocarcinoma cell adhesion to extracellular matrix components

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Abstract

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member with a diversity of extracellular ligands that is implicated in the initiation and progression of a variety of malignancies. We sought to characterize the effects of CEACAM6 crosslinking on pancreatic adenocarcinoma cellular interaction with the extracellular matrix (ECM) components fibronectin and vitronectin. Antibody-mediated CEACAM6 crosslinking was performed and the ability of BxPC3 cells, which inherently overexpress CEACAM6, to adhere to fibronectin and vitronectin was quantified. The roles of the archetypal fibronectin (α5β1 integrin) and vitronectin (αvβ3 integrin) receptors were determined. The effects of c-Src inhibition were investigated using the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and c-Src specific RNA interference. CEACAM6 crosslinking initiates c-Src-dependent cross-talk between CEACAM6 and αvβ3 integrin, leading to increased ECM component adhesion. CEACAM6-mediated signaling events may contribute to the invasive and metastatic potential of pancreatic adenocarcinoma cells by promoting their interaction with ECM components.

Section snippets

Materials and methods

Cell culture. BxPC3 pancreatic ductal adenocarcinoma cells were obtained from ATCC (Rockville, MD). BxPC3 was specifically chosen as these cells inherently overexpress CEACAM6 [14]. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco-BRL, Gaithersburg, MD). Cells were incubated in a humidified (37 °C, 5% CO2) incubator and passaged upon reaching 80% confluence.

CEACAM6 crosslinking. The By114 mouse monoclonal antibody (Innogenex, San

CEACAM6 crosslinking promotes pancreatic adenocarcinoma cellular adhesion to ECM components

Antibody-mediated crosslinking of CEACAM6 was performed by exposing BxPC3 cells to anti-CEACAM6 primary antibody, followed by anti-mouse IgG secondary antibody F(ab)2. F(ab)2 was used to exclude Fc-mediated effects, although use of intact secondary antibody produced identical results (data not shown). CEACAM6 crosslinking induced a marked increase in cellular adhesion to both fibronectin and vitronectin. The effect of anti-CEACAM6 antibody alone was minimal and secondary antibody F(ab)2

Discussion

In this study, we have shown that antibody-induced crosslinking of CEACAM6 enhances αvβ3 integrin-mediated adhesion to ECM components. We have also shown that c-Src is required for this cross-talk between CEACAM6 and αvβ3 integrin to occur. Despite lacking an intracellular domain, CEACAM6 is capable of independently activating neutrophil adhesion to human vascular endothelial cells (HUVECs) [23] and crosslinking of the GPI-linked CEACAM family members has been reported to induce a respiratory

Acknowledgements

The authors gratefully acknowledge the technical assistance of Jan Rounds. This work was supported by The National Pancreas Foundation and departmental funds from the Department of Surgery, Brigham and Women’s Hospital.

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