CEACAM6 Cross-linking Induces Caveolin-1-dependent, Src-mediated Focal Adhesion Kinase Phosphorylation in BxPC3 Pancreatic Adenocarcinoma Cells

Abstract

Despite lacking transmembrane or intracellular domains, glycosylphosphatidylinositol-anchored proteins can modulate intracellular signaling events, in many cases through aggregation within membrane "lipid raft" microdomains. CEACAM6 is a glycosylphosphatidylinositol-linked cell surface protein of importance in the anchorage-independent survival and metastasis of pancreatic adenocarcinoma cells. We examined the effects of antibody-mediated cross-linking of CEACAM6 on intracellular signaling events and anchorage-independent survival of the CEACAM6-overexpressing pancreatic ductal adenocarcinoma cell line, BxPC3. CEACAM6 cross-linking increased c-Src activation and induced tyrosine phosphorylation of p125 FAK focal adhesion kinase. Focal adhesion kinase phosphorylation was dependent on c-Src kinase activation, for which caveolin-1 was required. CEACAM6 cross-linking induced a significant increase in cellular resistance to anoikis. These observations represent the first characterization of the mechanism through which this important cell surface oncoprotein influences intracellular signaling events and hence malignant cellular behavior. CEACAM61 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein that is overexpressed in a variety of human malignancies, including pancreatic adenocarcinoma (1, 2). This immunoglobulin superfamily member is emerging as an important determinant of a variety of aspects of the malignant cellular phenotype (1-3). We reported previously that levels of CEACAM6 expression modulate the susceptibility of pancreatic adenocarcinoma cells to anoikis (2). Normal epithelial cells require anchorage to an extracellular matrix for growth, survival, and differentiation. Resistance to anoikis, a subset of apoptosis induced by inadequate or inappropriate cell-substrate contact in normal cells, is a property of transformed cells that is associated with enhanced tumorigenesis and metastatic ability. Despite clear indications of its important role in cancer cell biology, the mechanisms through which CEACAM6 influences intracellular signal transduction remain poorly understood.Despite lacking transmembrane and intracellular domains, several GPI-anchored proteins, including CEACAM family members, are able to influence intracellular events and have been implicated in transmembrane signaling via tyrosine kinases (4 -7). GPI-anchored proteins are known to exist in clusters and form microdomains at the surface of the plasma membrane, referred to as "lipid rafts" (8). These microdomains appear to be important for GPI-anchored protein signal transduction. A number of signal transduction components, including Src family tyrosine kinases, are associated with lipid rafts, and cross-linking of cell surface molecules by a variety of techniques has proven to be a useful tool for exploring the functional roles of raft-associated proteins (9 -14).Here, we examine the effects of antibody-mediated crosslinking of CEACAM6 in pancreatic adenocarcinoma cells. We determine the effects of CEACAM6 cross-linking on ...

The human carcinoembryonic antigen (CEA) GPI anchor mediates anoikis inhibition by inactivation of the intrinsic death pathway

“…Recently, the increase in anoikis susceptibility of CEACAM6-expressing human pancreatic carcinoma cells produced by CEACAM6 siRNA gene silencing was shown to be associated with an increase in caspase-8 and -3 activities (Duxbury et al, 2004). Our findings indicate that, in the case of CEA expression, there is also an associated caspase-mediated anoikis resistance, since we observed a partial inactivation of caspase-3 (Figure 1c) which correlated with anoikis inhibition by CEA (Figure 1a).…”

“…In the case of anoikis inhibition, although it is well documented that CEA/CEACAM6 expression can inhibit the anoikis of human colorectal carcinoma cells (Ordonez et al, 2000;Soeth et al, 2001) and human pancreatic carcinoma cells (Duxbury et al, 2004), an effect that it is not produced by the transmembraneanchored CEA-family member CEACAM1 or the GPIanchored variant of the immunoglobulin superfamily member adhesion molecule, NCAM, (Ordonez et al, Molecular mechanism of CEA-mediated anoikis inhibition M del Pilar Camacho-Leal and CP Stanners 2000), the molecular mechanism implicated in this inhibitory effect was unclear. This study was focused on the molecular mediators associated with the anoikis resistance conferred by CEA expression.…”

“…It has been demonstrated that essentially all CEA family members, including GPIanchored CEA and CEACAM6, function as homotypic intercellular adhesion molecules in vitro (Benchimol et al, 1989;Oikawa et al, 1989;Stanners and Fuks, 1998). In contrast with the transmembrane member CEACAM1 (Eidelman et al, 1993;Rojas et al, 1996;Ilantzis et al, 2002;Taheri et al, 2003), CEA and CEACAM6 overexpression inhibits cell differentiation and anoikis in vitro (Eidelman et al, 1993;Ordonez et al, 2000;Screaton et al, 2000;Soeth et al, 2001;Taheri et al, 2003;Duxbury et al, 2004) and in vivo (Chan et al, unpublished results). The structural requirements for the inhibitory effects of CEA on differentiation were demonstrated to depend critically on the CEA-specific GPI anchor attached to self-binding extracellular domains Taheri et al, 2003).…”

CEACAM6 attenuates adenovirus infection by antagonizing viral trafficking in cancer cells

“…Surprisingly, we found that overexpression of CEACAM6 downregulated Src activity and that the Src-selective inhibitor PP2 can disturb the cytoskeleton and interfere with adenovirus infection. This is contrary to a previous report that CEACAM6 cross-linking increased Src activity (28). It is known that several GPI-anchored proteins, including CEACAM family members, have no transmembrane or intracellular domains, but they are still able to modulate intracellular signaling events and to influence cell activities.…”

“…There is accumulating evidence that CEACAM6 has functional importance in tumorigenesis, including disrupting tissue architecture and affecting cell polarity, differentiation, anti-anoikis activity, tumor invasion, metastasis, and even chemoresistance (17,19,20,22,27,28). Inhibition of CEACAM6 expression by RNA interference or antibody targeting can inhibit tumor adhesion, growth, invasion, and metastasis, resulting in improved survival of mice with metastases (18,29,30).…”