Molecular mechanisms of CD99-induced caspase-independent cell death and cell–cell adhesion in Ewing's sarcoma cells: actin and zyxin as key intracellular mediators

Abstract

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspaseindependent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

Immediate early gene X-1 interacts with proteins that modulate apoptosis

“…Recent findings suggest that zyxin may play a role in CD99 associated signaling in Ewing's sarcoma cells. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a role for zyxin in CD99 function [64]. Additional data suggest that zyxin like domains may be involved in VASP and semaphorin cell signaling [65].…”

“…Remarkably, the first four proteins (out of a total of eleven) are involved in the regulation of apoptosis (see Table 1 for a summary of the actions of these proteins). A fifth protein, zyxin, has also been implicated in the apoptotic response [64,65]. Since IEX-1 has been shown to modulate apoptosis, this is of great interest as it points to mechanisms by which IEX-1 may function in this regard.…”

CD44 ligation induces caspase-independent cell death via a novel calpain/AIF pathway in human erythroleukemia cells

“…Interestingly, caspase-independent cell death has also been described for two other mAb-activated cell surface molecules, CD47 and CD99, in leukemic cells. However, CD47-and CD99-induced cell death differs from CD44-induced HEL cell death as it occurs very rapidly after antigen ligation, involves an early reorganization of cytoskeleton and is AIF-independent (Mateo et al, 2002;Roue et al, 2003;Cerisano et al, 2004).…”

CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity

“…Anchorage-independent growth and Poly-HEMA (2-Hydroxyethylmethacrylate) Assay experiments were performed as previously described (Manara et al, 2006). Homotypic adhesion, ECM adhesion and motility assay Homotypic adhesion assay was performed as previously described (Cerisano et al, 2004). The adhesive ability was analysed by using the CytoMatrix cell adhesion strips coated with human collagen type IV, vitronectin, fibronectin, laminin or collagen type I (Chemicon International, Temecula, CA, USA) according to the manufacturer's instructions.…”

“…In pathological conditions, the triggering of CD99 results in the induction of cell-cell adhesion and apoptosis of Ewing's sarcoma cells (Sohn et al, 1998;Scotlandi et al, 2000;Cerisano et al, 2004). Moreover, it has recently been found that CD99 downregulation is essential in osteosarcoma to express full malignancy (Manara et al, 2006).…”