Resistance of Tumor Cells to Cytolytic T Lymphocytes Involves Rho-GTPases and Focal Adhesion Kinase Activation

Abdelali Jalil,Claudine Kieda,Meriem Hasmim,Fathia Mami-Chouaib,Jocelyne Hamelin,Bassam Janji,Soraya Abouzahr-Rifai,Jacques Bertoglio,Habib Boukerche,Salem Chouaib

doi:10.1074/jbc.m800078200

Abstract

Tumor cells evade adaptive immunity by a variety of mechanisms, including selection of variants that are resistant to specific cytotoxic T lymphocyte (CTL) pressure. Recently, we have reported that the reorganization of the actin cytoskeleton can be used by tumor cells as a strategy to promote their resistance to CTL-mediated lysis. In this study, we further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways. Our data indicate that although the resistant cells do not display an increased migratory potential, an alteration of adhesion to the extracellular matrix was observed. When Rho-GTPases were activated in cells by the bacterial CNF1 (cytotoxic necrotizing factor 1), striking changes in the cell morphology, including actin cytoskeleton, focal adhesions, and membrane extensions, were observed. More importantly, such activation also resulted in a significant attenuation of resistance to CTL-induced cell death. Furthermore, we demonstrate that FAK signaling pathways were constitutively defective in the resistant cells. Silencing of FAK in the sensitive target cells resulted in the inhibition of immune synapse formation with specific CTLs and their subsequent lysis. Expression of the FAK mutant (Y397F) resulted in an inhibition of IGR-Heu cell adhesion and of their susceptibility to specific lysis. These results suggest that FAK activation plays a role in the control of tumor cell susceptibility to CTL-mediated lysis.

Highlights

Cytotoxic T lymphocytes (CTLs)3 are important effector cells in tumor rejection and have been described to play a crucial role in host defense against malignancies in both mouse and human [1]
We further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways
Our results suggest that besides actin content [4] that might act as a molecular switch in the control of tumor susceptibility to CTL killing, the activation of FAK may orchestrate a dynamic cross-talk between actin filaments and signaling pathways that play a key role in the control of tumor target sensitivity to CTL-induced cell death

Summary

EXPERIMENTAL PROCEDURES

Toxins—Cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli was used as a GST fusion protein and purified as described previously [14]. Activated Rho-GTPases were revealed by immunoblotting using specific anti-Rac and anti-Cdc antibodies (BD Transduction Laboratories) and an anti-RhoA antibody described previously by Lang et al [18]. Confocal Microscopy—Tumor cells alone or co-cultured with autologous CTL at a 1:2 target/effector ratio were plated on glass coverslips or poly-L-lysine slides (Sigma) (for conjugate formation study), fixed with 3% formaldehyde/PBS for 10 min, permeabilized with 0.1% Triton X-100/PBS for 5 min, followed by blocking with 1% bovine serum albumin/PBS for 20 min. Activation of Rho-GTPases Induces Morphological Changes in IGR-HeuR8-resistant Cells and Increases Their Adhesion to Type I and IV Collagens—Integrins are receptors that mediate dynamic linkage between extracellular matrix molecules and the actin cytoskeleton [21]. Z-projection Rho family in particular, because these regulatory molecules are of slices was performed using LSM Image Examiner software involved in the control of the actin cytoskeleton organization (Zeiss)

RESULTS

Findings

DISCUSSION