Focal adhesion kinase (FAK) is up-regulated in a variety of cancers, including breast cancer, in association with poor disease prognosis. In the present study, we examined the role of FAK in the control of anticancer drug-induced apoptosis of mammary adenocarcinoma MTLn3 cells. Doxorubicin caused the formation of well defined focal adhesions and stress fibers early after treatment, which was later followed by their loss in association with the onset of apoptosis. Phosphorylation of FAK on tyrosine 397 decreased only during the onset of doxorubicin-induced apoptosis in a Bcl-2 and caspase-independent manner. Doxorubicin also caused an early activation of protein kinase B (PKB). Expression of the dominant-negative acting focal adhesion kinase-related nonkinase (FRNK) sensitized MTLn3 cells to apoptosis caused by doxorubicin. FRNK inhibited the doxorubicin-induced activation of PKB. In addition, inhibition of phosphatidylinositide-3 (PI-3) kinase with wortmannin inhibited the activation of PKB by doxorubicin. Both wortmannin and transient overexpression of the dual lipid/protein phosphatase and tensin homolog deleted on chromosome 10 enhanced doxorubicin-induced cell death. Altogether, these data fit with a model wherein FAK is involved in the doxorubicin-induced activation of the PI-3 kinase/PKB signaling route, thereby suppressing the onset of apoptosis caused by doxorubicin.Adhesion to the ECM is essential for normal functioning of epithelial cells and provides survival signaling cues (Giancotti and Ruoslahti, 1999;Frisch and Screaton, 2001). In normal cells, detachment generally results in apoptosis, also termed anoikis (Frisch and Francis, 1994;Frisch and Ruoslahti, 1997). However, tumor cells are often resistant to anoikis. This resistance is linked to constitutive activation of survival signaling pathways by oncogenic transformation that otherwise depend on cell adhesion (Streuli and Gilmore, 1999;McFall et al., 2001). These survival signals may confer resistance not only to anoikis but also to apoptosis induced by anticancer drugs due to an overlap in signaling pathways that control cell death. Indeed, adhesion to ECM proteins via the ␤1 integrin subunit protected small cell lung cancer cells against doxorubicin-and etoposide-induced apoptosis and protected breast cancer cells against paclitaxel-induced apoptosis (Sethi et al., 1999;Aoudjit and Vuori, 2001). This dysregulation of cell-ECM interaction-mediated signaling in tumor cells followed by the suppression of or resistance to apoptosis may well contribute to metastasis formation. Likewise, metastatic tumor cells may already be (partly) resistant to anticancer agent-induced apoptosis. Therefore, it is important to know how focal adhesion-dependent signaling is linked to protection against anticancer agent-induced apoptosis. ABBREVIATIONS: ECM, extracellular matrix; AV, Annexin V; CLSM, confocal laser scanning microscopy; FAK, focal adhesion kinase; FRNK, focal adhesion kinase-related nonkinase; GFP, green fluorescent protein; eGFP, enhanced gree...