The emerging paradigm of ''oncogene addiction'' has been called an Achilles' heel of cancer that can be exploited therapeutically. Here, we show that integrin-linked kinase (ILK), which is either activated or overexpressed in many types of cancers, is a critical regulator of breast cancer cell survival through the protein kinase B (PKB)/Akt pathway but is largely dispensable for the survival of normal breast epithelial cells and mesenchymal cells. We show that inhibition of ILK activity with a pharmacologic ILK inhibitor, QLT-0267, results in the inhibition of PKB/Akt Ser 473 phosphorylation, stimulation of apoptosis, and a decrease in mammalian target of rapamycin (mTOR) expression in human breast cancer cells. In contrast, QLT-0267 treatment has no effect on PKB/Akt Ser 473 phosphorylation or apoptosis in normal human breast epithelial, mouse fibroblast, or vascular smooth muscle cells. The inhibition of PKB/Akt Ser 473 phosphorylation by QLT-0267 in breast cancer cells was rescued by a kinase-active ILK mutant but not by a kinasedead ILK mutant. Furthermore, a dominant-negative ILK mutant increased apoptosis in the MDA-MB-231 breast cancer cell line but not in normal human breast epithelial cells. The inhibitor was active against ILK isolated from all cell types but did not have any effect on cell attachment and spreading. Our data point to an ''ILK addiction'' of breast cancer cells whereby they become dependent on ILK for cell survival through the mTOR-PKB/Akt signaling pathway and show that ILK is a promising target for the treatment of breast cancer.

The annexins are a family of more than 160 unique annexin proteins that are present in more than 65 different species ranging from fungi and protists to plants and higher vertebrates (1). ANXA2

Annexin II heterotetramer (AIIt) is a multifunctional Ca 2؉ -binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains the binding sites for anionic phospholipids, heparin, and F-actin, whereas the p11 subunit provides a regulatory function. The F-actin-binding site is presently unknown. In the present study we have utilized site-directed mutagenesis to create annexin II mutants with truncations in the C terminus of the molecule. Interestingly, a mutant annexin II lacking its C-terminal 16, 13, or 9 amino acids was unable to bind to F-actin but still retained its ability to interact with both anionic phospholipids and heparin. Recombinant AIIt, composed of wild-type p11 subunits and the mutant annexin II subunits, was also unable to bundle F-actin. This loss of F-actin bundling activity was directly attributable to the inability of mutant AIIt to bind F-actin. These results establish for the first time that the annexin II Cterminal amino acid residues, LLYLCGGDD, participate in F-actin binding.

Cell spreading and migration are regulated in a Rho family GTPase-dependent manner by growth factors and integrin-mediated cell-extracellular matrix (ECM) interactions. The molecular mechanisms involved in the ECM-and growth factor-mediated activation of these small GTPases remain unclear. In the present study, we demonstrate that integrin-linked kinase (ILK), which is a focal adhesion protein activated by both ECM and growth factors, is required for the activation of Rac and Cdc42 in epithelial cells. Ectopic expression of active ILK in mammary epithelial cells induces dramatic reorganization of the actin cytoskeleton and promotes rapid cell spreading on fibronectin. These effects are associated with constitutive activation of both Rac and Cdc42, but not Rho. The use of ILK siRNA or small molecule inhibitors to inhibit ILK expression and kinase activity, respectively, results in diminished cell spreading and actin cytoskeleton reorganization, concomitant with a reduction in Rac and Cdc42 activation. Studies into the mechanism of ILK-mediated Rac activation suggest an important role for the ILK-b-parvin interaction and the activity of the Rac/Cdc42-specific guanine nucleotide exchange factor a-PIX downstream of ILK. Taken together, these data demonstrate an essential role of ILK kinase activity in Rac-and Cdc42-mediated actin cytoskeleton reorganization in epithelial cells, further solidifying a role for ILK in the regulation of cancer cell motility and invasiveness.

The annexin A2-S100A10 heterotetramer (AIIt) is a multifunctional Ca 2؉ -dependent, phospholipid-binding, and F-actin-binding phosphoprotein composed of two annexin A2 subunits and two S100A10 subunits. It was reported previously that oxidative stress from exogenous hydrogen peroxide or generated in response to tumor necrosis factor-␣ results in the glutathionylation of Cys 8 of annexin A2. In this study, we demonstrate that AIIt is an oxidatively labile protein whose level of activity is regulated by the redox status of its sulfhydryl groups. Oxidation of AIIt by diamide resulted in a timeand concentration-dependent loss of the ability of AIIt to interact with phospholipid liposomes and F-actin. The inhibitory effect of diamide on the activity of AIIt was partially reversed by dithiothreitol. In addition, incubation of AIIt with diamide and GSH resulted in the glutathionylation of AIIt in vitro. Mass spectrometry established the incorporation of 2 mol of GSH/mol of annexin A2 subunit at Cys 8 and Cys 132 . Glutathionylation potentiated the inhibitory effects of diamide on the activity of AIIt. Furthermore, AIIt could be deglutathionylated by glutaredoxin (thiol transferase). Thus, we show for the first time that AIIt can undergo functional reactivation by glutaredoxin, therefore establishing that AIIt is regulated by reversible glutathionylation.The molecular mechanisms by which the cell alleviates oxidative stress and achieves redox homeostasis are still a matter of considerable debate. However, the modulation of the thiol disulfide status of critical cysteine residues on proteins is being recognized as a critical mechanism of oxidative signal transduction as well as a cellular response to protect key regulatory molecules from oxidative insult (1-3). Recent evidence suggests that the reversible covalent modification of cysteine residues by the tripeptide glutathione (␥-Glu-Cys-Gly) plays a significant role in the antioxidant network of the cells and is involved in regulating individual aspects of cellular function (3, 4). Although proteins can bind cysteine, GSH, and homocysteine to generate mixed disulfides, GSH is the dominant ligand, as it occurs in the cell at concentrations between 1 and 10 mM (5, 6). S-Glutathionylation has been shown to alter the function of a number of discrete proteins under oxidant stress (7,8). Furthermore, the formation of a mixed disulfide with glutathione precludes the irreversible oxidation of the cysteine thiol to a sulfinic or sulfonic acid and enables reactivation of the protein by cellular thioreductases.Annexins compose a large multigene family of water-soluble proteins that can bind to negatively charged phospholipids and cellular membranes in a Ca 2ϩ -dependent fashion (9 -11). The annexin family is structurally characterized by two domains: a highly conserved ␣-helical protein core consisting of four 70-amino acid repeats (eight repeats in the case of annexin VI) and a variable N-terminal segment (12). Annexin A2 is unique among the annexins, for its N-terminal tail poss...