1: Cancer Res. 2004 Aug 15;64(16):5683-92. Overexpression of hyperactive integrin-linked kinase leads to increased cellular radiosensitivity. Cordes N. Bundeswehr Institute of Radiobiology, Munich, Germany. cordes@radiation-biology.de Integrin-linked kinase (ILK), bound to the cytoplasmic tails of integrin beta1, beta2, and beta3, is thought to signal through AKT and glycogen synthase kinase-3beta (GSK-3beta) for survival and proliferation regulation. To determine the role of ILK in the cellular radiation response, stably transfected A549 lung cancer cells overexpressing either wild-type (ILK-wk) or hyperactive ILK (ILK-hk) were studied for survival, signaling, proliferation, and examined in immunofluorescence and adhesion assays. Strong radiosensitization was observed in ILK-hk in contrast to ILK-wk mutants and empty vector controls. ILK small interfering RNA transfections showed radioresistance similar to irradiation on fibronectin. AKT, GSK-3beta-cyclin D1, mitogen-activated protein kinase kinase 1/2-mitogen-activated protein kinase, and c-Jun NH2-terminal kinase signaling was dysregulated in irradiated ILK-hk mutants. Immunofluorescence stainings of ILK-hk cells indicated disturbed ILK and paxillin membrane localization with concomitant decrease in focal adhesions. Profound ILK-hk-dependent changes in morphology were characterized by spindle-like cell shape, cell size reduction, increased cell protrusions, strong formation of membranous f-actin rings, and significantly reduced adhesion to matrix proteins. Additionally, ILK-wk and ILK-hk overexpression impaired beta1-integrin clustering and protein Tyr-phosphorylation. Taken together, the data provide evidence that ILK signaling modulates the cellular radiation response involving diverse signaling pathways and through changes in f-actin-based processes such as focal adhesion formation, cell adhesion, and spreading. Identification of ILK and its signaling partners as potential targets for tumor radiosensitization might promote innovative anticancer strategies by providing insight into the mechanism of cell adhesion-mediated radioresistance, oncogenic transformation, and tumor growth and spread. PMID: 15313908 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Biol Chem. 2004 Oct 15;279(42):43893-9. Epub 2004 Aug 6. Integrin-linked kinase regulates the nuclear entry of the c-Jun coactivator alpha-NAC and its coactivation potency. Quelo I, Gauthier C, Hannigan GE, Dedhar S, St-Arnaud R. Genetics Unit, Shriners Hospital for Children Montreal, Quebec H3G 1A6, Canada. Overexpression of the integrin-linked kinase (ILK) was shown to increase c-Jun-dependent transcription. We now show that this effect of ILK involves the c-Jun transcriptional coactivator, nascent polypeptide-associated complex and coactivator alpha (alpha-NAC). ILK phosphorylated alpha-NAC on residue Ser-43 upon adhesion of cells to fibronectin. Co-expression of constitutively active ILK with alpha-NAC led to the nuclear accumulation of the coactivator. Conversely, alpha-NAC remained in the cytoplasm of cells transfected with a dominant-negative ILK mutant, and a mutated alpha-NAC at phosphoacceptor position Ser-43 (S43A) also localized outside of the nucleus. The S43A alpha-NAC mutant could not potentiate the effect of ILK on c-Jun-dependent transcription. We conclude that ILK-dependent phosphorylation of alpha-NAC induced the nuclear accumulation of the coactivator and that phosphorylation of alpha-NAC by ILK is required for the potentiation of c-Jun-mediated responses by the kinase. The results represent one of the rare examples of a transcriptional coactivator shuttling between the cytosol and the nucleus. PMID: 15299025 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Clin Cancer Res. 2003 Mar;9(3):1155-60. Increased resistance of tumor cells to hyperthermia mediated by integrin-linked kinase. Zhang X, Li Y, Huang Q, Wang H, Yan B, Dewhirst MW, Li CY. Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA. PURPOSE: Integrin-linked kinase (ILK) is a serine-threonine kinase associated with anchorage-independent growth and tumorigenic transformation. Previous studies indicate that overexpression of ILK is common among several types of tumors, and it is involved in the regulation of tumor cell survival under stress. In this study, we examined the effects of ILK expression on tumor cellular response to hyperthermia. EXPERIMENTAL DESIGN: We used an adenovirus-mediated approach to overexpress the ILK gene in a prostate cancer cell line and examine its effects on heat stress-induced cell death. Clonogenic survival, as well as apoptosis, was evaluated in cells that overexpress ILK. In addition, the ability to form tumors in vivo was examined in syngeneic hosts. Finally, potential molecular mechanisms of ILK-mediated resistance to heat were examined by determining the status of a variety of signal transduction pathways. RESULTS: ILK overexpression made tumor cells significantly more resistant to the cell-killing effects of hyperthermia. This was correlated at the molecular level with the down-regulation of hyperthermia-induced activation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase, p38 mitogen-activated protein kinase activities, and caspase 9. The overexpression of ILK was also shown to induce a more rapid tumor growth in a murine prostate cancer cell line CONCLUSION: ILK plays an important role in tumor growth and tumor response to hyperthermia treatment. PMID: 12631621 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Biochem Pharmacol. 2000 Oct 15;60(8):1115-9. Integrin-linked kinase (ILK): a "hot" therapeutic target. Yoganathan TN, Costello P, Chen X, Jabali M, Yan J, Leung D, Zhang Z, Yee A, Dedhar S, Sanghera J. Kinetek Pharmaceuticals Inc., Vancouver, BC V6P6P2, Canada. nathan@kinetekpharm.com Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy. Publication Types: Review Review, Tutorial PMID: 11007949 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Mol Cell Biol. 1999 Nov;19(11):7420-7. Cell-extracellular matrix interactions stimulate the AP-1 transcription factor in an integrin-linked kinase- and glycogen synthase kinase 3-dependent manner. Troussard AA, Tan C, Yoganathan TN, Dedhar S. BC Cancer Agency and Vancouver Hospital, Jack Bell Research Centre, Vancouver, British Columbia V6H 3Z6. Integrin-mediated interactions of cells with components of the extracellular matrix regulate cell survival, cell proliferation, cell differentiation, and cell migration. Some of these physiological responses are regulated via activation of transcription factors such as activator protein 1 (AP-1). Integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase whose activity is rapidly and transiently stimulated by cell-fibronectin interactions as well as by insulin stimulation. ILK activates protein kinase B and inhibits the glycogen synthase kinase 3 (GSK-3) activity in a phosphatidylinositol-3-kinase (PI 3-kinase)-dependent manner. We now show that cell adhesion to fibronectin results in a rapid and transient stimulation of AP-1 activity. At the same time, the kinase activity of ILK is stimulated whereas that of GSK-3 is inhibited. This fibronectin-dependent activation of AP-1 activity is inhibited in a dose-dependent manner if the cells are transfected with wild-type GSK-3, and also by inhibitors of PI 3-kinase. Stable or transient overexpression of ILK results in a stimulation of AP-1 activity which is inhibited by cotransfection with wild-type GSK-3 and kinase-deficient ILK. Transient transfection of ILK in HEK-293 cells stimulates complex formation between an AP-1 consensus oligonucleotide and nuclear proteins containing c-jun. The formation of this complex is inhibited by cotransfection with active GSK-3 or kinase-deficient ILK, suggesting that ILK may regulate AP-1 activation by inhibiting GSK-3, which has previously been shown to be a negative regulator of AP-1. In the presence of serum, ILK has no effect on the phosphorylation of Ser-73 in the N-terminal transactivation domain of c-jun. These results demonstrate a novel signaling pathway for the adhesion-mediated stimulation of AP-1 transcriptional activity involving ILK and GSK-3 and the subsequent regulation of the c-jun-DNA interaction. PMID: 10523630 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------