Gregory E. Hannigan

Integrin-linked kinase: a cancer therapeutic target unique among its ILK.

Cancer development requires the acquisition of several capabilities that include increased replicative potential, anchorage and growth-factor independence, evasion of apoptosis, angiogenesis, invasion of surrounding tissues and metastasis. One protein that has emerged as promoting many of these phenotypes when dysregulated is integrin-linked kinase (ILK), a unique intracellular adaptor and kinase that links the cell-adhesion receptors, integrins and growth factors to the actin cytoskeleton and to a range of signalling pathways. The recent findings of increased levels of ILK in various cancers, and that inhibition of ILK expression and activity is antitumorigenic, makes ILK an attractive target for cancer therapeutics.

Expression Profiling Reveals Novel Pathways in the Transformation of Melanocytes to Melanomas

Affymetrix and spotted oligonucleotide microarrays were used to assess global differential gene expression comparing normal human melanocytes with six independent melanoma cell strains from advanced lesions. The data, validated at the protein level for selected genes, confirmed the overexpression in melanoma cells relative to normal melanocytes of several genes in the growth factor/receptor family that confer growth advantage and metastasis. In addition, novel pathways and patterns of associated expression in melanoma cells not reported before emerged, including the following: (a) activation of the NOTCH pathway; (b) increased Twist expression and altered expression of additional transcriptional regulators implicated in embryonic development and epidermal/mesenchymal transition; (c) coordinated activation of cancer/testis antigens; (d) coordinated down-regulation of several immune modulation genes, in particular in the IFN pathways; (e) down-regulation of several genes implicated in membrane trafficking events; and (f) down-regulation of growth suppressors, such as the Prader-Willi gene NECDIN, whose function was confirmed by overexpression of ectopic Flag-necdin. Validation of differential expression using melanoma tissue microarrays showed that reduced ubiquitin COOH-terminal esterase L1 in primary melanoma is associated with worse outcome and that increased expression of the basic helix-loop-helix protein Twist is associated with worse outcome. Some differentially expressed genes reside on chromosomal regions displaying common loss or gain in melanomas or are known to be regulated by CpG promoter methylation. These results provide a comprehensive view of changes in advanced melanoma relative to normal melanocytes and reveal new targets that can be used in assessing prognosis, staging, and therapy of melanoma patients.

Integrin cytoplasmic interactions and bidirectional transmembrane signalling.

Integrins are heterodimeric integral plasma membrane proteins containing extracellular, transmembrane, and cytoplasmic domains. These highly versatile receptors mediate not only cell adhesion and migration, but also the bidirectional transfer of information across the plasma membrane. The cytoplasmic domains of integrins are required for the transduction of this bidirectional information, and have recently been shown to participate in direct interactions with some novel cytoplasmic proteins, such as an ankyrin repeat containing serine/threonine protein kinase (integrin-linked kinase) and beta3 endonexin. New evidence also suggests that, via interactions with focal adhesion kinase, the integrin cytoplasmic domains can coordinate actin cytoskeletal organization and responses to growth factors. The elucidation of the signal transduction pathways activated by integrins is an intense area of investigation that has shown that integrins have some unique properties as signal transducing receptors.

Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling

Interaction of cells with the extracellular matrix (ECM) results in the regulation of cell growth, differentiation and migration by coordinated signal transduction through integrins and growth-factor receptors. Integrins achieve signalling by interacting with intracellular effectors that couple integrins and growth-factor receptors to downstream components. One well-studied effector is focal-adhesion kinase (FAK), but recently another protein kinase, integrin-linked kinase (ILK), has been identified as a receptor-proximal effector of integrin and growth-factor signalling. ILK appears to interact with and be influenced by a number of different signalling pathways, and this provides new routes for integrin-mediated signalling. This article discusses ILK structure and function and recent genetic and biochemical evidence about the role of ILK in signal transduction.

Integrin-Linked Kinase at the Heart of Cardiac Contractility, Repair, and Disease

Recent advances in cardiac physiology identify the integrin-linked kinase (ILK) as an essential molecule regulating cardiac growth, contractility, and repair. A key transducer of biochemical signals initiated at the plasma membrane by cell–matrix interactions, ILK now emerges as a crucial player in mechanotransduction by integrins. Animal models have been particularly instructive in dissecting the cardiac functions of ILK and its associated proteins, such as parvins and PINCH, and have clearly established ILK as a major contributor to cardiac health. ILK gene knockouts in mice, flies, and worms result in early embryonic lethality because of cell adhesion defects and cytoskeletal disorganization. Although widely distributed in mammalian tissues, ILK expression is highest in the heart, and cardiac-specific ablation of ILK causes cardiomyopathy and sudden death in mice. ILK protein complexes are found in the sarcomere, which is the basic contractile unit of myocytes. A natural inactivating mutation in the kinase domain of ILK disrupts ILK protein interactions in the sarcomere, causing a contractile defect in the zebrafish heart. The relatively subtle phenotype of mutant ILK hearts, compared with ILK-ablated hearts, suggests multiple cardiac ILK functions. Cardiac-specific expression of ILK in transgenic mice induces a hypertrophic program, pointing to ILK as a proximal regulator of multiple hypertrophic signal transduction pathways. ILK protein interactions may also be important in mediating postinfarct cell migration and myocardial repair.

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin‐linked kinase, ILK1

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two‐hybrid screen baited with integrin‐linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co‐precipitation and two‐hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM‐ and growth factor‐stimulated ILK1 activity, but did not inhibit Raf‐1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 β (GSK3β) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP‐mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin‐like growth factor‐1‐stimulated GSK3β phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK‐mediated GSK3β signalling. Consistent with this, active, but not H154D mutant or the related PP2Cα, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3β phosphorylation.

Interferon-induced binding of nuclear factors to promoter elements of the 2-5A synthetase gene.

Fragments of the 5′‐flanking sequence of a human 2‐5A synthetase gene were assayed for their ability to respond to interferon‐alpha (IFN). Transient transfection assays in monkey cells demonstrated that the 5′ boundary of the sequence required for IFN‐regulated transcription is, at most, 155 nucleotides upstream from the presumed translational initiation codon. The 3′ boundary of this sequence lies within a region of multiple transcription start sites preceded by no obvious TATA box. Binding assays, using a 40‐bp probe derived from this IFN‐responsive sequence, demonstrated the presence of three IFN‐modulated, DNA‐factor band shifts using nuclear extracts prepared from human and monkey cells. The induction of these complexes in human cells by IFN occurs with kinetics which closely parallel those previously observed for the transcriptional activation of the 2‐5A synthetase gene by IFN. In vivo competition assays showed that the same 40‐bp region which bound IFN‐modulated factors could decrease the IFN‐induced activity of a co‐transfected 2‐5A synthetase promoter; this fragment, regardless of its orientation, could confer IFN‐inducibility on a heterologous promoter.

Integrin-linked kinase: Not so 'pseudo' after all

Integrin-linked kinase (ILK) is a highly evolutionarily conserved intracellular protein that was originally identified as an integrin-interacting protein, and extensive genetic and biochemical studies have shown that ILK expression is vital during both embryonic development and tissue homeostasis. At the cellular and tissue levels, ILK regulates signaling pathways for cell adhesion-mediated cell survival (anoikis), apoptosis, proliferation and mitosis, migration, invasion, and vascularization and tumor angiogenesis. ILK also has central roles in cardiac and smooth-muscle contractility, and ILK dysregulation causes cardiomyopathies in humans. ILK protein levels are increased in several human cancers and often the expression level predicts poor patient outcome. Abundant evidence has accumulated suggesting that, of the diverse functions of ILK, some may require kinase activity whereas others depend on protein–protein interactions and are, therefore, independent of kinase activity. However, the past several years have seen an ongoing debate about whether ILK indeed functions as a protein serine/threonine kinase. This debate centers on the atypical protein kinase domain of ILK, which lacks some amino-acid residues thought to be essential for phosphotransferase activity. However, similar deficiencies are present in the catalytic domains of other kinases now known to possess protein kinase activity. Numerous studies have shown that ILK phosphorylates peptide substrates in vitro, corresponding to ILK-mediated phosphorylations in intact cells, and a recent report characterizing in vitro phosphotransferase activity of highly purified, full-length ILK, accompanied by detailed enzyme kinetic analyses, shows that, at least in vitro, ILK is a bona fide protein kinase. However, several genetic studies suggest that, not all biological functions of ILK require kinase activity, and that it can function as an adaptor/scaffold protein. Here, we review evidence for and against ILK being an active kinase, and provide a framework for strategies to further analyze the kinase and adaptor functions of ILK in different cellular contexts.

Hedgehog Overexpression Is Associated with Stromal Interactions and Predicts for Poor Outcome in Breast Cancer

Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial-stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers.

ILKAP regulates ILK signaling and inhibits anchorage-independent growth.

ILKAP is a protein phosphatase 2C that selectively associates with integrin linked kinase, ILK, to modulate cell adhesion and growth factor signaling. We investigated the role of endogenous cellular ILKAP in antagonizing ILK signaling of two key targets, PKB and GSK3β. Silencing of endogenous ILKAP by short interfering RNA (siRNA) stimulated GSK3β phosphorylation at S9, with no effect on PKB S473 phosphorylation. In LNCaP prostate carcinoma cells, transient or stable expression of ILKAP suppressed ILK immune complex kinase activity, demonstrating an interaction between ILKAP and ILK. Consistent with the silencing data, ILKAP inhibition of ILK selectively inhibited S9 phosphorylation of GSK3β without affecting S473 phosphorylation of PKB. The ILKAP-mediated inhibition of S9 phosphorylation was rescued by overexpression of ILK, but not by a dominant-negative ILK mutant. The expression level of cyclin D1, a target of ILK-GSK3β signaling, was inversely correlated with ILKAP protein levels, suggesting that antagonism of ILK modulates cell cycle progression. ILKAP expression increased the proportion of LNCaP cells in G1, relative to vector control cells, and siRNA suppression of ILKAP increased entry of cells into the S phase, consistent with ILK antagonism. Anchorage-independent growth of LNCaP cells was inhibited by ILKAP, suggesting a critical role in the suppression of cellular transformation. Taken together, our results indicate that endogenous ILKAP activity inhibits the ILK-GSK3β signaling axis, and suggest that ILKAP activity plays an important role in inhibiting oncogenic transformation.