The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion.

Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidence proteins identified include metabolic transporters, ß1 integrins, integrin-associated protein CD98hc and matrix metalloprotease 14 (MMP14). Biochemical studies validate the association of CAIX with α2ß1 integrin, CD98hc and MMP14, and immunofluorescence microscopy demonstrates colocalization of CAIX with α2ß1 integrin and MMP14 in F-actin/cofilin-positive lamellipodia/pseudopodia, and with MMP14 to cortactin/Tks5-positive invadopodia. Modulation of CAIX expression and activity results in significant changes in cell migration, collagen degradation and invasion. Mechanistically, we demonstrate that CAIX associates with MMP14 through potential phosphorylation residues within its intracellular domain, and that CAIX enhances MMP14-mediated collagen degradation by directly contributing hydrogen ions required for MMP14 catalytic activity. These findings establish hypoxia-induced CAIX as a novel metabolic component of cellular migration and invasion structures, and provide new mechanistic insights into its role in tumor cell biology.

Induction of the intestinal stem cell signature gene SMOC-2 is required for L1-mediated colon cancer progression.

Overactivation of Wnt-ß-catenin signaling, including ß-catenin-TCF target gene expression, is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin family of cell-adhesion receptors member L1 as a ß-catenin-TCF target gene preferentially expressed at the invasive edge of human CRC tissue. L1 can confer enhanced motility and liver metastasis when expressed in CRC cells. This ability of L1-mediated metastasis is exerted by a mechanism involving ezrin and the activation of NF-κB target genes. In this study, we identified the secreted modular calcium-binding matricellular protein-2 (SMOC-2) as a gene activated by L1-ezrin-NF-κB signaling. SMOC-2 is also known as an intestinal stem cell signature gene in mice expressing Lgr5 in cells at the bottom of intestinal crypts. The induction of SMOC-2 expression in L1-expressing CRC cells was necessary for the increase in cell motility, proliferation under stress and liver metastasis conferred by L1. SMOC-2 expression induced a more mesenchymal like phenotype in CRC cells, a decrease in E-cadherin and an increase in Snail by signaling that involves integrin-linked kinase (ILK). SMOC-2 was localized at the bottom of normal human colonic crypts and at increased levels in CRC tissue with preferential expression in invasive areas of the tumor. We found an increase in Lgr5 levels in CRC cells overexpressing L1, p65 or SMOC-2, suggesting that L1-mediated CRC progression involves the acquisition of a stem cell-like phenotype, and that SMOC-2 elevation is necessary for L1-mediated induction of more aggressive/invasive CRC properties.

Role of the integrin-linked kinase (ILK)/Rictor complex in TGFß-1-induced epithelial-mesenchymal transition (EMT).

Epithelial-to-mesenchymal transition (EMT) causes fibrosis, cancer progression and metastasis. Integrin-linked kinase (ILK) is a focal adhesion adaptor and a serine/threonine protein kinase that regulates cell proliferation, survival and EMT. Elucidating the molecular mechanisms necessary for development and progression of human malignancies is critical to predict the most appropriate targets for cancer therapy. Here, we used transforming growth factor beta-1 (TGFß-1) to promote EMT and migration in mammary epithelial cells. We demonstrate a requirement of ILK activity for TGFß-1-mediated EMT in mammary epithelial cells. In addition to nuclear translocation of Snail and Slug, TGFß-1 treatment also induced expression of the mammalian target of rapamycin complex 2 component Rictor and its phosphorylation on Thr1135. Interestingly, TGFß-1 treatment also induced an interaction between ILK and Rictor. All of these TGFß-1-induced processes were significantly suppressed by inhibiting ILK activity or by disrupting the ILK/Rictor complex using small-interfering RNA-mediated knockdown. Furthermore, we identified ILK/Rictor complex formation in cancer but not in normal cell types, and this was accompanied by ILK-dependent phosphorylation of Rictor on residue Thr1135. Inhibition of ILK partially reversed the basal mesenchymal phenotype of MDA-MB-231 cells and prevented EMT in MCF10A cells after TGFß-1 treatment. These data demonstrate a requirement for ILK function in TGFß-1-induced EMT in mammary epithelial cells and identify the ILK/Rictor complex as a potential molecular target for preventing/reversing EMT.

Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche.

The sub-population of tumor cells termed 'cancer stem cells' (CSCs) possess the capability to generate tumors, undergo epithelial-mesenchymal transition (EMT) and are implicated in metastasis, making treatments to specifically target CSCs an attractive therapeutic strategy. Tumor hypoxia plays a key role in regulating EMT and cancer stem cell function. Carbonic anhydrase IX (CAIX) is a hypoxia-inducible protein that regulates cellular pH to promote cancer cell survival and invasion in hypoxic microenvironments and is a biomarker of poor prognosis for breast cancer metastasis and survival. Here, we demonstrate that inhibition of CAIX expression or activity with novel small-molecule inhibitors in breast cancer cell lines, or in primary metastatic breast cancer cells, results in the inhibition of breast CSC expansion in hypoxia. We identify the mTORC1 axis as a critical pathway downstream of CAIX in the regulation of cancer stem cell function. CAIX is also required for expression of EMT markers and regulators, as well as drivers of 'stemness', such as Notch1 and Jagged1 in isolated CSCs. In addition, treatment of mice bearing orthotopic breast tumors with CAIX-specific small-molecule inhibitors results in significant depletion of CSCs within these tumors. Furthermore, combination treatment with paclitaxel results in enhanced tumor growth delay and eradication of lung metastases. These data demonstrate that CAIX is a critical mediator of the expansion of breast CSCs in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells, making CAIX an important therapeutic target for selectively depleting breast CSCs.

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.

A critical role of integrin-linked kinase, ch-TOG and TACC3 in centrosome clustering in cancer cells.

Many cancer cells contain more than two centrosomes, which imposes a potential for multipolar mitoses, leading to cell death. To circumvent this, cancer cells develop mechanisms to cluster supernumerary centrosomes to form bipolar spindles, enabling successful mitosis. Disruption of centrosome clustering thus provides a selective means of killing supernumerary centrosome-harboring cancer cells. Although the mechanisms of centrosome clustering are poorly understood, recent genetic analyses have identified requirements for both actin and tubulin regulating proteins. In this study, we demonstrate that the integrin-linked kinase (ILK), a protein critically involved in actin and mitotic microtubule organization, is required for centrosome clustering. Inhibition of ILK expression or activity inhibits centrosome clustering in several breast and prostate cancer cell lines that have centrosome amplification. Furthermore, cancer cells with supernumerary centrosomes are significantly more sensitive to ILK inhibition than cells with two centrosomes, demonstrating that inhibiting ILK offers a selective means of targeting cancer cells. Live cell analysis shows ILK perturbation leads cancer cells to undergo multipolar anaphases, mitotic arrest and cell death in mitosis. We also show that ILK performs its centrosome clustering activity in a focal adhesion-independent, but centrosome-dependent, manner through the microtubule regulating proteins TACC3 and ch-TOG. In addition, we identify a specific TACC3 phosphorylation site that is required for centrosome clustering and demonstrate that ILK regulates this phosphorylation in an Aurora-A-dependent manner.

Suppression of Her2/neu expression through ILK inhibition is regulated by a pathway involving TWIST and YB-1.

In a previous study it was found that the therapeutic effects of QLT0267, a small molecule inhibitor of integrin-linked kinase (ILK), were influenced by Her2/neu expression. To understand how inhibition or silencing of ILK influences Her2/neu expression, Her2/neu signaling was evaluated in six Her2/neu-positive breast cancer cell lines (LCC6(Her2), MCF7(Her2), SKBR3, BT474, JIMT-1 and KPL-4). Treatment with QLT0267 engendered suppression (32-87%) of total Her2/neu protein in these cells. Suppression of Her2/neu was also observed following small interfering RNA-mediated silencing of ILK expression. Time course studies suggest that ILK inhibition or silencing caused transient decreases in P-AKT(ser473), which were not temporally related to Her2/neu downregulation. Attenuation of ILK activity or expression was, however, associated with decreases in YB-1 (Y-box binding protein-1) protein and transcript levels. YB-1 is a known transcriptional regulator of Her2/neu expression, and in this study it is demonstrated that inhibition of ILK activity using QLT0267 decreased YB-1 promoter activity by 50.6%. ILK inhibition was associated with changes in YB-1 localization, as reflected by localization of cytoplasmic YB-1 into stress granules. ILK inhibition also suppressed TWIST (a regulator of YB-1 expression) protein expression. To confirm the role of ILK on YB-1 and TWIST, cells were engineered to overexpress ILK. This was associated with a fourfold increase in the level of YB-1 in the nucleus, and a 2- and 1.5-fold increase in TWIST and Her2/neu protein levels, respectively. Taken together, these data indicate that ILK regulates the expression of Her2/neu through TWIST and YB-1, lending support to the use of ILK inhibitors in the treatment of aggressive Her2/neu-positive tumors.

Integrin-linked kinase has a critical role in ErbB2 mammary tumor progression: implications for human breast cancer.

Elevated expression of the integrin-linked kinase (ILK) has been observed in a variety of cancers and has been further correlated with poor clinical outcome. Here, we show that mammary epithelial disruption of ILK results in a profound block in mammary tumor induction. Consistent with these observations, inhibition of ILK function in ErbB2-expressing cells with small molecule inhibitor or RNA interference resulted in profound block in their in vitro invasive properties due to the induction of apoptotic cell death. The rare ILK-deficient tumors that eventually arose overcame this block in tumor induction by an upregulation of ErB3 phosphorylation. These observations provide direct evidence that ILK has a critical role in the initiation phase of ErbB2 tumor induction.

Integrin-linked kinase regulates cell proliferation and tumour growth in murine colitis-associated carcinogenesis.

BACKGROUND: Integrins are transmembrane cell surface receptors that mediate cell-cell and cell-matrix contacts. Integrin-linked kinase (ILK) is the binding partner of beta1 and beta3 integrins, and has been ascribed essential roles in development, angiogenesis and tumourigenesis. However, in vivo evidence for the latter is currently lacking. AIM: The hypothesis that epithelial cell-specific deletion of ILK would impact on murine tumourigenesis was tested using a colitis-associated cancer model. METHODS: To create intestinal epithelial cell ILK knockout animals, Fabp/Cre mice (Cre recombinase expressed under the control of a modified Fabp promoter) were used, and they were mated with mice carrying a loxP-flanked (floxed) ILK gene (ILK(flox/flox)). RESULTS: ILK intestinal knockout mice exhibited a reduction in the size of the caecum, and reduced crypt height in the colon. Immunohistochemical analysis confirmed that there was diminished ILK expression, and bromodeoxyuridine (BrdU) staining was significantly reduced in the knockout animals as compared with the wild-type animals in both the caecum and colon (p<0.001 for both). Following azoxymethane and dextran sodium sulfate (DSS) treatment, fewer total tumours were observed in the ILK knockout animals, which were mosaic with respect to ILK expression. Cyclin D1, Snail, fibronectin and matrix metalloproteinase 9 (MMP9) were all reduced, and active caspase 3 increased, in tumours from ILK knockout mice, as compared with wild-type mice, on immunohistochemical analysis. Using small interfering RNA (siRNA) to knock down ILK in colonic cancer cell lines, it was confirmed that it is capable of regulating cyclin D1, Snail, MMP9 and fibronectin transcription. CONCLUSIONS: From these findings, it is concluded that ILK plays an important role in intestinal epithelial cell proliferation, and that it influences the development of colitis-associated cancer, through modulation of cyclin D1, the extracellular matrix and MMP9.

Development and assessment of conventional and targeted drug combinations for use in the treatment of aggressive breast cancers.

Combination chemotherapy has been at the forefront of cancer treatment for over 40 years. However, the rationale for selecting drug combinations and the process used to demonstrate clinical effectiveness has primarily followed trial and error methodology. Typically, the selection and assessment of combined drug therapies has been based on the effectiveness of each agent as monotherapy in treating the neoplasm and avoiding overlapping toxicities, followed by clinical trials to establish dose scheduling, toxicity, and efficacy. Unfortunately, this scheme is inefficient in terms of the time required to complete and revise these clinical trials based on the outcome to optimize the drug combination. A more rational approach for the development of combination oncology products should consider (i) in vitro assays for assessing therapeutic effects of drug combinations (antagonistic, additive or synergistic interactions) when added simultaneously; (ii) methods for measuring these interactions in vivo; (iii) the importance of understanding pharmacokinetic and biodistribution parameters when using drug combinations; (iv) the need to assess pathways known to contribute to cancer cell survival as well as metastasis; and (iv) the need to assess the fate of different cell populations (cancer and stroma) contributing to the development of cancer. Therefore, the goal of this article is to provide a road map for the preclinical development of drug combination products that will have improved therapeutic activity and a high likelihood of providing beneficial therapeutic outcomes in patients with aggressive cancers with a specific focus on patients with breast cancer.

Modulation of Wnt3a-mediated nuclear beta-catenin accumulation and activation by integrin-linked kinase in mammalian cells.

The Wnt gene family encodes secreted signaling molecules that play important roles in tumorgenesis and embryogenesis. The canonical Wnt signaling pathway regulates target gene expression via the stabilization and nuclear translocation of the cytoplasmic pool of beta-catenin. The activation of integrin-linked kinase (ILK) is also known to regulate the stabilization and subsequent nuclear translocation of beta-catenin in several epithelial cell models. We now report that molecular and pharmacological inhibition of ILK activity in mammalian cells directly modulates Wnt signaling by suppressing the stabilization and nuclear translocation of beta-catenin, as well as beta-catenin/Lef-mediated transcription. Inhibition of ILK activity, but not phosphatidylinositol-3 kinase (PI3K) or MEK activities suppresses nuclear beta-catenin stabilization in cells stably expressing Wnt3a as well as in cells exposed to either Wnt3a conditioned media or purified Wnt3a. Furthermore, ILK inhibition reverses the Wnt3a-induced suppression of beta-catenin phosphorylation that accompanies beta-catenin stabilization. In addition, we show that ILK can be identified in a complex with Wnt pathway components such as adenomatous polyposis coli and GSK-3. Upon treatment of L cells with Wnt3a-CM, glycogen synthase kinase-3 (GSK-3beta) becomes highly phosphorylated on Ser 9, which is completely abolished upon inhibition of ILK activity. However, acute exposure of L cells to purified Wnt3a does not result in the stimulation of GSK-3beta Ser 9 phosphorylation, despite beta-catenin stabilization. Together our data demonstrate that ILK activity can modulate acute Wnt3a mediated beta-catenin phosphorylation, stabilization and nuclear activation in a PI3K-independent manner, as well as the more prolonged PI3K-dependent secondary effects of Wnt signaling on GSK-3 phosphorylation. Finally, we suggest that a novel small molecule inhibitor of ILK, QLT-0267, may be a useful tool in the regulation of pathological Wnt signaling.

PTEN and GSK3beta: key regulators of progression to androgen-independent prostate cancer.

Prostate cancer (PrCa) is characterized by progression from an androgen-dependent phenotype to one that is inevitably androgen independent (AI) and lethal. Recent evidence strongly suggests that the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and androgen receptor (AR) signalling pathways provide prostatic epithelium with the necessary signalling events to escape the apoptotic response associated with androgen withdrawal therapy. Silencing of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and glycogen synthase kinase beta (GSK3beta) are frequently associated with advanced PrCa systems and likely serve critical roles in promoting AR and PI3K/Akt gain-of-function. That PTEN negatively regulates AR and is sufficient to promote metastatic PrCa in murine models strongly implies its role as a gatekeeper of progressive PrCa. In human PrCa, PTEN loss is correlated with substantial increases in Akt(Ser473) and integrin-linked kinase expression, both of which promote Ser(9) phospho-inhibition of GSK3beta and inactivation of apoptotic factors. Sufficient evidence also suggests that GSK3beta is not only a critical regulator of proproliferative signalling but also a promiscuous one as PI3K/Akt pools of GSK3beta are, at least in part, functionally interchangeable with those of the Wnt/beta-catenin pathway. Thus, GSK3beta may serve not only as a mediator of PI3K/Akt activation but may also regulate the potent transactivation and proproliferative effects that Wnt3a and beta-catenin confer upon AR. These data suggest that prostate-specific activation of GSK3beta may serve as a viable pharmacological option. Thus, in this review, we emphasize that temporal changes in GSK3beta and PTEN expression during progression to AI PrCa are important factors when considering the potential for therapies targeting the oncogenic contributions of PI3K/Akt and AR signalling pathways.

Characterisation of integrin-linked kinase signalling in sporadic human colon cancer.

The putative oncogene, integrin-linked kinase (ILK) is a protein serine/threonine kinase that has been reported to regulate a number of biological properties including anchorage-independent cell cycle progression, tumour cell invasion and apoptosis. Overexpression of ILK has been documented in a wide variety of human malignancies including Ewing's sarcoma (ES), primitive neural ectodermal tumours (PNETs) and prostate tumours (PT). We recently reported that ILK signalling was also dysregulated in patients with the genetic condition familial adenomatous polyposis (FAP), a precursor to colon cancer. In this study, we extended our previous work by investigating the ILK-signalling pathway in sporadic human colon cancer and representative lymph node metastases. The data indicate that the ILK protein is significantly hyperexpressed in malignant acini in relation to normal crypts. Moreover, overexpression of ILK not only coincided with increased MBP phosphotransferase activity but as well with effects on downstream targets like GSK3beta. Based upon the presented data, we propose that ILK signalling is dysregulated early during the development of human colon cancer, and that selective inhibition of this molecule alone or in combination with the standard therapeutic modality might be a more effective means of treating colon cancer.

Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties.

Integrin-linked kinase (ILK) is an ankyrin repeat-containing Ser/Thr kinase that interacts with the cytoplasmic domains of beta(1) and beta(3) integrins. ILK is widely expressed in tissues throughout the body, and, as might be expected, appears to mediate a diversity of functions relating to its role in coupling integrins and growth factor receptors to downstream signaling pathways. Through its downstream targets protein kinase B/Akt and glycogen synthase kinase-3beta, ILK appears to be involved in several oncogenesis-related events, including suppression of apoptosis and promotion of cell survival, as well as cell migration and invasion. Over-expression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression. Inoculation of nude mice with ILK over-expressing cells leads to tumor formation. Furthermore, increased ILK expression and activity have been correlated with malignancy in several human tumor types, including breast, prostate, brain, and colon carcinomas. Based on these findings, ILK represents an excellent therapeutic target for the prevention of tumor progression. Here, we provide an overview of the physical and biochemical properties of ILK, and present data describing the impact of small-molecule ILK inhibitors on several ILK-mediated cellular functions.

Mammary epithelial-specific expression of the integrin-linked kinase (ILK) results in the induction of mammary gland hyperplasias and tumors in transgenic mice.

The integrin linked kinase (ILK) is a cytoplasmic effector of integrin receptors, involved in the regulation of integrin binding properties as well as the activation of cell survival and proliferative pathways, including those involving MAP kinase, PKB/Akt and GSK-3beta. Overexpression of ILK in cultured intestinal and mammary epithelial cells has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth, invasiveness, suppression of anoikis and tumorigenicity in nude mice. In order to determine if ILK overexpression can result in the formation of mammary tumors in vivo, we generated transgenic mice expressing ILK in the mammary epithelium, under the transcriptional control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). By the age of 6 months, female MMTV/ILK mice developed a hyperplastic mammary phenotype, which was accompanied by the constitutive phosphorylation of PKB/Akt, GSK-3beta and MAP kinase. Focal mammary tumors subsequently appeared in 34% of the animals at an average age of 18 months. Given the focal nature and long latency of the tumors, however, additional genetic events are likely required for tumor induction in the MMTV/ILK mice. These results provide the first direct demonstration of a potential oncogenic role for ILK, which is upregulated in human tumors and tumor cell lines.

Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.

How intracellular cytoskeletal and signaling proteins connect and communicate with the extracellular matrix (ECM) is a fundamental question in cell biology. Recent biochemical, cell biological, and genetic studies have revealed important roles of cytoplasmic integrin-linked kinase (ILK) and its interactive proteins in these processes. Cell adhesion to ECM is an important process that controls cell shape change, migration, proliferation, survival, and differentiation. Upon adhesion to ECM, integrins and a selective group of cytoskeletal and signaling proteins are recruited to cell matrix contact sites where they link the actin cytoskeleton to the ECM and mediate signal transduction between the intracellular and extracellular compartments. In this review, we discuss the molecular activities and cellular functions of ILK, a protein that is emerging as a key component of the cell-ECM adhesion structures.

Dysregulation of integrin-linked kinase (ILK) signaling in colonic polyposis.

Mutation of the adenomatous polyposis coli (APC) gene and the subsequent dysregulation of beta-catenin are well-documented abnormalities in familial adenomatous polyposis (FAP), as well as sporadic polyposis. Intriguingly, overexpression of the integrin-linked kinase (ILK) has been shown to modulate beta-catenin subcellular localization and function. However, the significance of this finding for human carcinogenesis remains unclear. Here, we report the increased biochemical activity and expression of ILK protein in polyps from FAP patients. Furthermore, dramatic increases in ILK immunoreactivity were observed in all abnormal crypts from sporadic polyps, when compared with the normal appearing crypts within the same resected specimens. As sulindac and aspirin are the two most important therapeutic/chemopreventative agents demonstrated in colorectal carcinogenesis, in both humans and animals, further investigation revealed that these non-steroidal anti-inflammatory drugs (NSAIDs) target ILK and ILK-mediated events in vivo. These include inhibition of, both the biochemical activation of ILK, inhibition of serine 9 GSK3beta phosphorylation and the enhancement of TCF-4 transcriptional activity. In conclusion, ILK protein hyperexpression appears to be an early event in colonic polyposis. Additionally, ILK signaling is shown to undergo modulation by sulindac (and aspirin) for the first time, indicating that it is likely to be one of the targets affected by these agents in vivo.

Inhibition of tumor cell invasion and angiogenesis by motuporamines.

Tissue invasion is an important determinant of angiogenesis and metastasis and constitutes an attractive target for cancer therapy. We have developed an assay to identify agents that inhibit invasion by mechanisms other than inhibition of cell attachment or cytotoxicity. A screen of marine sponge extracts identified motuporamines as micromolar inhibitors of invasion of basement membrane gels by MDA-231 breast carcinoma, PC-3 prostate carcinoma, and U-87 and U-251 glioma cells. Motuporamine C inhibits cell migration in monolayer cultures and impairs actin-mediated membrane ruffling at the leading edge of lamellae. Motuporamine C also reduces beta1-integrin activation, raising the possibility that it interferes with "inside-out" signaling to integrins. In addition, motuporamine C inhibits angiogenesis in an in vitro sprouting assay with human endothelial cells and an in vivo chick chorioallantoic membrane assay. The motuporamines show little or no toxicity or inhibition of cell proliferation, and they are structurally simple and easy to synthesize, making them attractive drug candidates.

Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343.

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB- serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.