Penny Costello

Inhibition of integrin linked kinase (ILK) suppresses beta-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor, snail, in APC-/-human colon carcinoma cells

Loss of functional adenomatous polyposis coli (APC) protein results in the stabilization of cytosolic β-catenin and activation of genes that are responsive to Lef/Tcf family transcription factors. We have recently shown that an independent cell adhesion and integrin linked kinase (ILK)-dependent pathway can also activate β-catenin/LEF mediated gene transcription and downregulate E-cadherin expression. In addition, ILK activity and expression are elevated in adenomatous polyposis and colon carcinomas. To examine the role of this pathway in the background of APC mutations we inhibited ILK activity in APC−/− human colon carcinoma cell lines. In all cases, inhibition of ILK resulted in substantial inhibition of TCF mediated gene transcription and inhibition of transcription and expression of the TCF regulated gene, cyclin D1. Inhibition of ILK resulted in decreased nuclear beta-catenin expression, and in the inhibition of phosphorylation of GSK-3 and stimulation of its activity, leading to accelerated degradation of β-catenin. In addition, inhibition of ILK suppressed cell growth in culture as well as growth of human colon carcinoma cells in SCID mice. Strikingly, inhibition of ILK also resulted in the transcriptional stimulation of E-cadherin expression and correlated with the inhibition of gene transcription of snail, a repressor of E-cadherin gene expression. Overexpression of ILK caused a stimulation of expression of snail, but snail expression was found not to be regulated by β-catenin/Tcf. These data demonstrate that ILK can regulate β-catenin/TCF and snail transcription factors by distinct pathways. We propose that inhibition of ILK may be a useful strategy in the control of progression of colon as well as other carcinomas.

Regulation of tumor angiogenesis by integrin-linked kinase (ILK)

We show that integrin-linked kinase (ILK) stimulates the expression of VEGF by stimulating HIF-1alpha protein expression in a PKB/Akt- and mTOR/FRAP-dependent manner. In human prostate cancer cells, knockdown of ILK expression with siRNA, or inhibition of ILK activity, results in significant inhibition of HIF-1alpha and VEGF expression. In endothelial cells, VEGF stimulates ILK activity, and inhibition of ILK expression or activity results in the inhibition of VEGF-mediated endothelial cell migration, capillary formation in vitro, and angiogenesis in vivo. Inhibition of ILK activity also inhibits prostate tumor angiogenesis and suppresses tumor growth. These data demonstrate an important and essential role of ILK in two key aspects of tumor angiogenesis: VEGF expression by tumor cells and VEGF-stimulated blood vessel formation.

The integrin linked kinase (ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 (MMP-9)

Overexpression of Integrin Linked Kinase (ILK) in intestinal and mammary epithelial cells results in a highly invasive phenotype, associated with increased levels of expression of the matrix metalloproteinase MMP-9. This increase was at the transcriptional level as determined by MMP-9 promoter-CAT reporter assays. Mutations in the two AP-1 binding sites within the MMP-9 promoter completely inhibited the reporter activity. We have previously shown that ILK inhibits glycogen synthase kinase-3 (GSK-3) activity. Transient transfection of wild-type GSK-3β in ILK-overexpressing cells decreased MMP-9 promoter activity and AP-1 activity, indicating that ILK can stimulate MMP-9 expression via GSK-3β and AP-1 transcription factor. A small molecule inhibitor of the ILK kinase reduced the in vitro invasiveness of ILK-overexpressing cells as well as the invasiveness of several human brain tumor cell lines. Furthermore, both MMP-9 promoter and AP-1 activities were inhibited by the ILK inhibitor. Invasiveness of ILK-overexpressing cells was also reduced by inhibition of MMP-9. These data demonstrate that ILK can induce an invasive phenotype via AP-1-dependent upregulation of MMP-9.

Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types

SummaryPresumptive astrocytes isolated from 10-day white Leghorn chick embryos, Factor VIII-positive human brain capillary endothelial cells, meningeal fibroblasts from 10-day chick embryos, Swiss mouse 3T3 cells, and human astrocytoma cell lines, SKMG-1 and U373, were rendered quiescent when placed in culture medium that contained 0 or 0.2% serum for 48 h; their proliferation was markedly reduced and they incorporated [3H]thymidine at a low rate. [3H]Thymidine incorporation and cell proliferation were induced in all types of cells by addition of guanosine, GMP, GDP, GTP, and to a lesser extent, adenosine, AMP, ADP or ATP to the culture medium. The stimulation of proliferation by adenosine and guanosine was abolished by 1,3-dipropyl-7-methylxanthine (DPMX), an adenosine A2 receptor antagonist, but not by 1,3,-dipropyl-8-(2-amino-4-chorophenyl)xanthine (PACPX), an A1 antagonist. Stimulation of proliferation by the nucleotides was not abolished by either DPMX or PACPX. The P2 receptor agonists,α,β-methyleneATP and 2-methylthioATP, also stimulated [3H]thymidine incorporation into the cells with peak activity at approximately 3.5 and 0.03 nM, respectively. These data imply that adenosine and guanosine stimulate proliferation of these cell types through activation of an adenosine A2 receptor, and the stimulation of cell proliferation by the nucleotides may be due to the activation of purinergic P2y receptors. As the primary cultures grew older their growth rate slowed. The capacity of the purine nucleosides and nucleotides to stimulate their growth diminished concomitantly. The 3T3 cells showed neither decreased growth with increased passages nor reduced response to the purines. In contrast, although the doubling time of the immortalized human astrocytoma cell lines SKMG-1 and U373 remained constant, the responsiveness to purinergic stimulation of the U373 cells decreased but that of the SKMG-1 did not. These data are compatible with a decrease in the number, or the ligand-binding affinity of the purinergic receptors, or a decreased coupling of purinergic receptors to intracellular mediators in primary cells aged in tissue culture.

Integrin-linked kinase (ILK): a “hot” therapeutic target

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.

Isolation and culture of cells derived from human cerebral microvessels

SummaryMicrovessels were isolated from non-neoplastic human cerebral cortical fragments resected for treatment of intractable seizure disorder. The microvessels were incubated in modified Lewis medium with 20 or 30% fetal bovine serum. Within 1–2 weeks, two cell populations emerged from the isolates. One type of cells had polygonal morphology, showed density-dependent contact inhibition at confluence in vitro, showed lectin-binding characteristics of endothelium (but only moderate positivity for factor VIII antigen), demonstrated induction of γ-glutamyl trans-peptidase when exposed to astrocyte-conditioned media, and responded to insulin by a pronounced increase in DNA synthesis. The other variety of cells grew in vitro more slowly in irregular strands separated by clear zones, showed ultrastructural features of smooth muscle, and isoelectric focusing of cell proteins revealed the presence of smooth-musclespecific α-isoactin. Both types of cells could be serially subcultured. The ability to isolate and grow the two cell types, tentatively identified as human cerebral microvascular endothelium and smooth muscle, may facilitate studies of human blood-brain barrier function as well as the pathogenesis of cerebral microangiopathies unique to the human brain.

Integrin-Linked Kinase Inhibitor KP-392 Demonstrates Clinical Benefitsin an Orthotopic Human Non-small Cell Lung Cancer Model

INTRODUCTION The overexpression of integrin-linked kinase (ILK) has been implicated in the promotion of tumor invasion and metastasis. We studied the anticancer effects of KP-392, a potent selective inhibitor of ILK in the NCI-H460 cell line. In vitro, KP-392 inhibited ILK activity of H460 cells. In vivo, the effect of KP-392 was investigated in a metastatic H460 orthotopic lung cancer model. METHODS Intraperitoneal KP-392 (5 mg/day per animal) was administered both alone and in combination with cisplatin (5 mg/kg per week for 3 weeks). In group I, all treated animals were followed until death to assess therapeutic effect on survival. In group II, tumor growth and metastasis were evaluated by sacrificing one animal from each treatment when a control animal died. RESULTS Both cisplatin and KP-392 significantly enhanced survival (37.8 +/- 3.7 and 34.9 +/- 5.2 days) compared with the control (30.2 +/- 3.6 days, p < 0.0001 and p = 0.0418, respectively), and the survival benefit from combination treatment was greater than that of either agent alone (45.8 +/- 3.9 days, p < 0.0001). Although KP-392 alone did not impact the incidence of metastasis, in combination with cisplatin a consistent trend of inhibition was seen for metastases in the kidney, bone, and the contralateral lung. KP-392 was well tolerated throughout the study. KP-392 demonstrated increased tumor necrosis and decreased nuclear phospho-protein kinase/Akt but did not change the levels of phospho-extracellular signal-regulated kinase 1/2. CONCLUSIONS ILK inhibitor does not enhance the toxicity of standard chemotherapy and may have a beneficial therapeutic effect in lung cancer.

Human cerebral endothelium: isolation and characterization of cells derived from microvessels of non-neoplastic and malignant glial tissue.

Human microvessels were isolated and cultured from non-neoplastic cerebral tissue specimens resected for the treatment of seizure disorders and from malignant glial tumors. After 1–2 weeks, cobblestone patterned plaques of cells were isolated and cultured from these microvessels. Cell lines positive for Factor VIII antigen and negative for glial fibrillary acidic protein were designated as endothelium. Endothelium from both tissue sources produced γ-glutamyl transpeptidase in response to glial cell conditioned media. Tumor derived microvessel endothelium had decreased longevity in culture when compared to normal microvessel endothelium. Tumor derived endothelium also formed less extensive intercellular junctional complexes in vitro. The isolation and characterization of human cerebral microvessel endothelium derived from non-neoplastic tissue and glial tumors may lead to a further understanding of the role of endothelium in tumor growth and vascular permeability alterations.

In vitro three dimensional culture of hepatocellular carcinoma to measure prognosis and responsiveness to chemotherapeutic agents

BACKGROUND Understanding the prognosis of hepatocellular carcinoma (HCC) informs plans for care. Tumor morphology and molecular markers have been correlated with outcomes. Three-dimensional tissue culture (3DTC) allows for direct in vitro measurement of a tumors ability to grow and metastasize. The impact of chemotherapeutic agents, alone or in combinations, may also be measured. METHODS All patients with a presumed diagnosis of HCC were eligible for this study including those undergoing resection, chemoembolization and transplantation. Concomitant diseases and outcomes were recorded. One mm(3) HCC specimens were grown in multiwell plates containing gel media, without and with chemotherapeutic agents. RESULTS Tumors were sampled from 17 patients. Only 13 had HCC, all of whom had liver transplantation. Of the confirmed HCC patients, 6 (46%) are alive and disease free 82 months following transplantation, 1 (7%) is alive with recurrence of disease and 6 (46%) died, with a mean survival of 12 months post liver transplant. Ten of thirteen 3DTC samples grew, having an average migration distance of 108.3µm in the first 24 hours. Two of three patients who had prior chemoembolization had successful 3DTC. Migration distances (µm) were 188.8±104.3, 104.5±111.7 and 39.6±32.4 for tumors categorized as high, intermediate and low grade, respectively. Tumor migration was inhibited by irinotecan, paclitaxel and docetaxel (-68%±7%, -61%±19% and -60%±21%, respectively) whereas the effect was variable with 5 fluorouracil (5FU) and doxorubicin (-12%±51% and 9%±76%, respectively). CONCLUSIONS It is feasible to grow tissue from HCC in 3DTC to study the tumors capacity to grow and migrate and its responsiveness to commonly used chemotherapeutic protocols.