Integrin-linked kinase activity modulates the pro-metastatic behavior of ovarian cancer cells

Abstract

Epithelial ovarian cancer (EOC) is the most fatal gynecologic cancer in the U.S., resulting in >14,000 deaths/year. Most women are diagnosed at late stage with widely disseminated intra-peritoneal metastatic disease, resulting in a 5-year survival rate of <30%. EOCs spread via direct extension and exfoliation into the peritoneal cavity, adhesion to peritoneal mesothelial cells, mesothelial cell retraction to expose sub-mseothelial matrix and anchoring in the type I collagen-rich matrix to generate secondary lesions. As a molecular-level understanding of EOC metastasis may identify novel therapeutic targets, the current study evaluated the expression and activity of integrin-linked kinase (ILK), a Ser/Thr protein kinase activated upon integrin-mediated adhesion. Results show that ILK is co-expressed in EOC with the pro-metastatic enzyme membrane type 1 matrix metalloproteinase (MT1-MMP) and catalyzed phosphorylation of the cytoplasmic tail of the proteinase. Downregulation of ILK expression or activity reduced adhesion to and invasion of collagen gels and organotypic meso-mimetic cultures. As an initial early event in EOC metastasis is integrin-mediated adhesion, these results suggest that further evaluation of ILK inhibitors as anti-metastatic agents in EOC is warranted.

Keywords: integrin; integrin linked kinase; membrane type 1 matrix metalloproteinase; metastasis; ovarian cancer.

Figure 1. Expression of ILK and MT1-MMP in human ovarian carcinoma tissues and cells

A, B. Microarrayed cores of ovarian adenocarcinoma were subjected to immunohistochemical analyses for ILK (A) or MT1-MMP (B) as described in Materials and Methods. 20x magnification. C-F. Fluorescence co-localization of ILK and MT1-MMP in DOV13 human ovarian cancer cells. Subconfluent monolayers of cells were seeded onto collagen type I coated coverslips and processed for immunofluorescence. (C) ILK (red), (D) MT1-MMP (green), (E, F) representative merged images. G. Quantitative real time PCR analysis of ILK and MT1-MMP in ovarian cancer cells. The comparative C_T method was used to determine average relative quantitation. Results represent the mean of a minimum of three independent experiments. H. Immunoprecipitation of MT1-MMP. Cell lysates were subjected to immunoprecipitation with anti-MT1-MMP antibody (designated ‘MT1’) or control IgG and protein G beads. Immunoprecipitates were electrophoresed on 9% polyacrylamide gels and subject to western blotting using anti-MT1-MMP (upper panel), anti-phospho-TXR antibodies (middle panel) or anti-ILK antibodies (lower panel), followed by secondary antibodies and enhanced chemiluminescent detection.

Figure 2. Regulation of ILK expression and activity

A. siRNA-mediated silencing of ILK expression as confirmed using quantitative real time PCR analysis of ILK levels as described in Materials and Methods. Results are expressed relative to non-targeting control siRNA (NTC) and represent the mean of a minimum of three independent experiments. A Mann-Whitney U test was employed to determine statistical significance between NTC siRNA and ILK-siRNA as indicated. No difference between untreated and NTC siRNA was detected at any time point (0 h, p=0.479; 48 h, p=0.487). A significant difference is observed comparing either untreated or NTC-siRNA with ILK-siRNA at 48 h (p<.001 in both cases). B. Western blot analysis showing expression of ILK (upper panel) in cells transfected with NTC-siRNA or ILK-siRNA as indicated. In the middle panel, cell lysate was probed with the ILK downstream mediator phospho-AKT-Ser⁴⁷³. Lower panel is probed with GAPDH as a loading control. C. Western blot analysis showing inhibition of ILK activity using QLT0267, as determined by evaluating phosphorylation of AKT. Cells were treated with the ILK inhibitor QLT0267 or vehicle control, as indicated. Cell lysates were probed with antibodies directed against phospho-AKT-Ser473 (upper panel), total AKT (middle panel), or GAPDH (lower panel) as indicated.

Figure 3. Reduced ILK modulates MCA formation and cell spreading

A. MCAs were generated using the hanging drop method as described in Materials and Methods. Representative examples of two MCAs formed from NTC-siRNA control cells (control, upper panels) and ILK-knockdown cells (ILK-siRNA, lower panels) are shown. Scale bar, 400 um. B. Cells were plated on coverslips and incubated with vehicle (control, upper panel) or the ILK inhibitor QLT0267 (+QLT, lower panel, 25 uM) for 12 h prior to visualization with light microscopy.

Figure 4. Effect of modified ILK expression and activity on adhesion to and invasion of type I collagen

A. Adhesion to type I collagen. Untreated control cells (control, black bar), cells treated with QLT0267 (25 uM, dark grey bar), cells transfected with non-targeting control siRNA (NTC-siRNA, light grey bar) or cells transfected with ILK-targeting siRNA (ILK-siRNA, white bar) were incubated in wells coated with type I collagen for 30 min prior to washing to remove unbound cells and quantitation of adherent cells. Results are depicted as the percentage of total cells seeded allowed to adhere overnight and represent the mean of three independent experiments. Statistical significance was determined using Mann-Whitney U tests and p values are indicated. B. Invasion of 3-dimensional collagen gels. Cells, defined as in (A), were seeded into transwells containing a 8 um pore filter overlaid with a 3-dimensional collagen gel as described in Materials and Methods. After incubation for 24 h, non-invading cells were scraped from the top of the filter, the filter was removed and stained. Invading cells adherent to the underside of the filter were enumerated. Results represent the percentage of cells migrated relative to total cells seeded and represent the mean of three independent experiments. Statistical significance was determined using Mann-Whitney U tests and p values are indicated.

Figure 5. Effect of modified ILK expression and activity on adhesion to and invasion of live mesothelial cell monolayers or meso-mimetic cultures

A. Adhesion to mesothelial monolayers. Untreated control cells (control, black bar), cells treated with QLT0267 (25 uM, dark grey bar), cells transfected with non-targeting control siRNA (NTC-siRNA, light grey bar) or cells transfected with ILK-targeting siRNA (ILK-siRNA, white bar) were fluorescently labeled with CMFDA and incubated in wells containing a confluent monolayer of LP9 human peritoneal mesothelial cells for 30 min prior to washing to remove unbound cells and quantitation of adherent cells. Results are depicted as the percentage of total cells seeded allowed to adhere overnight and represent the mean of three independent experiments. Statistical significance was determined using Mann-Whitney U tests and p values are indicated. B. Invasion of meso-mimetic cultures. Control cells transfected with non-targeting siRNA (NTC-siRNA, black bar), NTC-siRNA control cells treated with QLT0267 (NTC-siRNA+QLT, dark grey bar), or cells transfected with ILK-targeting siRNA (ILK-siRNA, white bar) were fluorescently labeled with CMFDA and incubated in transwells containing organotypic meso-mimetic cultures comprised of a confluent layer of LP9 human peritoneal mesothelial cells cultured atop a 3-dimensional type I collagen gel on a porous membrane [57-58]. After incubation for 24 h, cells were scraped from the top of the filter, the filter was removed and stained. Invading cells adherent to the underside of the filter were enumerated. Results represent the percentage of cells migrated relative to total cells seeded and represent the mean of three independent experiments. A Kruskal-Wallis test was used to find a significant mean difference among all three groups (p=0.007). Statistical significance between 2 groups was determined using Mann-Whitney U tests and p values are indicated.