Interact CardioVasc Thorac Surg 2008;7:107-110. doi:10.1510/icvts.2007.160473
© 2008 European Association of Cardio-Thoracic Surgery
Institutional report - Experimental |
Reactivity of integrin-linked kinase in human mesothelial cell proliferation
Stefan B. Watzkaa,*,
Ulrike Setineka,
Monika Hubera,
Heidi Cantonatia,
Franz Laxa,
Sonja Watsona,
Günter Weigelb and
Michael R. Müllera
a Division of Thoracic Surgery, Otto Wagner Hospital, Sanatoriumstraße 2, 1140 Vienna, Austria
b Vienna General Hospital, Vienna, Austria
Received 31 May 2007;
received in revised form 5 November 2007;
accepted 6 November 2007
 Presented at the 15th European Conference on General Thoracic Surgery, Leuven, Belgium, June 3–6, 2007.
*Corresponding author. Tel.: (+431) 91060-44008; fax: (+431) 4023570.
E-mail address: s.watzka{at}utanet.at (S.B. Watzka).
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Abstract
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Integrin-linked kinase (ILK) is a protein kinase that links integrins and growth factors to a range of signalling pathways. ILK expression and activity are increased in a variety of human cancers. However, little is known regarding the role of ILK in malignant pleural mesothelioma (MPM). In this study, we assessed the expression of ILK in samples of human MPM, and compared it with the expression of epidermal growth factor receptor (EGFR). Thirty-four samples of human malignant mesothelioma were stained with a polyclonal antibody against ILK. Two independent observers evaluated the morphological pattern and intensity of staining. The findings have been compared with the patient's characteristics. Most MPM and mesothelial cell proliferation samples (87.9%) showed cytoplasmic ILK staining of varying intensity. Normal mesothelial cells and normal lung parenchyma did not stain for ILK at all. Conversely, the percentage of positive EGFR staining was somewhat lower (75.8%). The ILK-positive patients were significantly older than the ILK-negative patients. Here we report for the first time that ILK is indeed expressed in malignant mesothelioma. For further validation of a causal association between ILK and neoplastic mesothelial transformation, these immunohistochemical results should be supplemented with clinical and molecular biological data.
Key Words: Mesothelioma; Integrin-linked kinase; Immunohistochemistry; Epidermal growth factor receptor
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1. Introduction
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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 signaling pathways. The recent findings of increased levels of ILK in various cancers, and that inhibition of ILK expression and activity is anti-tumorigenic, makes ILK an attractive target for cancer therapeutics.
ILK is a β1-integrin cytoplasmic domain interacting protein, and functions as a scaffold in forming multi-protein complexes connecting integrins to the actin cytoskeleton and signaling pathways [1]. ILK is essential for embryonic development. ILK activity is stimulated by adhesion to the extracellular matrix and by growth factors in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Overexpression of ILK in epithelial cells induces epithelial–mesenchymal transition (EMT) by inhibiting E-cadherin expression, activates nuclear β-catenin and induces a transformed, tumorigenic phenotype. Mammary epithelial cell specific expression of ILK in transgenic mice induces hyperplasia and tumor formation. Overexpression or constitutive activation of ILK promotes cell survival by stimulating the phosphorylation of AKT on Ser473, and inhibiting ILK in cancer cells inhibits AKT phosphorylation and cell survival.
ILK activity is constitutively activated in PTEN-negative tumors, and inhibiting ILK induces apoptosis and cell-cycle arrest. ILK regulates tumor angiogenesis. ILK activation promotes vascular endothelial growth factor (VEGF) expression in tumor cells, and ILK has an essential role in VEGF-mediated endothelial activation and angiogenesis. ILK expression and activity are increased in many types of cancer, such as prostate, colon, gastric, and ovarian cancers, malignant melanomas (for a summary see Hannigan et al. [2]), and, as recently described, non-small cell lung cancer [3, 4]. Small-molecule inhibitors of ILK have been identified and shown to inhibit cancer-cell growth, survival and invasion in vitro, and tumor growth and angiogenesis in vivo [5].
Malignant pleural mesothelioma (MPM) is a tumor of mesothelial cells that is increasing in incidence in most countries. The disease is predominantly associated with exposure to carcinogenic asbestos fibers, in particular crocidolite. Despite recent improvements in the therapeutic management of MPM through surgery, chemotherapy, radiotherapy and supportive measures, MPM has still a poor prognosis. The median overall survival is approximately 6–12 months. Several clinicopathological scoring systems have been proposed, but they are not able to predict accurately the individual patient's prognosis, and therefore specific therapy is possibly deferred. Furthermore, in order to better define the biological profile of the disease, a number of biological disease markers (EGFR, SV-40 large T-antigen, and Telomerase) has been described, but in multivariate analyses they failed to represent independent prognostic markers.
Besides one experimental study showing an up-regulation of ILK in rat mesothelioma [6], little is known regarding the role of ILK in MPM. In this study, we analyze for the first time the presence and distribution of ILK in samples of human MPM retrieved at surgical interventions at our institution. In addition, we compare ILK expression with epidermal growth factor receptor (EGFR) expression, in order to validate our findings with already established knowledge.
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2. Materials and methods
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Surgical specimens of a total of 34 patients with MPM operated on at our institution between 1999 and 2006 have been retrieved from the archive of the Division of Pathology.
For immunohistochemical investigations, 2-µm tissue sections were used on coated slides. The staining procedure was performed in an automated slide stainer (Benchmark, Ventana). Antigen retrieval was done by heating in the stainer. First antibodies used were a polyclonal rabbit anti-ILK (MBL) antibody in dilution 1:25 and pre-diluted EGFR (clone3C6, Ventana). The staining procedure of EGFR was a fully automated staining run in the Benchmark. For ILK staining, incubation with the primary antibody was performed overnight at 4 °C. The detection step was done in the stainer with a streptavidin Biotin system. Visualization was performed with FastRed chromogen. In two cases, there was not enough material left for both ILK and EGFR staining; therefore, in those two cases each one has been stained for ILK or EGFR.
The intensity of immunohistochemical staining was assessed by two authors (U.S and M.H). The number of positively stained tumor cells was estimated as <50 or >50% positive cells. The staining intensity was scored from weak (+), moderate (++) to strong (+++); in addition, the percentage of positive tumor cells was evaluated. As internal positive control, smooth musculature of pulmonary vessels was used. In those cases where the observers differed in their assessment of ILK and EGFR expression, a consensus was determined using a dual-headed microscope. However, both histological observers were blinded against the clinical follow-up of the individual cases.
In addition, the charts of these patients were reviewed with regard to age and gender distribution, tumor histology, and overall survival. Overall or actuarial survival was defined as the time from surgery to death or last follow-up. The clinical data have been compared with the expression profiles of ILK and EGFR.
All summary statistics are presented as mean±standard error of the mean (S.E.M.) for continuous variables and as percentage for categorical variables. Differences of continuous variables between the clusters were evaluated performing a series of one-way analyses of variance (ANOVA). Cross-tabulation and  2-test were used for comparison of categorical variables. Results were considered to be significant at P<0.05. All statistical calculations were performed with SPSS 9.0 for Windows.
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3. Results
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A total of 34 MPM samples has been investigated. The mean patient age was 68.1±1.6 years (range 43–87 years); they were predominantly males. More than a half of the cases were of the epithelioid subtype; conversely, the pure sarcomatoid subtype was present only in two cases (Table 1). Only in 10 out of 34 cases, a curative surgical approach has been tried; the majority of patients have been treated either by chemotherapy or purely palliative measures (Table 1).
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Table 1 Patient demographic characteristics and distribution of MPM histological subtypes, as well as therapeutic approaches
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In 29 of 33 available MPM cases (87.9%), we could observe a positive ILK staining of variable degree. In most positive cases, more than 50% of the tumor tissue was ILK-positive. In addition, more than 50% of all positive cases were judged as having strong staining intensity, while approximately one-third were considered as either weak or moderately stained (Table 2). All ILK-positive cases showed cytoplasmic ILK staining, while the nuclei remained unstained. In the adjacent tissue, proliferating but not yet transformed mesothelial cells, as well as vascular and bronchial smooth musculature showed also cytoplasmic ILK staining of varying intensity. However, normal mesothelial cells and normal lung parenchyma did not stain for ILK at all (Fig. 1).
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Fig. 1. ILK staining patterns (x200 magnification). (a) Epithelioid subtype of MPM (x200 magnification); (b) sarcomatoid subtype of MPM (x200 magnification); (c) reactive mesothelial cell proliferation (x400 magnification); (d) ILK staining of smooth musculature of vessels and bronchi (x200 magnification).
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EGFR was positive in only 25 of 33 available MPM cases (75.8%). In positive EGFR cases, a membranous staining was seen. In the slight majority of cases, EGFR staining encompassed <50% of tumor area on the histological slides; in addition, the positive cases have been judged to nearly equal parts as having either weak, moderate, or strong staining intensity. Thus, the sensitivity of ILK staining in MPM has been calculated as 87.9%, as compared to a sensitivity of 75% observed in EGFR staining (Table 2).
The ILK-positive patients tended to be older than the ILK-negative patients (69±1.6 vs. 59.2±6.4 years); this difference was marginally statistically significant (P=0.052). All female patients were ILK-positive, while only about 85% of male MPM patients had a positive ILK result. There was no significant difference in ILK expression between the histological MPM subtypes: between 80 and 100 percent of them all were ILK-positive. The mean age of EGFR-negative and positive patients was nearly identical; again, all female patients were EGFR-positive. Similar to ILK findings, the EGFR expression was identical in epithelioid, biphasic, and sarcomatoid subtype (Table 3).
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4. Discussion
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In this study we report for the first time that ILK is expressed in human MPM. The membranous and cytoplasmic staining pattern is consistent with the membrane-bound position of the kinase. Most importantly, normal mesothelial cells, and normal lung parenchyma was always and consistently ILK-negative. Only smooth musculature in vascular and bronchial structures of normal parenchyma did also show some positive ILK staining; this is also to be expected from the known physiological over-expression of ILK in smooth vasculature [7]. In our series, both ILK and EGFR failed to reveal some characteristic staining distribution pattern in accordance with histological subtypes. We had the impression, though, that there was a much stronger, but not necessarily more homogenous staining of both ILK and EGFR in the sarcomatoid subtype. However, as we evaluated only two sarcomatoid cases, any possible difference to epithelioid and biphasic subtypes would anyway have failed to yield a statistically significant result. Interestingly, in contrast to a recently published study, which showed an average EGFR positivity in 44% of investigated MPM cases [8], we observed a positive EGFR reaction in more than 75% of our cases.
ILK is for two reasons an attractive molecule for the investigation of MPM: as shown, ILK is expressed to a high degree in MPM; its sensitivity is nearly 90%. This compares favorably with the sensitivity of other diagnostic modalities, like PET scan [9]. Nevertheless, immunohistochemical ILK expression seems not to be able to discriminate between reactive mesothelial cell proliferation and frank MPM. It must be mentioned, though, that in many cases the discrimination between reactive mesothelial cell proliferation, pulmonary adenocarcinoma, and epithelioid MPM is not possible on pure histological grounds either, and can thus be made only using a panel of several different antibodies, or on a clinical basis. However, regarding the potential capability of ILK to discriminate between pulmonary adenocarcinoma and epithelioid MPM, in a recent study assessing ILK expression in NSCLC, only 28% of investigated adenocarcinoma cases were ILK-positive [4]. In contrast, 83.3% of our epithelioid MPM cases were ILK-positive. In practical terms and following this reasoning, an ILK-negative tumor of either NSCLC or MPM character would be less likely an MPM.
Finally, small molecular inhibitors can selectively inhibit ILK. For example, in an orthotopic human NSCLC model, the compound KP-392 significantly enhanced survival, and inhibited metastasis formation [10]. Furthermore, in a human orthotopic pancreatic cancer model, another compound, QLT0254, produced significant tumor growth inhibition [11]. These experiences are potentially interesting for the treatment of MPM, as the vast majority of it does indeed express ILK. Thus, we can harbor the hope that in the future, using ILK-inhibiting pharmacological compounds, the progression of MPM can be stopped or at least delayed.
We have no clinical explanation of the older age of ILK-positive patients. However, this finding seems to confirm the multiple in vitro observations of increased ILK expression in aging rat mesangial and tubular epithelial cells [12], as well as in aging rat primary cardiac fibroblasts [13]. Intriguingly, these results showed that, in younger cells, ILK overexpression induced larger cell shapes, lower proliferation capacity, and higher levels of enzymatic beta-galactosidase activity, whereas knock-down of ILK prevented phenotypic changes typical of senescence in aging cells. As MPM is a neoplastic disease of older ages, there may be a common pathogenetic principle behind.
In conclusion, we report here for the first time the presence of ILK in a vast majority of MPM cases. As this is a pilot study with only a few cases, many questions remain still open, but ongoing molecular biological, histological, and clinical work will better define the pathogenetic and prognostic function of ILK in MPM, and maybe establish a role for selective ILK inhibition in the management of this disease.
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Abstract
1. Introduction
