Virchows Arch (2008) 453:511–519 DOI 10.1007/s00428-008-0668-8
ORIGINAL ARTICLE
Integrin-linked kinase cytoplasmic and nuclear expression in laryngeal carcinomas Anastasios K. Goulioumis & Vasiliki Bravou & John Varakis & Panos Goumas & Helen Papadaki
Received: 26 May 2008 / Revised: 27 August 2008 / Accepted: 27 August 2008 / Published online: 24 September 2008 # Springer-Verlag 2008
Abstract Integrin-linked kinase (ILK) has been implicated in the development and progression of several human malignancies. Previous in vitro studies also implicate ILK in the activation of Akt and β-catenin as well as in the regulation of E-cadherin expression. However, the role of ILK in human laryngeal cancer and its possible in vivo downstream effectors in the disease are currently unknown. We examined by immunohistochemistry the protein expression of ILK, phosphorylated-Akt (p-Akt), E-cadherin, and β-catenin in 97 invasive squamous laryngeal carcinomas. Increased cytoplasmic and nuclear expression of ILK and p-Akt decreased membranous expression of E-cadherin and nuclear accumulation of β-catenin was found in 87.6%, 85.6%, 71.1%, and 43.3% of cases, respectively. Our results suggest that ILK expression may be implicated in human laryngeal carcinoma and its localization in the nucleus possibly proposes novel nuclear functions of this molecule. In addition, enhanced ILK expression correlates with activation of Akt but not with downregulation of Ecadherin and activation of β-catenin. Finally, in our material while activated Akt seems to characterize welldifferentiated tumors, loss of E-cadherin and activation of β-catenin correlated with high grade carcinomas. Keywords ILK . p-Akt . E-cadherin . β-catenin . Laryngeal carcinoma A. K. Goulioumis : V. Bravou : J. Varakis : H. Papadaki (*) Department of Anatomy, School of Medicine, University of Patras, Patras 26500, Greece e-mail:
[email protected] P. Goumas Otorhinolaryngology–Head and Neck Surgery Department, School of Medicine, University of Patras, Patras, Greece
Introduction Laryngeal carcinoma, classified as squamous cell carcinoma in 90% of the cases, is a common cause of morbidity and mortality worldwide, representing 2.4% of new cancer diagnoses annually in men [1]. As in other malignancies, identification of molecular mechanisms involved in laryngeal cancer progression contributes to the better understanding of its biological behavior and perhaps to the development of novel targeted anticancer therapies. Integrin-linked kinase (ILK) which is normally located in the focal adhesion plaques is a cytoplasmic serine/ threonine kinase implicated in integrin, growth factor, and Wnt signaling pathways [2–6]. ILK is known to have a nodal role in bidirectional cell–extracellular matrix signaling and to regulate several important biological processes such as cell proliferation, survival, cell adhesion, and angiogenesis [5]. Overexpression of ILK has been shown to induce tumor formation in nude mice [6]. Increased expression of ILK has also been reported in many types of tumors including melanoma, colon, prostate, anaplastic thyroid carcinoma, nonsmall cell lung cancer, and head and neck squamous cell carcinomas [5, 7–9]. To the best of our knowledge, there are no reports of ILK expression in human laryngeal carcinoma. Several in vitro studies have implicated ILK in the regulation of E-cadherin expression as well as in the activation of Akt and β-catenin [3, 4, 10] signaling pathways. It has been well documented that downregulation of Ecadherin and activation of β-catenin, represent key molecular events in the development and progression of several human malignancies including laryngeal cancer [11, 12]. It is also known that the protein kinase Akt is involved in several processes thought to be critical in carcinogenesis, including aberrant cell proliferation, evasion from apoptosis, promotion of angiogenesis, and tumor cell invasiveness [13].
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In a series of 97 invasive human laryngeal squamous cell carcinomas, we studied by immunohistochemistry the expression of ILK, phosphorylated-Akt (p-Akt; Ser 473), E-cadherin, and β-catenin and correlated our results with clinicopathological parameters such as tumor grade, stage, and location.
Materials and methods Tissue specimens The study was performed in accordance with the institutional ethical guidelines and has been approved by the Committee on Research and Ethics and the Scientific Committee of the University Hospital of Patras, Greece. Formalin-fixed paraffin-embedded tissue samples from 97 primary human invasive squamous laryngeal carcinomas (63 glottic and 34 supraglottic) were obtained from the Department of Pathology, Agios Andreas General Hospital, Patras, Greece. Adjacent nonneoplastic laryngeal tissue was used as control. Two of the patients were women and 95 were men. Ages ranged from 40 to 86 years, with an average age of 60 years. The WHO classification of tumors was used to determine the histological grade: 25/97 tumors (25.8%) were classified as grade I, 40/97 (41.2%) as grade II, and 32/97 (33%) as grade III [14]. Thirty-two out of 97 (33%) tumors were stage I, 26/97 (26.8%) stage II, 17/97 (17.5%) stage III, and 22/97 (22.7%) stage IV A according to tumor–node–metastasis (TNM) staging. Immunohistochemistry Immunohistochemistry was performed as previously described [15]. Antigen retrieval was enhanced by microwaving the slides in 0.01 M citrate buffer (pH=6). Primary antibody used were rabbit anti-ILK (Santa Cruz Biotechnology, CA, USA; dilution 1:500), rabbit anti-p-Akt (Ser473; Cell Signaling, Beverly, MA, USA; dilution 1:40), mouse anti-β-catenin (BD Biosciences, CA, USA; dilution 1:2,000), and mouse anti-E-cadherin (BD Biosciences, CA, USA; dilution 1:2,000). The antibody used for detection of p-Akt detects Akt1, Akt2, and Akt3 only when phosphorylated at Ser 473. Bound primary antibodies were detected with the biotin–streptavidin–peroxidase method (Envision detection kit, DAKO, Hamburg, Germany). Immunohistochemical evaluation All slides were assessed by one pathologist (H.P.) and one investigator (A.G.) independently and blinded to the case. Cytoplasmic, membranous, and nuclear staining for each protein was evaluated separately. Cytoplasmic staining of
Virchows Arch (2008) 453:511–519
ILK, p-Akt, and β-catenin as well as membranous staining of β-catenin was homogenously distributed among tumor cells and it was scored based on the intensity of staining as follows: score 0—negative staining, 1—weak staining, 2— moderate staining, and 3—strong staining. Negative staining corresponds to complete absence of staining, strong corresponds to staining that can be easily recognized with light microscope at magnification (×4), weak corresponds to staining that can be recognized only at magnification (×20), and moderate is the staining with intensity values intermediate of weak and strong. Nuclear expression of ILK, p-Akt, and β-catenin and membranous expression of E-cadherin showed no significant variations in intensity of staining and the following scoring system was applied based on the percentage of positive cells: score 0—staining in less than 10% of tumor cells, 1—staining in 10-40% of tumor cells, score 2—staining in 40–70% of tumor cells, and score 3—staining in more than 70% of tumor cells. Cases with score 0 were considered negative and cases with scores 1, 2, or 3 were considered positive. In case of cytoplasmic β-catenin, increased cytoplasmic expression was defined as score >1 since in adjacent nonneoplastic laryngeal mucosa, expression of cytoplasmic β-catenin had a score 1. In case of membranous β-catenin and membranous E-cadherin, decreased membranous expression was defined as score
