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Co-Expression of KLK6 and KLK10 As Prognostic Factors for Survival in Pancreatic Ductal Adenocarcinoma

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Co-Expression of KLK6 and KLK10 As Prognostic Factors for Survival in Pancreatic Ductal Adenocarcinoma British Journal of Cancer (2008) 99, 1484 – 1492 & 2008 Cancer Research UK All rights reserved 0007 – 0920/08 $32.00 www.bjcancer.com Co-expression of KLK6 and KLK10 as prognostic factors for survival in pancreatic ductal adenocarcinoma *,1 1 2 1 1 1 3 3 FRu¨ckert , M Hennig , CD Petraki , D Wehrum , M Distler , A Denz , M Schro¨der , G Dawelbait , 4 1 5 1,6 1,6 H Kalthoff , H-D Saeger , EP Diamandis , C Pilarsky and R Gru¨tzmann 1Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technical University of Dresden, Fetscherstrasse 74, Dresden 01307, 2 3 Germany; Department of Nephropathology, Evangelismos Hospital, Phedriadon 109, Athens 11364, Greece; Bioinformatics Group, Biotechnological 4 Centre, Technical University Dresden Tatzberg 47/49, Dresden 01307, Germany; Division of Molecular Oncology, Clinic for General Surgery and Thoracic 5 Surgery, Schleswig–Holstein University Hospitals, Arnold-Heller-Str. 7, Kiel 24105, Germany; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada Kallikreins play an important role in tumour microenvironment and as cancer biomarkers in different cancer entities. Previous studies suggested an upregulation of KLK10 and KLK6 in pancreatic ductal adenocarcinoma (PDAC). Therefore, we evaluated the clinicopathological role of these kallikreins and their value as biomarkers in PDAC. Differential expression was validated by DNA-microarrays and immunohistochemistry in normal and malignant pancreatic tissues. Sera concentrations of both kallikreins were evaluated using ELISA. In silico analysis of possible protein interactions and gene silencing of KLK10 in vitro using siRNAs gave further insights in the pathomechanisms. Gene expression analysis and immunohistochemistry demonstrated a strong expression for KLK10 and KLK6 in PDAC. Statistical analysis showed that co-expression of these kallikreins correlated with an R1-resection status (P ¼ 0.017) and worse outcome for overall survival (P ¼ 0.031). Multivariate analysis proofed that co-expression is an independent prognostic factor for survival (P ¼ 0.043). Importantly, KLK10 knockdown in AsPC-1 cells significantly reduced cell migration, whereas computational analysis suggested interaction of KLK6 with angiogenetic factors as an important mechanism. Molecular Diagnostics Co-expression of KLK10 and KLK6 plays an unfavourable role in PDAC. Our results suggest that this effect is likely mediated by an interaction with the factors of the extracellular matrix and enhancement of cancer cell motility. British Journal of Cancer (2008) 99, 1484–1492. doi:10.1038/sj.bjc.6604717 www.bjcancer.com Published online 14 October 2008 & 2008 Cancer Research UK Keywords: pancreatic cancer; KLK10; KLK6; DNA-microarray; microenvironment Pancreatic ductal adenocarcinoma (PDAC) is one of the most KLK10 and KLK6 are members of the kallikrein family of 15 aggressive cancers with an incidence rate of 6.3/100 000 (Lowenfels known proteases in humans, which play an emerging role in and Maisonneuve, 2006; Jemal et al, 2008). It is characterised by tumour microenvironment, invasion and angiogenesis (Borgono early metastasising and aggressive, infiltrative growth along the and Diamandis, 2004). Kallikreins exert this function as secreted endothelium basement membrane and neurons (Eccles and Welch, trypsin and chymotrypsin-like proteases by degradation of the 2007). This leaves more than 85% of the patients inoperable at the extracellular matrix, which is an important reservoir for cytokines time of diagnosis. Unfortunately pancreatic carcinoma also shows and growth factors such as VEGF, TGF-b and kininogens (Borgono an unsatisfactory response to oncological treatment (Wolff et al, and Diamandis, 2004). Moreover, KLK3, also known as prostate- 2000). This demonstrates the need for new therapeutic approaches specific antigen, is of great clinical value as a serological marker in and also for biomarkers, which make early diagnosis possible. prostate cancer (Borgono and Diamandis, 2004). Other members Recently, we (Grutzmann et al, 2003) and others (Iacobuzio- of the kallikrein family might also have a utility in screening of Donahue et al, 2003; Yousef et al, 2004) showed that human malignancies, like KLK11 and KLK6 for ovarian cancer (Diamandis kallikrein 10 and human kallikrein 6 are among the most highly et al, 2003; McIntosh et al, 2007). This leaves kallikreins as and specifically overexpressed genes in pancreatic cancer com- advantageous candidate genes for diagnosis and therapy in pared with normal and benign pancreas tissues. pancreatic cancer. Therefore, the aim of this study was to evaluate the clinicopathological role of these kallikreins and their value as *Correspondence: Dr F Ru¨ckert, Visceral, Thoracic and Vascular Surgery, biomarkers in PDAC. University Hospital Carl Gustav Carus, Technical University of Dresden, KLK10, also known as the normal epithelial cell-specific 1 is one Haus 91, MTZ, Fetscherstr. 74, 1099 Dresden; of the newly identified members of the kallikrein family. Its role in E-mail: [email protected] carcinogenesis is unsolved as it is downregulated in some tumours 6 These two authors shares the senior authorship. such as breast cancer and acute lymphoblastic leukaemia, whereas Received 27 June 2008; revised 5 September 2008; accepted overexpressed in ovarian, prostate or renal cancer (Luo et al, 2003; 17 September 2008; published online 14 October 2008 Petraki et al, 2003, 2006; Zhang et al, 2006). Unfortunately, the Kallikreins in pancreatic cancer FRu¨ckert et al 1485 function of KLK10 protein remains poorly documented, neither omitting the primary antibody (KLK6 and KLK10) or by replacing the activators nor the substrates for KLK10 are actually known it by non-immune serum (dilution 1 : 500) (see Supplementary (Zhang et al, 2006). KLK6, or protease M, is highly expressed in Data 1). several malignancies like ovarian, breast, colon or gastric cancer. All sections were examined by one observer (CDP) blinded to It is correlated with lymphatic invasion and poor prognosis in both clinical and pathological data. Protein expression in PDACs gastric cancer (Yousef et al, 2004; Nagahara et al, 2005). KLK6 was quantified using a visual grading system with a range between might exert this effect by the degradation of matrix proteins 0 and 2, based on the intensity and the proportion of positive and thereby the augmentation of cancer cell motility and tumour cells on the studied section: 0 ¼ no immunoexpression or proliferation (Ghosh et al, 2004). weak staining in any proportion of the cancerous tissue, or In this study, we show that kallikrein 10 and 6 demonstrate a moderate expression in o/ ¼ 5% of the cancerous tissue, strong protein expression in pancreatic carcinoma and are 1 ¼ moderate staining in 5–50% of the cancerous tissue, associated with poor patient prognosis and R1 resection status 2 ¼ moderate staining in 450% of the cancerous tissue or strong and thereby might contribute to the aggressive character of this staining in any proportion of the cancerous tissue. According to malignancy. staining intensity, cancers were classified as low, medium or high The results indicate that this effect is most likely mediated by the expressing tumours. Coexpression of both KLKs was defined as interaction of KLK6 with factors of the extracellular matrix and the strong if the sample showed strong expression for both KLKs (2/2) enhancement of cancer cell motility by KLK10. and/or at least showed moderate expression for one KLK (2/1). KLK6 and 10 immunoexpression were also screened in the normal pancreatic parenchyma (acinar, ductal and endocrine cells) and in MATERIALS AND METHODS the ampulla of Vater region of the small intestine. Patients and demographic data Patient serum selection and ELISA for kallikrein For immunohistochemical analysis we used samples from 54 measurement in serum patients, operated from July 1996 until December 2003. None of the patients received adjuvant chemotherapy prior to operation. ELISA-type immunofluorometric procedures developed in-house The eligibility criterion was a histologically confirmed PDAC. A were used to measure KLK6 and 10 levels in these sera. Assays used positive microscopic resection margin (R1) was operationally in this study were of the ‘sandwich’ type with the primary antibody defined as at least one cancer cell within 1 mm of any surface of the used for capture and the secondary one for detection. Mono- resected specimen. We included patients with the finding of clonal–monoclonal combinations were used in this study. All metastases to the intra-aortocaval lymph nodes. Previous reports ELISAs were tested negative for cross-reactivity against other have shown that these metastases should not exclude patients in kallikreins. Assay precision within the dynamic range was o10%. good condition from oncologic resection of the PDAC (Shrikhande These assays were standardised with recombinant proteins et al, 2007). Relevant patient characteristics are summarised in produced in yeast or mammalian expression systems. More details Table 1. about the kallikrein ELISA have recently been published (Shaw and For serum-ELISA several panels of sera were selected from 130 Diamandis, 2007). healthy donors, eight patients with benign tumours, 34 with inflammatory diseases and 28 patients with malignant diseases (Table 2). All sera were from patients treated at the University Protein interaction prediction Hospital
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