ADAM8 expression is associated with increased invasiveness and reduced patient survival in pancreatic cancer

Abstract

ADAM8 belongs to a family of transmembrane proteins implicated in cell-cell interactions, proteolysis of membrane proteins, and various aspects of carcinogenesis. In the present study, we aimed to evaluate the expression and function of ADAM8 in pancreatic cancer. ADAM8 mRNA levels were analysed by quantitative RT-PCR and correlated to patient survival. Immunohistochemistry was performed to localize ADAM8 in pancreatic tis-sues. Silencing of ADAM8 expression was carried out by transfection with specific siRNA oligonucleotides. Cell growth and invasion assays were used to assess the functional consequences of ADAM8 silencing. SELDI-TOF-MS was performed to detect the proteolytic activity of ADAM8 in pancreatic cancer cells. ADAM8 mRNA was significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissues (5.3-fold increase; P= 0.0008), and high ADAM8 mRNA and protein expression levels correlated with reduced survival time of PDAC patients (P= 0.048 and P= 0.065, respectively). Silencing of ADAM8 expression did not significantly influence pancreatic cancer cell growth but suppressed invasiveness. In addition, decreased proteolytic activity was measured in cell culture supernatants following silencing of ADAM8. In conclusion, ADAM8 is overexpressed in PDAC, influences cancer cell invasiveness and correlates with reduced survival, suggesting that ADAM8 might be a potential target in pancreatic cancer therapy.

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(A) Domain organization and proposed model for ADAM8 processing. The pro-form of ADAM8 is processed by auto-catalytic prodomain removal into two active forms: the processed form and the remnant protein form. Additional proteolytic cleavage results in two soluble forms: the ectodomain and the MP domain (adapted according to ref.[11]). Pro:prodomain;MP: metalloprotease;DI:disintegrin; CRD: cysteine-rich domain; ELD: EGF-like domain; TM: transmembrane; CTD: cyto-plasmic domain. (B) ADAM8 protein expression in pancreatic tissues. Immunoblot analysis of ADAM8 in normal, chronic pancreatitis and pancreatic cancer tissues. Equal loading of the protein samples was confirmed using an ERK-2 anti-body. Size markers are indicated on the right.

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ADAM8 expression in pancreatic tissues and serum release. (A) Box and whisker diagram showing ADAM8 mRNA levels in the normal pancreas (n = 20), chronic pancreatitis (n = 28) and pancreatic cancer tissues (n = 68) were assessed by real-time QRT-PCR as described in the ‘Materials and methods’ section. The values are normalized to housekeeping genes (cyclophilin B and HRPT). (B) Survival curves of PDAC patients (n = 64) with low (<30 copies/μl cDNA) and high (≥30 copies/μl cDNA). (C) Serum levels of ADAM8 in patients and healthy controls. The ELISA analysis was performed for 28 serum samples in each examined group, and the results are presented as single values and median (horizontal line).

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ADAM8 expression and localization in the normal pancreas, CP and PDAC tissues: Immunohistochemistry was performed as described in the ‘Materials and methods’ section. (A) ADAM8 expression in normal pancreatic islets (arrows) and acinar cells and ductal cells (lower inset). Consecutive section stained with CK19 confirming ductal cells (upper inset). (B) ADAM8 expression in CP tissues (arrows indicate tubular complexes). Consecutive section stained with CK19 confirming ductal cells/tubular complexes (inset). (C, D) ADAM8 (C) and CK19 (D) staining in pancreatic cancer cells of PDAC tissues. (E, F) Strong ADAM8 staining in pancreatic cancer cells of PDAC tissues. Note the absent staining in a consecutive negative control tissue section (inset). Horizontal lines represent the scale bar of 50 μm. (G) Survival curves of PDAC patients (n = 99) with weak/moderate ADAM8 staining versus strong ADAM8 staining (P= 0.065).

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ADAM8 expression under normoxic and hypoxic conditions. ADAM8 mRNA (A) and protein (B) expression in pancreatic cancer cell lines under normal and hypoxic conditions (24 hrs) was determined by qRT-PCR and immunoblotting, respectively. The response towards hypoxic conditions was monitored by HIF-1α expression levels. Equal loading of the protein samples was confirmed using an -tubulin antibody. Size markers are indicated on the left (in kDa). (C) ELISA: ADAM8 expression in the cell lysates (grey bars) and cell culture supernatants (white bars). The values are presented as mean ⁺/⁻ SEM.

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Invasion and growth capacity of ADAM8 siRNA transfected ASPC-1 cells. (A) Real-time quantitative PCR and (B) immunoblot analysis were performed to analyse the effect of four different ADAM8 siRNA oligonucleotides (#1–#4) compared with control trans-fected ASPC-1 cells. Equal loading of the protein samples was confirmed using an -tubulin antibody. Size markers are indicated on the left (in kDa). (C) Cell proliferation and invasion assays were performed as described in the ‘Materials and methods’ section. ADAM siRNA oligonu-cleotides #1 were utilized. The values are expressed as % of control transfected cells and shown as mean ± SEM obtained from four independent experiments.

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SELDI-TOF-MS analysis. Proteolytic activity of ADAM8 in cell culture super-natants assessed by SELDI-TOF-MS. (A) Trace-view and gel view of the mass spectra between 30 and 50 kD of the control siRNA and ADAM8 siRNA #1 transfected cells. Arrow points to the 39.2 kD protein and its suppression in siRNA transfected cells. (B) Decreased intensities of protein peak (arrows) in control siRNA transfected cells.