Co-expression of KLK6 and KLK10 as prognostic factors for survival in pancreatic ductal adenocarcinoma

Abstract

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=0.017) and worse outcome for overall survival (P=0.031). Multivariate analysis proofed that co-expression is an independent prognostic factor for survival (P=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.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.

Figure 1

Results of the GeneChip analysis. KLK10 showed a marked upregulation in pancreatic cancer samples compared with normal individuals (P=0.009).

Figure 2

Immunohistochemical staining of PDAC samples. Moderate KLK6 immunoexpression in pancreatic ducts (arrow) and strong expression in Langerhans' islets (arrowhead) no staining in acini ( × 100) (A). Strong KLK10 immunoexpression in the crypts of the intestinal epithelium of the ampulla of Vater ( × 100) (B). Strong KLK6 immunoexpression in pancreatic adenocarcinomas ( × 200) (C and D). Moderate KLK10 immunoexpression in pancreatic adenocarcinomas ( × 200) (E). Strong KLK10 immunoexpression in Langerhans' islets (arrow), absence of expression in pancreatic adenocarcinoma (arrowhead) ( × 200) (F).

Figure 3

The survival curve shows a lower medium survival time of 20 months (15.0–24.0) in the subgroup of patients with strong KLK6 and KLK10 co-expression compared with patients without/weak expression of these kallikreins (29 months (22.8–35.8)) (P=0.031).

Figure 4

A qRT–PCR of different established pancreatic cancer cells lines showed Capan-2 and AsPC-1 with relevant KLK10-expression (A). KLK6-expression was high in nearly all measured cell lines (B).

Figure 5

AsPC-1 cells were transfected with two KLK10-sequences, KLK10.1 and KLK10.2. Transfection resulted in a strong downregulation of KLK10 protein synthesis in the western blot. Cell lysates of KLK10.1- and KLK10.2-transfected cells showed nearly no staining (lanes 1 and 2) compared with the controls (lanes 3–5) (A). Statistical analysis showed a statistical downregulation of the transfected group compared with the control group (P=0.025) (B). The same holds true for gene expression in RT–PCR (P<0.001) (C). The Boyden chamber migration assay: Invasion in vitro was measured as described in ‘Materials and methods’. Statistical analysis showed that KLK10.1-transfected cells had a significant decrease in cell motility compared with the controls (P=0.05) ( × 200) (D). Only few KLK10.1-transfected AsPC-1 cells migrated through the membrane (arrow) (E). The control group displayed normal cell migration (arrows) (F).