Immune-complex level of cofilin-1 in sera is associated with cancer progression and poor prognosis in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. To improve its outcome, reliable biomarkers are urgently needed. In this study, we aimed to elucidate the key molecules involved in PDAC progression using proteomics approaches. First, we undertook 2-D electrophoresis to identify the proteins overexpressed in PDAC tissues. Following the analysis of agarose gel spots, cofilin-1 was identified and verified as a candidate protein commonly upregulated in PDAC tissues. In immunohistochemistry, cofilin-1 was strongly expressed in the cytoplasm of PDAC cells. Samples were divided into two groups based on the level of cofilin-1 expression. The high expression group showed significantly higher incidence of hematogenous dissemination in relapsed patients than the low expression group (P = 0.0083). In in vitro experiments, knockdown of cofilin-1 significantly decreased chemotaxis in PDAC cell lines. After we confirmed that cofilin-1 was secreted from PDAC cells, we established a detection system for the immune-complex of cofilin-1 in sera. Using this system, we measured the IC levels of cofilin-1 in sera and observed that the IC levels of cofilin-1 in PDAC patients were higher than those in healthy volunteers and patients with pancreatitis (PDAC vs. healthy volunteers, P < 0.0001; PDAC vs. patients with pancreatitis, P < 0.026). Notably, the IC levels of cofilin-1 showed a stepwise increase during PDAC progression (P = 0.0034), and high IC levels of cofilin-1 indicated poor prognosis of patients after surgery (P = 0.039). These results suggest that the IC of cofilin-1 in sera is a potentially attractive serum biomarker for the prognosis of PDAC.

Keywords: Cofilin-1; immune-complex; pancreatic cancer; prognosis; serum biomarker.

Figure 1

Comparison of protein profiling in 2‐D electrophoresis (2‐DE) patterns and Western blot analysis. (a) Comparison of 2‐DE patterns of normal and tumor pancreatic tissues. The 2‐DE gel was stained with Coomassie brilliant blue. (b) Expanded views of six overexpressed protein spots (p20, p51, p39, p31, p53, and p49) in tumor tissues. Protein spots are named according to their apparent molecular mass. Basic end of the gel is to the right. (c) Western blot analysis of overexpressed proteins in tumor tissues. Proteins were identified by mass spectrometry. Total protein lysates were prepared from nine paired samples of normal (N) and tumor tissue (T). Anti‐GAPDH antibody was used as a loading control. PD‐ECGF, platelet‐derived endothelial cell growth factor. (d) Image analysis of Western blot data of cofilin‐1. The intensity and area of each band was measured, and these protein levels between normal and tumor tissue, normalized with GAPDH, were calculated. The expression of cofilin‐1 was increased in all tumor tissues.

Figure 2

Immunohistochemistry for cofilin‐1 in pancreatic ductal adenocarcinoma tissues. (a) Cofilin‐1 is weakly expressed in normal pancreatic ductal cells (×100). (b) Cofilin‐1 is weakly expressed in acinar cells (×200), and strongly overexpressed in the cytoplasm of tumor cells. (c–f) Immunohistochemical staining patterns of cofilin‐1 in resected pancreatic cancer tissues. Representative staining of low expression (c,d) and high expression (e,f) are indicated (×100).

Figure 3

Knockdown of cofilin‐1 decreases the chemotaxis in PANC‐1 and Capan‐1 pancreatic ductal adenocarcinoma cells. (a) Expression of cofilin‐1 in PANC‐1 and Capan‐1 cells using control siRNA (GL2) or cofilin‐1 siRNA, as determined by Western blot analysis. GAPDH was used as a control for internal protein loading. Cofilin‐1 siRNA dose‐dependently inhibits cofilin‐1 protein expression in PANC‐1 and Capan‐1 cells. (b) Proliferation assay of PANC‐1 and Capan‐1 cells using GL2 or cofilin‐1 siRNA at 3 days after siRNA transfection. (c) Chemotaxis assay of two cell lines using a Boyden chamber. Four randomly selected fields were photographed and the number of migrated cells on the back of the filter was counted. *P < 0.05, **P < 0.01, control siRNA versus cofilin‐1 siRNA. N.S., not significant.

Figure 4

Detection and clinical utility of the cofilin‐1 immune‐complex in sera of patients with pancreatic ductal adenocarcinoma (PDAC). (a) Left lane, cell lysates were applied as a loading control. Middle lane, supernatant of the medium (containing 10% FBS) in which no cells were cultured was used as a negative control. Cofilin‐1 was detected very weakly. Right lane, supernatant of the medium in which PANC‐1 and Capan‐1 cells were cultured for 24 h. Cultured supernatant was incubated in new medium for 24 h. Intense expression of cofilin‐1 was detected. (b) Schema of three detection systems of sandwich ELISA (left, cofilin‐1 antigen; middle, cofilin‐1 autoantibody; right: cofilin‐1 immune‐complex [IC]). (c) Measurement of IC to cofilin‐1 in sera of healthy volunteers (HV), patients with pancreatitis (PT), and PDAC patients. (d) Comparison of receiver operating characteristic curves for classification of cancer (PDAC) and non‐cancer (HV and PT) patients. Dashed line, cancer antigen 19‐9 (CA19‐9); dotted line, carcinoembryonic antigen (CEA); black line, cofilin‐1 IC; bold gray line, combination score of cofilin‐1 IC and CA19‐9 estimated by the logistic regression model. Thin gray line represents the random classification. (e) IC levels in HVs, PT, and each stage of PDAC (according to Union for International Cancer Control criteria). (f) Gray and black lines indicate cofilin‐1 IC low and high level groups, respectively. Kaplan–Meier survival curves show unfavorable prognosis in the cofilin‐1 IC high level group (P = 0.0397).