Quantitative proteomics investigation of pancreatic intraepithelial neoplasia

Abstract

Patients with pancreatic cancer are usually diagnosed at late stages, when the disease is incurable. Pancreatic intraepithelial neoplasia (PanIN) 3 is believed to be the immediate precursor lesion of pancreatic adenocarcinoma, and would be an ideal stage to diagnose patients, when intervention and cure are possible and patients are curable. In this study, we used quantitative proteomics to identify dysregulated proteins in PanIN 3 lesions. Altogether, over 200 dysregulated proteins were identified in the PanIN 3 tissues, with a minimum of a 1.75-fold change compared with the proteins in normal pancreas. These dysregulated PanIN 3 proteins play roles in cell motility, the inflammatory response, the blood clotting cascade, the cell cycle and its regulation, and protein degradation. Further network analysis of the proteins identified c-MYC as an important regulatory protein in PanIN 3 lesions. Finally, three of the overexpressed proteins, laminin beta-1, galectin-1, and actinin-4 were validated by immunohistochemistry analysis. All three of these proteins were overexpressed in the stroma or ductal epithelial cells of advanced PanIN lesions as well as in pancreatic cancer tissue. Our findings suggest that these three proteins may be useful as biomarkers for advanced PanIN and pancreatic cancer if further validated. The dysregulated proteins identified in this study may assist in the selection of candidates for future development of biomarkers for detecting early and curable pancreatic neoplasia.

Figure 1. Histology of PanIN and pancreatic cancer

A. PanIN 2 or low grade ductal dysplasia; B. PanIN 3 or high grade ductal dysplasia; C. Pancreatic ductal adenocarcinoma with neural invasion; D. Normal pancreatic duct and acinar tissue.

Figure 2. PanIN 3 protein expression profiling by ICAT

A. Protein ratio distribution. B. An example of MS/MS spectrum for peptide identification. C. An example of peptide quantification by ICAT labeling. Note that 4.54 ± 1.04 is the ICAT ratio of a single peptide VVQC*SDLGLDKVPK, one of six peptides derived from protein decorin.

Figure 3. Distribution of the over-expressed proteins analyzed by iTRAQ analysis

The distribution of the proteins can be divided into four regions based on their abundance ratio compared to the normal control; I. the proteins with expressional ratio <1.75 compared with normal control; II. the proteins overly expressed in pancreatic cancer sample only; III. the proteins overly expressed in both cancer and PanIN3 samples; IV. the proteins overly expressed in PanIN3 samples only.

Figure 4. Biological network analysis of the dysregulated proteins in PanIN3 tissue

The network was generated using the Analyze Network algorithm to map the interactions between the dysregulated proteins. Nodes represent proteins; lines between the nodes indicate direct protein-protein interactions. A small red circle denotes an over-expressed protein, whereas a small blue circle denotes an under-expressed protein. The most prominent regulatory protein in the network was c-MYC, which directly interacts with 18 of the dysregulated proteins in the network.

Figure 5. IHC analysis in advanced PanIN tissue and pancreatic cancer

Laminin (A–C): note the overexpression in the stromal tissue in PanIN3 (B) and pancreatic cancer (C) compared to normal pancreas (A). Actinin-4 (D–F): note the overexpression in the ductal epithelium of PanIN2 (E), and in both the stroma and ductal epithelium of pancreatic cancer (F). Galectin-1 (G–I): note the overexpression in the stroma of PanIN2 (H) and pancreatic cancer (I) while it is absent in normal pancreas (G).