Reg proteins promote acinar-to-ductal metaplasia and act as novel diagnostic and prognostic markers in pancreatic ductal adenocarcinoma

Qing Li, Hao Wang, George Zogopoulos, Qin Shao, Kun Dong, Fudong Lv, Karam Nwilati, Xian-Yong Gui, Adeline Cuggia, Jun-Li Liu, Zu-Hua Gao, Qing Li, Hao Wang, George Zogopoulos, Qin Shao, Kun Dong, Fudong Lv, Karam Nwilati, Xian-Yong Gui, Adeline Cuggia, Jun-Li Liu, Zu-Hua Gao

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor. Acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) are both precursor lesions that lead to the development of PDAC. Reg family proteins (Reg1A, 1B, 3A/G, 4) are a group of calcium-dependent lectins that promote islet growth in response to inflammation and/or injuries. The aim of this study was to establish a role for Reg proteins in the development of PDAC and their clinical value as biomarkers. We found that Reg1A and Reg3A/G were highly expressed in the ADM tissues by immunohistochemistry. In the 3-dimensional culture of mouse acinar cells, Reg3A promoted ADM formation with concurrent activation of mitogen-acitvated protein kinase. Upregulation of Reg1A and Reg1B levels was observed as benign ductal epithelium progresses from PanIN to invasive PDAC. Patients with PDAC showed significantly higher serum levels of Reg1A and Reg1B than matching healthy subjects. These results were further validated by the quantification of Reg 1A and 1B mRNA levels in the microdissected tissues (22- and 6-fold increases vs. non-tumor tissues). Interestingly, patients with higher levels of Reg1A and 1B exhibited improved survival rate than those with lower levels. Furthermore, tissue expressions of Reg1A, Reg1B, and Reg4 could differentiate metastatic PDAC in the liver from intrahepatic cholangiocarcinoma with 92% sensitivity and 95% specificity. Overall, our results demonstrate the upregulation of Reg proteins during PDAC development. If validated in larger scale, Reg1A and Reg1B could become clinical markers for detecting early stages of PDAC, monitoring therapeutic response, and/or predicting patient's prognosis.

Keywords: acinar-to-ductal metaplasia; cholangiocarcinoma; pancreatic ductal adenocarcinoma; pancreatic intraepithelial neoplasia; Reg family proteins.

Conflict of interest statement

CONFLICTS OF INTEREST

There are no conflicts of interest to disclose.

Figures

Figure 1. Reg1A and Reg3A/G (pancreatitis-associated proteins)…
Figure 1. Reg1A and Reg3A/G (pancreatitis-associated proteins) were associated with ADM (A) Increased Reg1A and Reg3A/G protein expressions in acinar cells undergoing ADM, compared to normal tissues
a, d) normal tissues, b, e) areas undergoing ADM, c, f) magnified pictures showing ADM clusters, positive for Reg1A and Reg3A/G. Arrows: duct-like structure in tumor adjacent acini B. Co-localization of Reg1A and CK19 in cancer epithelium and duct-like structures. a) arrows: cancer cells, as positive controls, b, c) arrows: duct-like structures. Blue: DAPI, Green: CK19, Red: Reg1A. C. Reg3A (100 nM) promoted the formation of duct-like structure in primary acinar cells in 3-D culture. Arrows: duct-like structures. Bottom: blue DAPI showing cell nuclei. The graphic illustrates the model of ADM formation. D. Quantification of the cysts formation from primary acinar cells. Data was presented as cysts formation per imaging field. The images were taken under 100x magnification, and the analysis was performed using Image J software. E. Western blotting of the phosphorylated and total Erk1/2 in primary acinar cells after 30 min treatment of Reg3A or TGF-α. The upper panel showed the representative membrane selected from 3 repeats of the experiment. The lower panel was the quantification of phosphorylated Erk corrected by total Erk levels (Image Lab). N=3, *p<0.05, **p<0.01 vs. vehicle treated cells.
Figure 2. Association of Reg1A and 1B…
Figure 2. Association of Reg1A and 1B tissue expressions with the histological grades of PanIN lesions and invasive PDACs
A-C. Representative illustrations of the different grades of PanIN lesions, from PanIN-I, II to III. D. Invasive cancer adjacent to a PanIN-II lesion. I-III: PanIN I, II and III; CA: cancer (100x). E-H. The staining intensity of Reg1A was positively associated with different grades of PanIN, marked as I, II, III. I-L. Reg1B staining was strongly positive with different grades of PanIN seen in ducts. Representative images were selected from at least 10 fields, each at 100x magnification.
Figure 3. Upregulation of Reg1A and Reg1B…
Figure 3. Upregulation of Reg1A and Reg1B in the sera and tissues of PDAC patients
A. Serum Reg1A and 1B levels in PDAC patients (n=41), healthy controls (n=61) and chronic pancreatitis pateints (n=9). The concentrations of Reg protein isoforms were determined using ELISA. The comparisons were performed using One-Way ANOVA. *P<0.05, **P<0.01, ****p<0.0001. B. Immunohistochemistry profiles of Reg proteins in pancreatic ducts and stroma in PDAC patients. The color brown represents positive staining of various Reg protein isoforms. D: ducts, S: stroma. Representative images were selected from at least 10 fields each. C. Microdissection of tissues from cancer and adjacent normal areas. Representative images showing how the microdissections were done. ADM areas were excluded for this study. D. Relative mRNA levels of Reg1A and 1B in microdissected cancer tissues vs. adjacent tumor-free tissues. N=5; 7. Levels of mRNA in cancer tissues were calculated as fold changes, compared to those in their paired adjacent tissues. *P<0.05, **p<0.01.
Figure 4. Higher levels of Reg1A and…
Figure 4. Higher levels of Reg1A and Reg1B were associated with lower differentiation grades of cancer cells and predicted better prognosis
A. Serum levels of Reg1A and Reg1B were negatively correlated with cell differentiation grades in PDAC patients. Correlation analysis was done by Spearman's test *P<0.05, **P<0.01. B. Immunohistochemistry showing Reg1A and Reg1B expression in different differentiation grades. G1: well-differentiated cancer, G2: medium-differentiated cancer, G3: poorly-differentiated cancer. C. Positivity of Reg1A and 1B in low and high grades of PDAC. Cases were divided into high and low expression groups, based on their immunohistochemical staining intensity. IRS≥9 was considered as high expression. G1-2 was defined as a low differentiation grade; G3 was defined as a high differentiation grade. D. Survival rate of patients with low and high Reg expression levels and differentiation grades. Data were analyzed in GraphPad Prism 6.0. ***P<0.001, ****P<0.0001.
Figure 5. Tissue expression of Reg1A, Reg1B,…
Figure 5. Tissue expression of Reg1A, Reg1B, and Reg4 could differentiate PDAC from ICA with high specificity and sensitivity
A. Immunoreactive scores (IRS) of Reg1A, Reg1B and Reg4 in PDAC were significantly higher than those in ICA. a-c) Reg1A, Reg1B and Reg4 expression in PDAC; d-f) Reg1A, Reg1B, and Reg4 expression in ICA; g-i) comparisons of Reg1A, Reg1B, and Reg4 IRS among PDAC, ECA, and ICA. **P<0.01, ****P<0.0001, NS: no significance. B. The sensitivity and specificity of combining Reg1A, Reg1B, and Reg4 immunohistochemical staining in distinguishing PDAC from ICA. Positive and negative cases of PDAC and ICA for Reg1A, Reg1B, and Reg4 were plotted on column graphs. Combined positivity was recorded when Reg1A, Reg1B, and Reg4 were all positive. Sensitivity, specificity and predictive values were calculated by using GraphPad Prism.
Figure 6. Summary of the roles of…
Figure 6. Summary of the roles of Reg proteins in the progression of ADM/PanIN to invasive cancer and metastasis in liver
The expression levels of Reg1A and Reg3A/G were elevated in ADM. Reg3A seems to directly promote ADM formation in vitro. The effects of additional mutations such as in Kras and TP53 transform ADM further into PanIN lesions and PDAC [43]. Serum levels of Reg1A and 1B as well as their tissue expressions were all elevated in PDAC patients. As such, increased Reg proteins may be used to differentiate PDAC from ICA.

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