Aberrant Wnt/beta-catenin signaling in pancreatic adenocarcinoma

Abstract

Wnt/beta-catenin signaling plays an important role in normal development. However, its aberrant activation is associated with several cancers. The aim of this study is to examine the Wnt/beta-catenin pathway in patients with advanced pancreatic adenocarcinoma (n = 31). Paraffin sections from tumors (n = 16) and normal pancreata (n = 3) were used to determine the localization of beta-catenin. An additional 15 frozen tumors, adjacent normal pancreata (n = 5), or normal pancreata (n = 4) were utilized for protein isolation. Tumors were also examined for mutations in exon 3 of the CTNNB1 gene. More than 65% of the tumors showed an increase in total beta-catenin, consistent with its enhanced membranous, cytoplasmic, and nuclear localization, but only two showed mutations in CTNNB1. The majority of the remaining tumors demonstrated concurrent increases in Wnt-1 and frizzled-2 (positive regulators) and a decrease in Ser45/Thr41-phospho-beta-catenin. Electrophoretic mobility shift assay demonstrated beta-catenin-T-cell factor binding in tumors only. Adenomatous polyposis coli and axin, which are both negative regulators, remained unchanged. Unexpectedly, total glycogen synthase kinase-3beta protein was elevated in these tumors. Elevated levels of E-cadherin were also observed, although E-cadherin-beta-catenin association in tumors remained unaffected. Thus, Wnt/beta-catenin activation was observed in 65% of pancreatic adenocarcinomas, independently of beta-catenin gene mutations in most tumors.

Figure 1

β-Catenin undergoes redistribution in the majority of pancreatic adenocarcinomas. (A) A representative section from a normal pancreas, obtained through autopsy, shows predominantly membranous localization in tall columnar acinar cells. Inset: Higher magnification highlights membranous localization (white arrowhead). (B–G) Representative tumors from six patients show aberrant β-catenin localization. Insets: Higher magnification clearly demonstrates increased nuclear (arrow) and cytoplasmic (arrowhead) β-catenin localization in pancreatic adenocarcinoma, while still maintaining membranous localization (white arrowhead). (H) A representative tumor from the minor subset of tumors shows normal membranous localization of β-catenin. Inset: Higher magnification shows membranous localization (white arrowhead) of β-catenin in this tumor.

Figure 2

An approximately five-fold increase in arbitrary immunohistochemical score was observed in pancreatic tumors (n = 16) versus normal pancreata (n = 3), which was statistically significant (P < .05).

Figure 3

Activation of the Wnt/β-catenin pathway in the majority of pancreatic adenocarcinomas. (A) Western blot analysis from four representative tumors that display increased total β-catenin protein compared to their adjacent normal pancreata (upper panel) or independent normal pancreata (lower panel). β-Actin demonstrates comparable loading of proteins. The c-myc protein remains unchanged between tumors and adjacent normal pancreata in the upper panel. (B) A representative Western blot analysis using nuclear lysates from a tumor that displayed increased total levels of β-catenin shows increased nuclear β-catenin as well. EMSA detects the β-catenin-TCF complex (arrow) in tumors and not in adjacent normal pancreas in a representative analysis. (C) Representative Western blot analyses for the status of canonical Wnt pathway in two tumors that display elevated total β-catenin reveal elevated Wnt-1 and frizzled-2 in tumors compared to normal adjacent (left set) or true normal pancreata (right set). No change in axin orAPC was detected; however, an unexpected increase in GSK3β was evident in tumors only. Although Ser33,37/Thr41-phosphorylated β-catenin showed either no change (left set) or an increase in tumor (right set) compared to their respective controls, Ser45/Thr41-phosphorylated β-catenin levels were decreased in tumors, again suggesting the stabilization of β-catenin as a mechanism of its increase and activation. β-Actin blot shows equal protein loading.

Figure 4

Missense point mutations in the β-catenin gene in pancreatic tumors in two patients FT2 (upper panel) and FT14 (lower panel). Sequence analysis shows S33C (A) and S37F (B) mutations.

Figure 5

Continued and increased E-cadherin localization at the membrane in pancreatic tumors. (A) A representative section from normal pancreata shows membranous localization (arrow) of β-catenin with some subcellular stainings. (B and C) Representative analysis from two tumors reveals continued membranous localization (arrow) of E-cadherin with increased intensity and some cytoplasmic staining as well. (D) A representative section from a tumor shows comparable E-cadherin localization or intensity at the membrane (arrow) to the control.

Figure 6

Increased E-cadherin level and its continued interaction with β-catenin is observed in pancreatic adenocarcinoma. (A) Two pairs of tumors and normal pancreata atthe upper left panel show increased levels of total E-cadherin in this representative Western blot analysis. The right pair shows no change. The bottom panels show β-actin as a loading control. (B) A representative coprecipitation study shows continued β-catenin-E-cadherin association (middle panel) in pancreatic adenocarcinomas in most tumors, which was a function of immunoprecipitated β-catenin in lysates (bottom panel). No tyrosine phosphorylation of β-catenin was detectable (top panel).

Figure 7

Summary of changes in the Wnt/β-catenin pathway in pancreatic adenocarcinoma. β-Catenin activation in the form of cytoplasmic stabilization and nuclear translocation occurs secondary to increased Wnt-1 or frizzled-2 proteins or mutations in β-catenin gene (CTNNB1). This is complemented by failure of increases in β-catenin's degradation complex proteins, such as axin and APC. GSK3β increase appears inadequate as reflected by decreased phosphorylation of β-catenin at Ser45 and Thr41. The E-cadherin-β-catenin complex's contribution to cytoplasmic or nuclear β-catenin appears highly unlikely due to continued association and absent tyrosine phosphorylation. A more protective response of this complex to sequester some of the stabilized cytoplasmic β-catenins, especially in nonmetastatic tumors, could be an alternative explanation.