Canonical wnt signaling is required for pancreatic carcinogenesis - PubMed (original) (raw)

Canonical wnt signaling is required for pancreatic carcinogenesis

Yaqing Zhang et al. Cancer Res. 2013.

Abstract

Wnt ligand expression and activation of the Wnt/β-catenin pathway have been associated with pancreatic ductal adenocarcinoma, but whether Wnt activity is required for the development of pancreatic cancer has remained unclear. Here, we report the results of three different approaches to inhibit the Wnt/β-catenin pathway in a established transgenic mouse model of pancreatic cancer. First, we found that β-catenin null cells were incapable of undergoing acinar to ductal metaplasia, a process associated with development of premalignant pancreatic intraepithelial neoplasia lesions. Second, we addressed the specific role of ligand-mediated Wnt signaling through inducible expression of Dkk1, an endogenous secreted inhibitor of the canonical Wnt pathway. Finally, we targeted the Wnt pathway with OMP-18R5, a therapeutic antibody that interacts with multiple Frizzled receptors. Together, these approaches showed that ligand-mediated activation of the Wnt/β-catenin pathway is required to initiate pancreatic cancer. Moreover, they establish that Wnt signaling is also critical for progression of pancreatic cancer, a finding with potential therapeutic implications.

©2013 AACR.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: Timothy Hoey and Austin Gurney are employees of OncoMed Pharmaceuticals. Funding from OncoMed Pharmaceutical funded the portion of this study that included the OMP-18R5 monoclonal antibody.

Figures

Figure 1

Figure 1. Inhibition of the Wnt/β-catenin signaling by Dkk1 prevents PanIN formation

(A) Genetic makeup of the KDC model. (B) Experimental design. (C) H&E staining of KC and KDC pancreata at 1 month, 2 months, 5 months and 8 months following induction of Dkk1 expression. Scale bar 100μm. (D) Pathological analysis. Data represent mean ± SEM, n=4. The statistical difference was determined by two-sided Student's t-test.

Figure 2

Figure 2. Dkk1 expression inhibits Wnt/β-catenin signaling

(A) Experimental design. (B) Western blot for Dkk1, β-catenin and phospho-β-catenin in KC and KDC pancreas; quantification of the fold change of phospho-β-catenin/β-catenin. Data represent mean ± SEM, n=3. (C) Immunohistochemistry for β-catenin and phospho-β-catenin (insert). Scale bar 20μm. (D) RT-qPCR analysis of Dkk1, Wnt/β-catenin target genes and component. Each dot represents one mouse. Data represent mean ± SEM. The statistical difference was determined by two-sided Student's t- test.

Figure 3

Figure 3. Inhibition of the Wnt/β-catenin signaling by OMP-18R5 prevents PanIN formation

(A) Experimental design for OMP-18R5 treatment; n=5 mice per cohort. (B) H&E staining and immunohistochemical analysis for β-catenin, phospho-β-catenin, phospho-ERK1/2 and ki67. Scale bar 50μm. (C) Quantification of the lesions. (D) RT-qPCR analysis of Wnt/β-catenin target genes. (E) RT-qPCR analysis of non-canonical Wnt signaling components. Each dot represents one mouse. Data represent mean ± SEM. The statistical difference was determined by two-sided Student's t- test.

Figure 4

Figure 4. Cross-talk of Wnt and MAPK signaling pathways

(A) Experimental design; n=3 to 5 mice per time point. (B) Western blot for ERK1/2 and phospho-ERK1/2; quantification of the fold change of phospho-ERK/Total ERK. Data represent mean ± SEM, n=3. (C) Immunohistochemistry for phospho-ERK1/2. Scale bar 50μm. (D) Co-immunofluorescence for Dkk1 (green), CK19 (red), pERK1/2 (magenta) and DAPI (blue). Arrows: Dkk1 positive cells. Scale bar 25μm. Primary pancreatic cancer cells 65671 were treated with rDkk1: (E) RT-qPCR for Wnt/β-catenin target genes Axin2 and Lef1. Data represent mean ± SEM, n=3. The statistical difference was determined by two-sided Student's t- test. (F) Western blot for phospho-ERK1/2, total ERK, phospho-Akt, total Akt and GAPDH.

Figure 5

Figure 5. Wnt signaling is required for Kras-driven acinar-ductal metaplasia and PanIN proliferation

(A) Transmitted light images, H&E staining and Amylase immunofluorescent staining of KC and KDC pancreatic cell clusters in 3D culture. Arrows: duct structures, asterisks: acinar clusters. Scale bar 20μm. (B) Quantification of duct-like structures at day 3. Data represent mean ± SEM, n=5. The statistical difference was determined by two-sided Student's t- test. (C) PAS staining. Arrows: PAS positive cells. Scale bar 20μm. (D) Experimental design; n=4 mice. (E) Pancreas size (mean ± SEM, n=4). The statistical difference was determined by two-sided Student's t-test. (F) Proliferation index in epithelial and stromal compartments. Data represent mean ± SEM, n=4. The statistical difference was determined by two-sided Student's t-test. (G) Gross morphology of KC and KDC pancreata (scale bar 2mm); H&E and Ki67 staining (scale bar 50μm).

Figure 6

Figure 6. Wnt/β-catenin signaling in Kras-driven pancreatic carcinogenesis

A physiological level of Wnt signaling (low Wnt signaling, magenta arrow) is maintained during the formation of pancreatic cancer precursor lesions. If Wnt signaling levels are altered, either by overexpression (red arrow) or by inactivation (blue arrow), pancreatic carcinogenesis is prevented.

References

    1. Katz MH, Wang H, Fleming JB, Sun CC, Hwang RF, Wolff RA, et al. Long-term survival after multidisciplinary management of resected pancreatic adenocarcinoma. Ann Surg Oncol. 2009;16:836–47. -PMC -PubMed
    1. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011;11:761–74. -PMC -PubMed
    1. Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 2008;321:1801–6. -PMC -PubMed
    1. Morris JP, Wang SC, Hebrok M. KRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma. Nat Rev Cancer. 2010;10:683–95. -PMC -PubMed
    1. Angers S, Moon RT. Proximal events in Wnt signal transduction. Nat Rev Mol Cell Biol. 2009;10:468–77. -PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources