A murine tumor progression model for pancreatic cancer recapitulating the genetic alterations of the human disease

Abstract

This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.

Figure 1

(A) Ras affinity precipitation assay shows increased levels of GTP-bound Ras in pancreatic lysates of transgenic mice (TGF-α) compared with littermate controls (wild type) at the age of 180 d. Immunohistochemical staining for activated Erk1/2 protein in pancreatic tissue sections of TGF-α transgenic mice (C,D) and littermate controls (B) is shown. The brown color indicates immunoreactivity for activated (phosphorylated) Erk1/2. Bar, 100 μm (B,C) or 20 μm (D).

Figure 2

(A) Relative mRNA levels for cyclin D1, cyclin D3, and cyclin E were evaluated in transgenic animals (TGF-α) by Real Time PCR and normalized to endogenous cyclophilin as described in Materials and Methods. Controls were performed with wild-type pancreas for each time point and target individually. One representative control is shown (WT). Immunostains were performed for cyclin D (B,E,H), Cdk4 (C,F,I), and cyclin E (D,G,J) in TGF-α transgenic mice with developing tubular complexes (at 28 d, B–D), or with developed complexes (at 180 d, E–G) and compared with wild-type controls (at 180 d, H–J). (B–D) Cyclin D, Cdk4, and cyclin E localize in small foci of developing tubular complexes and the surrounding fibroblasts. (E–G) Developed tubular complexes show strong immunoreactivity for cyclin D and Cdk4, whereas only single nuclei are positive for cyclin E. (H–J) Littermate controls show only occasional staining for either cyclin D, Cdk4, or cyclin E. (B–F) Bar, 50 μm. Autoradiograph of a representative immunocomplex kinase assay for cyclin D1/Cdk4 activity indicates increased phosphorylation of the *GST-Rb (769–921) substrate in pancreatic lysates from transgenic mice TGF-α at 180 d as compared with littermate controls (wild type).

Figure 3

Proliferating cells are detected by immunofluorescence analysis of BrdU labeling in the pancreas of TGF-α transgenic mice with developing tubular complexes (A) or developed complexes (B) and in littermate controls (C). Arrowheads indicate BrdU-positive nuclei. (D,E) Comparison of immunohistochemical staining for p53 in the premalignant lesions in 180-day-old TGF-α transgenic mice (D) with littermate controls (E). (F) Relative mRNA levels of p21^Cip1, p16^Ink4a, p19^Arf, and p27^Kip1. Controls were performed with wild-type pancreas for each time point and target individually. One representative control is shown (WT). (G) Cumulative tumor incidence of TGF-α, TGF-α/p53^+/−, TGF-α/p53^−/−, p53^+/−, and p53^−/−. Details are described in Materials and Methods.

Figure 3

Proliferating cells are detected by immunofluorescence analysis of BrdU labeling in the pancreas of TGF-α transgenic mice with developing tubular complexes (A) or developed complexes (B) and in littermate controls (C). Arrowheads indicate BrdU-positive nuclei. (D,E) Comparison of immunohistochemical staining for p53 in the premalignant lesions in 180-day-old TGF-α transgenic mice (D) with littermate controls (E). (F) Relative mRNA levels of p21^Cip1, p16^Ink4a, p19^Arf, and p27^Kip1. Controls were performed with wild-type pancreas for each time point and target individually. One representative control is shown (WT). (G) Cumulative tumor incidence of TGF-α, TGF-α/p53^+/−, TGF-α/p53^−/−, p53^+/−, and p53^−/−. Details are described in Materials and Methods.

Figure 4

Histological analysis of two representative pancreatic tumors (A–F) is compared with thymic lymphoma (G–I) and wild-type pancreas (J–L). (A,D,G,J) Each tissue was stained with hematoxylin and eosin. (B,E,H,K) Expression of CD45-LCA (red fluorescence) and cytokeratin 8/18 (green fluorescence) was analyzed by double-immunostaining. (C,F,I,L) Expression of vimentin (red fluorescence) and cytokeratin 19 (green fluorescence) was analyzed by double-immunostaining. Note the histological appearance of pancreatic tumors with ductal structures (asterisk in A) and frequent mitotic figures (arrowhead in D). Pancreatic tumors express cytokeratin 8/18 (B,E) and 19 (C,F) in contrast with the lymphoma, which stains for CD45-LCA (H). Vimentin staining is restricted to fibroblasts and lymphoma (C,F,I). Acinar and duct cells stain for cytokeratin 8/18 in the normal pancreas (K), whereas cytokeratin 19 is restricted to ducts (asterisk in I,L). Pancreatic tumors metastasize to the liver (M) and lung (N) and infiltrate the duodenal wall (O). Metastasis (M,N) originates from the tumor shown in D–F. Bars, 100 μm in H&E stains, 50 μm in immunostains; (O) bar, 160 μm.