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. 2016 Nov;18(11):666-673.
doi: 10.1016/j.neo.2016.08.009. Epub 2016 Oct 18.

Fbxw7 Deletion Accelerates KrasG12D-Driven Pancreatic Tumorigenesis via Yap Accumulation

Qiang Zhang  1 , Yaqing Zhang  2 , Joshua D Parsels  1 , Ines Lohse  1 , Theodore S Lawrence  1 , Marina Pasca di Magliano  2 , Yi Sun  3 , Meredith A Morgan  4
Affiliations

Fbxw7 Deletion Accelerates KrasG12D-Driven Pancreatic Tumorigenesis via Yap Accumulation

Qiang Zhang et al. Neoplasia. 2016 Nov.

Abstract

Pancreatic cancers driven by KRAS mutations require additional mutations for tumor progression. The tumor suppressor FBXW7 is altered in pancreatic cancers, but its contribution to pancreatic tumorigenesis is unknown. To determine potential cooperation between Kras mutation and Fbxw7 inactivation in pancreatic tumorigenesis, we generated P48-Cre;LSL-KrasG12D;Fbxw7fl/fl (KFCfl/fl) compound mice. We found that KFCfl/fl mice displayed accelerated tumorigenesis: all mice succumbed to pancreatic ductal adenocarcinoma (PDA) by 40 days of age, with PDA onset occurring by 2 weeks of age. PDA in KFCfl/fl mice was preceded by earlier onset of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions, and associated with chromosomal instability and the accumulation of Fbxw7 substrates Yes-associated protein (Yap), c-Myc, and Notch VSports手机版. Using KFCfl/fl and FBXW7-deficient human pancreatic cancer cells, we found that Yap silencing attenuated growth promotion by Fbxw7 deletion. Our data demonstrate that Fbxw7 is a potent suppressor of KrasG12D-induced pancreatic tumorigenesis due, at least in part, to negative regulation of Yap. .

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Figures

Figure 1
Figure 1
Deletion of mouse Fbxw7 accelerates KrasG12D-driven pancreatic tumorigenesis. (A) Genetic makeup of the KFC model. (B) Western blot analysis of Fbxw7 protein levels in KC, KFCfl/+, and KFCfl/fl pancreata specimens from 1-week-old mice. (C) The survival of KC, FC, KFCfl/+, and KFCfl/fl mice is expressed using the Kaplan-Meier method, and statistically significant differences from KC mice are shown (P < .0001****). (D) H&E staining of pancreata at 1 and 6 months. Scale bar, 100 μm. Each image is representative of at least three independent animals.
Figure 2
Figure 2
Pancreatic tumor progression in KFCfl/fl mice. (A) H&E staining of KC, KFCfl/+, and KFCfl/fl pancreata at 1, 2, and 3 weeks. Scale bar, 100 μm. (B) Pathologic analysis of the lesions of KC, KFCfl/+, and KFCfl/fl pancreata at 1, 2, and 3 weeks. Data represent at least three independent mice for each genotype and time point. (C) Deletion of Fbxw7 accelerates ADM. Transmitted light images of control, KC, and KFCfl/fl pancreatic cell clusters in 3D culture from day 0 to 3 are shown. Scale bar, 100 μM. (D) Quantification of ductlike structures. Statistically significant differences were determined by a two-sided, unpaired Student's t test and are indicated (P < .01**).
Figure 3
Figure 3
Analysis of Fbxw7 substrates in pancreata. (A) Western blot analysis of the indicated proteins in lysates obtained from pancreata of 1-week-old mice for each of the indicated genotypes. (B) Immunohistochemical assessment of Yap, c-Myc, Notch, and Ki67 in KC, KFCfl/+, and KFCfl/fl pancreata at 1 week. Scale bar, 100 μm.
Figure 4
Figure 4
Depletion of Yap rescues growth in Fbxw7-deficient mouse and human pancreatic cancer cells. (A) Western blot analysis of c-Myc, Yap, and Notch proteins in KFC primary tumor cells with depletion of Yap. FLAG-FBXW7 overexpression in KFC tumor cells was used as a control. (B) The growth of KFC primary cells was measured with or without Yap depletion by colony forming assays. (C) Western blot detection of YAP, c-MYC, Notch, and FBXW7 levels in Panc-1 cells with or without FBXW7 and/or YAP depletion. (D) The growth of Panc-1 cells with or without FBXW7 and/or YAP depletion was assessed by colony formation assays. Images are representative of at least two independent experiments (A, C). Data are expressed as the mean ± SE of three independent experiments, and statistical significance is indicated (P < .05*, < 0.01**, or <0.001***) (B, D).
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