Vav1 and mutant K-Ras synergize in the early development of pancreatic ductal adenocarcinoma in mice

Hematoxylin and eosin (H&E)–stained pancreata of control, Vav1, K-Ras^G12D, and K-Ras^G12D/Vav1 mice, at 1, 2, 3.5, 5, and 12 mo after the onset of transgene induction were analyzed for the appearance of malignant lesions. (A) Representative pictures of H&E–stained sections taken 3.5 mo after the onset of transgene induction are shown. Scale bar represents 20 μm. (B) The extent of malignant lesions generated in K-Ras^G12D and K-Ras^G12D/Vav1 mice at the indicated time points after onset of transgene induction was calculated. The sum of area occupied by ADM, PanINs, and PDAC lesions (APPD) was measured as a fraction of the total area of the pancreas (APPD%). Numbers of K-Ras^G12D and K-Ras^G12D/Vav1 mice used, respectively, were n = 4 and n = 8 at 1 mo; n = 6 and n = 8 at 2 mo; n = 13 and n = 17 at 3.5 mo; n = 9 and n = 9 at 5 mo; and n = 7 and n = 5 at 12 mo. Significant differences between the two analyzed groups (P < 0.05; t test) are indicated. N.S. refers to statistical nonsignificant differences. SEM are shown.

(A) Representative K-Ras^G12D (left) and K-Ras^G12D/Vav1 (right) stained with anti–pan-cytokeratin Abs are shown. The presence of PDAC is shown in a representative pancreatic section from a K-Ras^G12D/Vav1 mouse 1 mo after transgene induction (square, right panel). Scale bar represents 20 μm. (B) The extent of PDAC present in K-Ras^G12D and K-Ras^G12D/Vav1 mice at the different time points after transgene induction was assessed. The histogram summarizes the incidence of PDAC development in K-Ras^G12D mice and in K-Ras^G12D/Vav1 mice. Two of 26 K-Ras^G12D mice (7.7%) and 12 of 39 K-Ras^G12D/Vav1 mice (30.8%) had developed PDAC lesions. PDAC developed in K-Ras^G12D/Vav1 ranging from 1 to 12 mo post transgenes induction, whereas PDAC in K-Ras^G12D developed in mice of 3.5 and 5 mo post transgene induction. Significance of the difference between them (P < 0.05) was calculated using a two-tailed chi-squared test (Fisher’s exact test). (C) In some K-Ras^G12D/Vav1 mice (see numbers below), before completion of 3.5 mo of transgene induction, their Vav1 expression was discontinued for 20 d by removal of Dox from their drinking water (−Dox). Hematoxylin and eosin (H&E) staining of the pancreata of K-Ras^G12D and K-Ras^G12D/Vav1 either treated with Dox and tamoxifen (+Dox) or in which Dox was removed from their drinking water for 20 d before completion of 3.5 mo of transgene induction (−Dox) was performed. All the mice were then analyzed for the appearance of malignant lesions. The number of malignant lesions generated in these mice was calculated as APPD%, both for those treated with Dox (+Dox; n = 13 and n = 17, respectively) and for those in which Dox was omitted for 20 d (−Dox; n = 3 and n = 7, respectively). Significant differences between the two analyzed groups (P < 0.05; t test) are indicated. N.S. refers to statistical nonsignificant differences. SEM are shown. (D) Pancreatic sections from K-Ras^G12D/Vav1 mice after 3.5 mo post transgene induction either treated with Dox (+Dox) or in which Dox treatment was discontinued for 20 d (−Dox) were either stained by H&E (upper panel) or anti-GFP Abs (lower panel). Representative pictures are shown. Scale bar represents 200 μm.

Proliferation of acinar and ductal pancreatic cells obtained, at different times after initiation of transgene induction, from control, Vav1, K-Ras^G12D, and K-Ras^G12D/Vav1 mice was assessed by anti-Ki-67 staining. (A) Representative pictures of Ki-67–stained pancreatic sections taken from mice 5 mo after the start of transgene induction. Scale bar represents 50 μm. (B) Proliferation was quantified by counting 10 fields from each pancreatic section and calculating the mean value. The numbers of mice used for Ki-67 staining calculations are recorded in Table S1. To obtain the Ki-67 staining ratio in each case, the result at each time point was divided by the result obtained for the control at the same time point. Significant differences between the two analyzed groups (P < 0.05; t test) are indicated. N.S. refers to statistical nonsignificant differences. SEM are shown.

(A) Pancreatic sections from 4 K-Ras^G12D and 8 K-Ras^G12D/Vav1 mice treated with tamoxifen and Dox for 5 mo were stained for pEGFR (upper panel) and total EGFR (lower panel). Representative pictures at two magnifications are shown. The scale bars represent either 50 μm (left pictures in each group) or 20 μm (right pictures in each group) as indicated. (B) Pancreatic sections from K-Ras^G12D and K-Ras^G12D/Vav1 mice treated with tamoxifen and Dox for 5 mo were stained for pERK. Representative pictures are shown. Scale bar represents 20 μm. (C) Lysates from pancreatic tissues of 5-mo post-transgene induction from 3 control, 2 Vav1, 3 K-Ras^G12D, and 2 K-Ras^G12D/Vav1 mice were Western blotted using anti–phospho-Erk and anti-Erk (left panel). A representative blot from four independent experiments is shown. Relative Erk phosphorylation in the pancreata of control, Vav1, K-Ras^G12D, and K-Ras^G12D/Vav1 mice at 1, 2, 3.5, and 5 mo after the onset of transgene induction was calculated by quantifying their Western blots using ImageJ 1.49V software (right panel). Number of mice in these experiments are outlined in Table S1. SEM are shown.

Rac1–GTP activation in the pancreata of control, Vav1, K-Ras^G12D, and K-Ras^G12D/Vav1 mice was analyzed. (A) Western blotting using anti–Rac1–GTP Abs, anti-Rac1, and anti-actin were used to evaluate Rac1–GTP levels in protein lysates from pancreatic tissues of control, Vav1, K-Ras^G12D, and K-Ras^G12D/Vav1 mice (two mice in each group) at 5 mo after transgene induction. The vertical black lines delineate sliced images that juxtapose lanes that were nonadjacent in the gel. (B) The levels of Rac1–GTP versus total Rac1 at the pancreata of control, Vav1, K-Ras^G12D, and K-Ras^G12D/Vav1 mice at 1, 2, 3.5, and 5 mo after initiation of transgene induction were calculated. Each Rac1–GTP/Rac1 was then divided by the control ratio. Band intensity of the Western blots was quantified using ImageJ 1.49V software. Number of mice in these experiments are outlined in Table S1. SEM are shown. (C) Immunofluorescence of Rac1–GTP of the pancreata from 2 control, 3 Vav1, 3 K-Ras^G12D, and 4 K-Ras^G12D/Vav1 mice, 3.5 mo post-transgene induction was performed. Representative pictures are depicted. Scale bar represents 25 μm. All sections were also stained with anti–Alexa Flour 594 dye for background analysis and found negative.

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