YAP1 and TAZ Control Pancreatic Cancer Initiation in Mice by Direct Up-regulation of JAK-STAT3 Signaling

Abstract

Background & aims: Pancreatitis is the most important risk factor for pancreatic ductal adenocarcinoma (PDAC). Pancreatitis predisposes to PDAC because it induces a process of acinar cell reprogramming known as acinar-to-ductal metaplasia (ADM)-a precursor of pancreatic intraepithelial neoplasia lesions that can progress to PDAC. Mutations in KRAS are found at the earliest stages of pancreatic tumorigenesis, and it appears to be a gatekeeper to cancer progression. We investigated how mutations in KRAS cooperate with pancreatitis to promote pancreatic cancer progression in mice.

Methods: We generated mice carrying conditional alleles of Yap1 and Taz and disrupted Yap1 and Taz using a Cre-lox recombination strategy in adult mouse pancreatic acinar cells (Yap1fl/fl;Tazfl/fl;Ela1-CreERT2). We crossed these mice with LSL-KrasG12D mice, which express a constitutively active form of KRAS after Cre recombination. Pancreatic tumor initiation and progression were analyzed after chemically induced pancreatitis. We analyzed pancreatic tissues from patients with pancreatitis or PDAC by immunohistochemistry.

Results: Oncogenic activation of KRAS in normal, untransformed acinar cells in the pancreatic tissues of mice resulted in increased levels of pancreatitis-induced ADM. Expression of the constitutive active form of KRAS in this system led to activation of the transcriptional regulators YAP1 and TAZ; their function was required for pancreatitis-induced ADM in mice. The JAK-STAT3 pathway was a downstream effector of KRAS signaling via YAP1 and TAZ. YAP1 and TAZ directly mediated transcriptional activation of several genes in the JAK-STAT3 signaling pathway; this could be a mechanism by which acinar cells that express activated KRAS become susceptible to inflammation.

Conclusions: We identified a mechanism by which oncogenic KRAS facilitates ADM and thereby generates the cells that initiate neoplastic progression. This process involves activation of YAP1 and TAZ in acinar cells, which up-regulate JAK-STAT3 signaling to promote development of PDAC in mice.

Keywords: Inflammation; Mouse Model; PanINs; Pancreatic Cancer Progression.

Figure 1

YAP1 and TAZ are up-regulated in pancreatitis. (A) Gene set enrichment analysis of transcription data from caerulein-treated pancreata identified enrichment of the conserved YAP1 signatures reported by Yimlamai et al and Cordenonsi et al. Normalized enrichment score (NES) and nominal (NOM) P values are shown. (B) Scheme showing model of caerulein-induced acute pancreatitis in C57BL/6 mice. (C) Quantitative reverse transcription polymerase chain reaction of YAP1/TAZ target genes in caerulein-treated vs PBS-treated pancreata. n = 3 mice each group; means ± SEM are shown; ^∗∗∗P < .005, Student t test. (D) Immunoblots of pancreas lysates from caerulein- and PBS-treated animals. p-Lats1, phosphorylated Lats1 (Ser909); p-Yap1, phosphorylated Yap1 (Ser112). (E) Immunohistochemical stains of YAP1 and TAZ in pancreata of caerulein- and PBS-treated mice. Scale bars = 50 μm. n = 5–7 mice analyzed. (F) Triple immunofluorescence of pancreata from caerulein- and PBS-treated animals showing the duct marker CK19, the acinar marker amylase and either YAP1 or TAZ. Arrowheads indicate ADM cells. Scale bars = 20 μm. n = 5–7 mice analyzed. DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.

Figure 2

YAP1 and TAZ are up-regulated in ADM lesions induced by KRas^G12D. (A) Immunohistochemical analysis of YAP1 and TAZ in normal (LSL-KRas^G12D) pancreas and pancreas from 6-month-old LSL-KRas^G12D;Pdx1-Cre mice. Scale bars = 50 μm. Dotted lines highlight mouse PanIN lesions. n = 4–5 mice analyzed. (B) Immunoblot analysis of pancreas lysates from control LSL-KRas^G12D and LSL-KRas^G12D;Pdx1-Cre mice. p-Lats1, phosphorylated Lats1 (Ser909); p-Yap1, phosphorylated Yap1 (Ser112). (C) Immunohistochemical analysis of YAP1 and TAZ on human pancreatic tissue microarrays. Scale bars = 50 μm. (D, E) Quantification of immunohistochemical staining intensity, ranging from absent (0) to highest (6) for YAP1 (D) and TAZ (E) on human pancreatic tissue microarrays. n = number of tissue cores. Means ± SD are shown. ^∗∗P < .01; ^∗∗∗P < .005, Student t test.

Figure 3

YAP1 and TAZ are necessary and sufficient for ADM induction. (A) Scheme showing in vitro assay for KRas^G12D-induced ADM. (B) Brightfield images of pancreatic acinar cell clusters on day 5 after isolation from LSL-KRas^G12D and LSL-KRas^G12D;Yap1^fl/fl;Taz^fl/fl mice. Cells were infected with adenoviruses encoding either GFP (Ad-GFP) or GFP plus Cre (Ad-Cre) on day 0. White arrows: tubular ductal structures. Scale bars = 50 μm. Quantification of tubular ductal structures of the indicated genotypes with n = 3–4 mice per group; means ± SEM are shown; ^∗∗∗P < .005, n.s., not significant, Student t test. (C) Experimental design of caerulein-induced acute pancreatitis in the acinar-specific and tamoxifen-inducible Ela1-Cre^ERT2 mouse model combined with the R26-LSL-YFP lineage tracer. (D) Triple immunofluorescence of pancreata from R26-LSL-YFP;Ela1-Cre^ERT2 and R26-LSL-YFP;Yap1^fl/fl;Taz^fl/fl;Ela1-Cre^ERT2 mice treated with caerulein as indicated in (C). Scale bars = 20 μm. Dotted white lines indicate ADM lesions. Quantification of GFP-positive ADM cells (CK19/amylase double-positive) as a percentage of total GFP-positive cells in caerulein-treated mice of the indicated genotypes. n = 3 mice per group; means ± SEM are shown; ^∗∗P < .01, Student t test. (E) Scheme showing in vitro assay for YAP1 and TAZ-induced ADM. (F) Brightfield images (upper panels) and GFP signal (lower panels) of acinar cells after 5 days in culture, infected on day 0 with Ad-GFP, Ad-YAP1-5SA-GFP, or Ad-TAZ-S89A-GFP. Scale bars = 25 μm. Quantification of GFP-positive tubular ductal structures at day 5, in n = 3 experiments; means ± SEM are shown; ^∗∗∗P < .005, Student t test. DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.

Figure 4

YAP1/TAZ activation controls JAK-STAT3 signaling via transcription of STAT3. (A) Quantitative reverse transcription polymerase chain reaction (RT-PCR) of the indicated genes and immunoblots of lysates from PANC1 cells expressing short-hairpin RNAs (shRNAs) against YAP1 and TAZ (shYAP/TAZ-#1 and -#2) or non-targeted shRNA control (shNT). n = 3; means ± SEM shown. (B) Immunoblots of primary acinar cell lysates 2 days after isolation and infection with Ad-GFP, Ad-YAP1-5SA-GFP, or Ad-TAZ-S89A-GFP. (C) Quantitative chromatin immunoprecipitation PCR of the LIFR, GP130, and STAT3 loci (promoter and intron regions) using either IgG or an antibody against TEAD4. ZFP37 intron = negative control region; CTGF promoter = positive control region. n = 3 experiments; means ± SEM shown. (D) Scheme showing luciferase expression construct with human STAT3 promoter region. Base pair (bp) numbers indicate position relative to STAT3 transcription start site. Red box indicates GT-IIc TEAD binding motif; genomic sequence of WT GT-IIc shown below. Mutant STAT3 promoter construct with a 6-bp deletion of the GT-IIc site (mut GT-IIc) was used as control. Luciferase expression analysis using wild-type and mutant STAT3 promoter regions in AR42J acinar cells 1 day after transfection with empty vector, YAP1-5SA, or TAZ-S89A. An artificial TEAD luciferase reporter consisting of 8 GT-IIc motifs was used as positive control. n = 3–5 experiments; means ± SEM shown. (E) Immunohistochemistry showing phosphorylated STAT3 (Tyr705) in pancreata of caerulein- and PBS-treated C57BL/6 mice. Scale bars = 50 μm. n = 3 mice analyzed. (F) Immunoblots of pancreas lysates from PBS- and caerulein-treated animals on day 2 and day 7 after treatment. p-Stat3, phosphorylated Stat3 (Tyr705). Quantitative RT-PCR of JAK–STAT3 pathway genes in caerulein-treated vs PBS-treated pancreata 2 days after treatment. n = 3–4 mice per group; means ± SEM shown. ^∗P < .05; ^∗∗P < .01; ^∗∗∗P < .005, n.s., not significant, Student t test.

Figure 5

YAP1 and TAZ are required for KRas^G12D-induced ADM in vivo in response to pancreatitis. (A) Scheme showing experimental design of caerulein-induced acute pancreatitis in cooperation with acinar-specific KRas^G12D mutation. (B) H&E stain and amylase and CK19 antibody stains of pancreata from mice of the indicated genotypes. Scale bars: H&E (top row) = 2 mm; (2nd row) = 200 μm; (3rd row) = 50 μm; amylase = 200 μm; CK19 (5th row) = 200 μm; (6th row) = 50 μm; double immunofluorescence = 50 μm. (C) Quantification of CK19⁺amylase⁺ ADM pancreatic area in pancreatic sections of the genotypes shown in (B). n = 3 mice per genotype; means ± SEM are shown; ^∗∗∗P < .005, Student t test. (D) Immunoblots of pancreas lysates from mice of the indicated genotypes treated as shown in (A). DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.

Figure 6

KRas^G12D- and caerulein-mediated activation of STAT3 depends on YAP1 and TAZ. (A) Quantitative reverse transcription polymerase chain reaction of the YAP1/TAZ target genes Ctgf, Cyr61, and Amotl2 and the STAT3 pathway genes Stat3, Lifr, and Gp130 in pancreas lysates from Ela1-Cre^ERT2, LSL-KRas^G12D;Ela1-Cre^ERT2 and LSL-KRas^G12D;Yap1^fl/fl;Taz^fl/fl;Ela1-Cre^ERT2 mice 7 days after the last tamoxifen injection. n = 3–5 mice per genotype; means ± SEM are shown; ^∗P < .05; ^∗∗P < .01; n.s., not significant, Student t test. (B) Immunoblot analysis of primary acinar cells from wild-type (WT), LSL-KRas^G12D and LSL-KRas^G12D;Yap1^fl/fl;Taz^fl/fl mice, 2 days after isolation and infection with Ad-Cre adenovirus. pStat3, phosphorylated Stat3 (Tyr705). (C) Immunohistochemistry with antibodies against phosphorylated STAT3 (Tyr705) and STAT3 in pancreata of the indicated genotypes. Mice were treated with tamoxifen and caerulein as indicated in Figure 5A. Scale bars = 50 μm. n = 3–5 mice analyzed.

Figure 7

YAP1 and TAZ are required for KRas^G12D-induced PanIN. (A) Scheme showing experimental design of caerulein-induced PanIN formation in the KRas^G12D background. (B) H&E, amylase, and CK19 antibody, and Alcian Blue/periodic acid–Schiff (AB/PAS) stains of pancreata from mice of the indicated genotypes. Scale bars: H&E = 200 μm; CK19 = 200 μm; double immunofluorescence = 50 μm; AB/PAS (4th row) = 200 μm; AB/PAS (5th row) = 50 μm. (C) Quantification of CK19⁺amylase⁺ ADM lesions in control, LSL-KRas^G12D;Ela1-Cre^ERT2 and LSL-KRas^G12D;Yap1^fl/fl;Taz^fl/fl;Ela1-Cre^ERT2 mice. n = 3–4 mice per genotype; means ± SEM are shown; ^∗P < .05, Student t test. (D) Quantification of AB/PAS-positive PanIN lesions in control, LSL-KRas^G12D;Ela1-Cre^ERT2, and LSL-KRas^G12D;Yap1^fl/fl;Taz^fl/fl;Ela1-Cre^ERT2 mice. n = 3–4 mice per group; means ± SEM are shown; ^∗P < .05, Student t test. (E) Scheme showing the roles of YAP1 and TAZ in pancreatic cancer initiation by inducing ADM in response to oncogenic KRas^G12D and inflammation. See text for details.