APC Haploinsufficiency Coupled with P53 Loss Sufficiently Induces

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ORIGINAL ARTICLE APC haploinsufﬁciency coupled with p53 loss sufﬁciently induces mucinous cystic neoplasms and invasive pancreatic carcinoma in mice

T-L Kuo1, C-C Weng1, K-K Kuo2,3, C-Y Chen3,4, D-C Wu3,5,6, W-C Hung7 and K-H Cheng1,3,8

Adenomatous polyposis coli (APC), a tumor-suppressor gene critically involved in familial adenomatous polyposis, is integral in Wnt/β-catenin signaling and is implicated in the development of sporadic tumors of the distal gastrointestinal tract including pancreatic cancer (PC). Here we report for the first time that functional APC is required for the growth and maintenance of pancreatic islets and maturation. Subsequently, a non-Kras mutation-induced premalignancy mouse model was developed; in this model, APC haploinsufficiency coupled with p53 deletion resulted in the development of a distinct type of pancreatic premalignant precursors, mucinous cystic neoplasms (MCNs), exhibiting pathomechanisms identical to those observed in human MCNs, including accumulation of cystic fluid secreted by neoplastic and ovarian-like stromal cells, with 100% penetrance and the presence of hepatic and gastric metastases in 430% of the mice. The major clinical implications of this study suggest targeting the Wnt signaling pathway as a novel strategy for managing MCN.

Oncogene (2016) 35, 2223–2234; doi:10.1038/onc.2015.284; published online 28 September 2015

INTRODUCTION polyposis who presented concurrent solid pseudopapillary tumor, Pancreatic cancer (PC) is the fourth most common cause of adult a large encapsulated pancreatic mass with cystic and solid 12 cancer mortality and among the most lethal human cancers. components. Somatic mutations in APC are frequently observed The 5-year survival is only 6%.1–3 Unlike other malignancies, no in sporadic colon and rectal tumor, however, only rare mutations marked improvement has been achieved in PC survival. (o4%) are reported in pancreatic ductal adenocarcinomas – The signature molecular alterations in PC include multiple (PDACs).6,13 15 It is notable that somatic mutations in the APC evolutionary steps of the precursor lesions of which progression gene are found more commonly in rare types of pancreatic involves the acquisition of mutations in Kras, Ink4a, p53, SMAD4 tumors, such as solid pseudopapillary tumors acinar carcinomas and adenomatous polyposis coli (APC) or β-catenin.2,4–6 Recently, and pancreatoblastomas.16,17 Alternatively, several independent studies have recognized that PC can develop from three distinct studies revealed that the activation of Wnt pathway through types of precursor lesion that affect the pancreatic ducts: epigenetic downregulation of APC or secreted frizzled-related pancreatic intraepithelial neoplasms (PanINs), which are small protein (SFRP) genes or increased Wnt ligand secretion are often and focal; intraductal papillary mucinous neoplasms, which are found associated with more advanced human PanINs and – moderate-sized, papillary cystic lesions lined by mucin-producing PDAC.18 21 tall columnar epithelium; and mucinous cystic neoplasms (MCNs), APC negatively regulates the Wnt/β catenin pathway and 22,23 comprising oligomegacysts with a single thin layer of cuboidal promotes cytosolic β-catenin polyubiquitination and degradation. and flattened epithelium and associated progesterone receptor During embryogenesis, the repression of embryonic Wnt signaling (PR)+ as well as estrogen receptor (ER)+ ovarian-like stroma.7–9 is required for gastrointestinal and hepatopancreas progenitor These lesions exhibit distinct histopathological characteristics and specifications and development.24 Wnt⧸β-catenin is reactivated in clinical significance but share a common mutation profile. The PanINs, and its expression levels gradually increase during disease basis of these biological differences is unknown but may be progression.21,25 In addition to targeting β-catenin for degrada- associated with the cell of origin, various mutation combinations, tion, APC is also involved in microtubule dynamics, cell polarity – the order of the mutational events or other factors.5 and chromosome segregation.26 28 Studies of genetically mod- The APC gene was first characterized as a crucial tumor- ified APC-deficient mouse strains demonstrated that APC is crucial suppressor gene of the distal gastrointestinal tract, and germline in colon, skin, thymus and nervous system development, – mutations in APC cause familial adenomatous polyposis.10,11 as well as neoplasia.29 31 To understand APC-mediated tumor Le Borgneeta and Farahmand et al. recently described a 14-year- suppression in pancreatic tumorigenesis, we first assessed its old girl and a 29-year-old man with familial adenomatous role in pancreatic organogenesis and whether APC loss affects

1Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; 2Division of Hepatobiliopancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 3Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Department of Medical Imaging, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 5Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 6Division of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; 7National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan and 8Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan. Correspondence: Dr K-H Cheng, Institute of Biomedical Sciences, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung, 80424, Taiwan. E-mail: [email protected] Received 27 October 2014; revised 3 June 2015; accepted 22 June 2016; published online 28 September 2015 APC haploinsufficiency in the mouse pancreas T-L Kuo et al 2224 pancreatic development or homeostasis. Subsequently, a non-Kras (Po0.01; Figure 2c). Macroscopic examination and histological mutation-induced premalignancy mouse model was developed; in analysis revealed that the pancreatic lesions arising from the mice this model, heterozygous loss of one APC allele coupled with p53 were MCN (Figures 2d and e). Hyperlipidemia frequently occurs in deletion markedly accelerated pancreatic tumor progression patients with pancreatic disease and this MCN mouse model also in mice. exhibited elevated plasma cholesterol and triglyceride levels (Supplementary Figure 4). RESULTS For serial kinetic histopathological analysis of the pancreas, the Pdx1-CreAPCCKO/+p53L/L mice were killed and autopsied Conditional APC deficiency lethally impairs fetal islet development at 7, 14 and 24 weeks (Figure 2d). As shown in Figure 2e, in newborn mice histopathological analysis revealed that the pancreatic tissue of To assess the effects of APC deletion on pancreatic morphogen- the Pdx1-CreAPCCKO/+p53L/L mice demonstrated the full spectrum CKO CKO esis, APC / mice were crossed with the Pdx1-Cre transgenic of MCNs lesions, including cystic lesions that increased in size strain, which directs the expression of Cre recombinase to the (42 cm) at later time points. All of these mice (100%) exhibited epithelial lineages of the pancreas during embryogenesis. The large cystic pancreata namely mucinous cystadenomas, character- allele was engineered to sustain Cre-mediated deletion of exon ized by the presence of unilocular megacystic lesions with 14, resulting in loss of the APC protein (Supplementary Figure 1). mucoid/watery cyst content, and nodules or peripheral calcifica- The control and heterozygous mice exhibited no discernible CKO tion on the cyst wall resembling human MCN (Supplementary phenotypes (Supplementary Figure 2). APC homozygous mice Figure 5). Alcian blue and periodic acid-Schiff staining revealed harboring Pdx1-Cre transgenic strains were documented using fi mucin secreting in murine MCNs (Figure 2f). Immunohistochem- allele-speci c PCR genotyping and western blot analysis to istry (IHC) assessment of the murine MCNs stained positive for delete the locus and eliminate the truncated APC protein in the Mucin 4 (Figure 2g). Most importantly, the MCN lesions in these pancreas, separately (Supplementary Figure 1 and data not mice exhibited typical human MCN features with high levels of PR shown). and ER immunostaining around the stromal cells (Figure 2h). When the Pdx-1CreApcCKO/+ and APCCKO/CKO mice were interbred, no Pdx1-CreAPCCKO/CKO null pups were obtained after weaning (none of the 62 neonatally genotyped mice were Immunopathological characterization of MCN in mice with Pdx1-CreAPCCKO/CKO positive), strongly suggesting in utero or conditionally inactivated APC and p53 neonatal lethality. During embryogenesis, the various living Subsequently, the accessory signaling pathways in the murine genotypes of the mouse embryos were recovered at a Mendelian MCN lesions were assessed, IHC analysis of the lesions at 14 and ratio. No gross phenotypic differences were observed between 24 weeks implicated cellular differentiation and early develop- the embryos during dissection on embryonic day (E)10, E12, E14 ment of signaling pathways during the formation of MCNs and E16 (Figures 1a, i and q; data not shown). APC-positive revealing positive staining for the epithelial ductal markers cells were detected in a sub-population of pancreatic progenitor Dolichos biflorus lectin and E-cadherin, transforming growth wt cells in developing pancreata of Pdx1-CreAPC mouse embryos, factor β1, bone morphogenetic protein 4 (BMP4), Wnt1, Notch1/ CKO/CKO but not in prenatal pancreas of Pdx1-CreAPC or Pdx1- Hes1, epidermal growth factor receptor (EGFR) and phospho-Akt, CKO/CKO L/L CreAPC p53 embryos (Figures 1b, c, j, k, r and s). and lack of acinar (amylase) and islet (insulin) marker expression CKO/CKO Remarkably, the Pdx1-Cre APC mice exhibited reduction in (Figure 3 and data not shown). Tumor microenvironment analysis beta-cell mass and islet number disrupting the maturation of the revealed intense collagen type I (col-1), α-smooth muscle actin prenatal pancreas (Figures 1d and f, l-n, t-v and Supplementary and vimentin immunostaining predominantly localized in the Figure 3). Mechanistic analyses showed that the Pdx-1Cre- fibroblast compartment of the MCNs, indicating abundant tumor- fi mediated APC-de cient mice exhibited altered gastrointestinal reactive stromal elements. Ki67 staining revealed that the development associated with duodenal atresia causing neonatal proliferative activity in these MCNs was similar to that in Pdx1- death (Figures 1g, h, o, p). CreLSLKrasG12Dp53L/L mice. However, these lesions revealed more intense terminal deoxynucleotidyl transferase-mediated Heterozygous loss of APC and p53 inactivation promote MCN dUTP nick end labeling (TUNEL) staining than did the Pdx1- progression in mice CreLSLKrasG12Dp53L/Llesions (Supplementary Figure 6). A previous study using APCMin/+ (Min, multiple intestinal neoplasia) mice reported that the increased multiplicity and invasiveness of Metastatic potential of murine MCNs in Pdx1-CreAPCCKO/+p53L/L fi 32,33 intestinal adenomas were associated with p53 de ciency. P53 mice is involved in the transcriptional upregulation of APC gene Although most Pdx1-CreAPCCKO/+p53L/L mice had to be killed at expression in response to DNA damage, and p53 is inactivated 34 24–26 weeks of age, this genotype could survive for up to in 450% of PDAC, particularly late-stage tumors. To evaluate the 32 weeks. The overall incidence of pancreatic carcinoma association between APC and p53 defects in PC, the phenotypes 36 CKO/CKO associated with MCN in human is 15 ~ 20%. The Pdx1- of the mice with Pdx1-Cre-mediated deletion of the APC CKO/+ L/L allele on a p53lopx/loxp background were compared. PCR genotyp- CreAPC p53 tumors exhibited a higher incidence of meta- ing of the offspring was performed as described in the detailed stasis and invasion (Figure 4a and Supplementary Table 1). The Materials and methods section (Figure 2a). The expression levels murine MCNs became malignant PCs with nuclear anaplastic of the altered APC and p53 alleles and β-catenin were confirmed features (Figures 4b and c), and demonstrated stomach, duodenal using western blot analysis (Figure 2b). As reported previously, or intestinal invasion or liver or lung metastasis (Figures 4a i,ii and CKO/+ Pdx1-Crep53L/L mice with germline wild-type APC were generally d and Supplementary Table 1). APC expression plus p53 healthy until the age of 50 weeks.35 In this setting, we still knockout tumors were substantially invasive and metastatic observed that homozygous APC deletion results in neonatal death. and, thus, consistent with aggressive human pancreatic cystic Strikingly, all Pdx1-CreAPCCKO/+p53L/Lmice (n = 51) developed a adenocarcinoma. IHC analysis revealed persistence of high nuclear swollen abdomen with a palpable abnormal mass between 16 and c-myc and active-β catenin expression levels in invasive and 24 weeks. Pairwise log rank tests revealed that the average metastatic lesions (Figures 4e i-viii), as well as high levels of the survival of Pdx1-CreAPCcko/+p53L/L mice was significantly shorter metastatic protein S100A4, a target gene of the Wnt/β-catenin than that of the Pdx1-Crep53L/L and Pdx1-CreAPCCKO/+ mice pathway37 (Figures 4e ix-xii).

Figure 1. Pdx1-Cre-mediated conditional deletion of APC suppresses islet and pancreatic development and leads to fatal duodenal obstruction. Hematoxylin and eosin (H&E) staining of sagittal sections through the abdominal cavity of E14 mouse embryos of the indicated genotypes (a, i, q); scale bar is 2mm. H&E sections of developing pancreas from Pdx-1CreAPCCKO/CKO (b), Pdx-1CreAPCCKO/CKOp53L/L (j) and control Pdx-1Crep53L/L (r) embryos. Immunohistochemical staining shows an APC-positive sub-population in the developing pancreata of Pdx-1Crep53L/L mouse embryo (r, red arrow in s), but not in the Pdx-1CreAPCCKOCKO (b, c) and Pdx-1Cre APCCKOCKOp53L/L (j, k) embryos. Immunoﬂuorescent co-staining for insulin (red) and amylase (green) or glucagon (green) alone were performed on sections of the pancreas from Pdx-1CreAPCCKO/CKO (d, e, f), Pdx-1CreAPCCKO/CKOp53L/L (l, m, n) and control Pdx-1Crep53L/L (t, u, v) mouse embryos at E14. Nuclear DNA stained with 4′,6-diamidino -2-phenylindole (DAPI). Arrow in (e, m) points to β cell with reduced insulin staining. Low and higher magniﬁcation sections of the duodenum mucosa from Pdx-1CreAPCCKO/CKO (g, h), Pdx-1CreAPCCKO/CKOLp53L/L (o, p) and control Pdx-1Crep53L/L (w, x) embryos. Arrowheads indicate the duodenal atresia (g, o). Scale bar, 50 μm.

Identification of APC haploinsufficiency and p53 loss-induced protein 1, of which the expression was significantly increased cytokines by using mouse cytokine array system compared with that in normal pancreatic tissue. The expression of Next, we investigated which cytokines are responsible for the all other cytokines was similar (Figures 5b i, ii). rapid MCN tumor progression in Pdx1-CreAPCCKO/+p53L/L mice, To validate the cytokine array data, we investigated the protein using a mouse cytokine array system to detect 26 mouse expression levels of FasL, tumor necrosis factor receptor 1, cytokines (Figure 5a). Potential APC haploinsufficiency and macrophage inflammatory protein 1γ, IL-6 or monocyte chemoat- p53-loss-induced cytokines in the cystic fluid of the pancreata tractant protein 1 by using western blotting, which revealed that included FasL, soluble tumor necrosis factor receptor 1, IL-6, FasL, monocyte chemoattractant protein 1, tumor necrosis interleukin-1β (IL-1β), IL-6, macrophage inflammatory protein 1γ, factor receptor 1, macrophage inflammatory protein 1γ and keratinocyte chemoattractant and monocyte chemoattractant transforming growth factor β1 proteins were highly overexpressed

Figure 2. Concomitant APC haploinsufficiency and p53 loss drive cystic tumors of the pancreas in mice. (a) Specific PCR analyses to detect APC and p53 wild types and loxp alleles from wild-type, APCCKO/+, APCCOK/CKO, p53L/+, p53L/L offspring. (b) Western blot analyses for detection of p53, β-catenin and APC protein expression in pancreatic lysates from Pdx-1Cre control, Pdx1-Crep53L/L and Pdx1-CreAPCCKO/+ p53L/L mice. β-Actin is shown as a loading control. (c) Kaplan–Meier curve showing significantly reduced survival time of Pdx1-CreAPCCKO/+p53L/L mice compared with Pdx1-CreAPCCKO/+ wild-type and Pdx1-Crep53L/L mice. (d) Gross pathology of murine MCN lesions in Pdx1-CreAPCCKO/+p53L/L mice at different ages. (e) Histological analysis of pancreas from wild-type and Pdx1-CreAPCCKO/+p53L/L mice at different ages by hematoxylin and eosin (H&E) staining. (f) Periodic acid–Schiff (PAS) and Alcian blue staining revealed mucin content in cystic lesions of murine MCNs. Scale bar is 10 μm (insets), 50 μm (all other images). (g) IHC for Mucin4 revealed very strong expression in murine MCNs compared with normal pancreas. (h) IHC analysis using anti-PR and anti-ER antibodies show strong PR and ER expression levels in the stroma of murine MCNs. IHC staining for PR and ER confirmed the nuclear localization as well. Scale bar, 50 μm.

in the mouse MCN tumor samples (Figure 5c). Immunoblotting communication and adhesion. Gene Ontology (GO) revealed the analysis also revealed increased induction of cleaved caspase 3 in upregulation of 220 genes and the downregulation of 562 genes the Pdx1-Cre APC/CKO/+p53L/L pancreata (Figure 5c). (42-fold change, Po0.05) compared with normal Pdx1-Crep53L/L ductal cells. The analysis revealed the top 10 gene sets shown in Figure 6a were the most significantly upregulated or down- Concurrent heterozygous loss of APC and p53 affects the Wnt regulated in the Pdx1-Cre APCCKO/+p53L/L cells, suggesting their pathway to induce MCN progression in mice potential involvement in MCN pathogenesis (Supplementary Table 2). To investigate the molecular mechanisms underlying MCN forma- To validate the microarray analysis results, APC/p53-loss- tion and progression mediated by APC/P53 loss in the pancreas, mediated regulation of the top selected and various known Wnt microarray analysis was used to compare the gene expression in target genes (TBX15, FGF7, IGFBP4, TWIST2, TCF4 and SNAIL2) CKO/+ L/L early-passage PC cell lines from the Pdx1-CreAPC p53 and was verified using quantitative reverse transcriptase–PCRs. All L/L Pdx1-Crep53 models (Supplementary Figure 7). The APC/P53 analyzed genes showed significantly higher mRNA levels in the tumor cells were characterized by distinct gene expression Pdx1-CreAPCCKO/+p53L/L cells than in the Pdx-1Crep53L/L cells signatures mainly annotating the transmembrane receptor (Po0.01, 11-, 7.2-, 36-, 8-, 10.6- and 8.3-fold for TBX15, FGF7, tyrosine kinase signaling pathway for regulating of cell IGFBP4, TWIST2, GCNT4 and B-CAT, respectively) (Figure 6b).

Figure 3. IHC characterization of MCNs in Pdx1-CreAPCCKO/+p53L/L mice. Premalignant MCN lesions from Pdx1-CreAPCCKO/+p53L/L mice collected at 7 and 24 weeks and normal pancreas control were stained with Dolichos biﬂorus lectin (DBA) (red), anti-E-cadherin, anti-transforming growth factor β1(TGFβ1), anti-BMP4, anti-Wnt1, anti-Notch1, anti-Hes1, anti-α-smooth muscle actin (SMA), anti-collagen type 1 (col-1), anti- Vimentin, anti-EGFR and anti-pAkt antibodies. Epithelial cells lining the cysts showed positive staining for E-cadherin and DBA. Note the increasingly intensive immunoreactivity of TGFβ1, BMP4, Wnt1, Notch1 and EGFR, and strong nuclear expression of Hes1 and pAkt in cystic lesions and columnar epithelium following MCN progression, and also high expression of SMA, Vimentin and collagen type 1 in surrounding stromal cells. Scale bar is 50 μm.

The epithelial-mesenchymal transition-related genes involving role of APC in mitosis and to demonstrate the underlying causes increased cellular invasiveness such as Snail2, Twist2, Vimentin of chromosome aneuploidy, cycling and nocodazole-arrested and Col-1 were upregulated, whereas the epithelial marker, Pdx1-CreAPCCKO/+P53L/L cells and Pdx1-CreP53L/L control cells were including E-cadherin and Cldn 1 exhibited significantly decreased stained with propidium iodide and their DNA content was mRNA expression levels compared with those in the Pdx-1Crep53L/L analyzed by fluorescence-activated cell sorting (FACS). A high cells (Figure 6b). In addition, upregulation of several micro- percentage of cycling Pdx1-CreAPCCKO/+p53L/L cells contained 4N RNAsmiRNA), such as miR-19b, miR-125 and miR145 associated DNA content (18% compared with 5% in the Pdx1- with the Wnt signaling pathway, were confirmed as well38,39 CreLSLKrasG12Dp53L/L cells) (Figure 6d). Next, cells were then (Figure 6b). In conclusion, the data highlight the importance of arrested at the G0 phase through serum starvation, and both Wnt signaling pathway implicated in development of MCNs. spindle formation and cytokinesis were analyzed during the initial Moreover, IHC analysis conducted using anti-FGF7, anti-Twist2 and mitotic phase 8 h after release from the arrest. Immunofluorescent anti-Tbx15 confirmed that FGF7, Tbx15 and Twist2 protein co-staining for α-tubulin as well as NuMA revealed that numerous expression was predominantly increased in premalignant MCN Pdx1-CreAPCCKO/+p53L/L, but not Pdx1-CreLSLKrasp53L/L cells, with and carcinoma lesions (Figure 6c). multiple centrosomes displayed multipolar spindle formation and nucleation (Figure 6e). APC haploinsufficiency and p53 loss promotes mitotic chromosome instability in murine MCNs IWP-2 Wnt pathway inhibitor reduces MCN formation CKO/+ L/L APC was found to interact with the plus ends of the microtubules in Pdx1-CreAPC p53 mice and to modulate the kinetochore–microtubule attachments. The To determine whether MCNs formation and progression are mutated forms of APC can alter the mitotic spindle axis Wnt signaling pathway dependent, the Wnt inhibitor IWP-2 orientation, resulting in chromosome missegregation.26,40 Intrigu- was selected to treat primary PDAC mouse cells, as IWP-2 ingly, several mitotic spindle checkpoint genes associated with can prevent Wnt/β-catenin/Tcf signaling activation following chromosomal instability and aneuploidy showed significantly APC loss.44 IWP-2 strongly inhibited the proliferation of the higher mRNA levels in the Pdx1-CreAPCCKO/+p53L/L cells than in primary Pdx1-CreAPCCKO/+p53L/L cells in vitro compared with the the Pdx1-Crep53L/L cells (Po0.05; Figure 6b).41–43 To evaluate the IWP-2-treated Pdx1-Crep53L/L, Pdx1-CreLSLKrasG12Dp53L/L and

Figure 4. Tumor invasion and metastasis of cystic neoplasms in Pdx1-CreAPCCKO/+ p53L/L mice. (a) Gross photograph of a malignant MCN with invasive carcinoma arising in a Pdx1-CreAPCCKO/+ p53L/L mouse. The primary malignant MCN directly invades and compresses the proximal duodenum; also note liver metastasis and stomach invasion. L, liver; G, gallbladder; D, duodenum; S, stomach. Insets i, hepatic metastasis; ii, lung metastasis. (b) Histological features of malignant MCN in Pdx1-CreAPCCKO/+p53L/L mice with high-grade lesions containing intratumoral malignant epithelium and mucin. Scale bar is 50 μm. (c) High magnification of the abnormal anaphase figures in tumor sections. Scale bar, 20 μm. (d) A 6-month-old Pdx1-CreAPCCKO/+ p53L/L mouse with well-differentiated hepatic metastasis (i). PDAC invaded the duodenal (ii) and gastric wall (iii) in a 7.5-month-old Pdx1-CreAPCCKO/+ p53L/L mouse. Lung metastasis was observed in an 8-month-old Pdx1-CreAPCCKO/+ p53L/L mouse (iv). Scale bar is 50μm. (e) IHC analysis detected intensive c-myc (i–iv), active-β catenin (ABC) (v–viii) and S100A4 (ix–xii) nuclear staining in MCN lesions (arrows in i, v, ix), malignant MCN with associated invasive carcinoma (iii, ix) and hepatic (iii, vii), lung (iv, viii, xii) and stomach metastasis (xi). The arrows represent the areas shown at higher magnification in ii, vi and x. Scale bar is 50 μm.

dimethylsulfoxide-treated control cells, indicating that active Wnt decreased total and activated protein levels of β-catenin signaling is essential for the growth of PC cells derived from the compared with the dimethylsulfoxide control groups (Figure 7e). Pdx1-CreAPCCKO/+p53L/L MCN model (Figure 7a). Western blot and fluorogenic cleaved caspase 3 activity assays confirmed that IWP-2 treatment effectively inhibited Wnt signaling and induced caspase DISCUSSION 3-mediated apoptosis in the Pdx1-CreAPCCKO/+P53L/L cells The molecular mechanisms through which APC inactivation (Figure 7b and Supplementary Figure 8). FACS analysis also contributes to PC pathogenesis remain unclear, and a mouse demonstrated that the SubG1 apoptotic population significantly model for studying APC dependency in PC has not been increased in the IWP-2-treated Pdx1-CreAPCCKO/+p53L/L cells developed. This is the first report stating that APC is required for after 48 h, but not in the IWP-2-treated Pdx1-Crep53L/L, Pdx1-Cre islet development (β-cell development and maturation) and LSL-KrasG12Dp53L/L cells (Po 0.01; Figure 7c). To assess the pancreas maturation, and loss of APC function results in the apoptotic response to this compound in vivo, we treated induction of MCN formation in the context of p53 loss. The defects Pdx1-CreAPCCKO/+p53L/L mice (~6 weeks old) with IWP-2 for in islet maturation and normal pancreatic homeostasis in the 12 weeks (Figure 7d). Non-invasive image analysis using a 3.0 T Pdx1-Cre APCCKO/CKO mice contradict the findings reported by MRI scanner (GE, Sigma HDXt, Milwaukee, WI, USA) with a high- Strom et al.45 that APC loss induces postnatal pancreatomegaly resolution animal coil (3.0 cm diameter) performed on the during early pancreatic development, but does not impede dimethylsulfoxide-treated Pdx1-CreAPCCKO/+p53L/L mice clearly dis- pancreas neogenesis in aging mice. Presumably, the differences played hypertrophic pancreas with unilocular cysts (Figure 7e). Such in the flanking loxp sites for depleting the APC gene between two lesions were significantly reduced or absent in the pancreas of mutant mice explain the differences in findings. Our results Pdx1-Cre APCCKO/+p53L/L after IWP-2 treatment for 12 weeks revealed that Pdx-1CreAPCCKO/CKO mouse embryos exhibit a (Po0.01; Figure 7f). Gross autopsy examination and histological neonatally lethal duodenal stenosis, consistent with the expres- analysis using hematoxylin and eosin staining validated the anti- sion of Pdx-1 in the antral stomach and duodenum. Research MCN efficacy of IWP-2 (Figure 7e). IHC analysis confirmed that findings are limited and inconsistent regarding whether APC/β- treatment of Pdx1-Cre APCCKO/+p53L/L mice with IWP-2 resulted in catenin pathway does have a critical role in modulating

Figure 5. Cytokine antibody array analysis of pancreatic cyst fluid in Pdx1-CreAPCCKO/+p53L/L mice. (a) Template alignment of the mouse cytokines in the array. POS, positive; NEG, negative; IL, interleukin; SDF-1, stromal cell-derived factor 1; BLC, B-lymphocyte chemoattractant; TAC, protachykinin; TCA-3, small inducible cytokine A1; TIMP, tissue inhibitors of metalloproteinase; LIX, LPS-induced CXC chemokine; MCSF, macrophage colony-stimulating factor; MCP-1, monocyte chemotactic protein 1; MIG, mitogen-inducible gene; MIP-1, macrophage inflammatory protein 1. (b) Detection of mouse cytokine expression from the cyst fluid of two Pdx1-CreAPCCKO+p53L/L and one representative matched normal pancreatic tissue from Pdx1-Crep53L/L mice by the mouse cytokine array system. (c) Western blot analysis of pancreatic lysates from Pdx1-CreAPCCKO/+p53L/L mice at different time points for the expression of IL-6, FasL, MCP-1, MIP-γ,TGFβ1, TNFR1 and pro and cleavage caspase-3 protein compared with Pdx1-Crep53L/L control mice. GAPDH served as a loading control.

Kras-induced PanIN formation and progression.21,46,47 To elucidate The results obtained by applying a mouse cytokine array system the effects of APC loss in a mutant Krasdriven PC mouse model, demonstrated that proinflammatory cytokines, including IL-4, IL-6, Pdx-1CreAPC null mice were crossed with LSL-KrasG12D mice. Our soluble tumor necrosis factor receptor 1, keratinocyte chemoat- preliminary results confirmed that the inactivation of APC tractant, macrophage inflammatory protein 1γ, monocyte che- impeded Kras-induced PanINs progression (Supplementary moattractant protein 1 and the neutrophil-attracting chemokines Figure 9). Mechanistic studies dissecting how APC loss inhibits LIX (CXCL5) were upregulated in the cystic fluid of MCNs in our Kras-induced PanIN formation are underway. mouse model.49–51 These conclusions were confirmed using This study provided several lines of evidence demonstrating western blot analysis. MCN has been increasingly recognized as that in vivo APC haploinsufficiency and p53 loss sufficiently a crucial clinical condition because of its propensity to progress to promotes pancreatic cell transformation and induces oligocystic metastatic pancreatic carcinoma. Our MCN mouse model demon- pancreatic tumors. In contrast to our previous work demonstrated strated increased nuclear staining of c-Myc, active-β catenin and that Kras-Smad4 mutants develop cystic tumors more predomi- SA100A4 is associated with increased metastatic potential 52–56 nantly resembling intraductal papillary mucinous neoplasm, GeneGo pathway analysis depicted the top three molecular but can also present intraductal papillary mucinous neoplasm networks associated with altered gene expression from primary mixed with multiple small cysts (microcystic) lesions. The Pdx1-CreAPCCKO/+p53L/L PDAC cell lines versus Pdx1-Crep53L/L observation here was supported by a recent study from the Lewis controls were found to be involved in cell-gap adhesion dictating group, who elegantly showed that activated Wnt signaling in alterations in ECM cell adhesion/tight junction, the developmental the tumor stromal microenvironment contributes to the develop- Wnt signaling pathway and the developmental regulation of the ment of pancreatic MCNs in Ptf1a-Cre;LSL-Kras;elastase-tva mice epithelial-mesenchymal transition (Supplementary Figure 10). injected with replication-competent avian sarcoma-Wnt1 viruses.48 Upregulated genes included TBX15, a member of the T-box Immunohistochemical characterization of these MCN lesions family, which may be downstream of Wnt signaling, has an revealed the transforming growth factor β1, BMP4, Notch1, Wnt essential role in the DV patterning of the mouse coat. TBX15 null and EGFR signaling pathways were activated, implying involve- mice display defects in both intramembranous bone formation ment of multiple signaling pathways in the development of MCN. and endochondral ossification.57 Inaddition,DeanTangand

Figure 6. Complementary DNA microarray analysis of primary PDAC cells from Pdx1-CreAPCCKO/+p53L/L and Pdx1-Crep53 L/L mice. (a) Heat-map presentation of gene profiling of primary pancreatic cells established from three Pdx1-CreAPCCKO/+p53L/L and two Pdx1-Crep53L/L control mice showing genes with significantly increased (red), intermediate (black) and decreased (green) expression levels. The numerical values give the actual values on a log 2 scale associated with each color. The full gene name for the gene symbol is available in Supplementary Table 2. (b) Selected target genes that showed changes in the microarray analysis were picked for further verification by quantitative reverse transcriptase–PCR. RNA pools from three Pdx1-CreAPCCKO/+p53L/L primary PDAC cells and two control Pdx1-Crep53L/L primary ductal cells were compared and analyzed. The relative gene expression was normalized to GAPDH expression and compared with the Pdx1-Crep53L/L control. Data represent the means ± s.d. of triplicate samples. *Po0.05, t-test. (c) IHC analysis showed intense staining for FGF7, Tbx15 and Twist2 in premalignant MCN lesions and PDAC compared with normal pancreas. Scale bar, 50 μm. (d) FACS profiles showing DNA content of cell populations derived from Pdx1-CreAPCCKO/+p53L/L, Pdx1-Crep53L/L and Pdx1-CreLSLKrasp53L/L cells. Flow cytometry analysis showed increased aneuploidy (~18%) in Pdx1-CreAPCCKO/+p53L/L PDAC cells compared with Pdx1-Crep53L/L (~1.6%) and Pdx1-CreLSLKrasp53L/L (~3%) cells (Po0.01). (e) Centrosome amplification in Pdx-1CreAPCCKO/+p53L/L PDAC cell lines. Primary PDAC cells prepared from Pdx-1CreAPCCKO/+p53L/L mice exhibited greater than two centrosomes as demonstrated by co-staining with the anti-NuMA (green) and anti-α-tubulin (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm.

colleagues reported that the human prostate cancer periodic acid GCNT4, a glucosaminyl (N-acetyl) transferase 4 core 2, were shown -Schiff − /lo cancer cell population contains cancer stem cells that to have a major role in mucin glycan biosynthesis.62 Additional resist castration. They further demonstrated that the periodic acid experiments are required to investigate the precise role of these − -Schiff /lo cancer cell population contains cells that are relatively genes in APC/P53-loss-induced MCNs. quiescent and exhibit increased expression of several stem cell In conclusion, although the exact mechanism of the APC tumor regulatory genes, including NANOG, ASCL1, NKX3.1 and TBX15.58 The suppressor in PC is not entirely clear, the results of this study cross-talk of the FGF and Wnt pathways may accompany several clearly show that APC is a key regulator that modulates pancreatic biological processes including tumorigenesis.59 FGF7, also known as histopathology and the progression of PC to metastasis. Our data the keratinocyte growth factor, is reportedly related to cancer, suggest that molecular therapies targeting APC and the Wnt including PC. For example, Niu et al.60 reported that FGF7 induced signaling pathways may be novel effective strategies for control- vascular endothelial growth factor-A expression in PC cells. This ling MCN progression. study provides novel information implicating FGF7 in MCN formation and progression. In addition, IGFBP-4 is an antagonist of the Wnt/β-catenin signaling pathway and has been associated MATERIALS AND METHODS with cell growth and metastasis. Reportedly, IGFBP-4 expression in Genetically modiﬁed mice and mouse genotyping metastatic renal cell carcinoma is higher than that in primary renal Pdx-1Cre, LSLKrasG12D, p53Loxp/Loxp and APCCKO/CKO mice, obtained from the cell carcinoma and normal human kidney tissues.61 In regard to Mouse Models of Human Cancers Consortium (MMHCC) under material

Figure 7. Inhibition of Wnt signaling blocks MCN formation in Pdx1-CreAPCCKO/+p53L/L mice. (a) Cell proliferation rates were significantly reduced in Pdx1-CreAPCCKO/+p53L/L PDAC cells treated for 3 days with 20 μM of IWP-2 inhibitor, compared with Pdx1-CreLSLKrasL/+p53L/L PDAC, Pdx1-Crep53L/L cells and untreated groups. C: vehicle treatment; IWP-2: IWP-2 treatment. (b) Immunoblotting analysis revealed dramatically decreased c-myc, β-actenin and procaspase-3 protein levels in Pdx1-CreAPCCKO/+P53L/L PDAC cells following IWP-2 treatment. β-Actin served as a loading control. (c) Representative FACS profiles showing Pdx1-CreAPCCKO/+p53L/L PDAC cells contained a significantly higher (Po0.01) proportion of apoptotic cells followed by IWP-2 treatment than Pdx1-Crep53L/L and Pdx1-Cre LSLKrasp53L/L PDAC cells after IWP-2 treatment. (d) Schematic representation of IWP-2 treatment in Pdx1-CreAPCCKO/+p53L/L mice. The Pdx1-CreAPCCKO/+p53L/L mice aged 7 weeks old were given IWP-2 or vehicle (dimethylsulfoxide (DMSO)) treatment by intraperitoneal injection (10 mg/kg twice a week) for 12 weeks. (e) The treatment efficiency for IWP-2 was monitored by magnetic resonance imaging (MRI) at 24 weeks before killing. Representative MRI images of the abdomen of Pdx1-CreAPCCKO/+p53L/L mice treated intraperitoneally with IWP-2 or vehicle (DMSO) for 12 weeks showing the reduction of MCN formation in the IWP-2-treated Pdx1-CreAPCCKO/+p53L/L mice, but not in the mice receiving DMSO treatment. Macroscopic appearance, hematoxylin and eosin (H&E) histological analysis and anti-β-catenin immunostaining of murine MCNs after IWP-2 treatment or DMSO control groups. Scale bar, 50 μm. (f) Quantification of cyst size for IWP-2 treatment or DMSO control group. Mean ± s.e.m. *Po0.01 (n = 6 mice per groups).

transfer agreements, were generously made available by Drs Andrew M IHC and immunofluorescence 29,63,64 Lowy, Tyler Jacks, Anton Berns and Raju Kucherlapati, respectively. Hematoxylin and eosin staining followed the standard protocol. Periodic Mice were genotyped as described by the MMHCC PCR protocols for acid–Schiff stain and Alcian blue staining kits were purchased from Scy-Tek strains 01XL5, 01XJ6, 01XC2 and 01XAA. All studies were approved by the Laboratories (Logan, UT, USA) and performed according to the manu- Animal Care Committee of the University of Kaohsiung Medical University facturer's protocols. Standard procedure for IHC and immunofluorescence (animal permit number 10115) and all surgery and killing was performed analysis has been described in detail previously and antibodies used in 65 using isoflurane or avertin to ensure minimal suffering. Blood samples these studies are listed in Supplementary Table 3. Stained slides were were obtained from the cardiac puncture of isoflurane-anesthetized mice. captured using a Carl Zeiss. Axioskop 2 plus microscope (Carl Zeiss, fl The formed elements and plasma were separated through centrifugation Thornwood, NY, USA). Immuno uorescent images were captured using a (×3000 g, 15 min). Blood biochemistry parameters were measured with an Delta Vision Personal DV Imaging System (Personal DV Applied Precision, automatic chemistry analyzer (Hitachi 7170S, Hitachi Ltd., Tokyo, Japan). Issaquah, WA, USA). Mice embryos were harvested at E10, E12, E14 and E16. Embryos and pancreatic tissue samples were fixed in 10% buffered formalin overnight, Western blot analysis washed with 1 × phosphate-buffered saline, and transferred to 70% Standard procedures for immunoblotting analysis has been described in ethanol before paraffin embedding, sectioning, and hematoxylin and detail previously.66 The primary antibodies used in this study are listed in eosin staining. Supplementary Table 3.

© 2016 Macmillan Publishers Limited Oncogene (2016) 2223 – 2234 APC haploinsufficiency in the mouse pancreas T-L Kuo et al 2232 Collection of mouse pancreatic cystic fluid cultured in RPMI-1640 medium supplemented with 10% fetal bovine Cystic fluid was collected by placing the needle (21G) into the cystic cavity serum, nonessential amino acids, 100 units/ml penicillin and 100 μg/ml CKO/+ L/L of MCNs in Pdx1-CreAPC p53 mice before autopsy. Cystic fluid was streptomycin at 37 °C in a 5% CO2 incubator. Primary mouse pancreatic centrifuged at 2000 r.p.m. for 10 min at 4 °C to separate the fluid from ductal and PDAC cells were maintained for o6 passages and histopatho- cellular components. The protein levels of cystic fluid were determined by logically characterized through SCID mice xenograft studies before the BCA protein assay kit (Pierce, Rockford, IL, USA) and stored at – 80 °C. performing microarray expression profile analyses.

Mouse cytokine array analysis Xenograft SCID mice For cytokine analysis, the RayBio Mouse Inflammation Antibody Array I was Specific pathogen-free, 8-week-old female C.B17/lcr-SCID mice were purchased from RayBiotech, Inc., Norcross, GA, USA. Sample preparation purchased from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan) for the in vivo and hybridization to the array were performed according to the tumorigenicity study. The animals were maintained in the animal center at manufacturer's instructions. the Department of Medical Research, Kaohsiung Medical University Hospital under specific-pathogen-free conditions and treated according TUNEL staining to the institutional guidelines for the care and use of experimental animals. Fluorometric TUNEL staining was conducted according to the manufac- SCID mice subcutaneous injection were conducted as previously 66 turer’s protocol as described for the Dead End Fluorometric TUNEL system described (Promega, Madison, WI, USA), which identifies apoptotic cells by fl uorescein-12-dUTP labeling of fragmented DNA staining with analysis IWP-2 treatment performed as described previously.65 The in vitro cytotoxicity of the Wnt inhibitor IWP-2 (Sigma-Aldrich, St Louis, MO, USA) 20 μM was assessed by standard methyl tetrazolium cell RNA extraction and microarray detection proliferation assay in Pdx-1Crep53L/L normal ductal cells, Pdx-1Cre LSLKras Cells were scraped and collected by centrifugation, and total RNA was p53L/L and Pdx-1Cre APCL/+p53L/L PDAC cells. For in vivo treatment, IWP-2 subsequently isolated by RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA; P/ (5 mg) was dissolved in 100 μl of dimethylsulfoxide, which was then N 74104). RNA quantity and purity were assessed at 260 and 280 nm using diluted with phosphate-buffered saline buffer solution to a final a Nanodrop (ND-1000; Labtech International Ltd, Rigmer, UK). In all, 300 ng concentration of 1 mg/ml directly before use. Six-week-old Pdx-1Cre fi of each sample was ampli ed and labeled using the GeneChip WT Sense APCCKO/+p53L/L mice were administered twice weekly intraperitoneal Target Labeling and Control Reagents (900652) for Expression Analysis. injections of 10 mg/kg IWP-2 or the phosphate-buffered saline vehicle for 12 weeks (N = 6 per group). At the end of the experiment, mice were Complementary DNA microarray analysis killed by CO2 after magnetic resonance imaging, and their pancreata Hybridization was performed against the Affymetrix GeneChip MoGene 1.0 collected for pathohistological analysis. Randomization was done accord- ST array. The arrays were hybridized for 17 h at 45 °C and 60 r.p.m. Arrays ing to genotype and blinding was applied during histological analysis. were subsequently washed (Affymetrix Fluidics Station 450, Santa Clara, – CA, USA) and stained with streptavidin phycoerythrin (GeneChip Hybridi- Magnetic resonance imaging zation, Wash, and Stain Kit, Affymetrix, Santa Clara, CA, USA; 900720), and – scanned on an Affymetrix GeneChip Scanner 3000. The resulting data were Mice were anesthetized with 1 2 isolfurance/air, and body temperature analyzed using Expression Console software (Affymetrix) and Transcrip- was maintained by air conditioning through the bore of the magnet ring. tome Analysis Console software (Affymetrix) with default RMA parameters. Magnetic resonance imaging scans were performed using a 3 T MRI Genes regulated were determined with a 2.0-fold change; P-value o0.05. scanner (GE, HDXt Sigma; GE, Milwaukee, WI, USA) with a high-resolution animal coil (3.0 cm diameter). Mice were placed supine in the coil, taped below the thoracic cavity on the bed to reduce respiratory motion. T2 GeneGo analysis weighted images were acquired using a fast spin echo multi-slices fi The signi cant lists were uploaded from a Microsoft Excel spreadsheet sequence with TR/TE 2000/63.23 ms for coronal section and 5083/46.7 ms onto Metacore 6.13 software (GeneGo pathways analysis; http://www. for axial section, 16 echo trains, 4 averages, 2 dummy scans, field of genego.com). GeneGo recognizes the Affymetrix identifiers and maps the view = 8 × 4.8 cm3, for coronal section and 6 × 6 cm2 for axial section, tissues to the MetaCore data analysis suite, generating maps to describe matrix size = 256 × 192, slice thickness = 2 mm, number of slices = 20 common pathways or molecular connections between pancreatic tissues on the list. Graphical representations of the molecular relationships contiguous. Scans were captured every 10 min until the 90-min mark was between genes were generated using the GeneGo pathway analysis, based reached. A glass cylinder of pure water was positioned adjacent to each upon processes showing significant (Po0.05) association. mouse as a standard reference.

Real-time–quantitative PCR analysis (RT–qPCR) Statistical analysis RT–qPCR were carried out as described in detail previously, and the All experiments were repeated at least three times. One representative primers for RT–qPCR were listed in Supplementary Table 4.66 experiment is shown. RT–qPCR and cell proliferation assays are displayed as one representative experiment of three independent experiments, mean ± s.e.m. Data measured on continuous scale were analyzed using Cell proliferation assay Student’s t-test and categorical data were subjected to x2 test. P-value Standard methyl tetrazolium-based cell growth assay as described in detail o0.05 was considered signiﬁcant. previously.66

FACS analysis CONFLICT OF INTEREST In vitro caspase 3 activation assays were conducted according to the The authors declare no conflict of interest. manufacturer’s instructions. The protocol for FACS analysis as described in detail previously.67 ACKNOWLEDGEMENTS Primary pancreatic cell culture We gratefully thank Dr Sheau-Fang Yang, at Kaohsiung Medical University for helping The primary pancreatic ductal cells isolated from Pdx-1Crep53L/L mice were us to confirm the histologic features and pathology of MCN mice. This work was cultured in human pancreatic duct epithelial cell medium (Dulbecco’s supported by grants NSC 101-2314-B-110-001-MY2, 101-2628- B-110-001-MY2 (to KH modified Eagle’s medium/F12 serum-free medium supplemented with Cheng) and MOST 103-2314-B-037-062 (to DC Wu and KH Cheng) from the National 0.2 ng of epidermal growth factor, 30 μg/ml bovine pituitary extract and Science Council, Taiwan ROC, and grants KMU-TP103G00 and KMU-TP103G01 (to DC containing penicillin/streptomycin). The mouse primary PDAC cells were Wu and KH Cheng) from Kaohsiung Medical University, Kaohsiung, Taiwan.

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