Oncogene (2016) 35, 2223–2234 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc 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