Deficient Rnf43 potentiates hyperactive Kras‐mediated pancreatic preneoplasia initiation and malignant transformation

Abstract Background Largely due to incidental detection, asymptomatic pancreatic cystic lesions (PCLs) have become prevalent in recent years. Among them, intraductal papillary mucinous neoplasm (IPMN) infrequently advances to pancreatic ductal adenocarcinoma (PDAC). Conservative surveillance versus surgical intervention is a difficult clinical decision for both caregivers and PCL patients. Because RNF43 loss‐of‐function mutations and KRAS gain‐of‐function mutations concur in a subset of IPMN and PDAC, their biological significance and therapeutic potential should be elucidated. Methods Pancreatic Rnf43 knockout and Kras activated mice (Rnf43 −/−; KrasG12D ) were generated to evaluate their clinical significance in pancreatic pre‐neoplastic initiation and malignant transformation. Results Loss of Rnf43 potentiated the occurrence and severity of IPMN and PDAC in oncogenic Kras mice. The Wnt/β‐catenin signaling pathway was activated in pancreatic KrasG12D and Rnf43 knockout mice and the PORCN inhibitor LGK974 blocked pancreatic IPMN initiation and progression to PDAC accordingly. Conclusions Rnf43 is a tumor suppressor in the prevention of pancreatic malignant transformation. This genetically reconstituted autochthonous pancreatic Rnf43 −/−; KrasG12D preclinical cancer model recapitulates the pathological process from pancreatic cyst to cancer in humans and can be treated with inhibitors of Wnt/β‐catenin signaling. Since the presence of RNF43 and KRAS mutations in IPMNs predicts future development of advanced neoplasia from PCLs, patients with these genetic anomalies warrant surveillance, surgery, and/or targeted therapeutics such as Wnt/β‐catenin inhibitors.


| INTRODUC TI ON
With a pooled prevalence of 8% in adults, pancreatic cystic lesions (PCLs) are often asymptomatically and incidentally discovered on routine cross-sectional abdominal imaging. 1 PCLs are a heterogeneous group of benign tumors and precursor lesions of pancreatic ductal adenocarcinoma (PDAC). PDAC is a major type of pancreatic malignant tumor, with the worst prognosis among cancers. 2 Even though the malignant potential of PCL is very low, accurate diagnosis and correct assessment of malignant potential are often hard to achieve. Since it is a daunting task to distinguish tumors with or without malignant potentials, perceived dire prognosis of PDAC, evolved from incidentally detected PCLs, often forces both caregivers and patients to choose between major pancreatic resection, with substantial morbidity and mortality, and life-long surveillance, with associated financial burdens, anxiety, and the risk of missed malignancy.
Intraductal papillary mucinous neoplasm (IPMN) is a major type of PCL, which may progress from low-grade dysplasia (LGD) to highgrade dysplasia (HGD), and eventually to PDAC. 3 Therefore, IPMN is a precursor lesion of PDAC. [3][4][5] Progression from precancerous lesions to invasive pancreatic cancer may take about 20 years. 6 It is a curable disease if detected and treated before it proceeds to invasive carcinoma. In clinical practice, LGD can potentially be managed conservatively while HGD and invasive carcinoma would require surgical resection. 7,8 The surgical indication for these patients is based on the presence of indirect predictors in diagnostic images.
Given the lack of information on the natural history of IPMN malignant transformation, whether noninvasive carcinoma or HGD should be operated on or observed remains an unsolved practical issue. Therefore, analysis of these precancerous lesions is crucial to understand the earliest events of pancreatic tumorigenesis.
Recent studies have shown that the most frequently mutated genes in IPMNs are KRAS, GNAS, and RNF43. 9, 10 We and others identified that the pooled prevalence of RNF43 inactivating mutation in IPMN was 24% by next generation sequencing assessment. 9,[11][12][13] RNF43 inactivating mutation and KRAS activating mutation coexist in a subset of IPMN tumor tissues. [10][11][12] RNF43 is also altered in 7% of invasive PDACs with or without obvious IPMN. 14 RNF43 is a member of the RING finger protein family, 15

| Histological and immunohistochemical analyses
After fixing in 10% formalin for more than 24 hours, tissues were embedded in paraffin and cut to 4μm sections. H&E and immunohistochemical staining was performed according to standard procedures. Antibodies utilized are listed in Table 1. Photos were taken with Leica DMi8 microscope, with identical adjustments and exposure times between genotypes and treatment groups.
Immunohistochemical staining images were captured using CaseViewer software (3DHISTECH) and quantified with Image-Pro Plus 6.0 software (Media Cybernetics).

| cBioPortal analysis of gene mutations in human pancreatic adenocarcinoma
Pancreatic cancer genomics data deposited in ICGC, QCMG, UTSW and TCGA database were analyzed using the cBioPortal for Cancer Genomics (http://cbiop ortal.org) for genetic alterations of KRAS, TP53 and RNF43.

| Statistical analysis
The Kaplan-Meier log-rank test was used for analysis of mouse survival and body weight differentiation using GraphPad Prism software (Version 7). All quantitative data are reported as means ± SD unless otherwise indicated in the figure legends. A p-value of <0.05 was considered as significant.

| Pancreatic Rnf43-deficient mice have enlarged pancreata
We and others identified mutated RNF43 in tumor tissues of IPMN patients. 11,12 To check whether loss of RNF43 causes IPMN, we generated pancreatic Rnf43 knockout mice. First, conditional Rnf43 knockout mice were created by inserting LoxP and neo cassette into the introns flanking exon 7 and exon 8 of Rnf43 gene ( Figure 1A).

| Pancreatic Kras G12D and Rnf43 deficient mice develop intraductal papillary mucinous neoplasms
Oncogenic KRAS mutations are the earliest driver gene alterations in IPMNs. 21 Mutations of KRAS and RNF43 often concur in a subset of IPMNs. 11,12 To decipher the causative relationship between concomitant RNF43/KRAS mutations and pancreatic carcinogenesis, we generated pancreatic Rnf43 knockout and Kras activation mice

| Mutation co-occurrence of RNF43 and KRAS in human pancreatic adenocarcinoma
To determine the clinical relevance of our findings from mice, we ana-  Figure 6B). The body weight loss in LGK974 treated mice was not statistically significant ( Figure 6C). While ~50% control mice died, none of LGK974-treated mice died within the 14-day treatment period ( Figure 6D). After discontinuation of the treatment, mice from the experimental treatment group began to die, but higher mortality was still observed in shamtreated mice than in LGK974-treated mice ( Figure 6D). The surviving mice were sacrificed at the age of 6 months. No gender difference was observed in response to the treatment ( Figure 6D). Taken together, our data show that Wnt/β-catenin signaling activation is necessary for pancreatic IPMN development and malignant transformation.

| DISCUSS ION
Loss-of-function mutation of RNF43 coexists with gain-of-function mutation of KRAS in human IPMN and PDAC. 11,12,14 To study the  These mutations are evolutionally selected in a subset of IPMNs. [10][11][12] We observed development and progression of IPMN and PanIN in

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.