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^-/-; Kras^G12D) 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 Kras^G12D 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^-/-; Kras^G12D 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.

Keywords: KRAS; RNF43; Wnt; intraductal papillary mucinous neoplasms; pancreatic ductal adenocarcinoma.

FIGURE 1

Pancreatic Rnf43 ^−/− mice have larger pancreata. A, Targeted allele of Rnf43 conditional knockout mice. B, PCR analysis of mouse tail genomic DNA. #64 is Rnf43 ^LoxP−neo/+ mouse; #62,63 and 65 are Rnf43 ^+/+ mice; − is blank control; WT is negative control; + is positive cloned genomic DNA. C, Cross‐mating schedule for generating pancreatic Rnf43 knockout mice (Rnf43 ^−/−) and their control littermates (Rnf43 ^f/f). D, Identification of Rnf43 ^LoxP/LoxP mice by genomic DNA PCR. f/f: Rnf43 ^LoxP/LoxP; f/+: Rnf43 ^LoxP/+; +/+: Rnf43 ^+/+. E, Identification of Pdx1‐cre; Rnf43 ^LoxP/LoxP mice by genomic DNA PCR. F, Genomic DNA sequencing of Rnf43 ^−/− mouse. Gray: 5′ homologous arm; rose: LoxP sequence; blue: 3′ homologous arm. G, Ratio of pancreas weight to body weight of 1‐month‐old (left) and 6‐month‐old (right) Rnf43 ^f/f and Rnf43 ^−/− mice. Means ± SD. ***p < 0.001. H, H&E staining of pancreata from 1‐month‐old and 6‐month‐old Rnf43 ^f/f and Rnf43 ^−/− mice. Scale bar, 50 μm. I, Kaplan‐Meier curves of Rnf43 ^−/− mice (purple) (n = 42) and Rnf43 ^f/f mice (black) (n = 46)

FIGURE 2

Rnf43 ^−/−; Kras^G12D mice have shortened lifespan. A, Cross‐mating schedule for generating pancreatic Kras ^G12D and Rnf43 deficient mice. B, PCR genotyping of mouse tail genomic DNA. #2, 4, 6 and 9 are Rnf43 ^−/−; Kras ^G12D; #161 and 162 are Kras ^G12D; #3 is Rnf43 ^−/−; #1, 5 and 160 are Rnf43 ^f/f. C, Immunoblotting of pancreatic lysates from 6‐month‐old Rnf43^−/−; Kras ^G12D and control mice. D, Kaplan‐Meier curves of Rnf43 ^−/−; Kras ^G12D mice (red) (n = 42) comparing with Rnf43 ^f/f (black) (n = 46), Rnf43 ^−/− (purple) (n = 42) and Kras ^G12D (blue) (n = 42) mice. Log‐rank p < 0.0001

FIGURE 3

Rnf43 ^−/−; Kras ^G12D mice develop pancreatic intraductal papillary mucinous neoplasms and pancreatic intraepithelial neoplasia. H&E staining (scale bar, 100 μm) of pancreata from 16‐day‐old (left) and 6‐month‐old Rnf43 ^f/f, Kras ^G12D and Rnf43^−/−; Kras ^G12D mice

FIGURE 4

Rnf43 ^−/−; Kras ^G12D mice develop invasive and metastatic pancreatic ductal adenocarcinoma. A, B, Gross abdominal images of a 6.5‐month‐old Rnf43 ^−/−; Kras ^G12D mouse. A, Abdominal distension due to accumulation of ascetic fluid (black arrows). B, PDAC (yellow arrows) in pancreas and its invasion of adjacent organs. C, H&E Staining of invasive PDAC, bile duct, duodenum, and lymph node around the pancreas. Scale bar, left: 100 μm; right: 50 μm. D, Tumor nodules (red arrows) in the lungs of the mouse (A, B). H&E staining, IHC staining of CK19 and TTF‐1 (E) of lung tumor nodules (D). T, tumor; NT, non‐tumor. Scale bar, left, 200 μm; right, 50 μm

FIGURE 5

Biliary obstruction in pancreatic Kras^G12D and Rnf43 deficient mice. A, A 6‐month‐old Rnf43 ^−/−; Kras^G12D mouse with biliary obstruction and gallbladder distension. B, H&E staining of 6‐month‐old Rnf43 ^−/−; Kras ^G12D mouse and control mouse bile ducts. Scale bar, 200 μm. C, H&E staining of 6‐month‐old Rnf43 ^−/−; Kras ^G12D mouse and control mouse major duodenal papilla. Scale bar, 400 μm

FIGURE 6

Concurrent mutations of RNF43 and KRAS in human pancreatic adenocarcinoma. Landscape of KRAS, TP53 and RNF43 genetic alterations in pancreatic cancer deposited in ICGC, QCMG, UTSW and TCGA database of cBioPortal. A, Alteration co‐occurrence and mutual exclusivity between RNF43 and KRAS. B, Alteration co‐occurrence and mutual exclusivity between RNF43 and TP53. C, Alteration co‐occurrence and mutual exclusivity between KRAS and TP53

FIGURE 7

Wnt inhibitor blocks the development of pancreatic cystic neoplasms and prolongs the survival of the Rnf43 ^−/−; Kras ^G12D mice. A, IHC staining of pancreata from 6‐month‐old Rnf43 ^f/f, Rnf43 ^−/−, Kras ^G12D and Rnf43 ^−/−; Kras ^G12D mice (upper). Scale bar, 50 μm. Quantification of IHC stainings (lower). B, LGK974 treatment schedule. 20‐day‐old Rnf43 ^−/−; Kras ^G12D mice were divided into treatment group (LGK974, 22 in total, 14♂+8♀) and control group (Vehicle, 21 in total, 12♂+9♀). C, Body weights of LGK974‐treated and control mice during the treatment (means ±SEM). D, Kaplan‐Meier curves of Rnf43 ^−/−; Kras ^G12D mice with LGK974 (red) or vehicle (blue) treatment