Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma

Abstract

A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/β-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/β-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of β-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43-wild-type pancreatic cancer cells. LGK974 inhibited proliferation and induced differentiation of RNF43-mutant pancreatic adenocarcinoma xenograft models. Our data suggest that mutational inactivation of RNF43 in pancreatic adenocarcinoma confers Wnt dependency, and the presence of RNF43 mutations could be used as a predictive biomarker for patient selection supporting the clinical development of Wnt inhibitors in subtypes of cancer.T he evolutionarily conserved Wnt/β-catenin signaling pathway plays critical roles in embryonic development and adult tissue homeostasis (1, 2). Wnt signaling regulates the turnover of the transcription cofactor β-catenin and controls key developmental gene expression programs (3). In the absence of Wnt pathway activation, cytosolic β-catenin is degraded by the β-catenin destruction complex, consisting of adeomatous polyposis coli (APC), AXIN1/2, and glycogen synthase kinase 3α/β (GSK3α/β). Wnt ligand activates its two receptors, Frizzled and LRP5/6, and inactivates the β-catenin destruction complex. Stabilized β-catenin enters the nucleus, binds to the TCF family of transcription factors, and activates transcription. Secretion of Wnt proteins requires Porcupine (PORCN), a membrane bound O-acyltransferase dedicated to Wnt posttranslational acylation (4, 5). Precise regulation of Wnt signaling is critical and various feedback control mechanisms exist to ensure proper signaling output.Aberrant activation of Wnt/β-catenin signaling has been implicated in tumorigenesis, and many downstream components of the Wnt pathway are mutated in cancers (6). Truncation mutations of APC are found in 80% of colorectal cancer. Stabilization mutations of CTNNB1 (β-catenin) and loss of function mutations of AXIN1/2 are also fo...

Targeting the Wnt/β-catenin pathway in primary ovarian cancer with the porcupine inhibitor WNT974

“…The prevention of Wnt ligand secretion after treatment with WNT974 results in decreased phosphorylation of the surface receptor LRP6 and decreased expression of Wnt target genes like AXIN2. 16,17 WNT974 has shown anti-tumor activity in pancreatic adenocarcinoma and head and neck squamous cell cancer. Jiang et al 17 identified both WNT974 sensitive tumor cells and a potential predictive biomarker after demonstrating tumor growth inhibition through cell cycle arrest in in vivo pancreatic tumors treated with WNT974 with inactivating mutations in the RNF43 gene.…”

“…16 WNT974 has been shown to suppress growth of Wnt-driven tumors, such as pancreatic and head and neck squamous cell cancer with mutations in Wnt pathway modulators such as Ring finger 43 (RNF43). 17 The R-spondin (RSPO) protein family is comprised of four secreted growth factors. Secreted RSPO proteins activate Wnt/ β-catenin signaling, and RSPO mutations can further upregulate Wnt/β-catenin signaling.…”

Targeting Wnts at the Source—New Mechanisms, New Biomarkers, New Drugs

“…Recent studies (ref. 44; Madan and colleagues; unpublished data) have demonstrated that the proliferation of pancreatic cancer cell lines and patient-derived xenografts with inactivating RNF43 mutations is inhibited by PORCN inhibitors, including LGK974, Wnt-C59, and ETC-159. Hence, RNF43 and ZNRF3 mutations, by sensitizing cells to Wnts, may also be predictive of response to both PORCN inhibitors and Frizzled antibodies.…”

Clinicopathological significance of somatic RNF43 mutation and aberrant expression of ring finger protein 43 in intraductal papillary mucinous neoplasms of the pancreas

“…8 RNF43 mutations have also been shown to confer Wnt dependency in pancreatic cancer cells. 9 However, the clinicopathological relevance of RNF43 mutations in intraductal papillary mucinous neoplasms remains unclear.…”