RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer.

Wnt/ß-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce ß-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing ß-catenin turnover and propelling ligand-independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.

Associations with other cancer-related biomarkers might contribute to poor outcomes in RAS-altered, younger patients with colorectal cancer.

Colorectal cancer (CRC) is a common cancer in younger adults. In patients undergoing liver resection with RAS-altered CRCs, there is evidence suggesting younger patients have worse outcomes than older patients. To explain this pattern, differences in associations between RAS status and other cancer-related biomarkers in tumors from younger versus older patients with CRC were evaluated in a cohort of 925 patients with CRC, 277 (30.0%) of whom were ≤50 years old, and 454 (49.1%) who had RAS-altered tumors. For 3 biomarkers, RNF43, APC, and microsatellite instability (MSI), the association with RAS status was significantly modified by age after adjustment for multiple testing. Specifically, younger patients with RAS-altered tumors were more likely to be MSI-high, RNF43 mutated, and APC wild type. These differences might contribute to the observed pattern of diminished survival in younger versus older patients with CRC with RAS-mutated tumors undergoing liver metastasis resection.

RNF43 in cancer: Molecular understanding and clinical significance in immunotherapy.

Identifying biomarkers to predict immune checkpoint inhibitor (ICI) efficacy is warranted. Considering that somatic mutation-derived neoantigens induce strong immune responses, patients with a high tumor mutational burden reportedly tend to respond to ICIs. Therefore, the original function of neoantigenic mutations and their impact on the tumor microenvironment (TME) require attention. RNF43 is a type of RING E3 ubiquitin ligase, and long-term survivors in most cancers had conserved patterns of mutations of RNF43. Also, high microsatellite instability patients had a higher RNF43 mutation rate compared with microsatellite stability tumor patients, who were more sensitive to ICI treatment. Therefore, RNF43 has become a promising biomarker of immunotherapy in a wide range of cancers. This review focuses on the up-to-date knowledge of RNF43 mutation in cancer. We summarize the cancer hallmarks involving activities regulated by RNF43 and highlight its extremely sophisticated regulation of WNT signaling and tumor microenvironment. The key genes interacting with RNF43 have also been summarized and discussed. Additionally, we highlight and propose new strategies of targeting RNF43 and RNF43-based combinations with established immunotherapy and combination therapy. These efforts may provide new perspectives for RNF43-based target therapy in cancer.

RNF43 induces the turnover of protease-activated receptor 2 in colon cancer.

Post-translational modification of G-protein coupled receptors (GPCRs) plays a central role in tissue hemostasis and cancer. The molecular mechanism of post-translational regulation of protease-activated receptors (PARs), a subgroup of GPCRs is yet understudied. Here we show that the cell-surface transmembrane E3 ubiquitin ligase ring finger 43 (RNF43) is a negative feedback regulator of PAR2 , impacting PAR2 -induced signaling and colon cancer growth. RNF43 co-associates with PAR2 , promoting its membrane elimination and degradation as shown by reduced cell surface biotinylated PAR2 levels and polyubiquitination. PAR2 degradation is rescued by R-spondin2 in the presence of leucine-rich repeat-containing G-protein-coupled receptor5 (LGR5). In fact, PAR2 acts jointly with LGR5, as recapitulated by increased ß-catenin levels, transcriptional activity, phospho-LRP6, and anchorage-independent colony growth in agar. Animal models of the chemically induced AOM/DSS colon cancer of wt versus Par2/f2rl1 KO mice as also the 'spleen-liver' colon cancer metastasis, allocated a central role for PAR2 in colon cancer growth and development. RNF43 is abundantly expressed in the Par2/f2rl1 KO-treated AOM/DSS colon tissues while its level is very low to nearly null in colon cancer adenocarcinomas of the wt mice. The same result is obtained in the 'spleen-liver' model of spleen-inoculated cells, metastasized to the liver. High RNF43 expression is observed in the liver upon shRNA -Par2 silencing. "Limited-dilution-assay" performed in mice in-vivo, assigned PAR2 as a member of the cancer stem cell niche compartment. Collectively, we elucidate an original regulation of PAR2 oncogene, a member of cancer stem cells, by RNF43 ubiquitin ligase. It impacts ß-catenin signaling and colon cancer growth.

R-spondin substitutes for neuronal input for taste cell regeneration in adult mice.

Taste bud cells regenerate throughout life. Taste bud maintenance depends on continuous replacement of senescent taste cells with new ones generated by adult taste stem cells. More than a century ago it was shown that taste buds degenerate after their innervating nerves are transected and that they are not restored until after reinnervation by distant gustatory ganglion neurons. Thus, neuronal input, likely via neuron-supplied factors, is required for generation of differentiated taste cells and taste bud maintenance. However, the identity of such a neuron-supplied niche factor(s) remains unclear. Here, by mining a published RNA-sequencing dataset of geniculate ganglion neurons and by in situ hybridization, we demonstrate that R-spondin-2, the ligand of Lgr5 and its homologs Lgr4/6 and stem-cell-expressed E3 ligases Rnf43/Znrf3, is expressed in nodose-petrosal and geniculate ganglion neurons. Using the glossopharyngeal nerve transection model, we show that systemic delivery of R-spondin via adenovirus can promote generation of differentiated taste cells despite denervation. Thus, exogenous R-spondin can substitute for neuronal input for taste bud cell replenishment and taste bud maintenance. Using taste organoid cultures, we show that R-spondin is required for generation of differentiated taste cells and that, in the absence of R-spondin in culture medium, taste bud cells are not generated ex vivo. Thus, we propose that R-spondin-2 may be the long-sought neuronal factor that acts on taste stem cells for maintaining taste tissue homeostasis.

New Target(s) for RNF43 Regulation: Implications for Therapeutic Strategies.

Cancer cells depend on specific oncogenic pathways or present a genetic alteration that leads to a particular disturbance. Still, personalized and targeted biological therapy remains challenging, with current efforts generally yielding disappointing results. Carefully assessing onco-target molecular pathways can, however, potently assist with such efforts for the selection of patient populations that would best respond to a given drug treatment. RNF43, an E3 ubiquitin ligase that negatively regulates Wnt/frizzled (FZD) receptors by their ubiquitination, internalization, and degradation, controls a key pathway in cancer. Recently, additional target proteins of RNF43 were described, including p85 of the PI3K/AKT/mTOR signaling pathway and protease-activated receptor 2 (PAR2), a G-protein-coupled receptor that potently induces ß-catenin stabilization, independent of Wnts. RNF43 mutations with impaired E3 ligase activity were found in several types of cancers (e.g., gastrointestinal system tumors and endometrial and ovarian cancer), pointing to a high dependency on FZD receptors and possibly PAR2 and the PI3K/AKT/mTOR signaling pathway. The development of drugs toward these targets is essential for improved treatment of cancer patients.

BRAF V600E and RNF43 Co-mutations Predict Patient Outcomes With Targeted Therapies in Real-World Cases of Colorectal Cancer.

Anti-BRAF/EGFR therapy is approved for metastatic colorectal cancer (mCRC) with BRAFV600E mutations, although not all patients respond. Novel recent findings indicate the potential of RNF43 mutations to predict outcomes in patients with BRAF-mutated microsatellite stable (MSS) mCRC treated with anti-BRAF/EGFR therapy. This study aimed to independently and rapidly validate BRAFV600E/RNF43 co-mutations as predictive biomarkers of benefit to anti-EGFR/BRAF therapy. Clinical data were derived from electronic health record data from ~280 US cancer clinics between January 2011 and March 2022 from the Flatiron Health-Foundation Medicine real-world clinico-genomic mCRC database. Real-world cases of BRAFV600E-mutated mCRC, with patients receiving anti-BRAF/EGFR therapy (n = 49), were included. Patients who were MSS, with RNF43 mutations, had favorable progression-free survival (hazard ratio [HR] 0.29; 95% CI [CI], 0.13-0.65) and overall survival (HR 0.32, 95% CI, 0.12-0.84) compared with wild type. No difference in outcomes was observed between patient groups with RNF43-mutant versus wild-type receiving standard-of-care chemotherapy. BRAFV600E/RNF43 co-mutations predict mCRC anti-BRAF/EGFR outcomes in diverse clinical settings.

PD-L1 expression downregulation by RNF43 in gastric carcinoma enhances antitumour activity of T cells.

Ring finger protein 43 (RNF43), a transmembrane E3 ubiquitin ligase, has been indicated to be a potential biomarker for gastric cancer treatment, as this protein increases tumour cell apoptosis and suppresses cellular proliferation. The role of RNF43 in cellular immunotherapy remains unclear. Herein, we aimed to explore the expression level of RNF43 in gastric cancer cell lines and its role in cellular immunotherapy. The expression level of RNF43 and PD-L1 and their correlation in gastric cancer cell lines were analysed. The expression of PD-L1 was negatively correlated with that of RNF43 in gastric cancer cell lines. RNF43 interacted with PD-L1 to augment both K48- and K63-linked ubiquitination of PD-L1 in gastric cancer cell lines. In addition, RNF43 expression in gastric cancer cell lines could enhance the antitumour activity of T cells. In conclusion, this study reveals that RNF43 can inhibit PD-L1 expression to enhance the antitumour activity of cellular immunotherapy.

Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.

Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/ß-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active ß-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the ß-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Post-translational Wnt receptor regulation: Is the fog slowly clearing?: The molecular mechanism of RNF43/ZNRF3 ubiquitin ligases is not yet fully elucidated and still controversial.

Wnt signaling plays pivotal roles during our entire lives, from conception to death, through the regulation of morphogenesis in developing embryos and the maintenance of tissue homeostasis in adults. The regulation of Wnt signaling occurs on several levels: at the receptor level on the plasma membrane, at the ß-catenin protein level in the cytoplasm, and through transcriptional regulation in the nucleus. Several recent studies have focused on the mechanisms of Wnt receptor regulation, following the discovery that the Wnt receptor frizzled (Fzd) is a target of the ubiquitin ligases, RNF43 and ZNRF3. RNF43 and ZNRF3 are homologous genes that are mutated in several cancers. The details underlying their mechanism of action continue to unfold, while at the same time raising many new questions. In this review, we discuss advances and controversies in our understanding of Wnt receptor regulation.

Regulation of Wnt receptor activity: Implications for therapeutic development in colon cancer.

Hyperactivation of Wnt/ß-catenin (canonical) signaling in colorectal cancers (CRCs) was identified in the 1990s. Most CRC patients have mutations in genes that encode components of the Wnt pathway. Inactivating mutations in the adenomatous polyposis coli (APC) gene, which encodes a protein necessary for ß-catenin degradation, are by far the most prevalent. Other Wnt signaling components are mutated in a smaller proportion of CRCs; these include a FZD-specific ubiquitin E3 ligase known as ring finger protein 43 that removes FZDs from the cell membrane. Our understanding of the genetic and epigenetic landscape of CRC has grown exponentially because of contributions from high-throughput sequencing projects such as The Cancer Genome Atlas. Despite this, no Wnt modulators have been successfully developed for CRC-targeted therapies. In this review, we will focus on the Wnt receptor complex, and speculate on recent discoveries about ring finger protein 43regulating Wnt receptors in CRCs. We then review the current debate on a new APC-Wnt receptor interaction model with therapeutic implications.

RNF43 pathogenic Germline variant in a family with colorectal cancer.

The role of RNF43 as a cause of an inherited predisposition to colorectal cancer (CRC) is yet to be fully explored. This report presents our findings of two individuals with CRC from a single family carrying a likely-pathogenic inherited germline variant in RNF43. The proband (III:1) and the proband's mother (II:2) were diagnosed with mismatch repair proficient CRCs at the age of 50 years and 65 years, respectively. Both patients had BRAFV600E mutated colon tumours, indicating that the CRCs arose in sessile serrated lesions. The germline variant RNF43:c.375+1G>A was identified in both patients. RNA studies showed that this variant resulted in an aberrantly spliced transcript, which was predicted to encode RNF43:p.Ala126Ilefs*50 resulting in premature termination of protein synthesis and was classified as a likely-pathogenic variant. Our report adds further evidence to the hereditary role of RNF43 as a tumour suppressor gene in colorectal tumorigenesis and supports the inclusion of RNF43 as a gene of interest in the investigation of CRC predispositions outside the setting of serrated polyposis.

Mechanisms of development and progression of pancreatic neoplasms.

Pancreatic ductal adenocarcinoma (PDAC) develops via dysplastic changes in the epithelia graded as low- and high-grade with accumulation of molecular alterations. Constitutive activation of mitogen-activated protein kinase (MAPK) contributed by attenuation of DUSP6 plays a key role in sustaining PDAC. Active MAPK induces various molecules that function as effectors to sustain PDAC. AURKA and SON are downstream effectors that contribute substantially to the proliferation and survival of PDAC cells and are potentially useful as therapeutic targets. Active MAPK also promote microRNAs that modulate the proliferation of PDAC cells and are useful as diagnostic markers. Familial pancreatic cancer kindreds in Japan show various germline mutations supposed to increase a pancreatic cancer risk. Intraductal papillary mucinous neoplasms (IPMNs) consist of dilated ducts lined by papillary neoplastic epithelia of various shapes and varying grades of atypia. Various papillae of IPMNs are classified into four subtypes that are associated with clinicopathological features, including patient prognosis. GNAS is a specific driver gene for the development of IPMN through gain-of-function mutations. Tracing of molecular alterations has elucidated the mechanism of progression of IPMN from dysplasia to carcinoma, as well as one type of papillae. Intraductal tubulopapillary neoplasms belong to a distinct class of pancreatic neoplasms.

The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength.

Lgr5 was originally discovered as a common Wnt target gene in adult intestinal crypts and colon cancer. It was subsequently identified as an exquisite marker of multiple Wnt-driven adult stem cell types. Lgr5 and its homologs, Lgr4 and Lgr6, constitute the receptors for R-spondins, potent Wnt signal enhancers and stem cell growth factors. The Lgr5/R-spondin complex acts by neutralizing Rnf43 and Znrf3, two transmembrane E3 ligases that remove Wnt receptors from the stem cell surface. Rnf43/Znrf3 are themselves encoded by Wnt target genes and constitute a negative Wnt feedback loop. Thus, adult stem cells are controlled by an intricate interplay of potent Wnt agonists, antagonists, and anti-antagonists.

A DNA repair player, ring finger protein 43, relieves etoposide-induced topoisomerase II poisoning.

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase which is well-known for its role in negative regulation of the Wnt-signaling pathway. However, the function in DNA double-strand break repairs has not been investigated. In this study, we used a lymphoblast cell line, DT40, and mouse embryonic fibroblast as cellular models to study DNA double-strand break (DSB) repairs. For this purpose, we created RNF43 knockout, RNF43-/- DT40 cell line to investigate DSB repairs. We found that deletion of RNF43 does not interfere with cell proliferation. However, after exposure to various types of DNA-damaging agents, RNF43-/- cells become more sensitive to topoisomerase II inhibitors, etoposide, and ICRF193, than wild type cells. Our results also showed that depletion of RNF43 results in apoptosis upon etoposide-mediated DNA damage. The delay in resolution of γH2AX and 53BP1 foci formation after etoposide treatment, as well as epistasis analysis with DNAPKcs, suggested that RNF43 might participate in DNA repair of etoposide-induced DSB via non-homologous end joining. Disturbed γH2AX foci formation in MEFs following pulse etoposide treatment supported the notion that RNF43 also functions DNA repair in mammalian cells. These findings propose two possible functions of RNF43, either participating in NHEJ or removing the blockage of 5' topo II adducts from DSB ends.

RNF43 mutation analysis in serrated polyposis, sporadic serrated polyps and Lynch syndrome polyps.

AIMS: RNF43 is suggested to be involved in the serrated pathway towards colorectal cancer and encodes a transmembrane Ring-type E3 ubiquitin ligase that negatively regulates the Wnt pathway. This study aimed to elucidate the role of RNF43 gene variants in serrated polyposis syndrome (SPS) and serrated polyps. METHODS AND RESULTS: Three cohorts were tested. The first cohort included germline DNA of 26 SPS patients tested for pathogenic variants in RNF43 by Sanger sequencing all exons. In the second cohort we tested somatic DNA for RNF43 mutations from sporadic serrated lesions: 25 hyperplastic polyps, 35 sessile serrated lesions and 38 traditional serrated adenomas (TSA). In the third cohort we investigated RNF43 mutations in 49 serrated polyps and 60 conventional adenomas from 40 patients with Lynch syndrome. No germline RNF43 pathogenic variants were detected in our SPS cohort. In sporadic colorectal lesions we detected RNF43 deleterious frameshift mutations in three TSA and one SSL. The RNF43 mutations in previously described homopolymeric hot-spots were detected in microsatellite-instable (MSI) polyps and the other RNF43 mutations in microsatellite-stable (MSS) serrated polyps. RNF43 hot-spot mutations were discovered in seven serrated polyps and 12 conventional adenomas from Lynch patients. CONCLUSION: Truncating germline RNF43 mutations are uncommon in SPS patients. Somatic mutations in RNF43 were found in sporadic TSA and SSL and both serrated polyps and adenomas from Lynch syndrome patients, suggesting that they do not develop early in the pathway to CRC and are not specific for serrated polyp subtypes.

The structural basis of R-spondin recognition by LGR5 and RNF43.

R-spondins (RSPOs) enhance Wnt signaling, affect stem cell behavior, bind to leucine-rich repeat-containing G-protein-coupled receptors 4-6, (LGR4-6) and the transmembrane E3 ubiquitin ligases RING finger 43/zinc and RING finger 3 (RNF43/ZNRF3). The structure of RSPO1 bound to both LGR5 and RNF43 ectodomains confirms their physical linkage. RSPO1 is sandwiched by LGR5 and RNF43, with its rod module of the cysteine-rich domain (CRD) contacting LGR5 and a hairpin inserted into RNF43. LGR5 does not contact RNF43 but increases the affinity of RSPO1 to RNF43, supporting LGR5 as an engagement receptor and RNF43 as an effector receptor. Disease mutations map to the RSPO1-RNF43 interface, which promises therapeutic targeting.

Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles.

OBJECTIVE: Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. DESIGN: We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. RESULTS: We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time. CONCLUSIONS: These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

Protease associated domain of RNF43 is not necessary for the suppression of Wnt/ß-catenin signaling in human cells.

BACKGROUND: RNF43 and its homolog ZNRF3 are transmembrane E3 ubiquitin ligases frequently mutated in many human cancer types. Their main role relays on the inhibition of canonical Wnt signaling by the negative regulation of frizzled receptors and LRP5/6 co-receptors levels at the plasma membrane. Intracellular RING domains of RNF43/ZNRF3 mediate the key enzymatic activity of these proteins, but the function of the extracellular Protease Associated (PA) fold in the inhibition of Wnt/ß-catenin pathway is controversial up-to date, apart from the interaction with secreted antagonists R-spondin family proteins shown by the crystallographic studies. METHODS: In our research we utilised cell-based approaches to study the role of RNF43 lacking PA domain in the canonical Wnt signalling pathway transduction. We developed controlled overexpression (TetON) and CRISPR/Cas9 mediated knock-out models in human cells. RESULTS: RNF43ΔPA mutant activity impedes canonical Wnt pathway, as manifested by the reduced phosphorylation of LRP6, DVL2 and DVL3 and by the decreased ß-catenin-dependent gene expression. Finally, rescue experiments in the CRISPR/Cas9 derived RNF43/ZNRF3 double knock-out cell lines showed that RNFΔPA overexpression is enough to inhibit activation of LRP6 and ß-catenin activity as shown by the Western blot and Top flash dual luciferase assays. Moreover, RNF43 variant without PA domain was not sensitive to the R-spondin1 treatment. CONCLUSION: Taken together, our results help to understand better the mode of RNF43 tumor suppressor action and solve some discrepancies present in the field. Video Abstract.

Contribution to colonic polyposis of recently proposed predisposing genes and assessment of the prevalence of NTHL1- and MSH3-associated polyposes.

Technological advances have allowed the identification of new adenomatous and serrated polyposis genes, and of several candidate genes that require additional supporting evidence of causality. Through an exhaustive literature review and mutational screening of 177 unrelated polyposis patients, we assessed the involvement of MCM9, FOCAD, POLQ, and RNF43 in the predisposition to (nonserrated) colonic polyposis, as well as the prevalence of NTHL1 and MSH3 mutations among genetically unexplained polyposis patients. Our results, together with previously reported data and mutation frequency in controls, indicate that: MCM9 and POLQ mutations are not associated with polyposis; germline RNF43 mutations, with a prevalence of 1.5-2.5% among serrated polyposis patients, do not cause nonserrated polyposis; MSH3 biallelic mutations are highly infrequent among European polyposis patients, and the prevalence of NTHL1 biallelic mutations among unexplained polyposes is ~2%. Although nonsignificant, FOCAD predicted deleterious variants are overrepresented in polyposis patients compared to controls, warranting larger studies to provide definite evidence in favor or against their causal association with polyposis predisposition.