BackgroundLow molecular weight protein tyrosine phosphatase (LMWPTP), encoded by the ACP1 gene, has been implicated in tumor progression across multiple malignancies. While its oncogenic functions have been reported in colorectal cancer (CRC), its role in gastric cancer (GC) remains poorly defined. This study investigated the expression patterns, functional relevance, and prognostic impact of LMWPTP in GC, with comparative analyses in CRC.MethodsGene expression, immune infiltration, and survival analyses were performed using data from The Cancer Genome Altas (TCGA). LMWPTP protein expression was evaluated in a gastric cancer tissue microarray. Functional assays were conducted in GC and CRC cell lines with CRISPR-Cas9-mediated LMWPTP knockout.ResultsACP1 mRNA expression was significantly upregulated in both GC and CRC compared with adjacent normal tissues. In GC, high LMWPTP expression was associated with poor differentiation in intestinal-type tumors and reduced survival in diffuse-type cases. ACP1 overexpression correlated with an elevated tumor mutation burden but a decreased cytotoxic lymphocyte infiltration signature in GC, indicating a potential relationship between ACP1 expression, tumor mutational load, and tumor immune microenvironment. Functional assays in vitro showed that LMWPTP knockout reduced migration in both GC and CRC cells, whereas a decrease in invasion was observed only in CRC cells. These findings indicate context-dependent contributions of LMWPTP to gastrointestinal tumor biology.ConclusionLMWPTP is consistently upregulated in gastric and colorectal cancers and exhibits tumor-type-specific functional and immune associated features. These findings highlight its distinct oncogenic role and potential as a biomarker and therapeutic target in GC.

BACKGROUND & AIMS:Barrett's esophagus (BE), a metaplastic transformation driven by gastroesophageal reflux disease (GERD), induces oxidative stress but the underlying redox mechanisms remain poorly understood. Protein persulfidation (PSSH), a redox-sensitive, reversible, and antioxidative post-translational modification regulated by hydrogen sulfide (H2S) metabolism, has not been explored in this context. Here, we identify epithelial PSSH as a key regulator of this premalignant process. METHODS:We applied proteomics and chemoproteomics in 2 patient cohorts to map and validate PSSH and total proteome profiles across healthy (squamous), GERD-exposed, and metaplastic epithelium. Using in vitro and in vivo models of chronic GERD and BE, we modulated H2S levels genetically and pharmacologically. Mechanistic and functional effects were assessed using tissue biopsies or recombinant human proteins. RESULTS:GERD-induced oxidative loss of H2S and its enhanced catabolism initiated early PSSH proteome remodeling in squamous epithelium, which expanded in BE and affected >1300 proteins in clinical samples, indicating potential biomarkers. This also included altered persulfidation of enzymes regulating accumulation of prostaglandin E2 (PGE2), a well-established driver of BE development and progression. H2S depletion accelerated metaplastic transformation, whereas H2S donors reversed these effects in experimental models. PSSH of 15-hydroxyprostaglandin dehydrogenase reversibly suppressed its activity, protecting the enzyme and, unlike irreversible oxidation, allowing recovery of PGE2 degradation. CONCLUSIONS:These findings redefine the origin of PGE2 accumulation in metaplasia and establish sulfide loss and persulfidomic remodeling as central, druggable drivers of epithelial reprogramming and redox imbalance in BE pathogenesis.

Background & Aims Trimethylamine N-oxide (TMAO) is a host-microbial co-metabolite that significantly increases following Roux-en-Y gastric bypass (RYGB). While TMAO is associated with cardiovascular diseases, its role in colorectal cancer (CRC) remains unclear. Methods FabplCre;Apc15lox/+ mice, a genetically altered CRC model, together with murine macrophages and human colonic cancer cells (HCT-116) were used to investigate TMAO impact on colonic tumorigenesis. Results TMAO supplementation significantly reduced the colonic tumor load in male, but not female mice. Consistently, dietary TMAO resulted in higher retention of circulating TMAO in males, which was significantly and inversely correlated with tumor loads. Furthermore, TNF-α-expressing cell frequencies in the colonic intraepithelial lymphocytes (IEL) were significantly lower in TMAO-supplemented male mice compared to controls, suggesting that circulating TMAO could play a protective effect against colonic tumor growth via down-regulation of TNF-α-expressing cells. This was supported by in vitro observations that TMAO reduced lipopolysaccharides (LPS)-stimulated TNF-α production from macrophages. TMAO exerted no effects on cell proliferation (Ki67) and DNA damage (γH2AX) of HCT-116 cells. Conclusion Our study leads us to conclude that higher retention of circulating TMAO has a protective effect against CRC in a sex-dependent manner, highlighting the importance of understanding the complex relationship amongst the concentrations of host-microbial cometabolite TMAO, its systemic circulation, and its biological function in modulating CRC risk.

ZNRF3, a negative regulator of beta-catenin signaling, removes Wnt receptors from the membrane. Currently, it is unknown which tumor-associated variants can be considered driver mutations and through which mechanisms they contribute to cancer. Here we show that all truncating mutations analyzed at endogenous levels exhibit loss-of-function, with longer variants retaining partial activity. Regarding missense mutations, we show that 27/82 ZNRF3 variants in the RING and R-Spondin domain structures, lead to (partial) loss-of-function/hyperactivation. Mechanistically, defective R-Spondin domain variants appear to undergo endoplasmic-reticulum-associated degradation due to protein misfolding, leading to reduced protein levels. They fail to reach the membrane correctly, which can be partially restored for several variants by culturing cells at 27 degrees C. Although RING and R-Spondin domain mutations in RNF43/ZNRF3 are often considered to possess dominant-negative oncogene-like activity in cancers, our findings challenge this notion. When representative variants are heterozygously introduced into endogenous ZNRF3, their impact on beta-catenin signaling mirrors that of heterozygous knockout, suggesting that the supposed dominant-negative effect is non-existent. In other words, so-called "hyperactivating" ZNRF3/RNF43 mutations behave as classical loss-of-function mutations at endogenous levels.

RNF43 and ZNRF3 are recognized as important regulators of Wnt/β-catenin signaling by maintaining Wnt-receptors at minimal essential levels. In various cancer types, particularly gastrointestinal tumors, mutations in these genes lead to abnormal Wnt-dependent activation of β-catenin signaling. However, recent findings implicate RNF43/ZNRF3 also in the regulation of other tumor-related proteins, including EGFR, BRAF, and the BMP-signaling pathway, which may have important implications for tumor biology. Additionally, we describe in detail how phosphorylation and ubiquitination may finetune RNF43 and ZNRF3 activity. We also address the variety of mutations observed in cancers and the mechanism through which they support tumor growth, and challenge the prevailing view that specific missense mutations in the R-spondin and RING domains may possess dominant-negative activity in contributing to tumor formation.

BACKGROUND:Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 mutations contribute to HCC development remains unclear. METHODS:In this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines. RESULTS:For each cell line we successfully obtained 2-4 correctly repaired clones, which all show reduced β-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored β-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive β-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling. CONCLUSIONS:Our study provides insights into the effects of repairing AXIN1 mutations on β-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations.

Addition of an extra RGS/APC domain restores APC and β-catenin binding to selected RGS/APC domain variants, and (partially) restores β-catenin regulation

AbstractAXIN1 is a major component of the β-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to β-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3β, β-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper β-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected β-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on β-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the β-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect β-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other β-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients.Significance:Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its β-catenin regulatory function, whereas the majority are passenger mutations.

ZNRF3, a negative regulator of β-catenin signaling, removes Wnt receptors from the membrane. Currently, it is unknown which tumor-associated variants can be considered driver mutations and through which mechanisms they contribute to cancer. Here we show that all truncating mutations analyzed at endogenous levels exhibit loss-of-function, with longer variants retaining partial activity. Regarding missense mutations, we show that 27/82 ZNRF3 variants in the RING and R-Spondin domain structures, lead to (partial) loss-of-function/hyperactivation. Mechanistically, defective R-spondin domain variants appear to undergo endoplasmic-reticulum-associated degradation due to protein misfolding. They show reduced stability and fail to reach the membrane correctly, which can be partially restored for several variants by culturing cells at 27°C. Although RING and R-spondin domain mutations in RNF43/ZNRF3 are often considered to possess dominant-negative oncogene-like activity in cancers, our findings challenge this notion. When representative variants are heterozygously introduced into endogenous ZNRF3, their impact on β-catenin signaling mirrors that of heterozygous knockout, suggesting that the supposed dominant-negative effect is non-existent. In other words, so-called “hyperactivating” ZNRF3/RNF43 mutations behave as classical loss-of-function mutations at endogenous levels. Taken together, our findings provide valuable information on ZNRF3 mutation impact in tumorigenesis and clarify their mechanism of action.### Competing Interest StatementThe authors have declared no competing interest.

Supplementary Methods, Figures and Tables.

RNF43 is an important negative regulator of β-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of β-catenin. RNF43 has also been suggested to regulate β-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/β-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers.

β-Catenin (Ctnnb1) has been shown to play critical roles in the development and maintenance of epithelial cells, including the retinal pigment epithelium (RPE). Ctnnb1 is not only a component of intercellular junctions in the epithelium, it also functions as a transcriptional regulator in the Wnt signaling pathway. To identify which of its functional modalities is critically involved in mouse RPE development and maintenance, we varied Ctnnb1 gene content and activity in mouse RPE lineage cells and tested their impacts on mouse eye development. We found that a Ctnnb1 double mutant (Ctnnb1dm), which exhibits impaired transcriptional activity, could not replace Ctnnb1 in the RPE, whereas Ctnnb1Y654E, which has reduced affinity for the junctions, could do so. Expression of the constitutively active Ctnnb1∆ex3 mutant also suppressed the development of RPE, instead facilitating a ciliary cell fate. However, the post-mitotic or mature RPE was insensitive to the loss, inactivation, or constitutive activation of Ctnnb1. Collectively, our results suggest that Ctnnb1 should be maintained within an optimal range to specify RPE through transcriptional regulation of Wnt target genes in the optic neuroepithelium.

S1. Specificity of STRAP antibody tested on a STRAP knock-out Huh6 clone and control thereof. S2. Knockout of the STRAP gene by CRISPR/Cas9 technology in HCC cell lines. S3. Elevated expression of STRAP in DEN-induced mouse liver tumors (T) compared with flanking normal liver tissue (N). S4. Analysis of the TCGA liver cancer cohort reveals that no significant difference in average STRAP RNA levels is observed between normal and tumor samples. S5. Baseline levels of STRAP protein and RNA in a panel of 9 HCC cell lines. S6. Log2 fold change of liver stemness markers (A), liver differentiation related genes (B) and TGF-β signaling target genes (C) in STRAP knock-out clones compared to controls. Supplemental Table S1. Gene mutations of Wnt/β-catenin signaling components in HCC cell lines. Supplemental Table S2. Clinicopathologic characteristics of HCC TMA. Supplemental Table S3. Selected STRAP sgRNAs. Supplemental Table S4. Primer sequences of STRAP used for Sanger sequencing. Supplemental Table S5. Primer sequence used for Ion Torrent sequencing. Supplemental Table S6. Primer sequences used for qRT-PCR Supplemental Table S7. Gene information

Anti-BRAF/EGFR therapy was recently approved for the treatment of metastatic BRAF V600E colorectal cancer (mCRC BRAF-V600E ). However, a large fraction of patients do not respond, underscoring the need to identify molecular determinants of treatment response. Using whole-exome sequencing in a discovery cohort of patients with mCRC BRAF-V600E treated with anti-BRAF/EGFR therapy, we found that inactivating mutations in RNF43 , a negative regulator of WNT, predict improved response rates and survival outcomes in patients with microsatellite-stable (MSS) tumors. Analysis of an independent validation cohort confirmed the relevance of RNF43 mutations to predicting clinical benefit (72.7% versus 30.8%; P = 0.03), as well as longer progression-free survival (hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.12–0.75; P = 0.01) and overall survival (HR, 0.26; 95% CI, 0.10–0.71; P = 0.008), in patients with MSS- RNF43 mutated versus MSS- RNF43 wild-type tumors. Microsatellite-instable tumors invariably carried a wild-type-like RNF43 genotype encoding p.G659fs and presented an intermediate response profile. We found no association of RNF43 mutations with patient outcomes in a control cohort of patients with MSS-mCRC BRAF-V600E tumors not exposed to anti-BRAF targeted therapies. Overall, our findings suggest a cross-talk between the MAPK and WNT pathways that may modulate the antitumor activity of anti-BRAF/EGFR therapy and uncover predictive biomarkers to optimize the clinical management of these patients.

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.

Barrett's esophagus in gastrointestinal reflux patients constitutes a columnar epithelium with distal characteristics, prone to progress to esophageal adenocarcinoma. HOX genes are known mediators of position-dependent morphology. Here we show HOX collinearity in the adult gut while Barrett's esophagus shows high HOXA13 expression in stem cells and their progeny. HOXA13 overexpression appears sufficient to explain both the phenotype (through downregulation of the epidermal differentiation complex) and the oncogenic potential of Barrett's esophagus. Intriguingly, employing a mouse model that contains a reporter coupled to the HOXA13 promotor we identify single HOXA13-positive cells distally from the physiological esophagus, which is mirrored in human physiology, but increased in Barrett's esophagus. Additionally, we observe that HOXA13 expression confers a competitive advantage to cells. We thus propose that Barrett's esophagus and associated esophageal adenocarcinoma is the consequence of expansion of this gastro-esophageal HOXA13-expressing compartment following epithelial injury. Barrett's esophagus is a pro-oncogenic lesion in the proximal gastrointestinal tract, but with a distal colon-like morphology. Here the authors report that the distal HOX gene HOXA13 is expressed in Barrett's esophagus and in single cells of the physiological esophagus, and may underlie the phenotypic aspects of metaplasia and increase proliferation.

AXIN1 mutations are observed in 8–10% of hepatocellular carcinomas (HCCs) and originally were considered to support tumor growth by aberrantly enhancing β-catenin signaling. This view has however been challenged by reports showing neither a clear nuclear β-catenin accumulation nor clearly enhanced expression of β-catenin target genes. Here, using nine HCC lines, we show that AXIN1 mutation or siRNA mediated knockdown contributes to enhanced β-catenin signaling in all AXIN1-mutant and non-mutant lines, also confirmed by reduced signaling in AXIN1-repaired SNU449 cells. Both AXIN1 and AXIN2 work synergistically to control β-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels. The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of β-catenin-dependent cancers. At concentrations that inhibit tankyrase activity, some lines (e.g. HepG2, SNU398) were clearly affected in colony formation, but in most cases apparently independent from effects on β-catenin signaling. Overall, our analyses show that AXIN1 inactivation leads to enhanced β-catenin signaling in HCC cell lines, questioning the strong statements that have been made in this regard. Enhancing AXIN activity by tankyrase monotherapy provides however no effective treatment to affect their growth exclusively through reducing β-catenin signaling.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are thought to be the main drivers for disease progression and treatment resistance across various cancer types. Identifying and targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the enrichment of LGR5 expressing cells, a well-recognized stem cell marker, in mouse liver tumors, and the upregulation of LGR5 expression in human hepatocellular carcinoma. Isolated LGR5 expressing cells from mouse liver tumors are superior in initiating organoids and forming tumors upon engraftment, featuring candidate TICs. These cells are resistant to conventional treatment including sorafenib and 5-FU. Importantly, LGR5 lineage ablation significantly inhibits organoid initiation and tumor growth. The combination of LGR5 ablation with 5-FU, but not sorafenib, further augments the therapeutic efficacy in vivo. Thus, we have identified the LGR5+ compartment as an important TIC population, representing a viable therapeutic target for combating liver cancer.