Negative Regulator Of Wnt Signaling Research Articles

Abstract PR-18: RNF43 Loss Induces an IRE1-dependent Metabolic Reprogramming in Pancreatic Cystic Neoplasms

Abstract Background: RNF43 is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which are cystic precursor lesions of pancreatic ductal adenocarcinoma (PDAC). RNF43 encodes for an E3 ubiquitin ligase and was initially identified as a negative regulator of Wnt signaling in colorectal cancer. Clinical trials targeting ligand dependent Wnt signaling in RNF43-mutated cancers have failed to demonstrate any efficacy, suggesting other mechanisms may be involved. Methods: To elucidate the role of RNF43 in an organ specific context, we generated a genetically engineered mouse model with pancreas-specific loss of Rnf43 and co-expression of mutant Kras (referred to as Kras;Rnf43 mice). The Kras;Rnf43 mice develop pancreatic cystic lesions resembling human IPMNs, a subset of which progressive to invasive cancers. We generated derivative cell lines from the IPMN-associated cancers, referred to as Kras;Rnf43 lines. Single-cell RNA sequencing (scRNA-seq) was performed to compare murine PDAC arising in Kras;Rnf43 mice with that of the Kras;p53 (“KPC”) mice. To determine if observed perturbations were dependent on Rnf43, we also created isogenic Kras;Rnf43 cell lines with re-expression of full-length Rnf43 (Kras;Rnf43-RNF43OE). Results: Kras;Rnf43 tumors showed upregulation of several mitochondrial transcripts (Mt-Atp8, Atp5o.1, and Atp6v0c), as well as mitochondrial biogenesis markers (PPARGC1A and NFE2L2), compared to KPC mice. Concurrently, the oxidative phosphorylation (OXPHOS) pathway was enriched on proteomics analyses of Kras;Rnf43 cells, relative to isogenic cell lines with RNF43 restitution. Seahorse metabolic flux analyzer revealed enhanced mitochondrial OXPHOS activity in Kras;Rnf43 cell lines, along with increased mitophagy. Kras;Rnf43 cells harbored gene signatures indicative of endoplasmic reticulum (ER) stress compared to KPC lines. Endoplasmic reticulum-mitochondria contact sites (ERMCS) regulate ER stress and mitochondrial dynamics. Notably, Kras;Rnf43 cells had significantly increased ERMCS compared to KPC cells, and these were attenuated upon RNF43 re-expression. We further identified the ER stress sensor inositol-requiring protein 1 (IRE1) was markedly elevated in Kras;Rnf43 cells compared to KPC cells, and decreased upon RNF43 re-expression. Furthermore, IRE1 localized at the mitochondrial-associated membrane (MAM) and enhanced ERMCS formation via a non-canonical scaffolding function. Deletion of IRE1 by CRISPR/Cas9 editing in Kras;Rnf43 cells inhibited OXPHOS and significantly impaired cell viability. Conclusion: Our data suggests that bi-allelic loss of RNF43 caused increased mitochondrial function and OXPHOS dependency. This is largely attributed to upregulated level of IRE1, which localizes at MAM to promote ERMCS formation and OXPHOS in Kras;Rnf43 cells. IRE1-dependent reprogramming of mitochondrial bioenergetics serves as a cell survival mechanism. Therefore, targeting OXPHOS or the noncanonical function of IRE1 could be a potential strategy to inhibit the progression of RNF43-mutated IPMNs to PDAC. Citation Format: Akiko Sagara, Xiangdong Lv, Brandon Chen, Yuki Makino, Shui Ping So, Sonja M Woermann, Costas A Lyssiotis, Yatrik M Shah, Bidyut Ghosh, Xi Chen, Johannes F Fahrmann, Anirban Maitra. RNF43 Loss Induces an IRE1-dependent Metabolic Reprogramming in Pancreatic Cystic Neoplasms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-18.

Abstract P46: Small Extracellular Vesicles from Therapy-induced Senescent TNBC Elicit Paracrine Anti-tumour Effects

Abstract Triple-negative breast cancers (TNBC), associated with poor prognosis and high tumour recurrence, are often treated with taxanes. However, cells may bypass treatment-induced cell death via mitotic slippage, resulting in multinucleated polyploid cells and therapy-induced senescence (TIS). TIS cancer cells represent a population of residual disease and may modulate therapy resistance and recurrence through secretion of the senescence-associated secretory phenotype (SASP). While SASP have been heavily linked with pro-tumorigenic behaviour mediated through soluble cytokines, small extracellular vesicles (sEVs) derived from TIS cancer cells represent an underappreciated aspect of the senescent tumour secretome, as its mechanistic role in mediating paracrine effects remains poorly understood. In this study, we uncovered an unexpected role for TIS-derived sEVs within the secretome, which conferred paracrine anti-tumour activity in recipient TNBC cancer cells, in contrast to SASP that support pro-tumorigenic behaviour. SEVs profiling through mass-spectrometry revealed that DKK1, a negative regulator of WNT signalling, is enriched in TIS-derived sEVs. Further investigation into the tumour microenvironment with animal models and neoadjuvant chemo-treated patient samples revealed STING-dependent immune response. Hence, our study suggests that sEVs should be considered as distinct SASP entities within the senescent tumour secretome and provide an additional basis for combinatorial use of senomorphics with taxanes to potentially modulate senescence-associated sEVs while suppressing pro-tumorigenic SASP factors. Citation Format: Matius Robert, Rekha Jakhar, Bijin Veonice Au, Gracie Wee Ling Eng, Alvin Kunyao Gao, You Heng Chuah, Karishma Sachaphibulkij, Lina Hsiu Kim Lim, Derrick Sek Tong Ong, Elaine Hsuen Lim, Yoko Itahana, Koji Itahana, Jit Kong Cheong, John Edward Connolly, Karen Carmelina Crasta. Small Extracellular Vesicles from Therapy-induced Senescent TNBC Elicit Paracrine Anti-tumour Effects [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P46.

Selective epigenetic alterations in RNF43 in pancreatic exocrine cells from high-fat-diet-induced obese mice; implications for pancreatic cancer

ObjectivePancreatic cancer (PC) originates and progresses with genetic mutations in various oncogenes and suppressor genes, notably KRAS, CDKN2A, TP53, and SMAD4, prevalent across diverse PC cells. In addition to genetic mutations/deletions, persistent exposure to high-risk factors, including obesity, induces whole-genome scale epigenetic alterations contributing to malignancy. However, the impact of obesity on DNA methylation in the presymptomatic stage, particularly in genes prone to PC mutation, remains uncharacterized.ResultsWe analyzed the methylation levels of 197 loci in six genes (KRAS, CDKN2A, TP53, SMAD4, GNAS and RNF43) using Illumina Mouse Methylation BeadChip array (280 K) data from pancreatic exocrine cells obtained from high-fat-diet (HFD) induced obese mice. Results revealed no significant differences in methylation levels in loci between HFD- and normal-fat-diet (NFD)-fed mice, except for RNF43, a negative regulator of Wnt signaling, which showed hypermethylation in three loci. These findings indicate that, in mouse pancreatic exocrine cells, high-fat dietary obesity induced aberrant DNA methylation in RNF43 but not in other frequently mutated PC-related genes.

Abstract 762: The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer-associated mortality. Our recent study in Pirc rats revealed that the combination of eicosapentaenoic acid and sodium naproxen exhibited additive tumor protection. While the results from this preclinical study were impressive, showing tumor suppression in excess of 95%, potential mechanisms underlying this tumor protection remain unclear. The purpose of this study was to determine the transcriptomic changes resulting in the tumor suppression afforded by the combination of EPA and naproxen supplementation in the diet. Methods: Six week-old Pirc rats were fed either a control AIN-93G diet, or AIN-93G diet supplemented with 2% EPA combined with 200 ppm naproxen, for a total of 20 weeks. Global transcriptomic analysis was performed using RNA sequencing (RNA-Seq) of colon tumors and tumor-adjacent normal tissue from 26-week-old Pirc rats. 100-bp paired-end sequencing was performed on an Illumina NovaSeq to a depth of 25 million reads per sample. Reads were trimmed with Trimmomatic-v0.39, and reads were mapped to the mRatBN7.2.105 rat reference genome using HISAT2. Differentially expressed genes were calculated using DESeq2, and gene set enrichment analysis (GSEA) were performed using Rstudio. Results: DESeq2 identified 809 differentially expressed genes (DEGs, 2-fold change > 1, FDR < 0.05) between large tumors from AIN-93G fed rats versus suppressed tumors from rats fed the combination of EPA and naproxen (490 up-regulated, 319 down-regulated). GSEA analysis revealed activation of inflammatory pathways including IL-17 signaling, JAK-STAT signaling, complement activation, cytokine signaling, ribosome biogenesis, and cell cycle were enriched in untreated control. In contrast, suppressed tumors from animals supplemented with EPA and naproxen were enriched in pathways including negative regulation of Wnt signaling, cell fate specification, ion channel complexes, and nutrient absorption. Furthermore, suppressed tumors shared significant GSEA pathway overlap with normal tissue, leading us to believe the combination of EPA and naproxen may be preventing tumorigenesis in part by maintaining cells in a differentiated state. Conclusion and Future directions: Inflammation plays a key role in tumorigenesis and promoting cancer stem cell niches in tumors. According to our analysis, the combination of EPA and naproxen attenuated cancer-associated inflammation and reduced proliferation in part by augmenting tumor differentiation. Future studies will utilize single-nuclei RNA-Seq (snRNA-Seq) to determine at a single cell resolution the transcriptomic changes of epithelial and stromal populations within the TME. This work was supported by National Cancer Institute task order 75N91019F00132. Citation Format: Ryan Beach, Michael Martinez, Vignesh Gunasekharan, Daniel W. Rosenberg. The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 762.

Ezh2 promotes mammary tumor initiation through epigenetic regulation of the Wnt and mTORC1 signaling pathways

The regulation of gene expression through histone posttranslational modifications plays a crucial role in breast cancer progression. However, the molecular mechanisms underlying the contribution of histone modification to tumor initiation remain unclear. To gain a deeper understanding of the role of the histone modifier Enhancer of Zeste homology 2 (Ezh2) in the early stages of mammary tumor progression, we employed an inducible mammary organoid system bearing conditional Ezh2 alleles that faithfully recapitulates key events of luminal B breast cancer initiation. We showed that the loss of Ezh2 severely impairs oncogene-induced organoid growth, with Ezh2-deficient organoids maintaining a polarized epithelial phenotype. Transcriptomic profiling showed that Ezh2-deficient mammary epithelial cells up-regulated the expression of negative regulators of Wnt signaling and down-regulated genes involved in mTORC1 (mechanistic target of rapamycin complex 1) signaling. We identified Sfrp1, a Wnt signaling suppressor, as an Ezh2 target gene that is derepressed and expressed in Ezh2-deficient epithelium. Furthermore, an analysis of breast cancer data revealed that Sfrp1 expression was associated with favorable clinical outcomes in luminal B breast cancer patients. Finally, we confirmed that targeting Ezh2 impairs mTORC1 activity through an indirect mechanism that up-regulates the expression of the tumor suppressor Pten. These findings indicate that Ezh2 integrates the mTORC1 and Wnt signaling pathways during early mammary tumor progression, arguing that inhibiting Ezh2 or therapeutically targeting Ezh2-dependent programs could be beneficial for the treatment of early-stage luminal B breast cancer.

TCF7L1 Controls the Differentiation of Tuft Cells in Mouse Small Intestine.

Continuous and rapid renewal of the intestinal epithelium depends on intestinal stem cells (ISCs). A large repertoire of transcription factors mediates the correct maintenance and differentiation of ISCs along either absorptive or secretory lineages. In the present study, we addressed the role of TCF7L1, a negative regulator of WNT signalling, in embryonic and adult intestinal epithelium using conditional mouse mutants. We found that TCF7L1 prevents precocious differentiation of the embryonic intestinal epithelial progenitors towards enterocytes and ISCs. We show that Tcf7l1 deficiency leads to upregulation of the Notch effector Rbp-J, resulting in a subsequent loss of embryonic secretory progenitors. In the adult small intestine, TCF7L1 is required for the differentiation of secretory epithelial progenitors along the tuft cell lineage. Furthermore, we show that Tcf7l1 promotes the differentiation of enteroendocrine D- and L-cells in the anterior small intestine. We conclude that TCF7L1-mediated repression of both Notch and WNT pathways is essential for the correct differentiation of intestinal secretory progenitors.

Ginsenoside Rh2 attenuates the progression of non-small cell lung cancer by sponging miR-28-5p/STK4 axis and inactivating Wnt/β-catenin signaling.

Ginsenoside Rh2 (G-Rh2) exerts anti-tumor activity in non-small cell lung cancer (NSCLC). microRNAs (miRNAs, miRs) play pivotal roles in NSCLC. We aimed to investigate whether G-Rh2 inhibited NSCLC progression by targeting miRNA. Cell viability, apoptosis and cycle were determined by Cell Counting Kit-8, 6-diamidino-2-phenylindole (DAPI) staining and flow cytometry. The potential target miRNAs of G-Rh2 were screened by real-time quantitative polymerase chain reaction (RT-qPCR). The difference in miR-28-5p expression between lung adenocarcinoma (LUAD) tissues and normal tissues or lung squamous cell carcinoma (LUSC) tissues and normal tissues was retrieved from TCGA-LUAD and TCGA-LUSC, respectively. Kaplan-Meier Plotter was conducted to analyze the survival rate for different serine/threonine-protein kinase 4 (STK4) expressions with different prognostic risks. immunohistochemistry of STK4 expression in non-tumor and tumor tissues was analyzed from the HPA database. RT-qPCR and Western blot were adopted for detecting mRNA and protein expression. TargetScan V7.2, miRanda and PITA were adopted for predicting targets of miR-28-5p, overlapped genes were subjected to GO analysis. The interactions of miR-28-5p-Wnt and miR-28-5p-STK4 were detected by TOP/FOP luciferase reporter assay and dual luciferase reporter assay, respectively. Current study observed that G-Rh2 reduced miR-28-5p expression in NSCLC cells dose-dependently. miR-28-5p was upregulated in NSCLC tissues and cells. The target genes of miR-28-5p were enriched in negative regulation of Wnt signaling. miR-28-5p inhibitor inactivated Wnt signaling, inhibited cell viability and cell cycle, while enhanced cell apoptosis of NSCLC cells by targeting STK4. G-Rh2 exerted the similar effects with miR-28-5p inhibitor by reducing miR-28-5p. G-Rh2 and miR-28-5p inhibitor exerted a synergistic effect on inhibiting NSCLC tumor growth. In conclusion, G-Rh2 attenuates NSCLC development by affecting miR-28-5p/STK4 axis and inactivating Wnt signaling. Taken together, we project out a novel therapeutic target for NSCLC.

Pomegranate Extract Affects Gut Biofilm Forming Bacteria and Promotes Intestinal Mucosal Healing Regulating the Crosstalk between Epithelial Cells and Intestinal Fibroblasts

Background: Pomegranate (Punica granatum) can be used to prepare a bioactive extract exerting anti-inflammatory activities. Clinical studies demonstrated an improvement in clinical response in inflammatory bowel disease (IBD) patients when pomegranate extract (PG) was taken as a complement to standard medications. However, the molecular mechanisms underlying its beneficial effects are still scarcely investigated. This study investigates the effect of PG on bacterial biofilm formation and the promotion of mucosal wound healing. Methods: The acute colitis model was induced in C57BL/6N mice by 3% dextran sodium sulfate administration in drinking water for 5 days. During the recovery phase of colitis, mice received saline or PG (200 mg/kg body weight) by oral gavage for 11 days. Colitis was scored daily by evaluating body weight loss, bleeding, and stool consistency. In vivo intestinal permeability was evaluated by fluorescein isothiocyanate-conjugated dextran assay, bacterial translocation was assessed by fluorescence in situ hybridization on tissues, whereas epithelial and mucus integrity were monitored by immunostaining for JAM-A and MUC-2 markers. Bacterial biofilm formation was assessed using microfluidic devices for 24 or 48 h. Primary fibroblasts were isolated from healthy and inflamed areas of 8 IBD patients, and Caco-2 cells were stimulated with or without PG (5 μg/mL). Inflammatory mediators were measured at the mRNA and protein level by RT-PCR, WB, or Bio-plex multiplex immunoassay, respectively. Results: In vivo, PG boosted the recovery phase of colitis, promoting a complete restoration of the intestinal barrier with the regeneration of the mucus layer, as also demonstrated by the absence of bacterial spread into the mucosa and the enrichment of crypt-associated fibroblasts. Microfluidic experiments did not highlight a specific effect of PG on Enterobacterales biofilm formation, even though Citrobacter freundii biofilm was slightly impaired in the presence of PG. In vitro, inflamed fibroblasts responded to PG by downregulating the release of metalloproteinases, IL-6, and IL-8 and upregulating the levels of HGF. Caco-2 cells cultured in a medium supplemented with PG increased the expression of SOX-9 and CD44, whereas in the presence of HGF or plated with a fibroblast-conditioned medium, they displayed a decrease in SOX-9 and CD44 expression and an increase in AXIN2, a negative regulator of Wnt signaling. Conclusions: These data provide new insight into the manifold effects of PG on promoting mucosal homeostasis in IBD by affecting pathogen biofilm formation and favoring the regeneration of the intestinal barrier through the regulation of the crosstalk between epithelial and stromal cells.

Abstract 6134: Exploiting the oncogenic state of colorectal cancer for therapeutic development

Abstract Over the past two decades, only incremental progresses have been made in the therapeutic management of colorectal cancer (CRC). This is in part attributed to the fact that the Wnt pathway, the oncogenic pathway that initiates the transformation of CRC and sustains tumor maintenance, has been largely undruggable. While most efforts targeting Wnt signaling have revolved around interfering with Wnt target genes that regulate cell growth, we turned our attention to a subset of genes encoding for negative regulators of the pathway. Of particular interest are the cell-surface E3 ubiquitin ligases, ring finger protein 43 (RNF43) and ZNRF3, two known negative regulators of Wnt signaling that promote the turnover of Wnt receptors Frizzled/low-density lipoprotein receptor-related proteins (FZD/LRPs) via ubiquitination-mediated degradation. Given their exposed extracellular domains (ECDs), we set out to explore whether this intrinsic cellular degradative machinery could be repurposed for an antibody-based targeted protein degradation platform. More specifically, we have devised a technology that exploits the elevated expression of ZNRF3 and RNF43 in colon cancer by generating bispecific antibodies that can tether these E3 ubiquitin ligases to a variety of cell surface receptors to promote tumor receptor degradation. Most notably, these antibody degraders are highly active in vivo and can degrade receptors in a tumor specific manner while sparing normal tissues. This platform, that we have been dubbed Proteolysis Targeting Antibodies (PROTABs) describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins. Citation Format: Felipe de Sousa e Melo. Exploiting the oncogenic state of colorectal cancer for therapeutic development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6134.

P092 Punica Granatum affects gut biofilm-forming bacteria and promotes intestinal mucosal healing regulating the crosstalk between epithelial cells and intestinal fibroblasts

Abstract Background Punica Granatum (PG), is a bioactive compound of Pomegranate exerting anti-inflammatory activities. Clinical studies have demonstrated an improvement of clinical response in inflammatory bowel diseases (IBD) patients when PG was taken as a complement to standard medications. However, the molecular mechanisms underlying its beneficial effects are still scarcely investigated. In this study, we assessed the effects of PG on bacterial biofilm formation, and we investigated the properties of the PG to promote mucosal wound healing. Methods Bacterial biofilm formation was assessed using microfluidic devices for 24 or 48 hours. Acute colitis model was induced in C57BL/6N mice by 3% dextran sodium sulfate administration in their drinking water for 5 days. During the recovery phase of colitis, mice received saline or PG (200 mg/kg body weight) oral gavage for 11 days. . Colitis was scored daily evaluating the body weight loss, bleeding and consistency of stool. In vivo intestinal permeability was performed by fluorescein isothiocyanate-conjugated dextran assay, bacterial translocation by Fluorescence in situ hybridization on tissues, whereas epithelial and mucus integrity by immunostaining for JAM-A and MUC-2 markers. Primary fibroblasts, isolated from healthy and inflamed area of 6 IBD patients, and Caco-2 cells were stimulated with or without PG (15 ug/ml). Inflammatory mediators were measured at mRNA and protein levels by RT-PCR, WB or by Bio-plex multiplex immunoassay respectively. Results Microfluidic experiments revealed beneficial effects of PG to inhibit biofilm formation of GRAM- bacteria and to promote the expansion of some intestinal commensal Gram+ bacteria. In vivo, PG boosted the recovery phase of colitis promoting a complete restoration of intestinal barrier with the regeneration of the mucus layer compared to saline group as also demonstrated by no traces of bacteria spread into the mucosa. The PG enriched crypt-associated fibroblasts. In vitro, inflamed fibroblasts responded to PG by down-regulating the release of metalloproteinases, IL-6, IL-8 and up-regulating the levels of HGF. Caco-2 cells cultured in medium supplemented with PG increased the expression of Sox-9 and CD44, whereas in the presence of HGF or when plated with fibroblast-conditioned medium displayed a decrease expression of Sox-9 and CD44 and increase of AXIN2, a negative regulator of Wnt signaling. Conclusion These data provide new insight of the multiple effects of PG on promoting mucosal homeostasis in IBD by affecting biofilm formation of pathogens, promoting expansion of commensals and favoring the regeneration of intestinal barrier by regulating the crosstalk between epithelial cells and stromal cells.

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants

PurposeGermline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. MethodsGenetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. ResultsThe phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. ConclusionNEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN–CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

Co-expression patterns explain how a basic transcriptional role for MYC modulates Wnt and MAPK pathways in colon and lung adenocarcinomas

ABSTRACT A subset of proliferation genes that are associated with origin licensing, firing, and DNA synthesis has been compared to known drivers of colon (COAD) and lung (LUAD) adenocarcinomas using Spearman’s rank correlation coefficients. The frequency with which APC, CTNNB1, KRAS, MYC, Braf, TP53, Rb1, EGFR, and cell cycle components have direct or indirect co-expression with the proliferation factors permits identification of their expression relative to the G1-S phase of the cell cycle. Here, adenomatous polyposis coli (APC), a negative regulator of Wnt signaling known to function through MYC, indirectly co-expresses at the same frequency as proliferation genes in both COAD and LUAD, consistent with M phase expression. However, APC is indirectly co-expressed with MYC and is found mutated only in COAD. MYC is thought to function at the interface of transcription and replication, acting through the SWI/SNF chromatin remodeling complex, and increased or decreased expression of MYC can induce or repress tumorigenesis, respectively. These data suggest that transcription of APC during the M phase with low MYC co-expression contributes by an unknown mechanism to APC mutations and Wnt pathway deregulation in COAD and that upper and lower limits of MYC expression, enforced by the cell cycle, may influence cancer differentially. Other Wnt signaling components co-expressed in the low MYC context in COAD also have significantly higher mutation frequencies, supporting the hypothesis. Additionally, Braf is found here to have direct co-expression with multiple proliferation factors in non-EGFR activated LUAD, and EGFR-activated LUAD are completely deregulated with respect to E2F(s) 4/5/6 expression, potentially explaining the low proliferation rates seen in LUAD. Abbreviations: TCGA: The cancer genome atlas; PANCAN: Pan cancer; CDK: Cyclin-dependent kinase; COAD: Colon adenocarcinoma; LUAD: Lung adenocarcinoma; Wnt: Wingless/integrated signaling; MAPK: Mitogen-activated protein kinase; APC: Adenomatous polyposis coli gene; CIN: Chromosomal instability; GS: Genome stabile; MMR: Mismatch repair; mRNA: Messenger RNA; RPKM: Reads per kilobase of transcription per million mapped reads; RSEM: RNA Seq by expectation maximization; FDR: False discovery rate; GTEx: Genotype-Tissue Expression portal; MPF: Maturation-promoting factor; SCF: Skp1-Cul1-F-box; HH: Hedgehog; EMT: Epithelial mesenchymal transition; RB: Retinoblastoma; EGFR: Epidermal growth factor receptor; rs or ρ: Spearman’s correlation

UCHL5 controls \u03b2-catenin destruction complex function through Axin1 regulation

Wnt/β-catenin signaling is crucially involved in many biological processes, from embryogenesis to cancer development. Hence, the complete understanding of its molecular mechanism has been the biggest challenge in the Wnt research field. Here, we identified ubiquitin C-terminal hydrolase like 5 (UCHL5), a deubiquitinating enzyme, as a novel negative regulator of Wnt signaling, upstream of β-catenin. The study further revealed that UCHL5 plays an important role in the β-catenin destruction complex, as it physically interacts with multiple domains of Axin1 protein. Our functional analyses also elucidated that UCHL5 is required for both the stabilization and the polymerization of Axin1 proteins. Interestingly, although these events are governed by deubiquitination in the DIX domain of Axin1 protein, they do not require the deubiquitinating activity of UCHL5. The study proposes a novel molecular mechanism of UCHL5 potentiating the functional activity of Axin1, a scaffolder of the β-catenin destruction complex.

RNF43/ZNRF3 negatively regulates taste tissue homeostasis and positively regulates dorsal lingual epithelial tissue homeostasis.

SummaryTaste bud cells are renewed throughout life in a process requiring innervation. Recently, we reported that R-spondin substitutes for neuronal input for taste cell regeneration. R-spondin amplifies WNT signaling by interacting with stem-cell-expressed E3 ubiquitin ligases RNF43/ZNRF3 (negative regulators of WNT signaling) and G-protein-coupled receptors LGR4/5/6 (positive regulators of WNT signaling). Therefore, we hypothesized that RNF43/ZNRF3 may serve as a brake, controlled by gustatory neuron-produced R-spondin, for regulating taste tissue homeostasis. Here, we show that mice deficient for Rnf43/Znrf3 in KRT5-expressing epithelial stem/progenitor cells (RZ dKO) exhibited taste cell hyperplasia; in stark contrast, epithelial tissue on the tongue degenerated. WNT signaling blockade substantially reversed all these effects in RZ dKO mice. Furthermore, innervation becomes dispensable for taste cell renewal in RZ dKO mice. We thus demonstrate important but distinct functions of RNF43/ZNRF3 in regulating taste versus lingual epithelial tissue homeostasis.

Virtual Screening Directly Identifies New Fragment-Sized Inhibitors of Carboxylesterase Notum with Nanomolar Activity.

Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC50 < 500 nM. A critical selection process was then applied with two clusters and two singletons (1-4d) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/β-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the β-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

QSAR Docking on Azoles as Inhibitors of Notum Carboxylesterase

Background: Carboxylesterase Notum is a negative regulator of Wnt signaling. Notum carboxylesterase is a carboxylic ester hydrolase enzyme that functions as a negative feedback regulator of Wnt proteins by depalmitoylation reaction. It is of great importance to understand the pathway of Wnt regulation because, conversely, misregulation of Wnt signaling is a telltale sign of cancer and other degenerative diseases. The Wnt inhibition is important in the control of colorectal cancer. Objective: In the present study, we carried out a QSAR analysis of a series of reported compounds with carboxylesterase Notum inhibitory activity using multiple regression analysis. A series of 83 compound datasets of pyrrole derivatives with carboxy Notum inhibitory values were taken from the reported literature. Methods: The study was performed by conducting multiple linear regression analysis followed by validation of the model. The multiple linear regression (MLR) models with the highest coefficients of correlation (R2) and explained variance in leave-one-out (Q2 LOO) prediction and leave- manyout (Q2 LMO) were selected for the whole dataset. The developed models were subjected to internal and external validation. The reliability of the predicted model was checked by plotting the Williams plot. The docking methodology was performed using Autodock 4 for the designed compounds to study the interaction between the ligand and the receptor. Results: The best model generated exhibited an r2 value of 0.7413, Q2LOO =0.6379, Q2LMO =0.6368. Novel compounds of phenyl pyrrolidine were designed based on generated QSAR equations. The carboxylesterase Notum inhibitory activity was predicted using the QSAR equations. The docking studies were carried out for designed compounds using Autodock against Carboxylesterase Notum esterase. Conclusion: From the results, the designed compounds were found to inhibit Notum Carboxylase. Thus, the study led to the development of a novel lead compound for Carboxylesterase Notum.

Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids.

Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited because of the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles, we performed in vitro aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50 μmol/L aspirin or vehicle control for 72 hours and subjected to bulk RNA sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant (q < 0.10) DEGs prior to deconvolution. We provide replication of these findings in an independent population-based RNA-sequencing dataset of mucosal biopsies (BarcUVa-Seq), where a significant enrichment for overlap of DEGs was observed (P < 2.2E-16). Single-cell deconvolution revealed changes in cell composition, including a decrease in transit-amplifying cells following aspirin treatment (P = 0.01). Following deconvolution, DEGs included novel putative targets for aspirin such as TRABD2A (q = 0.055), a negative regulator of Wnt signaling. Weighted gene co-expression network analysis identified 12 significant modules, including two that contained hubs for EGFR and PTGES2, the latter being previously implicated in aspirin chemoprevention. In summary, aspirin treatment of patient-derived colon organoids using physiologically relevant doses resulted in transcriptome-wide changes that reveal altered cell composition and improved understanding of transcriptional pathways, providing novel insight into its chemopreventive properties. PREVENTION RELEVANCE: Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transit-amplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis.

EVIP2: Expression-based variant impact phenotyping to predict the function of gene variants.

While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type (WT) versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric, and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659 has been previously described as a passenger mutation that maintains the RNF43 WT function as a negative regulator of Wnt. Surprisingly, eVIP2 predicted G659fs to be a change-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF-α via NFκB signaling, KRAS signaling, and hypoxia, highlighting the benefit of a more comprehensive approach when determining the impact of gene variant function. To validate these predictions, we performed reporter assays and found that each pathway activated by expression of RNF43 G659fs, but not expression of RNF43 WT, was identified as impacted by eVIP2, supporting that RNF43 G659fs is a change-of-function mutation and its effect on the identified pathways. Pathway activation was further validated by Western blot analysis. Lastly, we show primary colon adenocarcinoma patient samples with R117fs and G659fs variants have transcriptional profiles similar to BRAF missense mutations with activated RAS/MAPK signaling, consistent with KRAS signaling pathways being GOF in both variants. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2.