Non-canonical Wnt Signaling Research Articles

\u0394Np63\u03b1 suppresses cells invasion by downregulating PKC\u03b3/Rac1 signaling through miR-320a

ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration, and invasion. ΔNp63α has been shown to regulate several microRNAs that are involved in development and cancer. We identified miRNA miR-320a as a positively regulated target of ΔNp63α. Previous studies have shown that miR-320a is downregulated in colorectal cancer and targets the small GTPase Rac1, leading to a reduction in noncanonical WNT signaling and EMT, thereby inhibiting tumor metastasis and invasion. We showed that miR-320a is a direct target of ΔNp63α. Knockdown of ΔNp63α in HaCaT and A431 cells downregulates miR-320a levels and leads to a corresponding elevation in PKCγ transcript and protein levels. Rac1 phosphorylation at Ser71 was increased in the absence of ΔNp63α, whereas overexpression of ΔNp63α reversed S71 phosphorylation of Rac1. Moreover, increased PKCγ levels, Rac1 phosphorylation and cell invasion observed upon knockdown of ΔNp63α was reversed by either overexpressing miR-320a mimic or Rac1 silencing. Finally, silencing PKCγ or treatment with the PKC inhibitor Gö6976 reversed increased Rac1 phosphorylation and cell invasion observed upon silencing ΔNp63α. Taken together, our data suggest that ΔNp63α positively regulates miR-320a, thereby inhibiting PKCγ expression, Rac1 phosphorylation, and cancer invasion.

Interplay of miR-137 and EZH2 contributes to the genome-wide redistribution of H3K27me3 underlying the Pb-induced memory impairment

Compromised learning and memory is a common feature of multiple neurodegenerative disorders. A paradigm spatial memory impairment could be caused by developmental lead (Pb) exposure. Growing evidence implicates epigenetic modifications in the Pb-mediated memory deficits; however, how histone modifications exemplified by H3K27me3 (H3 Lys27 trimethylation) contribute to this pathogenesis remains poorly understood. Here we found that Pb exposure diminished H3K27me3 levels in vivo by suppressing EZH2 (enhancer of zeste homolog 2) expression at an early stage. EZH2 overexpression in Pb-treated rats rescued the H3K27me3 abundance and partially restored the normal spatial memory, as manifested by the rat performance in a Morris water maze test, and structural analysis of hippocampal spine densities. Furthermore, miR-137 and EZH2 constitute mutually inhibitory loop to regulate the H3K27me3 level, and this feedback regulation could be specifically activated by Pb treatment. Considering genes targeted by H3K27me3, ChIP-chip (chromatin immunoprecipitation on chip) studies revealed that Pb could remodel the genome-wide distribution of H3K27me3, represented by pathways like transcriptional regulation, developmental regulation, cell motion, and apoptosis, as well as a novel Wnt9b locus. As a Wnt isoform associated with canonical and noncanonical signaling, Wnt9b was regulated by the opposite modifications of H3K4me3 (H3 Lys4 trimethylation) and H3K27me3 in Pb-exposed neurons. Rescue trials further validated the contribution of Wnt9b to Pb-induced neuronal impairments, wherein canonical or noncanonical Wnt signaling potentially exhibited destructive or protective roles, respectively. In summary, the study reveals an epigenetic-based molecular change underlying Pb-triggered spatial memory deficits, and provides new potential avenues for our understanding of neurodegenerative diseases with environmental etiology.

WNT5B governs the phenotype of basal-like breast cancer by activating WNT signaling

BackgroundBreast cancer is the leading cause of cancer-related death in women worldwide. Metastatic disease remains the primary cause of death in patients with breast cancer. Basal-like breast cancer (BLBC) is associated with aggressive behavior, stem-like phenotype, high histological grade, poor clinical features, and high rates of recurrences and/or metastasis. However, the mechanism of BLBC phenotype shaping remains obscure.MethodsSeventeen normal breast/breast cancer cell lines were used for evaluating the breast cancer subtype-markers, WNT targets and constitutive components, and epithelial mesenchymal transition (EMT) markers analysis by western blot. One hundred and twenty formalin-fixed breast cancer tissues were used for immunohistochemistry (IHC) staining. Nine online platforms (cBioPortal, CCLE, GEPIA, etc.) were used for related analyses.ResultsWe identified Wnt5b as a key regulatory factor that governs the phenotype of BLBC by activating canonical and non-canonical WNT signaling. Wnt5b exhibited basal-like specificity in cells and clinical samples both at the mRNA and protein levels and also showed good correlation with basal-like phenotype at the mRNA level. Besides, Wnt5b was also a promising therapeutic target for LGK-974 treatment. In addition, we identified that CK1α was expressed at low levels in BLBC and that the activation of CK1α by pyrvinium was an alternative strategy for BLBC treatment.ConclusionsWnt5b is not only a diagnostic biomarker but also a potential therapeutic target of BLBC.Graphical abstract

Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by Type II Spiral Ganglion Neurons.

Type II spiral ganglion neurons provide afferent innervation to outer hair cells of the cochlea and are proposed to have nociceptive functions important for auditory function and homeostasis. These neurons are anatomically distinct from other classes of spiral ganglion neurons because they extend a peripheral axon beyond the inner hair cells that subsequently makes a distinct 90 degree turn toward the cochlear base. As a result, patterns of outer hair cell innervation are coordinated with the tonotopic organization of the cochlea. Previously, it was shown that peripheral axon turning is directed by a nonautonomous function of the core planar cell polarity (PCP) protein VANGL2. We demonstrate using mice of either sex that Fzd3 and Fzd6 similarly regulate axon turning, are functionally redundant with each other, and that Fzd3 genetically interacts with Vangl2 to guide this process. FZD3 and FZD6 proteins are asymmetrically distributed along the basolateral wall of cochlear-supporting cells, and are required to promote or maintain the asymmetric distribution of VANGL2 and CELSR1. These data indicate that intact PCP complexes formed between cochlear-supporting cells are required for the nonautonomous regulation of axon pathfinding. Consistent with this, in the absence of PCP signaling, peripheral axons turn randomly and often project toward the cochlear apex. Additional analyses of Porcn mutants in which WNT secretion is reduced suggest that noncanonical WNT signaling establishes or maintains PCP signaling in this context. A deeper understanding of these mechanisms is necessary for repairing auditory circuits following acoustic trauma or promoting cochlear reinnervation during regeneration-based deafness therapies.SIGNIFICANCE STATEMENT Planar cell polarity (PCP) signaling has emerged as a complementary mechanism to classical axon guidance in regulating axon track formation, axon outgrowth, and neuronal polarization. The core PCP proteins are also required for auditory circuit assembly, and coordinate hair cell innervation with the tonotopic organization of the cochlea. This is a non-cell-autonomous mechanism that requires the formation of PCP protein complexes between cochlear-supporting cells located along the trajectory of growth cone navigation. These findings are significant because they demonstrate how the fidelity of auditory circuit formation is ensured during development, and provide a mechanism by which PCP proteins may regulate axon outgrowth and guidance in the CNS.

Wnt Binding Affinity Prediction for Putative Frizzled-Type Cysteine-Rich Domains.

Several proteins other than the frizzled receptors (Fzd) and the secreted Frizzled-related proteins (sFRP) contain Fzd-type cysteine-rich domains (CRD). We have termed these domains “putative Fzd-type CRDs”, as the relevance of Wnt signalling in the majority of these is unknown; the RORs, an exception to this, are well known for mediating non-canonical Wnt signalling. In this study, we have predicted the likely binding affinity of all Wnts for all putative Fzd-type CRDs. We applied both our previously determined Wnt‒Fzd CRD binding affinity prediction model, as well as a newly devised model wherein the lipid term was forced to contribute favourably to the predicted binding energy. The results obtained from our new model indicate that certain putative Fzd CRDs are much more likely to bind Wnts, in some cases exhibiting selectivity for specific Wnts. The results of this study inform the investigation of Wnt signalling modulation beyond Fzds and sFRPs.

Two Isoforms of the Guanine Nucleotide Exchange Factor, Daple/CCDC88C Cooperate as Tumor Suppressors

Previously, Aznar et al., showed that Daple/CCDC88C enables Wnt receptors to transactivate trimeric G-proteins during non-canonical Wnt signaling via a novel G-protein binding and activating (GBA) motif. By doing so, Daple serves two opposing roles; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial-to-mesenchymal-transition (EMT). We have identified and characterized two isoforms of the human Daple gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G-protein regulatory GBA motif as one of the minimal modules essential for Daple’s role as a tumor suppressor.

IL-36γ Promotes Killing of Mycobacterium tuberculosis by Macrophages via WNT5A-Induced Noncanonical WNT Signaling

Mycobacterium tuberculosis, which primarily infects mononuclear phagocytes, remains the leading bacterial cause of enormous morbidity and mortality because of bacterial infections in humans throughout the world. The IL-1 family of cytokines is critical for host resistance to M. tuberculosis As a newly discovered subgroup of the IL-1 family, although IL-36 cytokines have been proven to play roles in protection against M. tuberculosis infection, the antibacterial mechanisms are poorly understood. In this study, we demonstrated that IL-36γ conferred to human monocyte-derived macrophages bacterial resistance through activation of autophagy as well as induction of WNT5A, a reported downstream effector of IL-1 involved in several inflammatory diseases. Further studies showed that WNT5A could enhance autophagy of monocyte-derived macrophages by inducing cyclooxygenase-2 (COX-2) expression and in turn decrease phosphorylation of AKT/mTOR via noncanonical WNT signaling. Consistently, the underlying molecular mechanisms of IL-36γ function are also mediated by the COX-2/AKT/mTOR signaling axis. Altogether, our findings reveal a novel activity for IL-36γ as an inducer of autophagy, which represents a critical inflammatory cytokine that control the outcome of M. tuberculosis infection in human macrophages.

TET1-mediated DNA hydroxymethylation activates inhibitors of the Wnt/\u03b2-catenin signaling pathway to suppress EMT in pancreatic tumor cells

BackgroundTen-eleven translocation 1 (TET1) is a dioxygenase that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) to induce DNA demethylation. TET1 has been reported to be absent in cancers, and to influence various oncogenes and anti-oncogenes. However the function of TET1 in pancreatic tumor remains poorly understood. In this study, we investigated the role of TET1 in the progression of pancreatic tumor and its mechanism of tumor suppression.MethodsQuantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining and dot blot were performed to detect the TET1 and 5-hmC expression in pancreatic tumor tissues and its adjacent non-tumor tissues. The clinical parameters significance of pancreatic tumor tissues was determined statistically. TET1 over-expression and knock-out cell lines were built and confirmed in vitro. Cell proliferation assay, wound-healing assays, transwell migration assay and nude mice model of orthotopic pancreatic cancer implantation were performed to assess the function of TET1 in pancreatic tumor. Western blot, qRT-PCR, immunofluorescence (IF), bisulfate sequencing (BSP), Chromatin immunoprecipitation (ChIP) were used to uncover the mechanism.ResultsTET1 levels and 5-hmC content were downregulated in pancreatic tumor tissues and cell lines, and pancreatic tumor patients with low TET1 levels had a shorter overall survival than patients with high levels of TET1. TET1 suppressed pancreatic tumor proliferation and metastasis in vivo and in vitro. TET1 bound to the secreted frizzled-related protein 2 (SFRP2) promoter and catalyzed demethylation to activate transcription of SFRP2, inhibiting both the canonical and non-canonical Wnt signaling pathways, and ultimately obstructing epithelial-mesenchymal transition (EMT) in pancreatic tumors.ConclusionWe found TET1 plays as a suppressor in pancreatic tumor progression via obstructing Wnt signaling pathways.

Convergence of Canonical and Non-Canonical Wnt Signal: Differential Kat3 Coactivator Usage.

Background:The ancient and highly evolutionarily conserved Wnt signaling pathway is criti-cal in nearly all tissues and organs for an organism to develop normally from embryo through adult. Wnt signaling is generally parsed into ƒ?ocanonicalƒ?? or Wnt-Iý catenin-dependent or ƒ?onon-canonicalƒ?? Iý catenin-independent signaling. Even though designating Wnt signaling as either canonical or non-canonical allows for easier conceptual discourse about this signaling pathway, in fact canonical and non-canonical Wnt crosstalk regulates complex nonlinear networks.Objective:In this perspective, we discuss the integration of canonical and non-canonical Wnt signaling via differential Kat3 (CBP and p300) coactivator usage, thereby regulating and coordinating gene expression programs associated with both proliferation and cellular differentiation and morphogenesis. Methods: Pharmacologic inhibitors, cell culture, real-time PCR, chromatin immunoprecipitation, protein immuno-precipitation, Western blotting, reporter-luciferase, protein purification, site-directed mutagenesis, in vitro phosphorylation and binding assays, and immunofluorescence were utilized.Conclusion:Coordinated integration between both canonical and non-canonical Wnt pathways appears to be crucial not only in the control of fundamental morphologic processes but also in the regulation of nor-mal as well as pathologic events. Such integration between both canonical and non-canonical Wnt signal-ing is presumably effected via reversible phosphorylation mechanism (e.g., protein kinase C) to regulate differential Iý-catenin/Kat3 coactivator usage in order to coordinate proliferation with differentiation and adhesion.

Emerging Pathogenic and Prognostic Significance of Paired Box 3 (PAX3) Protein in Adult Gliomas

Gliomas present the most common type of brain tumors in adults, characterized by high morbidity and mortality. In search of potential molecular targets, members of paired box (PAX) family have been found expressed in neural crest cells, regulating their proliferation, apoptosis, migration and differentiation. Recently, PAX3 overexpression has been implicated in glioma tumorigenesis by enhancing proliferation, increasing invasiveness and inducing resistance to apoptosis of glioma cells, while maintaining brain glioma stem cells (BGSCs) stemness. Although the oncogenic potential of PAX3 in gliomas is still under investigation, experimental evidence suggests that PAX3 function is mainly mediated through the canonical and non-canonical Wnt signaling pathway as well as through its interaction with GFAP and p53 proteins. In addition, PAX3 may contribute to the chemoresistance of glioma cells and modulates the effectiveness of novel experimental therapies. Further evidence indicates that PAX3 may represent a novel diagnostic and prognostic biomarker for gliomas, facilitating personalized treatment. This review addresses the emerging role of PAX3 in glioma diagnosis, prognosis and treatment, aiming to shed more light on the underlying molecular mechanisms that could lead to more effective treatment approaches.

The pathological role of Wnt5a in psoriasis and psoriatic arthritis.

Psoriasis (PsO) is a chronic inflammatory skin disease with both local and systemic components. PsO‐associated arthritis, known as psoriatic arthritis (PsA), develops in approximately 13%‐25% of PsO patients. Various factors associated with both PsO and PsA indicate that these conditions are part of a single disease. Identification of novel targets for the development of drugs to treat both PsO and PsA is desirable to provide more patient‐friendly treatment regimens. Such targets will likely represent ‘common checkpoints’ of inflammation, for example key components or transduction cascades of the signalling pathways involved. Emerging evidence supports involvement of the non‐canonical Wnt signalling pathways in the development of both PsO and PsA, especially the Wnt5a‐activated signalling cascades. These, together with interlinked factors, are crucial in the interactions among keratinocytes, immune cells and inflammatory factors in PsO, as well as among chondrocytes, osteoblasts and osteoclasts that trigger both subchondral bone remodelling and cartilage catabolism in PsA. This review focuses on the pathological role of Wnt5a signalling and its interaction with other interlinked pathways in both PsO and PsA, and also on the main challenges for future research, particularly with respect to molecules targeting Wnt signalling pathways for the treatment of PsO and PsA.

Influence of Wilms' tumor suppressor gene WT1 on bovine Sertoli cells polarity and tight junctions via non-canonical WNT signaling pathway

Sertoli cells (SCs) are polarized epithelial cells and provide a microenvironment for the development of germ cells (GCs). The Wilms' tumor suppressor gene WT1 which support spermatogenesis is expressed explicitly in SCs. This study investigated the effect of WT1 on the polarity and blood-testis barrier (BTB) formation of bovine SCs in order to provide theoretical and practical bases for the spermatogenic process in mammals. In this study, newborn calf SCs were used as research material, and the RNAi technique was used to knockdown the endogenous WT1. The results show that WT1 knockdown did not affect the proliferation ability of SCs, but down-regulated the expression of polarity-associated proteins (Par3, Par6b, and E-cadherin), junction-associated protein (occludin) and inhibits transcription of downstream genes (WNT4, JNK, αPKC, and CDC42) in non-canonical WNT signaling pathway. WT1 also altered ZO-1 and occludin protein distribution. Overexpression of WNT1 did not affect the expression of Par6b, E-cadherin, and occludin, whereas the non-canonical WNT signaling pathway inhibitors wnt-c59, CCG-1423, and GO-6983 down-regulated the expression of Par6b, E-cadherin, and occludin respectively. This study indicates that WT1 mediates the regulation of several proteins involved in bovine SCs polarity maintenance and intercellular tight junctions (TJ) by non-canonical WNT signaling pathway.

Wnt Signaling in Inflammation in Tissue Repair and Regeneration.

Inflammation is the first response occurring after damage or infection, which is a defensive process for the body. It is well known that excessive inflammation can lead to further diseases such as fibrosis. But a regenerative inflammatory response can accelerate the process of repairing injury, in which a variety of cytokines, immune cells, and stem cells are involved. The Wnt signaling pathway was originally known in the field of development. Recently, its role in regenerative inflammation has gradually been established. Wnt signaling can regulate cell proliferation and differentiation through regulating participants of regenerative inflammation. Canonical and noncanonical Wnt signaling pathways are coordinated to maintain homeostasis. Based on the process of regenerative inflammation and recent research in this field, this paper reviews how the Wnt signaling pathway interact with other cells and pathways.

Identification and interaction analysis of key miRNAs inmedullary thyroid carcinoma by bioinformatics analysis.

Medullary thyroid carcinoma (MTC) is an endocrine tumor and comprises 5–10% of all primary thyroid malignancies. However, the biomechanical contribution to the development and progression of MTC remains unclear. In this study, To discover the key microRNAs (miRNAs or miRs) and their potential roles in the tumorigenesis of MTC, the microarray datasets GSE97070, GSE40807 and GSE27155 were analyzed. The datasets were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed miRNAs (DEMs) and genes (DEGs) were accessed by R. Targets of DEMs and predicted using starBase, and functional and pathway enrichment analyses were performed using Metascape. A protein-protein interaction (PPI) network and an analysis of modules were constructed using NetworkAnalyst. Finally, a network was constructed to show the regulatory association between transcription factors (TFs), DEMs and downstream genes. A total of 5 DEMs were found both in GSE97070 and GSE40807, including 3 upregulated DEMs and 2 downregulated DEMs. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses from Metascape revealed that the target genes of upregulated DEMs were significantly enriched in adherens junction, kinase and protein binding, while the target genes of downregulated DEMs were mainly involved in non-canonical Wnt signaling pathway and RNA transport. From the PPI network, 13 nodes were screened as hub genes. Pathway enrichment analysis revealed that the top 5 modules were mostly enriched in the neurotrophin signaling pathway, mRNA surveillance pathway and MAPK signaling pathway. In addition, the TF-DEMs-target gene and DEGs regulatory network revealed that 17 TFs regulated 2 miRNAs, including upregulated or downregulated DEMs, CREB1 regulated all upregulated DEMs, and TGFB1 was an activator of hsa-miR-199a-3p and a repressor of hsa-miR-429. Taken together, the present study identified several miRNAs and potential biological mechanisms involved in the tumorigenesis of MTC. This study identified the key DEMs and potential mechanisms underlying the development of MTC, and provided a series of biomarkers and targets for the management of MTC.

Abstract 2628: Non-canonical Wnt signaling in late-stage PDAC

Abstract Pancreatic cancer has a disproportionate death-to-incidence rate largely resultant from late stage diagnoses, leading to advanced cancers which have metastasized and have increased resistance to common chemotherapeutics. This malignant progression is characterized by the increase of many molecular markers, and developing novel therapeutics targeting these late stage markers is essential for improving patient prognoses. We have identified several proteins within the non-canonical Wnt pathway that are upregulated in advanced pancreatic ductal adenocarcinomas (PDAC), and we are exploring the mechanisms driving upregulation and the resulting alterations to cell signaling. Analysis of microarray data from human derived organoids (from 6 normal pancreas samples and 38 tumor samples) revealed that two receptors in the Wnt pathway, Frizzled 2 (FZD2) and Frizzled 6 (FZD6), have increased expression levels in late-stage PDAC. The temporal increase of these non-canonical, planar cell polarity altering receptors suggests a previously underappreciated role for Wnt signaling in the progression of pancreatic cancer. The increase of these two receptors in tumor tissue was confirmed through analysis of RNA-sequencing of patient samples, including 179 tumor and 171 normal samples. Interestingly, after analyzing patient overall survival and progression free survival we found that, out of the ten members of the Frizzled receptor family, higher levels of FZD2 and FZD6 result in the worst patient prognoses. Additionally, retrospective analysis of gene expression data from Moffitt et al. suggests that FZD2 and FZD6 are higher in the aggressive “basal-like” sub-category of pancreatic cancer. To begin to understand the mechanism for these changes we have used a panel of PDAC cell lines, grown in both traditional and 3-dimensional culture. We have found that decreasing levels of FZD2 and FZD6, through stable sh-RNA knockdown, results in cells which are less motile (observed via Transwell and scratch assays) and are also less capable of anchorage independent growth. Also, cell lines with increased levels of FZD2 result in cells migrating through 3D-matrix as small cell clusters rather than as single cells, which has also been observed in partial-epithelial to mesenchymal transition cell lines. Taken together, non-canonical Wnt signaling in PDAC, mediated by FZD2 and/or FZD6 receptors, results in phenotypic alterations which are consistent with those often observed during cancer progression. These changes may help explain why patients with increased expression of FZD2 and FZD6 have worse prognoses and may also warrant more exploration into specific Frizzled inhibitors. Citation Format: Payton D. Stevens, Adam Racette, Rochelle L. Tiedemann, Bart O. Williams. Non-canonical Wnt signaling in late-stage PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2628.

Abstract 2738: Wnt11 regulates migration and invasion in pancreatic ductal adenocarcinoma

Abstract Canonical Wnt/β-catenin signaling is strongly associated with cancer development and metastasis, but non-canonical Wnt signaling and its role in pancreatic ductal adenocarcinoma (PDAC) metastasis is not well understood. Wnt11 is implicated in cancer cell motility through non-canonical Wnt signaling pathways, however, previously described mechanisms are diverse and tissue specific. We investigated Wnt11 expression and its effects on PDAC cell motility/invasion and correlated these with gene expression signatures generated through single cell RNA-sequencing of PDAC tumors. Expression of Wnt11 was confirmed by RT-qPCR in PDAC cell lines derived from genetically engineered mouse models (KPC) and PDAC patient derived xenografts. Wnt11 was overexpressed in three PDAC cell lines following transfection with Wnt11 cDNA cloned into a pcDNA3.1 backbone and confirmed by RT-qPCR and western blot. siRNAs targeting Wnt11 were used to silence Wnt11 in PDAC cell lines relative to non-targeting controls and confirmed by RT-qPCR. Transwell migration/invasion assays were performed in triplicate with matched MTT proliferation assays in 3 PDAC cell lines with stable Wnt11 overexpression and 3 PDAC cell lines following Wnt11 siRNA knockdown. For single cell transcriptomic analysis, single cell suspensions were generated from 14 unique patient PDAC tumors. 10x GemCode microfluidics technology was used to isolate single cells into gel-bead in emulsion (GEM) units and were individually barcoded, lysed and reverse-transcribed. Barcoded cDNA libraries were then amplified and underwent next-generation sequencing. PDAC cell subpopulations with >4-fold levels of Wnt11 expression relative to all sequenced cells were identified and compared to the remaining tumor cell population to develop differentially expressed gene profiles. Pathways analysis of this gene set was performed using ENRICHR. Single cell transcriptomic analysis of PDAC cells derived from 14 different PDAC patients confirmed strong Wnt11 expression in subpopulations of PDAC cells. Wnt11 overexpression resulted in a 3.2-fold increase in migration and invasion (p<0.001) and RNAi-mediated depletion of Wnt11 resulted in a 6.6-fold reduction in migration and invasion (p<0.001). In PDAC cell populations with increased (>4-fold) Wnt11 expression, ERICHR analysis using the NCI pathway interaction database revealed significant enrichment in β-integrin signaling (p<0.001, z = -1.16). Wnt11 is strongly expressed in subpopulations of human PDAC cells and significantly enhances PDAC cell motility and invasion. Ongoing tail vein injection experiments with Wnt11-null PDAC cell lines and Wnt11 wildtype controls will test the in vivo role of Wnt11 in PDAC metastasis. β-integrin signaling is associated with Wnt11 expression in human PDAC tumors and may be involved in PDAC cell motility and invasion. Citation Format: Tara Hughes, Xinqun Li, Bingbing Dai, Jenying Deng, Christian Siangco, Kaberi Das, Shanshan Bai, Min Hu, Emi Sei, Tapsi Seth, Nicholas Navin, Michael Kim. Wnt11 regulates migration and invasion in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2738.

The MMTV-Wnt1 murine model produces two phenotypically distinct subtypes of mammary tumors with unique therapeutic responses to an EGFR inhibitor.

ABSTRACTThe Wnt gene family encodes an evolutionarily conserved group of proteins that regulate cell growth, differentiation and stem cell self-renewal. Aberrant Wnt signaling in human breast tumors has been proposed as a driver of tumorigenesis, especially in the basal-like tumor subtype where canonical Wnt signaling is both enriched and predictive of poor clinical outcomes. The development of effective Wnt-based therapeutics, however, has been slowed in part by a limited understanding of the context-dependent nature with which these aberrations influence breast tumorigenesis. We previously reported that MMTV-Wnt1 mice, an established model for studying Wnt signaling in breast tumors, develop two subtypes of tumors by gene expression classification: Wnt1-EarlyEx and Wnt1-LateEx. Here, we extend this initial observation and show that Wnt1-EarlyEx tumors exhibit high expression of canonical Wnt, non-canonical Wnt, and EGFR signaling pathway signatures. Therapeutically, Wnt1-EarlyEx tumors showed a dynamic reduction in tumor volume when treated with an EGFR inhibitor. Wnt1-EarlyEx tumors had primarily Cd49fpos/Epcamneg FACS profiles, but it was not possible to serially transplant these tumors into wild-type FVB female mice. Conversely, Wnt1-LateEx tumors had a bloody gross pathology, which was highlighted by the presence of ‘blood lakes’ identified by H&E staining. These tumors had primarily Cd49fpos/Epcampos FACS profiles, but also contained a secondary Cd49fpos/Epcamneg subpopulation. Wnt1-LateEx tumors were enriched for activating Hras1 mutations and were capable of reproducing tumors when serially transplanted into wild-type FVB female mice. This study definitively shows that the MMTV-Wnt1 mouse model produces two phenotypically distinct subtypes of mammary tumors that differ in multiple biological aspects including sensitivity to an EGFR inhibitor.

Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration.

Morphogenesis of hierarchical vascular networks depends on the integration of multiple biomechanical signals by endothelial cells, the cells lining the interior of blood vessels. Expansion of vascular networks arises through sprouting angiogenesis, a process involving extensive cell rearrangements and collective cell migration. Yet, the mechanisms controlling angiogenic collective behavior remain poorly understood. Here, we show this collective cell behavior is regulated by non-canonical Wnt signaling. We identify that Wnt5a specifically activates Cdc42 at cell junctions downstream of ROR2 to reinforce coupling between adherens junctions and the actin cytoskeleton. We show that Wnt5a signaling stabilizes vinculin binding to alpha-catenin, and abrogation of vinculin in vivo and in vitro leads to uncoordinated polarity and deficient sprouting angiogenesis in Mus musculus. Our findings highlight how non-canonical Wnt signaling coordinates collective cell behavior during vascular morphogenesis by fine-tuning junctional mechanocoupling between endothelial cells.

Primary cilia in murine palatal rugae development

Periodic patterning of iterative structures is a fundamental process during embryonic development, since these structures are diverse across the animal kingdom. Therefore, elucidating the molecular mechanisms in the formation of these structures promotes understanding of the process of organogenesis. Periodically patterned ridges, palatal rugae (situated on the hard palate of mammals), are an excellent experimental model to clarify the molecular mechanisms involved in the formation of periodic patterning of iterative structures. Primary cilia are involved in many biological events, including the regulation of signaling pathways such as Shh and non-canonical Wnt signaling. However, the role of primary cilia in the development of palatal rugae remains unclear. We found that primary cilia were localized to the oral cavity side of the interplacode epithelium of the palatal rugae, whereas restricted localization of primary cilia could not be detected in other regions. Next, we generated mice with a placodal conditional deletion of the primary cilia protein Ift88, using ShhCre mice (Ift88 fl/fl;ShhCre). Highly disorganized palatal rugae were observed in Ift88 fl/fl;ShhCre mice. Furthermore, by comparative in situ hybridization analysis, many Shh and non-canonical Wnt signaling-related molecules showed spatiotemporal expression patterns during palatal rugae development, including restricted expression in the epithelium (placodes and interplacodes) and mesenchyme. Some of these expression were found to be altered in Ift88 fl/fl;ShhCre mice. Primary cilia is thus involved in development of palatal rugae.

Frizzled-10 Extracellular Vesicles Plasma Concentration Is Associated with Tumoral Progression in Patients with Colorectal and Gastric Cancer.

Extracellular vesicles (EVs) are involved in intercellular communication during the carcinogenesis. Our attention has been focused on small EVs (sEVs) protein content in colorectal and gastric cancer (CRC and GC). Frizzled (FZD) proteins, a family of receptors comprised in the Wnt signaling pathway, play an important role in the carcinogenesis of CRC and GC. Here, the expression of a specific FZD protein, namely, FZD-10, was investigated in the sEVs extracted from plasma of patients affected by CRC and GC as involved in canonical and noncanonical Wnt signaling in cancer stem cells with a subsequent modification of cellular heterogeneity, omics reprogramming, and tumor plasticity. The expression of FZD-10 protein in the sEVs extracted from plasma of patients affected by CRC and GC and sEVs from plasma of healthy subjects was evaluated against the level of protein Hsp70, established as EVs specific markers along with CD63 and ALIX proteins. The FZD-10 extract from sEVs isolated from plasma of the controls and the CRC or GC subjects indicated that its expression in oncological patients was higher than in the control group, while, at the end of the treatment, it reached values comparable with the average level of controls. Furthermore, the level of FZD-10 in the whole plasma was found comparable with its level in the sEVs extract. The level of FZD-10 in the sEVs represents a potential reliable biomarker with a valuable prognostic function for the diagnosis of CRC and GC and for monitoring the treatment response.