Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain.

Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.

New insights in ubiquitin-dependent Wnt receptor regulation in tumorigenesis.

Wnt signaling plays a crucial role in embryonic development and homeostasis maintenance. Delicate and sensitive fine-tuning of Wnt signaling based on the proper timings and positions is required to balance cell proliferation and differentiation and maintain individual health. Therefore, homeostasis is broken by tissue hypoplasia or tumor formation once Wnt signal dysregulation disturbs the balance of cell proliferation. The well-known regulatory mechanism of Wnt signaling is the molecular reaction associated with the cytoplasmic accumulation of effector ß-catenin. In addition to ß-catenin, most Wnt effector proteins are also regulated by ubiquitin-dependent modification, both qualitatively and quantitatively. This review will explain the regulation of the whole Wnt signal in four regulatory phases, as well as the different ubiquitin ligases and the function of deubiquitinating enzymes in each phase. Along with the recent results, the mechanism by which RNF43 negatively regulates the surface expression of Wnt receptors, which has recently been well understood, will be detailed. Many RNF43 mutations have been identified in pancreatic and gastrointestinal cancers and examined for their functional alteration in Wnt signaling. Several mutations facilitate or activate the Wnt signal, reversing the RNF43 tumor suppressor function into an oncogene. RNF43 may simultaneously play different roles in classical multistep tumorigenesis, as both wild-type and mutant RNF43 suppress the p53 pathway. We hope that the knowledge obtained from further research in RNF43 will be applied to cancer treatment in the future despite the fully unclear function of RNF43.

The non-canonical Wnt receptor Ror2 is required for cartilage cell polarity and morphogenesis of the craniofacial skeleton in zebrafish.

Non-canonical/ß-catenin-independent Wnt signaling plays crucial roles in tissue/cell polarity in epithelia, but its functions have been less well studied in mesenchymal tissues, such as the skeleton. Mutations in non-canonical Wnt signaling pathway genes cause human skeletal diseases such as Robinow syndrome and Brachydactyly Type B1, which disrupt bone growth throughout the endochondral skeleton. Ror2 is one of several non-canonical Wnt receptor/co-receptors. Here, we show that ror2-/- mutant zebrafish have craniofacial skeletal defects, including disruptions of chondrocyte polarity. ror1-/- mutants appear to be phenotypically wild type, but loss of both ror1 and ror2 leads to more severe cartilage defects, indicating partial redundancy. Skeletal defects in ror1/2 double mutants resemble those of wnt5b-/- mutants, suggesting that Wnt5b is the primary Ror ligand in zebrafish. Surprisingly, the proline-rich domain of Ror2, but not its kinase domain, is required to rescue its function in mosaic transgenic experiments in ror2-/- mutants. These results suggest that endochondral bone defects in ROR-related human syndromes reflect defects in cartilage polarity and morphogenesis.

Cytotoxic synergism of Clostridioides difficile toxin B with proinflammatory cytokines in subjects with inflammatory bowel diseases.

Clostridioides difficile (C. difficile) is progressively colonizing humans and animals living with humans. During this process, hypervirulent strains and mutated toxin A and B of C. difficile (TcdA and TcdB) are originating and developing. While in healthy subjects colonization by C. difficile becomes a risk after the use of antibiotics that alter the microbiome, other categories of people are more susceptible to infection and at risk of relapse, such as those with inflammatory bowel disease (IBD). Recent in vitro studies suggest that this increased susceptibility could be due to the strong cytotoxic synergism between TcdB and proinflammatory cytokines the tumor necrosis factor-alpha and interferon-gamma (CKs). Therefore, in subjects with IBD the presence of an inflammatory state in the colon could be the driver that increases the susceptibility to C. difficile infection and its progression and relapses. TcdB is internalized in the cell via three receptors: chondroitin sulphate proteoglycan 4; poliovirus receptor-like 3; and Wnt receptor frizzled family. Chondroitin sulphate proteoglycan 4 and Wnt receptor frizzled family are involved in cell death by apoptosis or necrosis depending on the concentration of TcdB and cell types, while poliovirus receptor-like 3 induces only necrosis. It is possible that cytokines could also induce a greater expression of receptors for TcdB that are more involved in necrosis than in apoptosis. Therefore, in subjects with IBD there are the conditions: (1) For greater susceptibility to C. difficile infection, such as the inflammatory state, and abnormalities of the microbiome and of the immune system; (2) for the enhancement of the cytotoxic activity of TcdB +Cks; and (3) for a greater expression of TcdB receptors stimulated by cytokines that induce cell death by necrosis rather than apoptosis. The only therapeutic approach currently possible in IBD patients is monitoring of C. difficile colonization for interventions aimed at reducing tumor necrosis factor-alpha and interferon-gamma levels when the infection begins. The future perspective is to generate bacteriophages against C. difficile for targeted therapy.

A Spatial Atlas of Wnt Receptors in Adult Mouse Liver.

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, especially all 10 Frizzled (Fzd) receptors, has not been characterized in adult liver. To address this, the spatial expression of Fzd receptors was quantitatively mapped in adult mouse liver via multiplex fluorescent in situ hybridization. Although all 10 Fzd receptors were expressed within a metabolic unit, Fzd receptors 1, 4, and 6 were the highest expressed. Although most Wnt signaling occurs in zone 3, expression of most Fzd receptors was not zonated. In contrast, Fzd receptor 6 was preferentially expressed in zone 1. Wnt2 and Wnt9b expression was highly zonated and primarily found in zone 3. Therefore, the current results suggest that zonated Wnt/ß-catenin signaling at baseline occurs primarily due to Wnt2 and Wnt9b rather than zonation of Fzd mRNA expression. Finally, the study showed that Fzd receptors and Wnts are not uniformly expressed by all hepatic cell types. Instead, there is broad distribution among both hepatocytes and nonparenchymal cells, including endothelial cells. Overall, this establishment of a definitive mRNA expression atlas, especially of Fzd receptors, opens the door to future functional characterization in healthy and diseased liver states.

In Adult Skeletal Muscles, the Co-Receptors of Canonical Wnt Signaling, Lrp5 and Lrp6, Determine the Distribution and Size of Fiber Types, and Structure and Function of Neuromuscular Junctions.

Canonical Wnt signaling is involved in skeletal muscle cell biology. The exact way in which this pathway exerts its contribution to myogenesis or neuromuscular junctions (NMJ) is a matter of debate. Next to the common co-receptors of canonical Wnt signaling, Lrp5 and Lrp6, the receptor tyrosine kinase MuSK was reported to bind at NMJs WNT glycoproteins by its extracellular cysteine-rich domain. Previously, we reported canonical Wnt signaling being active in fast muscle fiber types. Here, we used conditional Lrp5 or Lrp6 knockout mice to investigate the role of these receptors in muscle cells. Conditional double knockout mice died around E13 likely due to ectopic expression of the Cre recombinase. Phenotypes of single conditional knockout mice point to a very divergent role for the two receptors. First, muscle fiber type distribution and size were changed. Second, canonical Wnt signaling reporter mice suggested less signaling activity in the absence of Lrps. Third, expression of several myogenic marker genes was changed. Fourth, NMJs were of fragmented phenotype. Fifth, recordings revealed impaired neuromuscular transmission. In sum, our data show fundamental differences in absence of each of the Lrp co-receptors and suggest a differentiated view of canonical Wnt signaling pathway involvement in adult skeletal muscle cells.

γ-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor.

Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. Here, we show that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development.

Cyclic activity of signal transduction pathways in fimbrial epithelium of the human fallopian tube.

INTRODUCTION: The local environment of the fallopian tube represents the optimal conditions for reproductive processes. To maintain tissue homeostasis, signal transduction pathways are thought to play a pivotal role. Enhancing our understanding of functional signal transduction pathway activity is important to be able to clarify the role of aberrant signal transduction pathway activity leading to female subfertility and other tubal diseases. Therefore, in this study we investigate the influence of the hormonal cycle on the activity of key signal transduction pathways in the fimbrial epithelium of morphologically normal fallopian tubes. MATERIAL AND METHODS: We included healthy pre- (n = 17) and postmenopausal (n = 8) patients who had surgical interventions for benign gynecologic conditions. Histologic sections of the fallopian tubes were reviewed by two pathologists and, for the premenopausal patients, hormone serum levels and sections of the endometrium were examined to determine the hormonal phase (early follicular [n = 4], late follicular [n = 3], early luteal [n = 5], late luteal [n = 5]). After laser capture microdissection, total mRNA was extracted from the fimbrial epithelium and real-time quantitative reverse transcription-PCR was performed to determine functional signal transduction pathway activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor-beta (TGF-ß) and canonical wingless-type MMTV integration site (Wnt) pathways. RESULTS: The early luteal phase demonstrated high AR and ER pathway activity in comparison with the late luteal phase (p = 0.016 and p = 0.032, respectively) and low PI3K activity compared with the late follicular phase (p = 0.036), whereas the late luteal phase showed low activity of HH and Wnt compared with the early follicular phase (both p = 0.016). Signal transduction pathway activity in fimbrial epithelium from postmenopausal patients was most similar to the early follicular and/or late luteal phase with regard to the AR, ER and PI3K pathways. Wnt pathway activity in postmenopausal patients was comparable to the late follicular and early luteal phase. We observed no differences in HH and TGF-ß pathway activity between pre- and postmenopausal samples. The cyclic changes in signal transduction pathway activity suggest a stage-specific function which may affect the morphology and physiology of the human fallopian tube. CONCLUSIONS: We demonstrated cyclic changes in activity of the AR, ER, PI3K, HH and Wnt pathways throughout the hormonal cycle.

The amyloid precursor protein is a conserved Wnt receptor.

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner.

A MET-PTPRK kinase-phosphatase rheostat controls ZNRF3 and Wnt signaling.

Zinc and ring finger 3 (ZNRF3) is a transmembrane E3 ubiquitin ligase that targets Wnt receptors for ubiquitination and lysosomal degradation. Previously, we showed that dephosphorylation of an endocytic tyrosine motif (4Y motif) in ZNRF3 by protein tyrosine phosphatase receptor-type kappa (PTPRK) promotes ZNRF3 internalization and Wnt receptor degradation (Chang et al 2020). However, a responsible protein tyrosine kinase(s) (PTK) phosphorylating the 4Y motif remained elusive. Here we identify the proto-oncogene MET (mesenchymal-epithelial transition factor) as a 4Y kinase. MET binds to ZNRF3 and induces 4Y phosphorylation, stimulated by the MET ligand HGF (hepatocyte growth factor, scatter factor). HGF-MET signaling reduces ZNRF3-dependent Wnt receptor degradation thereby enhancing Wnt/ß-catenin signaling. Conversely, depletion or pharmacological inhibition of MET promotes the internalization of ZNRF3 and Wnt receptor degradation. We conclude that HGF-MET signaling phosphorylates- and PTPRK dephosphorylates ZNRF3 to regulate ZNRF3 internalization, functioning as a rheostat for Wnt signaling that may offer novel opportunities for therapeutic intervention.

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

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

Ehrlichia chaffeensis TRP120 Is a Wnt Ligand Mimetic That Interacts with Wnt Receptors and Contains a Novel Repetitive Short Linear Motif That Activates Wnt Signaling.

Ehrlichia chaffeensis expresses the TRP120 multifunctional effector, which is known to play a role in phagocytic entry, on the surface of infectious dense-cored ehrlichiae, but a cognate host receptor has not been identified. We recently reported that E. chaffeensis activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled family of receptors. In this study, we used confocal immunofluorescence microscopy to demonstrate very strong colocalization between E. chaffeensis and Fzd2, 4, 5, 7, and 9 as well as coreceptor LRP5 at 1 to 3 h postinfection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. chaffeensis infection, demonstrating that complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. chaffeensis infection. This study reports the first example of bacterial mimicry of Wnt pathway ligands and highlights a pathogenic mechanism with potential for targeting by antimicrobial therapeutics.IMPORTANCE Upon infecting mammalian hosts, Ehrlichia chaffeensis establishes a replicative niche in microbe-eating immune system cells where it expertly orchestrates infection and spread. One of the ways Ehrlichia survives within these phagocytes is by activating evolutionarily conserved signaling pathways including the Wnt pathway; however, the molecular details of pathway hijacking have not been defined. This study is significant because it identifies an ehrlichial protein that directly interacts with components of the Wnt receptor complex, influencing pathway activity and promoting infection. Consequentially, Ehrlichia serves as a unique tool to investigate the intricacies of how pathogens repurpose human immune cell signaling and provides an opportunity to better understand many cellular processes in health and disease. Furthermore, understanding how this bacterium utilizes its small genome to survive within cells that evolved to destroy pathogens will facilitate the development of antibacterial therapeutics that could target Ehrlichia as well as other intracellular agents of human disease.

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.

Canonical and non-canonical Wnt signaling are simultaneously activated by Wnts in colon cancer cells.

The Wnt signaling pathway is a crucial regulator of the intestinal epithelium homeostasis and is altered in most colon cancers. While the role of aberrant canonical, ß-catenin-dependent Wnt signaling has been well established in colon cancer promotion, much less is known about the role played by noncanonical, ß-catenin-independent Wnt signaling in this type of cancer. This work aimed to characterize the noncanonical signal transduction pathway in colon cancer cells. To this end, we used the prototype noncanonical ligand, Wnt5a, in comparison with Wnt3a, the prototype of a canonical ß-catenin activating ligand. The analysis of the expression profile of Wnt receptors in colon cancer cell lines showed a clear increase in both level expression and variety of Frizzled receptor types expressed in colon cancer cells compared with non-malignant cells. We found that Wnt5a activates a typical Wnt/Ca++ - noncanonical signaling pathway in colon malignant cells, inducing the hyperphosphorylation of Dvl1, Dvl2 and Dvl3, promoting Ca++ mobilization as a result of phospholipase C (PLC) activation via pertussis toxin-sensitive G-protein, and inducing PLC-dependent cell migration. We also found that while the co-receptor Ror2 tyrosine kinase activity is not required for Ca++ mobilization-induced by Wnt5a, it is required for the inhibitory effects of Wnt5a on the ß-catenin-dependent transcriptional activity. Unexpectedly, we found that although the prototype canonical Wnt3a ligand was unique in stimulating the ß-catenin-dependent transcriptional activity, it also simultaneously activated PLC, promoted Ca++ mobilization, and induced Rho kinase and PLC-dependent cell migration. Our data indicate, therefore, that a Wnt ligand can activate at the same time the so-called Wnt canonical and noncanonical pathways inducing the formation of complex signaling networks to integrate both pathways in colon cancer cells.

Sustained Release of Decoy Wnt Receptor (sLRP6E1E2)-Expressing Adenovirus Using Gel-Encapsulation for Scar Remodeling in Pig Model.

An adenoviral vector (Ad) expressing a Wnt decoy receptor (sLRP6E1E2) is known to induce an anti-fibrotic effect by inhibiting Wnt signaling. We evaluated its effects in vivo using pig models and attempted to introduce an alginate gel-matrix system to prolong the effect of the Ad. Transduction efficiency as to the biological activity of Ad in different forms was evaluated. Then, 50 days after the formation of full-thickness skin defects on the backs of Yorkshire pigs, scars were treated with each form of Ad. Therapeutic efficacy and various factors influencing scar formation and collagen rearrangement were analyzed. Inflammatory cell infiltration within the scar tissues was also evaluated. Decoy Wnt receptor (sLRP6E1E2)-expressing adenovirus treatment improved scar quality in a pig model. Loading this construct in alginate gel allows sustained virus release into local tissues and prolongs Ad activity, thus maintaining its therapeutic effect longer in vivo.

Endosomal Wnt signaling proteins control microtubule nucleation in dendrites.

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.

The receptor tyrosine kinase Ror is required for dendrite regeneration in Drosophila neurons.

While many regulators of axon regeneration have been identified, very little is known about mechanisms that allow dendrites to regenerate after injury. Using a Drosophila model of dendrite regeneration, we performed a candidate screen of receptor tyrosine kinases (RTKs) and found a requirement for RTK-like orphan receptor (Ror). We confirmed that Ror was required for regeneration in two different neuron types using RNA interference (RNAi) and mutants. Ror was not required for axon regeneration or normal dendrite development, suggesting a specific role in dendrite regeneration. Ror can act as a Wnt coreceptor with frizzleds (fzs) in other contexts, so we tested the involvement of Wnt signaling proteins in dendrite regeneration. We found that knockdown of fz, dishevelled (dsh), Axin, and gilgamesh (gish) also reduced dendrite regeneration. Moreover, Ror was required to position dsh and Axin in dendrites. We recently found that Wnt signaling proteins, including dsh and Axin, localize microtubule nucleation machinery in dendrites. We therefore hypothesized that Ror may act by regulating microtubule nucleation at baseline and during dendrite regeneration. Consistent with this hypothesis, localization of the core nucleation protein γTubulin was reduced in Ror RNAi neurons, and this effect was strongest during dendrite regeneration. In addition, dendrite regeneration was sensitive to partial reduction of γTubulin. We conclude that Ror promotes dendrite regeneration as part of a Wnt signaling pathway that regulates dendritic microtubule nucleation.

Wnt5a Plays Controversial Roles in Cancer Progression.

Wnt5a is a representative Wnt ligand that regulates multiple cellular functions through the Wnt5a non-classical pathway. Although Wnt5a has been implicated in various pathological conditions, its role in cancer is ambiguous and might involve methyl modifications, distinct mRNA isoforms, as well as different downstream pathways. Therefore, it is an essential factor in cancers' progression (invasion, migration, proliferation, and epithelial-mesenchymal transition), and a potential biomarker for prognosis and treatment.

Wnt-3a Induces Cytokine Release in Human Mast Cells.

Mast cells are well known for their detrimental effects in allergies and asthma, and Wnt signaling has recently been implicated in asthma and other airway diseases. However, it is not known if or how Wnts affect human mast cells. Since Wnt expression is elevated in individuals with asthma and is linked to a Th2 profile, we hypothesized that mast cells could be affected by Wnts in the context of asthma. We therefore sought to investigate the role of Wnt signaling in human mast cell development and activation. We first examined the expression of the 10 main Wnt receptors, Frizzled 1-10 (FZD1-10), and found expression of several FZDs in human mast cells. Treatment with purified recombinant Wnt-3a or Wnt-5a did not affect the proliferation or maturation of CD34+ progenitors into mast cells, as indicated by cellular expression of CD117 and FcεRI, activation by FcεRI crosslinking, and histamine and tryptase release. Furthermore, Wnt treatment did not change the phenotype from MCT to MCTC, since MrgX2 expression, compound 48/80-mediated activation, and carboxypeptidase A3 content were not affected. However, Wnt-3a activated WNT/ß-catenin signaling in mature human mast cells, as revealed by stabilization of ß-catenin, upregulation of IL-8 and CCL8 mRNA expression, and release of IL-8 protein. Thus, our data suggest that Wnt-3a activation of mast cells could contribute to the recruitment of immune cells in conditions associated with increased Wnt-3a expression, such as asthma.

Oncolytic Ad co-expressing decorin and Wnt decoy receptor overcomes chemoresistance of desmoplastic tumor through degradation of ECM and inhibition of EMT.

Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. To overcome desmoplasia and chemoresistance of pancreatic cancer, we utilized an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor (HEmT-DCN/sLRP6). An orthotopic pancreatic xenograft tumor model was established in athymic nude mice using Mia PaCa-2 cells, and the antimetastatic and antitumor efficacy of systemically administered HEmT-DCN/sLRP6 was evaluated. Immunohistochemical analysis of tumor tissues was performed to assess ECM degradation, induction of apoptosis, viral dispersion, and inhibition of the Wnt/ß-catenin signaling pathway. HEmT-DCN/sLRP6 effectively degraded tumor ECM and inhibited EMT, leading to enhanced viral distribution, induction of apoptosis, and attenuation of tumor cell proliferation in tumor tissue. HEmT-DCN/sLRP6 prevented metastasis of pancreatic cancer. Importantly, HEmT-DCN/sLRP6 sensitized pancreatic tumor to gemcitabine treatment. Furthermore, HEmT-DCN/sLRP6 augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived tumor spheroids. Collectively, these results illustrate that HEmT-DCN/sLRP6 can enhance the dispersion of both oncolytic Ad and a chemotherapeutic agent in chemoresistant and desmoplastic pancreatic tumor, effectively overcoming the preexisting limitations of standard treatments.