Non-canonical Wnt Signaling Research Articles

Wnt7b signalling through Frizzled-7 receptor promotes dendrite development by coactivating CaMKII and JNK.

The formation of complex dendritic arbors is crucial for the assembly of functional networks as abnormal dendrite formation underlies several neurodevelopmental and psychiatric disorders. Many extracellular factors have been postulated as regulators of dendritic growth. Wnt proteins play a critical role in neuronal development and circuit formation. We previously demonstrated that Wnt7b acts through the scaffold protein dishevelled 1 (Dvl1) to modulate dendrite arborisation by activating a non-canonical Wnt signalling pathway. Here, we identify the seven-transmembrane frizzled-7 (Fz7, also known as FZD7) as the receptor for Wnt7b-mediated dendrite growth and complexity. Importantly, Fz7 is developmentally regulated in the intact hippocampus, and is localised along neurites and at dendritic growth cones, suggesting a role in dendrite formation and maturation. Fz7 loss-of-function studies demonstrated that Wnt7b requires Fz7 to promote dendritic arborisation. Moreover, in vivo Fz7 loss of function results in dendritic defects in the intact mouse hippocampus. Furthermore, our findings reveal that Wnt7b and Fz7 induce the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and JNK proteins, which are required for dendritic development. Here, we demonstrate that Wnt7b-Fz7 signals through two non-canonical Wnt pathways to modulate dendritic growth and complexity.

Cadmium Activates Noncanonical Wnt Signaling to Impair Hematopoietic Stem Cell Function in Mice.

Cadmium (Cd) is a toxic heavy metal that impairs the development of hematopoietic stem cells (HSCs) in mice, yet the mechanism of how Cd influences HSC remains elusive. Herein, we show that Cd activated non-canonical Wnt signaling pathway to impair HSC function in mice. After exposure to 10 ppm Cd chloride (CdCl2) via drinking water for 3 months, C57BL/6 mice displayed aberrant HSC function, in that HSC from Cd-treated mice were less efficient in rescue of lethally irradiated hosts and less competitive under mixed chimeric condition. Further analyses indicated that the small GTPase cdc42 was activated and its distribution pattern was depolarized in HSC by Cd exposure, and inhibition of cdc42 by casin, a selective chemical inhibitor, recovered the HSC capacity in rescue assay and their potential for lymphopoiesis under competitive mixed chimeric assay. Cd interaction with HSC was sufficient to promote noncanonical Wnt signaling pathway, but not canonical Wnt signaling pathway, to drive cdc42 activation and further increase the expression of C/EBPα and decrease the expression of Hhex. Moreover, Cd-induced activation of non-canonical Wnt signaling pathway in HSC did not persist long-termly in the presence of a normal niche without Cd, in that the elevated non-canonical Wnt signaling by Cd was diminished in HSC in the BM of normal recipients receiving purified HSC from Cd-treated mice after 6 months posttransplantation. Taken together, our study suggests that Cd activates cdc42 of noncanonical Wnt signaling pathway to impair HSC function, a previously unknown mechanism for Cd toxicity on HSC.

B-1 cells and B-1 cell precursors prompt different responses to Wnt signaling.

Recently several studies demonstrated a role for the Wnt pathway in lymphocyte development and self-renewal of hematopoietic stem cells (HSCs). B-1 cells constitute a separate lineage of B lymphocytes, originating during fetal hematopoiesis, expressing lymphoid and myeloid markers and possessing self-renewal ability, similar to early hematopoietic progenitors and HSCs. A plethora of studies have shown an important role for the evolutionary conserved Wnt pathway in the biology of HSCs and T lymphocyte development. Our previous data demonstrated abundant expression of Wnt pathway components by B-1 cells, including Wnt ligands and receptors. Here we report that the canonical Wnt pathway is activated in B-1 cell precursors, but not in mature B-1 cells. However, both B-1 precursors and B-1 cells are able to respond to Wnt ligands in vitro. Canonical Wnt activity promotes proliferation of B-1 cells, while non-canonical Wnt signals induce the expansion of B-1 precursors. Interestingly, using a co-culture system with OP9 cells, Wnt3a stimulus supported the generation of B-1a cells. Taking together, these results indicate that B-1 cells and their progenitors are differentially responsive to Wnt ligands, and that the balance of activation of canonical and non-canonical Wnt signaling may regulate the maintenance and differentiation of different B-1 cell subsets.

Decreased local and systemic levels of sFRP3 protein in osteosarcoma patients

Osteosarcoma is a malignant bone tumor that occurs mainly in children and adolescents. Because Wnt signaling has been implicated in the pathogenesis of osteosarcoma, we have investigated the circulating and local levels of the Wnt antagonist protein, Secreted Frizzled Related Protein (sFRP) 3, in osteosarcoma patients. Enzyme linked immunosorbent assay (ELISA) analysis of 67 osteosarcoma and age-matched non-diseased control sera showed that sFPR3 protein levels were significantly lower in osteosarcoma than in normal. Analysis of tumor and adjacent normal tissues (9 pairs) from osteosarcoma patients showed a decrease in sFRP3 expression in 5 out of 9 tumor samples compared to normal tissues. Furthermore, immunohistochemical analysis of tissue microarray revealed a significant decrease in sFRP3 levels in tumor compared to normal bone. RNA sequencing analysis in osteosarcoma cells shows suppression of sFRP3 and concomitant expression of multiple Wnt family members mediating canonical or non-canonical Wnt signaling. Taken together, our findings show that the systemic and local levels of sFRP3 protein are downregulated in osteosarcoma and sFRP3 levels could be explored further in the diagnosis and the care of osteosarcoma patients.

Essential role of ATP6AP2 enrichment in caveolae/lipid raft microdomains for the induction of neuronal differentiation of stem cells

BackgroundThe subcellular distribution of prorenin receptor and adaptor protein ATP6AP2 may affect neurogenesis. In this study, we hypothesized that ATP6AP2 expression and subcellular relocalization from caveolae/lipid raft microdomains (CLR-Ms) to intracellular sites may correlate with neuronal differentiation (Neu-Dif) of adipose-derived mesenchymal stem cells (ADSCs).MethodsHuman ADSCs isolated from 24 healthy donors and 24 patients with neurological disorders (ND) were cultured and induced for Neu-Dif. The mechanism of action of ATP6AP2 and the impact of its localization within the plasma membrane (particularly CLR-Ms) and intracellular sites on several pathways (mitogen-activated protein kinase, Wnt(s) signaling and others) and intracellular calcium and exosome release were evaluated. The impact of CLR-Ms on ATP6AP2 or vice versa was determined by pharmacological disruption of CLR-Ms or siATP6AP2 assays.ResultsIn patients with ND, loss of ATP6AP2 from CLR-Ms correlated with an inhibition of Neu-Dif and signaling. However, its relocalization in CLR-Ms was positively correlated to induction of Neu-Dif in healthy subjects. An apparent switch from canonical to noncanonical Wnt signaling as well as from caveolin to flotillin occurs concurrently with the increases of ATP6AP2 expression during neurogenesis. Stimulation by renin activates ERK/JNK/CREB/c-Jun but failed to induce β-catenin. Wnt5a enhanced the renin-induced JNK responsiveness. Gα proteins crosslink ATP6AP2 to caveolin where a switch from Gαi to Gαq is necessary for Neu-Dif. In ATP6AP2-enriched CLR-Ms, the release of exosomes was induced dependently from the intracellular Ca2+ and Gαq. Pharmacological disruption of CLR-M formation/stability impairs both ATP6AP2 localization and Neu-Dif in addition to reducing exosome release, indicating an essential role of ATP6AP2 enrichment in CLR-Ms for the induction of Neu-Dif. The mechanism is dependent on CLR-M dynamics, particularly the membrane fluidity. Knockdown of ATP6AP2 inhibited Neu-Dif but increased astrocytic-Dif, depleted ATP6AP2/flotillin/Gαq but accumulated caveolin/Gαi in CLR-Ms, and blocked the activation of JNK/ERK/c-Jun/CREB/exosome release. siATP6AP2 cells treated with sphingomyelinase/methyl-β-cyclodextrin reversed the levels of caveolin/flotillin in CLR-Ms but did not induce Neu-Dif, indicating the crucial relocalization of ATP6AP2 in CLR-Ms for neurogenesis. Treatment of ND-derived cells with nSMase showed reversibility in ATP6AP2 abundance in CLR-Ms and enhanced Neu-Dif.ConclusionsThis study gives evidence of the determinant role of CLR-M ATP6AP2 localization for neuronal and oligodendrocyte differentiation involving mechanisms of switches from Gαi/caveolin/canonical to Gαq/flotillin/PCP, the ERK/JNK pathway and Ca2+-dependent release of exosomes and as a potential target of drug therapy for neurodegenerative disorders.

The down-regulated Tumour suppressor Wnt Inhibitory Factor 1 (WIF1) regulates non-Canonical Wnt signalling in Pituitary Adenomas (PA)

The down-regulated Tumour suppressor Wnt Inhibitory Factor 1 (WIF1) regulates non-Canonical Wnt signalling in Pituitary Adenomas (PA)

Retinoid-Sensitive Epigenetic Regulation of the Hoxb Cluster Maintains Normal Hematopoiesis and Inhibits Leukemogenesis

Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes.

A GTPase-activating protein–binding protein (G3BP1)/antiviral protein relay conveys arteriosclerotic Wnt signals in aortic smooth muscle cells

In aortic vascular smooth muscle (VSM), the canonical Wnt receptor LRP6 inhibits protein arginine (Arg) methylation, a new component of noncanonical Wnt signaling that stimulates nuclear factor of activated T cells (viz NFATc4). To better understand how methylation mediates these actions, MS was performed on VSM cell extracts from control and LRP6-deficient mice. LRP6-dependent Arg methylation was regulated on >500 proteins; only 21 exhibited increased monomethylation (MMA) with concomitant reductions in dimethylation. G3BP1, a known regulator of arteriosclerosis, exhibited a >30-fold increase in MMA in its C-terminal domain. Co-transfection studies confirm that G3BP1 (G3BP is Ras-GAP SH3 domain-binding protein) methylation is inhibited by LRP6 and that G3BP1 stimulates NFATc4 transcription. NFATc4 association with VSM osteopontin (OPN) and alkaline phosphatase (TNAP) chromatin was increased with LRP6 deficiency and reduced with G3BP1 deficiency. G3BP1 activation of NFATc4 mapped to G3BP1 domains supporting interactions with RIG-I (retinoic acid inducible gene I), a stimulus for mitochondrial antiviral signaling (MAVS) that drives cardiovascular calcification in humans when mutated in Singleton-Merten syndrome (SGMRT2). Gain-of-function SGMRT2/RIG-I mutants increased G3BP1 methylation and synergized with osteogenic transcription factors (Runx2 and NFATc4). A chemical antagonist of G3BP, C108 (C108 is 2-hydroxybenzoic acid, 2-[1-(2-hydroxyphenyl)ethylidene]hydrazide CAS 15533-09-2), down-regulated RIG-I-stimulated G3BP1 methylation, Wnt/NFAT signaling, VSM TNAP activity, and calcification. G3BP1 deficiency reduced RIG-I protein levels and VSM osteogenic programs. Like G3BP1 and RIG-I deficiency, MAVS deficiency reduced VSM osteogenic signals, including TNAP activity and Wnt5-dependent nuclear NFATc4 levels. Aortic calcium accumulation is decreased in MAVS-deficient LDLR-/- mice fed arteriosclerotic diets. The G3BP1/RIG-I/MAVS relay is a component of Wnt signaling. Targeting this relay may help mitigate arteriosclerosis.

Regulation of neural crest development by the formin family protein Daam1

The neural crest (NC) multipotent progenitor cells form at the neural plate border and migrate to diverse locations in the embryo to differentiate into many cell types. NC is specified by several embryonic pathways, however the role of noncanonical Wnt signaling in this process remains poorly defined. Daam1 is a formin family protein that is present in embryonic ectoderm at the time of NC formation and can mediate noncanonical Wnt signaling. Our interference experiments indicated that Daam1 is required for NC gene activation. To further study the function of Daam1 in NC development we used a transgenic reporter Xenopus line, in which GFP transcription is driven by sox10 upstream regulatory sequences. The activation of the sox10:GFP reporter in a subset of NC cells was suppressed after Daam1 depletion and in embryos expressing N-Daam1, a dominant interfering construct. Moreover, N-Daam1 blocked reporter activation in neuralized ectodermal explants in response to Wnt11, but not Wnt8 or Wnt3a, confirming that the downstream pathways are different. In complementary experiments, a constitutively active Daam1 fragment expanded the NC territory, but this gain-of-function activity was eliminated in a construct with a point mutation in the FH2 domain that is critical for actin polymerization. These observations suggest a new role of Daam1 and actin remodeling in NC specification.

Pulmonary Colonization Resistance to Pathogens via Noncanonical Wnt and Interleukin-17A by Intranasal pep27 Mutant Immunization.

Previous studies have focused on colonization resistance of the gut microbiota against antibiotic resistant strains. However, less research has been performed on respiratory colonization resistance. Because respiratory colonization is the first step of respiratory infections, intervention to prevent colonization would represent a new approach for preventive and therapeutic measures. The Th17 response plays an important role in clearance of respiratory pathogens. Thus, harnessing the Th17 immune response in the mucosal site would be an effective method to design a respiratory mucosal vaccine. In this study, we show that intranasal Δpep27 immunization induces noncanonical Wnt and subsequent interleukin (IL)-17 secretion, and it inhibits Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae colonization. Moreover, IL-17A neutralization or nuclear factor of activated T-cell inhibition augmented bacterial colonization, indicating that noncanonical Wnt signaling is involved in pulmonary colonization resistance. Therefore, Δpep27 immunization can provide nonspecific respiratory colonization resistance via noncanonical Wnt signaling and IL-17A-related pathways.

TiO2 nanoparticles induce omphalocele in chicken embryo by disrupting Wnt signaling pathway

Titanium dioxide nanoparticles (TiO2 NPs) are among abundantly used metal oxide NPs but their interactions with biomolecules and subsequent embryonic toxicity in higher vertebrates is not extensively reported. Physicochemical interactions of TiO2 NPs with egg albumen reveals that lower doses of TiO2 NPs (10 and 25 µg/ml) accounted for higher friccohesity and activation energy but an increment in molecular radii was recorded at higher doses (50 and 100 µg/ml). FTIR analysis revealed conformational changes in secondary structure of egg albumen as a result of electrostratic interactions between egg albumen and TiO2 NPs. The morphometric data of chicken embryo recorded a reduction at all the doses of TiO2 NPs, but toxicity and developmental deformity (omphalocele and flexed limbs) were recorded at lower doses only. Inductively coupled plasma optical emission spectrometry (ICP-OES) confirmed presence of Ti in chicken embryos. mRNA levels of genes involved in canonical and non-canonical Wnt signaling were lowered following TiO2 NPs treatment resulting in free radical mediated disruption of lateral plate mesoderm and somite myogenesis. Conformational changes in egg albumen and subsequent developmental deformity in chicken embryo following TiO2 NPs treatment warrants detailed studies of NP toxicity at lower doses prior to their biomedical applications.

Casein kinase 1 is a therapeutic target in chronic lymphocytic leukemia

AbstractCasein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1–induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition.

Metabolic Reprogramming and the Recovery of Physiological Functionality in 3D Cultures in Micro-Bioreactors.

The recovery of physiological functionality, which is commonly seen in tissue mimetic three-dimensional (3D) cellular aggregates (organoids, spheroids, acini, etc.), has been observed in cells of many origins (primary tissues, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and immortal cell lines). This plurality and plasticity suggest that probably several basic principles promote this recovery process. The aim of this study was to identify these basic principles and describe how they are regulated so that they can be taken in consideration when micro-bioreactors are designed. Here, we provide evidence that one of these basic principles is hypoxia, which is a natural consequence of multicellular structures grown in microgravity cultures. Hypoxia drives a partial metabolic reprogramming to aerobic glycolysis and an increased anabolic synthesis. A second principle is the activation of cytoplasmic glutaminolysis for lipogenesis. Glutaminolysis is activated in the presence of hypo- or normo-glycaemic conditions and in turn is geared to the hexosamine pathway. The reducing power needed is produced in the pentose phosphate pathway, a prime function of glucose metabolism. Cytoskeletal reconstruction, histone modification, and the recovery of the physiological phenotype can all be traced to adaptive changes in the underlying cellular metabolism. These changes are coordinated by mTOR/Akt, p53 and non-canonical Wnt signaling pathways, while myc and NF-kB appear to be relatively inactive. Partial metabolic reprogramming to aerobic glycolysis, originally described by Warburg, is independent of the cell’s rate of proliferation, but is interwoven with the cells abilities to execute advanced functionality needed for replicating the tissues physiological performance.

Exploring the Wnt signaling pathway in schizophrenia and bipolar disorder

The Wnt signaling pathway plays a crucial role in neurodevelopment and in regulating the function and structure of the adult nervous system. Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders with evidence of subtle neurodevelopmental, structural and functional neuronal abnormalities. We aimed to elucidate the role of aberrant regulation of the Wnt system in these disorders by evaluating plasma levels of secreted Wnt modulators in patients (SCZ = 551 and BD = 246) and healthy controls (HCs = 639) using enzyme immune-assay. We also investigated the expression of 141 Wnt-related genes in whole blood in a subsample (SCZ = 338, BD = 241, and HCs = 263) using microarray analysis. Both SCZ and BD had dysregulated mRNA expression of Wnt-related genes favoring attenuated canonical (beta-catenin-dependent) signaling, and there were also indices of enhanced non-canonical Wnt signaling. In particular, FZD7, which may activate all Wnt pathways, but favors non-canonical signaling, and NFATc3, a downstream transcription factor and readout of the non-canonical Wnt/Ca2+ pathway, were significantly increased in SCZ and BD (p < 3 × 10−4). Furthermore, patients had lower plasma levels of soluble dickkopf 1 and sclerostin (p < 0.01) compared with HC. Our findings suggest that SCZ and BD are characterized by abnormal Wnt gene expression and plasma protein levels, and we propose that drugs targeting the Wnt pathway may have a role in the treatment of severe mental disorders.

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients

ABSTRACTSecreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.

A Conserved Role of the Unconventional Myosin 1d in Laterality Determination

Anatomical and functional asymmetries are widespread in the animal kingdom [1, 2]. In vertebrates, many visceral organs are asymmetrically placed [3]. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question [1]. Nodal signaling is widely used [4], and many, but not all, vertebrates use cilia for symmetry breaking [5]. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextralrotation of chiral organs [6, 7]. Here, we studied the role of myo1d in left-right (LR) axis formation in Xenopus. Morpholino oligomer-mediatedmyo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila [8], acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited inmyo1d morphants, and the ATF2 reporter genefor non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, our data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry.

Abstract P1-01-05: Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells

Abstract Background: ROR1is an evolutionarily conserved, oncoembryonic surface-antigen expressed in breast cancer. Previously, we found that ROR1 can serve as a receptor for Wnt5a, which can induce non-canonical Wnt signaling that enhances cancer-cell migration. Recently we found that Wnt5a could induce ROR1 to complex with a cytoskeletal protein designated HS1, which recruited ARHGEF1, enhanced activation of RhoA, and promoted leukemia-cell migration. These effects of Wnt5a on CLL cells could be inhibited by cirmtuzumab, a humanized, high-affinity monoclonal antibody (mAb) specific for ROR1. As expression of HS1 is limited to hematopoietic cells we examined breast cancer cell lines and primary breast cancer cells for expression of cortactin. Cortactin (also called EMS1), is a cytoskeletal protein, which is homologous in structure and function to HS1 and is broadly expressed in human cancers, including breast cancer. Methods: We performed immunoprecipitation studies using high-affinity mAb to ROR1 or cortactin and immunoblot analysis to examine for the association of ROR1 with cortactin and tyrosine phosphorylation of cortactin at Y421. We also assessed the expression levels of cortactin, ROR1, and ARHGEF1, in the MCF7 breast cancer cell line and in breast-cancer patient-derived xenografts (PDX). We also generated MCF7-ROR1 cells, which were stably transfected to express ROR1 or various mutant forms of ROR1 generated to study the structure-function requirements for effective ROR1-cortactin interactions. Results: We found that ROR1 associates with cortactin in freshly-isolated breast cancer PDX tumors or in PDX cells cultured with exogenous Wnt5a. We corroborated these results in breast cancer cell line MCF7-ROR1 cells, which were transfected to express ROR1 and found to migrate more effectively than the parental MCF7 lacking expression of ROR1. Wnt5a also induced cortactin tyrosine phosphorylation at Y421, recruitment of ARHGEF1, and activation of RhoA, which we found associated with enhanced breast-cancer cell migation. The capacity of Wnt5a to induce such changes could be blocked by treatment of the cells with cirmtuzumab. We generated truncated forms of ROR1 without a proline-rich domain (PRD) and found PRD was necessary for this association or Wnt5a-induced cortactin phosphorylation and enhanced cancer-cell migration. Accordingly, we introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→A mutants, ROR1P(841)A had impaired capacity to recruit cortactin and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin or activate ARHGEF1, and was unable to enhance the motility of the MCF7 cells transfected with this mutant form of ROR1. Conclusions: Collectively, these studies indicate the capacity of cortactin to complex with ROR1 plays an important role in ROR1-dependent Wnt5a-enhanced breast cancer cell migration. These studies also demonstrate that cirmtuzumab can inhibit the formation of cortactin-ROR1 complexes, cortactin phosphorylation, and cancer-cell migration, and metastasis. Citation Format: Hasan MK, Tripple V, Widhopf II GF, Yu J, Zhang S, Parker BA, Kipps TJ. Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-05.

Abstract P1-09-07: Breast cancer initiating cells express functional ROR1, which can be targeted by cirmtuzumab to potentially mitigate the risk of relapse after therapy

Abstract Although initially responsive to chemotherapy, patients with advanced breast cancer often relapse, generally with incurable metastatic disease. This may be due to a subpopulation of tumor cells, called cancer-initiating cells, or cancer stem cells (CSCs), which are relatively resistant to chemotherapy and have self-renewing and tumor-initiating capacities. Prior studies in our laboratory found that CSCs may express ROR1, an onco-embryonic, tyrosine-kinase-like orphan receptor, which we found could bind Wnt5a to activate non-canonical Wnt-signaling (Proc Nat Acad Sci USA 111:17266, 2014). Interrogation of the transcriptomes of breast-cancer cells obtained from patients before and after paclitaxel therapy revealed that chemotherapy treatment enhanced cancer-cell expression of ROR1, along with genes induced by activation of Rho-GTPases (e.g. RhoA, cdc42, and Rac1). We found that primary breast-cancer patient-derived xenografts with high-level expression of ROR1 were enriched for cells that had activated Rho-GTPases and stem-cell-like gene-expression signatures. Furthermore, we found that treatment of breast cancer cell lines with Wnt5a induced ROR1-dependent activation of Rho-GTPases andAKT and induced high-level protein expression of BMI1, also known as polycomb group RING finger protein 4 (PCGF4) or RING finger protein 51 (RNF51); Wnt5a also enhanced the capacity of breast cancer cell lines to form spheroids. All these effects could be inhibited by cirmtuzumab, a humanized high-affinity anti-ROR1 mAb, which can block Wnt5a signaling. We find that ROR1-positive breast cancer cells have a greater capacity to form spheroids or engraft immune-deficient mice than did ROR1-negative cancer cells isolated from the same PDX tumor. Treatment of immune-deficient mice bearing breast-cancer PDX with paclitaxel reduced tumor volumes but enhanced expression of ROR1 and other CSC markers, such as aldehyde dehydrogenase 1 (ALDH1). Moreover, the breast cancer cells surviving such paclitaxel treatment had increased activation of Rho-GTPases and AKT, and increased expression of BMI1, relative to that of breast cancer cells obtained from the same primary tumor prior to therapy. On the other hand, treatment of such mice with cirmtuzumab also reduced breast cancer PDX tumor volumes, but the remaining cells had reduced expression of ROR1 and CSC markers and had impaired capacity to re-engraft immune-deficient mice. Finally, therapy with cirmtuzumab and paclitaxel was more effective in eradicating breast-cancer PDX than treatment with either agent alone. Collectively, these findings support use of cirmtuzumab in combination with conventional anti-cancer drugs to improve the outcome of patients with advanced breast cancer. Citation Format: Zhang S, Zhang H, Ghia EM, Liu G, Tripple V, Xu S, Cui B, Widhopf G, Yu J, Schwab R, Messer K, Parker BA, Kipps TJ. Breast cancer initiating cells express functional ROR1, which can be targeted by cirmtuzumab to potentially mitigate the risk of relapse after therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-07.

Secreted AGR2 promotes invasion of colorectal cancer cells via Wnt11-mediated non-canonical Wnt signaling

Human anterior gradient-2 (AGR2), a member of protein disulfide isomerase (PDI) family, is present in both intracellular and extracellular compartments. Although AGR2 is overexpressed in various human cancers and reported to promote aggressive tumor features, little is known regarding AGR2′s extracellular functions during tumorigenesis. Here, we demonstrate that secreted AGR2 promotes cell migration and metastasis of colorectal cancer (CRC) in vitro and in vivo. Mechanistically, secreted AGR2 elevated Wnt11 expression, triggering non-canonical Wnt signaling: the Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and c-jun amino-terminal kinase (JNK) pathways. Knockdown of Wnt11 or pretreatment with CaMKII and JNK inhibitors reversed the secreted AGR2′s migration-promoting effect. Further studies revealed that AGR2 antagonized canonical Wnt/β-catenin signaling via activating CaMKII. Collectively, our study uncovers a critical role of Wnt11-mediated non-canonical Wnt signaling (CaMKII and JNK pathways) in secreted AGR2′s promoted migration of CRC cells. These results raise the possibility that secreted AGR2 may be a potential therapeutic target towards inhibiting CRC metastasis.

WNT5A promotes migration and invasion of human osteosarcoma cells via SRC/ERK/MMP-14 pathway.

WNT5A, a representative ligand of activating several non-canonical WNT signal pathways, plays significant roles in oncogenesis and tumor inhibition. It has been shown that the non-receptor tyrosine kinase SRC is required for WNT5A-induced invasion of osteosarcoma cells. However, the precise molecular mechanism underlying WNT5A/SRC-mediated osteosarcoma cells invasion remains poorly defined. The study was designed to explore the role of ERK1/2 in WNT5A/SRC-induced osteosarcoma cells invasion and the downstream target of the SRC/ERK1/2 signalings. We found that WNT5A (100 ng/mL) remarkably stimulated migration and invasion of human osteosarcoma MG-63 cells, whereas inhibiting either SRC kinase activity by siRNA-mediated SRC silence or ERK1/2 phosphorylation by PD98059 treatment suppressed these effects, which suggested that the activation of SRC and ERK1/2 is essential for WNT5A-induced MG-63 cells migration and invasion. Furthermore, ERK1/2 phosphorylation induced by WNT5A was dramatically blocked by SRC siRNA. Additionally, our study further demonstrated that MMP-14 was upregulated after exposure to WNT5A in MG-63 cells, and the increased expression was blocked by SRC siRNA or PD98059. Collectively, these results indicate that WNT5A activates SRC/ERK1/2 signal pathway, leading to the upregulation of MMP-14 expression and MG-63 cells migration and invasion.