FZD6 triggers Wnt-signalling driven by WNT10BIVS1 expression and highlights new targets in T-cell acute lymphoblastic leukemia.
Ontology highlight
ABSTRACT: Wnt/Fzd signaling has been implicated in hematopoietic stem cell maintenance and in acute leukemia establishment. In our previous work, we described a recurrent rearrangement involving the WNT10B locus (WNT10BR ), characterized by the expression of WNT10BIVS1 transcript variant, in acute myeloid leukemia. To determine the occurrence of WNT10BR in T-cell acute lymphoblastic leukemia (T-ALL), we retrospectively analyzed an Italian cohort of patients (n = 20) and detected a high incidence (13/20) of WNT10BIVS1 expression. To address genes involved in WNT10B molecular response, we have designed a Wnt-targeted RNA sequencing panel. Identifying Wnt agonists and antagonists, it results that the expression of FZD6, LRP5, and PROM1 genes stands out in WNT10BIVS1 positive patients compared to negative ones. Using MOLT4 and MUTZ-2 as leukemic cell models, which are characterized by the expression of WNT10BIVS1 , we have observed that WNT10B drives major Wnt activation to the FZD6 receptor complex through receipt of ligand. Additionally, short hairpin RNAs (shRNAs)-mediated gene silencing and small molecule-mediated inhibition of WNTs secretion have been observed to interfere with the WNT10B/FZD6 interaction. We have therefore identified that WNT10BIVS1 knockdown, or pharmacological interference by the LGK974 porcupine (PORCN) inhibitor, reduces WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. These results describe the molecular circuit induced by WNT10B and suggest WNT10B/FZD6 as a new target in the T-ALL treatment strategy.
Project description:Frizzled receptors are the mediators of the wnt canonical and non-canonical pathways, which play fundamental roles in cell differentiation and organism development. A large body of work indicates that dysregulation of wnt signalling is a feature of oncogenic transformation, but most of the studies published so far focus on the assessment of the consequences of aberrations of the canonical pathway in human cancer. In this review, we discuss the emerging role of the wnt non-canonical pathway regulated by frizzled receptor 6 (Fzd6) in the pathogenesis of different types of human malignancies. The function played by Fzd6 in the physiology of normal and cancer cells has been highlighted in the view that an increased knowledge of the signalling pathways upstream and downstream of this receptor could ultimately result in the identification of new targets for cancer therapy.
Project description:Molecularly defined subclassification is associated with phenotypic malignancy of glioblastoma (GBM). However, current understanding of the molecular basis of subclass conversion that is often involved in GBM recurrence remain rudimentary at best. Here we report that canonical Wnt signalling that is active in proneural (PN) but inactive in mesenchymal (MES) GBM, along with miR-125b and miR-20b that are expressed at high levels in PN compared with MES GBM, comprise a regulatory circuit involving TCF4-miR-125b/miR-20b-FZD6. FZD6 acts as a negative regulator of this circuit by activating CaMKII-TAK1-NLK signalling, which, in turn, attenuates Wnt pathway activity while promoting STAT3 and NF-κB signalling that are important regulators of the MES-associated phenotype. These findings are confirmed by targeting differentially enriched pathways in PN versus MES GBM that results in inhibition of distinct GBM subtypes. Correlative expressions of the components of this circuit are prognostic relevant for clinical GBM. Our findings provide insights for understanding GBM pathogenesis and for improving treatment of GBM.
Project description:Hypothesis and proof of concept:
Our hypothesis is that increase in cellular iron import proteins (TfR1, DMT1) occur early in the adenoma-carcinoma sequence through mutations in APC and lead to cellular iron loading. As demonstrated in our previous work the effects of this iron loading is to mediate increased Wnt signalling resulting in c-myc induction. This in turn serves to increase the expression of iron import proteins (TfR1, DMT1) and decrease the expression of iron export (ferroportin [FPN]) and storage (ferritin) proteins. Such a hypothesis explains how Wnt signalling controls iron metabolism and ensures that there is adequate cellular iron for ATP generation and cellular proliferation.
Experimental design:
To test such a hypothesis we aim to prospectively collect the following colorectal tissue from patients attending for colonoscopy:
1. Normal colonic mucosa in patients with no colorectal pathology (n = 30)
2. Polyps and matched normal colon (n = 30)
3. Colorectal cancers and matched normal colon (n = 30)
We also intend to collect serum and urine from the following patient groups:
4. Normal colonoscopy (n = 30)
5. Colorectal adenomas (n = 30)
6. Colorectal cancers (n = 30)
Primary outcome(s): Measured at baseline, using the expression of proteins in the tissue and serum to detect the cellular and systemic iron transport proteins. The techniques used will include mass spectrometry, western blotting, real time PCR and immunohistochemistry.
Project description:Gastric cancer persists as a frequent and deadly disease that claims over 700 000 lives annually. Gastric cancer is a multifactorial disease that is genetically, cytologically and architecturally more heterogeneous than other gastrointestinal cancers, making it therapeutically challenging. As such, and largely attributed to late-stage diagnosis, gastric cancer patients show only partial response to standard chemo and targeted molecular therapies, highlighting an urgent need to develop new targeted therapies for this disease. Wnt signalling has a well-documented history in the genesis of many cancers and is, therefore, an attractive therapeutic target. As such, drug discovery has focused on developing inhibitors that target multiple nodes of the Wnt signalling cascade, some of which have progressed to clinical trials. The collective efforts of patient genomic profiling has uncovered genetic lesions to multiple components of the Wnt pathway in gastric cancer patients, which strongly suggest that Wnt-targeted therapies could offer therapeutic benefits for gastric cancer patients. These data have been supported by studies in mouse models of gastric cancer, which identify Wnt signalling as a driver of gastric tumourigenesis. Here, we review the current literature regarding Wnt signalling in gastric cancer and highlight the suitability of each class of Wnt inhibitor as a potential treatment for gastric cancer patients, in relation to the type of Wnt deregulation observed.Linked articlesThis article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.
Project description:Synapse degeneration is an early and invariant feature of neurodegenerative diseases. Indeed, synapse loss occurs prior to neuronal degeneration and correlates with the symptom severity of these diseases. However, the molecular mechanisms that trigger synaptic loss remain poorly understood. Here we demonstrate that deficient Wnt signalling elicits synaptic degeneration in the adult striatum. Inducible expression of the secreted Wnt antagonist Dickkopf1 (Dkk1) in adult mice (iDkk1) decreases the number of cortico-striatal glutamatergic synapses and of D1 and D2 dopamine receptor clusters. Synapse loss occurs in the absence of axon retraction or cell death. The remaining excitatory terminals contain fewer synaptic vesicles and have a reduced probability of evoked transmitter release. IDkk1 mice show impaired motor coordination and are irresponsive to amphetamine. These studies identify Wnts as key endogenous regulators of synaptic maintenance and suggest that dysfunction in Wnt signalling contributes to synaptic degeneration at early stages in neurodegenerative diseases.
Project description:Wnt signaling is crucial for proper development, tissue homeostasis and cell cycle regulation. A key role of Wnt signaling is the GSK3β-mediated stabilization of β-catenin, which mediates many of the critical roles of Wnt signaling. In addition, it was recently revealed that Wnt signaling can also act independently of β-catenin. In fact, Wnt mediated stabilization of proteins (Wnt/STOP) that involves an LRP6-DVL-dependent signaling cascade is required for proper regulation of mitosis and for faithful chromosome segregation in human somatic cells. We show that inhibition of Wnt/LRP6 signaling causes whole chromosome missegregation and aneuploidy by triggering abnormally increased microtubule growth rates in mitotic spindles, and this is mediated by increased GSK3β activity. We demonstrate that proper mitosis and maintenance of numerical chromosome stability requires continuous basal autocrine Wnt signaling that involves secretion of Wnts. Importantly, we identified Wnt10b as a Wnt ligand required for the maintenance of normal mitotic microtubule dynamics and for proper chromosome segregation. Thus, a self-maintaining Wnt10b-GSK3β-driven cellular machinery ensures the proper execution of mitosis and karyotype stability in human somatic cells.
Project description:Wnt proteins control diverse biological processes through ?-catenin-dependent canonical signalling and ?-catenin-independent non-canonical signalling. The mechanisms by which these signalling pathways are differentially triggered and controlled are not fully understood. Dishevelled (Dvl) is a scaffold protein that serves as the branch point of these pathways. Here, we show that cholesterol selectively activates canonical Wnt signalling over non-canonical signalling under physiological conditions by specifically facilitating the membrane recruitment of the PDZ domain of Dvl and its interaction with other proteins. Single-molecule imaging analysis shows that cholesterol is enriched around the Wnt-activated Frizzled and low-density lipoprotein receptor-related protein 5/6 receptors and plays an essential role for Dvl-mediated formation and maintenance of the canonical Wnt signalling complex. Collectively, our results suggest a new regulatory role of cholesterol in Wnt signalling and a potential link between cellular cholesterol levels and the balance between canonical and non-canonical Wnt signalling activities.
Project description:Wnt/?-catenin signalling has been suggested to be active in basal-like breast cancer. However, in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of ?-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active ?-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ER?, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical ?-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/?-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.
Project description:BackgroundDeregulation of the Wnt/ beta-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in beta-catenin stabilization and accumulation, and the aberrant modulation of beta-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.MethodsGene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of beta-catenin was used to identify target genes of this pathway.ResultsThe differential expression in response to DeltaNbeta-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/beta-catenin tumour cells and between the tumour and its microenvironment. One of these genes, Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon DeltaNbeta-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.ConclusionsHIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer.
Project description:Concerted co-regulation of multiple signalling pathways is crucial for tissue homoeostasis and tumorigenesis. Here we report that VGLL4, a previously identified YAP antagonist, also functions as a regulator of Wnt/β-catenin signalling. The expression of VGLL4 is significantly downregulated in clinical colorectal carcinoma (CRC) specimens, positively associated with patient survival rate, and inversely correlated with the expression of Wnt target genes in CRCs. Knockdown of VGLL4 enhances proliferation and tumour formation of CRC cells. A designed peptide mimicking the function of VGLL4 effectively inhibits CRC progression in a de novo mouse model. Mechanistically, TEAD4 associates with TCF4 to form a complex and cobind target genes. VGLL4 targets this TEAD4-TCF4 complex to interfere the functional interplay between TEAD4 and TCF4, suppressing the transactivation of TCF4. Collectively, our study indicates that Wnt/β-catenin and Hippo-YAP signalling are directly linked at transcription factor-level, and VGLL4 can target a TEAD4-TCF4 complex to co-regulate both pathways.