Wnt Signaling Components Research Articles

S858 SUZ12 INACTIVATION COOPERATES WITH JAK3 MUTANT SIGNALING TO DRIVE T‐ALL DEVELOPMENT

Background:The PRC2 complex, with core components EZH2, SUZ12 and EED, is responsible for writing the H3K27me3 histone mark that is associated with gene repression. Sequence data analysis of 419 T‐cell acute lymphoblastic leukemia (T‐ALL) cases demonstrated that 14.3% of the cases harbored mutations in EZH2 (7.2%), SUZ12 (6.2%) and/or EED (3.8%). A significant positive association was found between SUZ12 loss‐of function mutations and JAK3 activating mutations.Aims:The goals of our study were to assess whether SUZ12 inactivation cooperates with JAK3 mutations in driving T‐ALL development, and to unravel the mechanism of oncogenic cooperation.Methods:We applied CRISPR gene editing technology to mutate endogenous Suz12 in mouse cells. For cell culture experiments, we used primary pro‐T‐cells derived from Cas9 transgenic mice that were retrovirally transduced with an activating JAK3(M511I) mutant, a Suz12 guide RNA (Suz12gRNA) or the combination of both. To generate mouse models, we performed bone marrow transplantations with hematopoietic stem/progenitor cells from Cas9 transgenic mice that were transduced with JAK3(M511I), Suz12gRNA or both. These cells were injected in wild type recipient mice and leukemia development was followed. RNA‐seq, ATAC‐seq and ChIP‐seq was performed on leukemia cells. We performed a drug screen with 189 inhibitors targeting a variety of epigenetic factors and signaling molecules on the mouse leukemia cells ex vivo.Results:The combined expression of JAK3(M511I) with Suz12gRNA conferred IL7‐independent growth to ex vivo cultured pro‐T cells, whereas JAK3(M511I) or Suz12gRNA alone did not, indicating direct cooperation between mutant JAK3 signaling and Suz12 inactivation. Moreover, in mouse bone marrow transplant leukemia models, combined expression of JAK3(M511I) and Suz12gRNA resulted in accelerated leukemia development with a mean latency of 74 days, compared to JAK3(M511I) or Suz12gRNA alone which generated leukemias with longer latency (112 to 150 days, p < 0.001). JAK3(M511I)+Suz12gRNA leukemias were CD8+ or CD4+CD8+ T‐cell leukemias and were transplantable. Effective Suz12 gene editing was confirmed by sequence analysis, Western blotting and intracellular flow cytometry documenting very low Suz12 and H3K27me3 levels. Both in vitro and in vivo data thus showed that inactivation of Suz12 cooperates with JAK3(M511I). Integration of gene expression data with ChIP‐seq data for Suz12 and H3K27me3, revealed increased expression of Wnt signaling components and Wnt target genes, as well as activation of the PI3K‐AKT‐mTOR signaling pathway in the JAK3(M511I)+Suz12gRNA leukemia cells compared to JAK3(M511I) leukemia cells. In addition, a drug screen on JAK3(M511I)+Suz12gRNA leukemia cells revealed increased sensitivity for PI3K, mTOR and Wnt signaling inhibitors, which is in line with upregulation of Pik3cb, Akt3 and Wnt signaling genes detected by RNA‐seq and ChIP‐seq. Surprisingly, this pharmacological approach also uncovered an increased sensitivity of the JAK3(M511I)+Suz12gRNA leukemia cells to HDAC6 inhibitors compared to JAK3(M511I) leukemia cells.Summary/Conclusion:We have shown that Suz12 inactivation cooperates with JAK3 mutant signaling to drive T‐ALL through upregulation of Wnt and PI3K/mTOR pathways. Furthermore, we were also able to identify specific vulnerabilities of the JAK3/Suz12 mutant leukemia cells, as they showed increased sensitivity to Wnt, PI3K/mTOR and HDAC6 inhibitors.

Wnt Signaling Deregulation in the Aging and Alzheimer's Brain.

Growing evidence suggests that synaptic signaling is compromised in the aging brain and in Alzheimer’s disease (AD), contributing to synaptic decline. Wnt signaling is a prominent pathway at the synapse and is required for synaptic plasticity and maintenance in the adult brain. In this review, we summarize the current knowledge on deregulation of Wnt signaling in the context of aging and AD. Emerging studies suggest that enhancing Wnt signaling could boost synaptic function during aging, and ameliorate synaptic pathology in AD. Although further research is needed to determine the precise contribution of deficient Wnt signaling to AD pathogenesis, targeting Wnt signaling components may provide novel therapeutic avenues for synapse protection or restoration in the brain.

Wnt Signaling Alterations in the Human Spinal Cord of Amyotrophic Lateral Sclerosis Cases: Spotlight on Fz2 and Wnt5a.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no cure, and elucidation of the mechanisms mediating neuronal death in this neuropathology is crucial to develop effective treatments. It has recently been demonstrated in animal models that the Wnt family of proteins is involved in this neuropathology, although its potential involvement in case of humans is almost unknown. We analyzed the expression of Wnt signaling components in healthy and ALS human spinal cords by quantitative RT-PCR, and we found that most Wnt ligands, modulators, receptors, and co-receptors were expressed in healthy controls. Moreover, we observed clear alterations in the mRNA expression of different components of this family of proteins in human spinal cord tissue from ALS cases. Specifically, we detected a significant increase in the mRNA levels of Wnt3, Wnt4, Fz2, and Fz8, together with several non-significant increases in the mRNA expression of other genes such as Wnt2b, Wnt5a, Fz3, Lrp5, and sFRP3. Based on these observations and on previous reports of studies performed in animal models, we evaluated with immunohistochemistry the protein expression patterns of Fz2 and Fz5 receptors and their main ligand Wnt5a in control samples and ALS cases. No substantial changes were observed in Fz5 protein expression pattern in ALS samples. However, we detected an increase in the amount of Fz2+ astrocytes in the borderline between gray and white matter at the ventral horn in ALS samples. Finally, Wnt5a expression was observed in neurons and astrocytes in both control and ALS samples, although Wnt5a immunolabeling in astroglial cells was significantly increased in ALS spinal cords in the same region where changes in Fz2 were observed. Altogether, these observations strongly suggest that the Wnt family of proteins, and more specifically Fz2 and Wnt5a, might be involved in human ALS pathology.

HIF-1α/Wnt signaling-dependent control of gene transcription regulates neuronal differentiation of glioblastoma stem cells.

HIF-1α has been suggested to interplay with Wnt signaling components in order to activate a neuronal differentiation process in both normal brain and glioblastoma (GBM). Based on these data, we explored the molecular mechanisms underlying the observed capability of GBM cells to acquire a neuronal phenotype upon Wnt signaling stimulation and how the microenvironment, particularly hypoxia, modulates this process.Methods: here, the employment of ChIP-seq techniques together with co-immunoprecipitation approaches allowed to reconstruct the molecular interactions responsible for activating specific pro-differentiating transcriptional programs in GBM cells. Moreover, gene silencing/over-expression approaches coupled with the functional analysis of cell phenotype were applied to confirm ChIP-driven hypotheses. Finally, we combined the use of publicly available gene expression datasets with protein expression data by immunohistochemistry to test the clinical relevance of obtained results.Results: our data clearly suggest that HIF-1α is recruited by the β-catenin/TCF1 complex to foster neuronal differentiation gene transcription in hypoxic GBM cells. Conversely, at higher oxygen levels, the increased expression of TCF4 exerts a transcriptional inhibitory function on the same genomic regions, thus counteracting differentiation. Moreover, we demonstrate the existence of a positive correlation between the expression levels of HIF-1α, TCF1 and neuronal phenotype in GBM tumors, accompanied by the over-expression of several Wnt signaling components, finally affecting patient prognosis.Conclusion: we unveiled a peculiar mechanism by which TCF1 and HIF-1α can induce a reminiscent neuronal differentiation of hypoxic GBM cells, which is hampered, in normoxia, by high levels of TCF4, thus not only de facto controlling the balance between differentiation and stemness, but also impacting on intra-tumoral heterogeneity and eventually patient outcome.

Wnt Signaling in vascular eye diseases.

The Wnt signaling pathway plays a pivotal role in vascular morphogenesis in various organs including the eye. Wnt ligands and receptors are key regulators of ocular angiogenesis both during the eye development and in vascular eye diseases. Wnt signaling participates in regulating multiple vascular beds in the eye including regression of the hyaloid vessels, and development of structured layers of vasculature in the retina. Loss-of-function mutations in Wnt signaling components cause rare genetic eye diseases in humans such as Norrie disease, and familial exudative vitreoretinopathy (FEVR) with defective ocular vasculature. On the other hand, experimental studies in more prevalent vascular eye diseases, such as wet age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and corneal neovascularization, suggest that aberrantly increased Wnt signaling is one of the causations for pathological ocular neovascularization, indicating the potential of modulating Wnt signaling to ameliorate pathological angiogenesis in eye diseases. This review recapitulates the key roles of the Wnt signaling pathway during ocular vascular development and in vascular eye diseases, and pharmaceutical approaches targeting the Wnt signaling as potential treatment options.

RETRACTED ARTICLE:Flavonoids modulate multidrug resistance through wnt signaling in P-glycoprotein overexpressing cell lines

BackgroundWnt signaling has been linked with P-glycoprotein (P-gp) overexpression and which was mainly mediated by β-catenin nuclear translocation. Flavonoids have already been reported as modulators of the Wnt/β-catenin pathway and hence they may serve as promising agents in the reversal of P-gp mediated cancer multi drug resistance (MDR).MethodsIn this study, we screened selected flavonoids against Wnt/β-catenin signaling molecules. The binding interaction of flavonoids (theaflavin, quercetin, rutin, epicatechin 3 gallate and tamarixetin) with GSK 3β was determined by molecular docking. Flavonoids on P-gp expression and the components of Wnt signaling in drug-resistant KBCHR8–5 cells were analyzed by western blotting and qRT-PCR. The MDR reversal potential of these selected flavonoids against P-gp mediated drug resistance was analyzed by cytotoxicity assay in KBCHR8–5 and MCF7/ADR cell lines. The chemosensitizing potential of flavonoids was further analyzed by observing cell cycle arrest in KBCHR8–5 cells.ResultsIn this study, we observed that the components of Wnt/β-catenin pathway such as Wnt and GSK 3β were activated in multidrug resistant KBCHR8–5 cell lines. All the flavonoids selected in this study significantly decreased the expression of Wnt and GSK 3β in KBCHR8–5 cells and subsequently modulates P-gp overexpression in this drug-resistant cell line. Further, we observed that these flavonoids considerably decreased the doxorubicin resistance in KBCHR8–5 and MCF7/ADR cell lines. The MDR reversal potential of flavonoids were found to be in the order of theaflavin > quercetin > rutin > epicatechin 3 gallate > tamarixetin. Moreover, we observed that flavonoids pretreatment significantly induced the doxorubicin-mediated arrest at the phase of G2/M. Further, the combinations of doxorubicin with flavonoids significantly modulate the expression of drug response genes in KBCHR8–5 cells.ConclusionThe present findings illustrate that the studied flavonoids significantly enhances doxorubicin-mediated cell death through modulating P-gp expression pattern by targeting Wnt/β-catenin signaling in drug-resistant KBCHR8–5 cells.

17β-Estradiol attenuates the influence of chronic activated microglia on SH-SY5Y cell proliferation via canonical WNT signaling pathway

The decline in circulating estrogen following menopause or aging is likely to initiate chronic inflammatory disorders, leading to neurodegenerative disease. Though, WNT1 paracrine molecules are crucial in embryonic neuroblastoma cell proliferation, very less is known about its role in adult brain that is associated with estrogen as preventive therapeutic strategy. The present study evidenced for the first time that 17β-estradiol (E2), a potent form of estrogen, could compensate the chronic neuroinflammation-associated loss of neurons by upregulating canonical WNT signaling pathway. Lipopolysaccharide was used to induce inflammatory responses in microglial cell line. The increased secretion of IL-6 cytokine was confirmed as a marker of chronic microglial activation. LPS-conditioned microglial media significantly reduced the viable cells and proliferative markers, BrdU and CyclinD1 in SH-SY5Y. It also decreased the expression of canonical WNT signaling components; WNT1 and β-catenin, which were significantly rescued with pre- and co-treatment of 10 nM E2. Furthermore, estrogen antagonist ICI 182,780 abolished the E2-mediated recovery in WNT1 expression. Whereas, canonical WNT receptor antagonist, Dkk1 was able to inhibit E2-mediated recovery in the expression of downstream component, β-catenin. It suggests a promising role of canonical WNT signaling pathway in estrogen mediated prevention of neuronal cell loss under chronic neuroinflammatory condition.

Dynamics of WNT signaling components in the human ovary from development to adulthood.

WNT signaling has been shown to play a pivotal role in mammalian gonad development and sex differentiation; however, its role in the developing human ovary has not been investigated. We analyzed a quantitative mass spectrometry dataset to determine the expression of WNT signaling components between 47 and 137 days of development and in adult ovarian cortex tissue. WNT signaling was identified within the top ten canonical pathways of proteins detected at every developmental stage examined. We further examined the specific localization of WNT signaling components glycogen synthase kinase 3 (GSK3B), frizzled 2 (FZD2), and β-catenin (CTNNB1) within ovarian tissue. GSK3B was nearly ubiquitously expressed during fetal development, while FZD2 was specific to germ cell nests during early development. β-catenin exhibited translocation from primarily membrane bound during early ovarian development to cytoplasmic and nuclear staining specifically in early primordial follicles in the fetal ovary. This cytoplasmic and nuclear β-catenin persisted in primordial follicles in adult ovarian tissue, but returned to membrane-bound localization in secondary follicles. We conclude that WNT signaling components are expressed in the human ovary from early to mid-gestation and remain in the adult ovary, and observed evidence for canonical WNT signaling only in the oocytes of primordial follicles. Together, these data are indicative of a role for canonical WNT signaling via β-catenin nuclear translocation during human follicle formation and follicle maintenance.

Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

SUMMARYWnt/β-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/β-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/β-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/β-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with β-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/β-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.

Hepatology Basic Science

Hepatology Basic Science

FGFR1-Activated Translation of WNT Pathway Components with Structured 5' UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation.

Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this cross-talk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multiomics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became premalignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in nontransformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upregulated by FGF signaling had long and structured 5' UTRs with a high frequency of polypurine sequences, several of which harbored (CGG)4 motifs that can fold into either stable G-quadruplexes or other stable secondary structures. The FGF-mediated increase in translation of WNT pathway components was compromised by silvestrol, an inhibitor of EIF4A that clamps EIF4A to polypurine sequences to block 43S scanning and inhibits its RNA-unwinding activity important for translation initiation. Moreover, silvestrol treatment significantly delayed FGF-WNT-driven tumorigenesis. Taken together, these results suggest that FGF signaling selectively enhances translation of structured mRNAs, particularly WNT signaling components, and highlight their vulnerability to inhibitors that target the RNA helicase EIF4A.Significance: The RNA helicase EIF4A may serve as a therapeutic target for breast cancers that require FGF and WNT signaling. Cancer Res; 78(15); 4229-40. ©2018 AACR.

Abstract B68: EGFR signaling mediated modulation of transcription and probable crosstalk with components of Wnt signaling

Abstract Introduction: The purpose of the study is to examine the crosstalk between HER2/EGFR and Wnt signaling in HER2-positive (HER2+) breast cancer cells. Human epidermal growth factor receptors (HER) constitute a family of four transmembrane receptors (HER1-HER4). Ligand binding to HER1 (EGFR), HER3, and HER4 results in heterodimerization with other HER family members, including HER2. HER2+ breast cancer is characterized by an amplification of the HER2 gene, resulting in an increase in HER2 protein presence on the surface of cells, magnification of downstream intracellular signaling, and enhanced responsiveness to ligand stimulation (e.g., EGF). Approximately 20-30% of breast cancers have HER2 amplifications. The Wnt pathway is highly conserved in mammals and overexpression of some Wnt family members results in cancer. Wnts are ligands that bind to Frizzled/LRP receptors to initiate downstream signaling that results in the stabilization and nuclear translocation of β-catenin. Once in the nucleus, β-catenin associates with activators such as TCF/LEF, SMADs, ATF2, and KLF4 to promote the transcription of many target genes. EGFR and Wnt crosstalk has been observed in various cell types following EGF treatment. As an example, EGF treatment of human epidermoid carcinoma cells results in β-catenin nuclear translocation and activation of TCF/LEF dependent reporters. Mitogen-Activated Protein Kinases (MAPKs), which are activated following EGF stimulation, have been demonstrated to inhibit GSK3β, a negative regulator of β-catenin. However, no genome-wide analysis has been conducted to determine what genes are regulated by β-catenin following EGF treatment in HER2+ breast cancer cells. We sought to determine the modulation of gene expression following the stimulation of HER2+ breast cancer cells with EGF and to investigate the mechanisms that underlie the changes observed in gene expression. Our studies have revealed exciting and novel findings that elucidate the effects of EGFR signaling on the epigenetic landscape. Specifically, we have identified putative β-catenin targets that become activated following EGFR stimulation. We hypothesize that EGFR signaling promotes the activation of specific β-catenin genes in order to alter cellular identity. Methods: RNA-seq and ChIP-seq for H3K18ac and H3K27ac was conducted following an EGF treatment time course in SKBR3 cells. The levels of several proteins of interest were determined by Western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by Western blot analysis. Results: RNA-seq analysis following an EGF treatment time course revealed that approximately 2,200 genes are either upregulated or downregulated compared to untreated cells. Moreover, the expression profiles clearly demonstrated waves of transcription. Next, we determined the status of H3K18ac and H3K27ac using ChIP-seq following an EGF time course. We found that H3K18ac and H3K27ac increased globally within 1h post-EGF treatment compared to untreated cells. We conducted a motif discovery search for transcription factor binding sites contained from -1000bp to +200bp for all activated genes and determined that each wave of transcription had some unique putative regulators. As expected, the genes activated at 1h and 2h post-EGF treatment contained c-Jun and JunD binding sites. Surprisingly, TCF3, TCF5, and LEF1 motifs were enriched in some genes that peaked in expression at 6h, 16h, and 24h post EGF treatment. Lastly, we biochemically fractionated the cellular compartments and detected an increase in chromatin associated β-catenin following EGF treatment, suggesting a crosstalk between EGFR and Wnt signaling components. We plan to determine the genome-wide localization of β-catenin following EGF treatment. Conclusions: Our data suggest that a crosstalk between EGFR and Wnt signaling components may regulate β-catenin target genes and lead HER2+ cells' resistance to therapeutic treatment. Citation Format: Miguel Nava, Nwamaka Amobi, Nathan Zemke, Arnold Berk, Robin Farias-Eisner, Jay Vadgama, Yanyuan Wu. EGFR signaling mediated modulation of transcription and probable crosstalk with components of Wnt signaling [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B68.

Abstract 385: HIF1A can regulate Wnt signaling in human colon cancer cells

Abstract Standard-of-care treatment for KRAS mutant metastatic colorectal cancer (mCRC) combines a cytotoxic chemotherapy backbone (FOLFOX) with an antiangiogenesis biologic agent (i.e., bevacizumab). Despite significant efforts to improve treatment, the prognosis for this type of cancer remains poor with an average 5-year overall survival of ~11%. Oncogenic KRAS mutations lead to the overactivation of RAS/ERK/MAPK pathwa,y driving many hallmarks of cancer such as cell proliferation, growth, and altered metabolism. In particular, several studies have shown that oncogenic activation of KRAS leads to normoxic stabilization of HIF1A protein, which can then reprogram cellular metabolism towards aerobic glycolysis. Our studies have discovered that overactive Wnt signaling can also reprogram cancer cell metabolism towards aerobic glycolysis. Of note, KRAS (~40%) and Wnt signaling (~90%) are very frequently coactivated in colorectal cancer. Interestingly, an important, key metabolic gene targeted by Wnt signaling, Pyruvate Dehydrogenase Kinase I (PDK1), is also directly regulated by HIF1A, suggesting there may be crosstalk between the HIF1A and Wnt pathways. Indeed, several published studies support the idea that HIF1A can influence Wnt signaling, and our data show this may occur through regulation of the Wnt mediating transcription factors LEF1 and TCF1 (TCF7 gene). Using luciferase reporters driven by LEF/TCF promoters, we observed significantly increased LEF1/TCF7 promoter activity in hypoxic colon cancer cells but not for other TCF family members (TCF7L1/TCF7L2). We identified putative hypoxia responsive elements (HREs) in the human LEF1 promoter, and deletion of these HREs reduced responsiveness to hypoxia. Furthermore, we have found that targeting glycolysis and Wnt can be synergistic in inhibiting colon cancer cell colony growth. We find that HIF1A and LEF/TCF/beta-catenin complexes coregulate gene expression of metabolic targets as well as Wnt signaling components (e.g., LEF1 and TCF7). These data suggest that cross-regulation between the HIF1A and Wnt signaling pathway is important and may be a potential Achilles heel for novel therapeutics in targeting KRAS mutant colon cancer. Current studies are under way to further investigate the mechanisms of crosstalk and joint regulation of metabolic reprogramming in KRAS mutant colon cancer cells. Citation Format: Yung Lyou, George Chen, Marian Waterman. HIF1A can regulate Wnt signaling in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 385.

SMYD3 controls a Wnt-responsive epigenetic switch for ASCL2 activation and cancer stem cell maintenance

Tumor growth is fueled by subset of cells with stem cell properties (Cancer stem cells, CSCs). While persistent activation of Wnt/β-catenin signaling confers CSC properties, it remains unclear how epigenetic modifications regulate Wnt target genes to dictate their self-renewal. Here, we report a novel Wnt-responsive epigenetic switch for CSC maintenance through activating the stem cell transcription factor ASCL2 in gastric carcinoma (GC). We characterize ASCL2-expressing (ASCL2+) GC cells as a subset of Wnt-responsive CSCs that depend on ASCL2 for self-renewal. High-throughput RNAi screening uncovers that the histone methyltransferase SMYD3 determines H3K4me3 status at the ASCL2 locus to promote ASCL2 expression. Moreover, SMYD3 may be transcriptionally activated by the β-catenin/TCF4 complex, indicating that the SMYD3-ASCL2 axis may be an integral component of Wnt signaling. Consistently, SMYD3 maintains self-renewal and tumorigenicity of ASCL2+ CSCs largely through inducing ASCL2. Clinically, overexpression of SMYD3 and ASCL2 are associated with malignant progression and poor patient outcomes in GC. Together, these findings define a Wnt-responsive CSC pathway that could be exploited to identify essential regulators of the signaling output, and reveal SMYD3 as an epigenetic target for eliminating CSCs in human cancers.

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.

NOX4 Deletion in Male Mice Exacerbates the Effect of Ethanol on Trabecular Bone and Osteoblastogenesis.

Chronic alcohol consumption increases bone resorption and decreases bone formation. A major component of ethanol (EtOH) pathology in bone is the generation of excess reactive oxygen species (ROS). The ROS-generating NADPH oxidase-4 (NOX4) is proposed to drive much of the EtOH-induced suppression of bone formation. Here, 13-week-old male wild-type (WT) and NOX4-/- mice were pair fed (PF) a high-fat (35%), Lieber-DeCarli liquid diet with or without EtOH at 30% of their total calories for 12 weeks. Micro-computed tomography analysis demonstrated significant decreases in trabecular bone volume/total volume (BV/TV) percentage and cortical thickness in WT, EtOH-fed mice compared with PF controls. EtOH-fed NOX4-/- mice also displayed decreased trabecular BV/TV and trabecular number compared with PF (P < 0.05). However, NOX4-/- mice were protected against EtOH-induced decreases in cortical thickness (P < 0.05) and decreases in collagen1 and osteocalcin mRNA expression in cortical bone (P < 0.05). In WT and NOX4-/- vertebral bone, EtOH suppressed expression of Wnt signaling components that promote osteoblast maturation. A role for NOX4 in EtOH inhibition of osteoblast differentiation was further demonstrated by protection against EtOH inhibition of osteoblastogenesis in ex vivo bone marrow cultures from NOX4-/-, but not p47phox-/- mice lacking active NADPH oxidase-2. However, bone marrow cultures from NOX4-/- mice formed fewer osteoblastic colonies compared with WT cultures (P < 0.05), suggesting a role for NOX4 in the maintenance of mesenchymal progenitor cell populations. These data suggest that NOX4 deletion is partially protective against EtOH effects on osteoblast differentiation, but may predispose bone to osteogenic impairments.

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.

CfDNA analysis of mCRPC patients expressing mutations in Wnt signaling.

256 Background: Alterations in Wnt signaling have been shown to play a role in the development of castrate resistant prostate cancer. Cell free DNA (cfDNA) isolated from patient plasma can provide a non-invasive way to further assess this role. The goal of this study was to identify patients with cfDNA alterations in major canonical Wnt signaling components (APC and/or beta-catenin), and relate the emergence of these mutations during treatment to alterations in other common mutations. Methods: 134 clinically progressive metastatic CRPC patients from Tulane Cancer Center underwent cfDNA analysis through Guardant360 test (Guardant Health, Redwood City, CA). This analysis consisted of exonic coverage of 70 genes as well as amplifications in 18 genes, with mutations categorized as either pathologic, non-pathologic or as variants of unknown significance (VUS). Clinical annotation of prior treatment history was recorded. Results: 21.6% (29/134) of the mCRPC patients evaluated had a canonical Wnt signaling (APC and/or CTNNB) alteration. Of these patients, 62.1% (18/29) had mutations identified as pathologic. 77.8% (14/18) of patients identified with a pathologic Wnt mutation were treated with abiraterone and/or enzalutamide prior to Guardant360 testing. To determine potential associations between Wnt signaling alterations and other detected changes in cfDNA, the relationship between Wnt mutations (APC and/or CTNNB1) with pathologic TP53 mutations, AR mutations, BRAF amplifications, and MYC amplifications was assessed using a patient’s latest Guardant360 test. A significant positive association was found between Wnt mutations (n = 18) and MYC amplifications (n = 22), (p = 0.0373). 33.3% (6/18) of patients with a pathologic Wnt mutation were found to have amplification in MYC. Other notable associations included Wnt mutations and AR mutations (n = 74), which approached statistical significance (p = 0.112). Conclusions: While the understanding of the role of Wnt signaling in the treatment of mCRPC is still evolving, co-segregation of Wnt-signaling alterations with other oncogenic alterations, particularly MYC, which has been identified as a target of canonical Wnt signaling, may provide insights with regards to future management of mCRPC.

CFTR mutation enhances Dishevelled degradation and results in impairment of Wnt-dependent hematopoiesis

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) with a multitude of clinical manifestations. Some CF patients develop clinically significant anemia, suggesting that CFTR may regulate hematopoiesis. Here, we report that cftr mutant zebrafish model exhibits primitive and definitive hematopoietic defects with impaired Wnt signaling. Cftr is found to interact, via its PDZ-binding domain (PDZBD), with Dishevelled (Dvl), a key component of Wnt signaling required for hematopoietic progenitor specification, thus protecting Dvl from Dapper1 (Dpr1)-induced lysosomal degradation. Defective hematopoiesis and impaired Wnt signaling in cftr mutant can be rescued by overexpression of wild-type or channel function-defective G551D mutant CFTR with an intact PDZBD, but not Cftr with mutations in the PDZBD. Analysis of human database (http://r2.amc.nl) shows that CFTR is positively correlated with DVL2 and Wnt-related hematopoietic factors in human blood system. The results reveal a previously unrecognized role of CFTR, which is independent of its channel function, in regulating DVL degradation and thus Wnt signaling required for hematopoiesis in both zebrafish and humans, providing an explanation for the anemic phenotype of CF patients.

Abstract P3-07-07: The pan-HER inhibitor, neratinib and wingless-type MMTVs (Wnt)/Wnt regulators in human breast cancer; a biological and clinical perspective

Abstract Background. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. Neratinib has been shown to have clinical activity in HER2-amplified or overexpressed breast cancers and those with HER2 mutations. However, there are indications that it may also work on other subtypes that are not strongly positive for the receptors. The present study first screened the effects of neratinib on a range of kinase targets and identified that the Wnt signalling components are key factors that allow neratinib to interact and targets. These targets were also validated in a cohort of human breast cancer. Methods. Human breast cancer cohorts (n=124) were tested for the transcript expression of HER family including EGFR, HER2, HER3 and HER4 and a number of Wnt family members and the Wnt signalling regulators, ie. GSK3, Axin-1, Axin-2 and β-catenin. The expression patterns were analysed against the clinicopathological and survival status of the patients. Neratinib was tested on a panel of breast cancer cell lines including triple negative cells for the effects on cytoxicity, cell growth, matrix adhesiveness and cellular migration. Signalling kinase pathways were screened using an antibody based kinase array. The effect of neratinib on multiple protein kinases was tested on the cell models, together with other kinase inhibitors. Results. Neratinib had an inhibitory effect on the cellular migration and cell-matrix adhesiveness of breast cancers at non-toxic concentrations, an effect more profound with MCF-7 and T47D cell lines than with BT20 and MDA MB-231 which are negative for the ER/EGFR/HER2 receptors. Of the multiple kinase inhibitors tested, neratinib was found to exert inhibition on cell function in synergy with the Wnt/β-catenin inhibitor (FK535) and GSK3 inhibitor (TWS119). The expression of the HER and Wnt family members, Wnt Inhibitory Factor-1 and Wnt regulators varied in mammary and breast cancer tissues and in their correlation with the clinicopathological factors. Of the aberrantly expressed receptors, Wnts and Wnt regulators, HER-2 and 4 were found to significantly correlate with Wnt10b (p&amp;lt;0.05), EGFR/HER1 was found significantly correlated with GSK3 (p&amp;lt;0.05) and HER-3 with Wnt5a (p&amp;lt;0.03). Furthermore, we identified that the integrated expression pattern of five of these factors, namely EGFR, HER2, HER4, Wnt5 and Wnt Inhibitory Factor-1 formed an expression signature that were significantly linked to the overall survival (survival time 148±3.7 vs 113.7 ±7.5 months for favourable and non-favourable pattern, respectively, p=0.002) and disease free survival (p=0.004) of the patients (median follow-up 120 months) Conclusion. Neratinib, at non-toxic concentration levels, is a profound inhibitor of the migration and matrix adhesion of breast cancer cells, cell functions linked to the aggressiveness and metastasis of breast cancer cells. Its synergistic effects with the Wnt and GSK3 inhibitor, together with the prognostic value of the HER family/Wnt, indicate that the Wnt pathway together with the HER family forms a new molecular indicator and target when considering neratinib in the treatment patients with breast cancer. Citation Format: Owen S, Ruge F, Lalani AS, Avogadri-Connors F, Bryce RP, Davies E, Jiang WG. The pan-HER inhibitor, neratinib and wingless-type MMTVs (Wnt)/Wnt regulators in human breast cancer; a biological and clinical perspective [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 P3-07-07.