Wnt Regulators Research Articles

A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells

Background: Wnt signaling plays an essential role in tumor cell growth, including the development of malignant mesothelioma (MM). Epigenetic silencing of negative Wnt regulators leading to constitutive Wnt signaling has been observed in various cancers and warrants further attention. We have reported that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic effects in MM cells. Thus, in this study, we investigated whether the anti-MM effect of T3E could be mediated via the epigenetic alteration of the Wnt antagonist gene, Dickkopf-1 (DKK1). Methods: WST-1 and cell analyzers were employed to analyze the effects of T3E on cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR and Western blot were performed to evaluate the expression at mRNA and protein levels. Methylation status and epigenetic modifications of DKK1’s promoter regions after T3E treatment in MM cells were studied using methylation-specific PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown ­(siRNA), and specific inhibitors, were used to validate DKK1 as a target of T3E. Results: T3E markedly impaired MM cell viability, increased the expression of phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, which promotes cell proliferation as a Wnt-independent DKK1 receptor was inhibited by T3E. Silencing CKAP4 by ­siRNA did not appear to directly affect MM cell viability, thereby indicating that expression of both DKK1 and CKAP4 is required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases (DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells leads to increased DKK1 expression, thereby promoting tumor growth inhibition. MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells following T3E treatment is positively correlated with the methylation status of its promoter; T3E decreased DNA methylation and increased histone acetylation. Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation activity, whereas no effects were observed on histone H3K9 and H3K27. Conclusions: Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.

Structural basis for tankyrase-RNF146 interaction reveals noncanonical tankyrase-binding motifs.

Poly(ADP-ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase-1 and -2; a.k.a. PARP-5a and -5b) is involved in mitosis, telomere length regulation, GLUT-4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase-binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C-terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate-binding region of TNKS, forming a robust one-to-one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide-binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase-binding proteins including those of IF4A1 and NELFE.

Ferric citrate and ferric EDTA but not ferrous sulfate drive amphiregulin-mediated activation of the MAP kinase ERK in gut epithelial cancer cells.

Ferric chelates may be used as oral iron supplements or phosphate binders but both ferric citrate and ferric EDTA have been shown to promote tumor burden in murine models of colon cancer. Here we studied their effects on cancer cell growth, at typical supplemental iron levels encountered in the gastrointestinal tract (0.01-0.2 mM). Caco-2 and/or Hutu-80 cells were exposed to these forms of chelated iron or to ferrous sulfate and outcomes were assessed using cell proliferation assays, proteome profiler arrays, western blot, and ELISA. Ferric EDTA and ferric citrate increased cellular levels of the onco-protein amphiregulin and its receptor (EGFr) which in turn stimulated the activation of the MAP kinase ERK. Simultaneously, the expression of the negative Wnt regulator, DKK-1, increased suggesting that cell proliferation through the Wnt pathway may be less pronounced in the presence of ferric EDTA and ferric citrate, unlike for ferrous sulfate. Moreover, ferrous sulfate did not increase levels of cellular amphiregulin or EGFr. We conclude that specific iron compounds affect cell signaling differently and some may increase the risk of colon cancer advancement in an amphiregulin-dependent fashion. Further scrutiny of safe oral iron use is merited.

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<0.05), EGFR/HER1 was found significantly correlated with GSK3 (p<0.05) and HER-3 with Wnt5a (p<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.

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent-offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10-11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

Negative regulators of Wnt signaling pathway SFRP1 and SFRP3 expression in preterm and term pathologic placentas

Objective: Since Wnt signaling pathway plays a pivotal role in the placental development, we explored the expression of its negative regulators, SFRP1 and SFRP3 proteins in placentas from pathological pregnancies and compared their levels with those in healthy placentas.Methods: Placentas (n = 79) were stained for SFRP1, and SFRP3 proteins by immunohistochemistry and their expression levels were quantified by stereological variable of volume density (Vv, mm°).Results: Significantly higher expressions of SFRP1 and SFRP3 were found in all investigated groups of term and preterm pathologic placentas as well as in preterm control placentas in comparison with normal-term placentas.Conclusions: Our findings indicate the active involvement of negative Wnt regulators SFRP1/SFRP3 in placental development and important role in pathology of pregnancy.

Exosomes Mediate Epithelium-Mesenchyme Crosstalk in Organ Development.

Organ development requires complex signaling by cells in different tissues. Epithelium and mesenchyme interactions are crucial for the development of skin, hair follicles, kidney, lungs, prostate, major glands, and teeth. Despite myriad literature on cell-cell interactions and ligand-receptor binding, the roles of extracellular vesicles in epithelium-mesenchyme interactions during organogenesis are poorly understood. Here, we discovered that ∼100 nm exosomes were secreted by the epithelium and mesenchyme of a developing tooth organ and diffused through the basement membrane. Exosomes were entocytosed by epithelium or mesenchyme cells with preference by reciprocal cells rather than self-uptake. Exosomes reciprocally evoked cell differentiation and matrix synthesis: epithelium exosomes induce mesenchyme cells to produce dentin sialoprotein and undergo mineralization, whereas mesenchyme exosomes induce epithelium cells to produce basement membrane components, ameloblastin and amelogenenin. Attenuated exosomal secretion by Rab27a/b knockdown or GW4869 disrupted the basement membrane and reduced enamel and dentin production in organ culture and reduced matrix synthesis and the size of the cervical loop, which harbors epithelium stem cells, in Rab27aash/ash mutant mice. We then profiled exosomal constituents including miRNAs and peptides and further crossed all epithelium exosomal miRNAs with literature-known miRNA Wnt regulators. Epithelium exosome-derived miR135a activated Wnt/β-catenin signaling and escalated mesenchymal production of dentin matrix proteins, partially reversible by Antago-miR135a attenuation. Our results suggest that exosomes may mediate epithelium-mesenchyme crosstalk in organ development, suggesting that these vesicles and/or the molecular contents they are transporting may be interventional targets for treatment of diseases or regeneration of tissues.

PRDM15 safeguards naive pluripotency by transcriptionally regulating WNT and MAPK-ERK signaling.

The transcriptional network acting downstream of LIF, WNT and MAPK-ERK to stabilize mouse embryonic stem cells (ESCs) in their naive state has been extensively characterized. However, the upstream factors regulating these three signaling pathways remain largely uncharted. PR-domain-containing proteins (PRDMs) are zinc-finger sequence-specific chromatin factors that have essential roles in embryonic development and cell fate decisions. Here we characterize the transcriptional regulator PRDM15, which acts independently of PRDM14 to regulate the naive state of mouse ESCs. Mechanistically, PRDM15 modulates WNT and MAPK-ERK signaling by directly promoting the expression of Rspo1 (R-spondin1) and Spry1 (Sprouty1). Consistent with these findings, CRISPR-Cas9-mediated disruption of PRDM15-binding sites in the Rspo1 and Spry1 promoters recapitulates PRDM15 depletion, both in terms of local chromatin organization and the transcriptional modulation of these genes. Collectively, our findings uncover an essential role for PRDM15 as a chromatin factor that modulates the transcription of upstream regulators of WNT and MAPK-ERK signaling to safeguard naive pluripotency.

The Wnt regulator SFRP4 inhibits mesothelioma cell proliferation, migration, and antagonizes Wnt3a via its netrin-like domain.

Secreted frizzled related proteins (SFRPs) are a family of Wnt regulators which are frequently downregulated in cancers. In malignant mesothelioma (MM), downregulation of SFRP4 has been reported as a mechanism which contributes to aberrant activation of oncogenic Wnt signaling. Here we investigated the biological consequences of SFRP4 in two mesothelioma cell models where this protein is downregulated. We used recombinant SFRP4 and transient overexpression to study changes in proliferation, migration and downstream signaling. We found that recombinant SFRP4 inhibited both proliferation and migration of MM cells as well as abrogating the stimulatory effect of recombinant Wnt3a. Morphologically SFRP4 induced a cytotoxic effect distinct from apoptosis and consistent with mitotic catastrophe. Overexpression of SFRP4 in these cell lines displayed similar effects as endogenous protein on cell viability, migration and nuclear morphology. We also used expression constructs to examine the role of the SFRP4 cysteine rich domain (CRD) and a netrin-like domain(NLD) in these effects. Interestingly, we found it was the NLD which mediated the biological effects of SFRP4 in these cells. Our results indicate that SFRP4 inhibits mesothelioma proliferation, migration and activates alternative cell death pathways. The finding that the NLD is responsible for these has broader implications for this protein family. Overall this study suggests that the Wnt pathway may prove a promising target for therapy in mesothelioma.

Role of GSK3β in breast cancer susceptibility.

Breast cancer is one of the principal causes of death among Brazilian women, so it is a challenge to find new and specific early diagnostic markers, using simple and fast procedures. GSK3β gene is an important Wnt signaling regulator involved in β-Catenin degradation. Wnt signaling is associated with initiation and progression process in many tumor types, and alterations in β-Catenin explain only a small proportion of aberrant signaling found in breast cancer, indicating that other Wnt signaling components and/or regulators as GSK3β may be involved. The aim of this study was to evaluate the genetic, epigenetic and transcriptional alterations of GSK3β in breast cancer. Peripheral blood samples from 204 breast cancer and healthy women were collected. Assessment of rs334558 polymorphism was performed by PCR-RFLP, promoter methylation profiles analysis by MS-PCR and qPCR was used to determine GSK3β expression levels. The rs334558 polymorphism showed a strong association with aggressive cancer. A significant increase was observed in GSK3β expression level respect to hormone receptors status and tumor size. The results indicated an inverse relationship between GSK3β performance and tumor progression. This is the first study to relate GSK3β gene with breast cancer in Brazilian population.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

MiRNA-194 activates the Wnt/β-catenin signaling pathway in gastric cancer by targeting the negative Wnt regulator, SUFU

Emerging evidence has shown that miRNA-194 is aberrantly upregulated in gastric cancer (GC); however, the biological mechanisms underlying its involvement are largely unknown. Wnt/β-catenin signaling has been implicated in gastric tumorigenesis; we therefore hypothesized that miRNA-194 promotes gastric carcinogenesis by activating Wnt/β-catenin signaling. MiRNA-194 was found to be overexpressed in GC cell lines and 43 paired GC tissues. Overexpression of miRNA-194 promoted cell proliferation and migration, while inhibition of miRNA-194 blocked these processes. Inhibition of miRNA-194 decreased tumor volumes in nude mice. Furthermore, miRNA-194 inhibitors promoted cytoplasmic localization of β-catenin, leading to repression of Wnt signaling. We also discovered that SUFU, a known negative regulator of Hedgehog and Wnt signaling, was a target of miRNA-194. Anti-SUFU siRNAs rescued the inhibitory effects of miRNA-194 antagonists on cell proliferation and migration and on colony formation. We also found that SUFU expression was downregulated in GC tissues and cell lines and negatively correlated with miRNA-194 expression in primary GC tissues. Moreover, SUFU expression was negatively correlated with tumor stage, supporting its potential as a diagnostic or prognostic marker in GC. Taken together, these findings suggest that miRNA-194 is oncogenic and promotes GC cell proliferation and migration by activating Wnt signaling, at least in part, via suppression of SUFU.

C-Cbl mediates the degradation of tumorigenic nuclear β-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors.

Despite the loss of Adenomatous Polyposis Coli (APC) in a majority of colorectal cancers (CRC), not all CRCs bear hallmarks of Wnt activation, such as nuclear β-catenin. This underscores the presence of other Wnt regulators that are important to define, given the pathogenic and prognostic roles of nuclear β-catenin in human CRC. Herein, we investigated the effect of Casitas B-lineage lymphoma (c-Cbl) on nuclear β-catenin, which is an oncoprotein upregulated in CRC due to loss-of-function APC or gain-of-function CTNNB1 mutations. Despite mechanistic rationale and recent discoveries of c-Cbl's mutations in solid tumors, little is known about its functional importance in CRC. Our study in a cohort of human CRC patients demonstrated an inverse correlation between nuclear β-catenin and c-Cbl. Further investigation showed that the loss of c-Cbl activity significantly enhanced nuclear β-catenin and CRC tumor growth in cell culture and a mouse xenograft model. c-Cbl interacted with and downregulated β-catenin in a manner that was independent of CTNNB1 or APC mutation status. This study demonstrates a previously unrecognized function of c-Cbl as a negative regulator of CRC.

Analysis of the expression of the Wnt family of proteins and its modulatory role on cytokine expression in non activated and activated astroglial cells

Despite the essential functions of astrocytes and the emerging relevance of the Wnt family of proteins in the CNS under physiological and pathological conditions, the astroglial expression of this family of proteins and its potential modulatory role on astroglial activation is almost unknown. Thus, we have evaluated the expression of all Wnt ligands, receptors and regulators, and the activation state of Wnt-related signaling pathways in non-activated and differentially activated astroglial cultures. We found that numerous Wnt ligands, receptors and regulators were expressed in non-activated astrocytes, while the Wnt-dependent pathways were constitutively active. Moreover, the expression of most detectable Wnt-related molecules and the activity of the Wnt-dependent pathways suffered post-activation variations which frequently depended on the activation system. Finally, the analysis of the effects exerted by Wnt1 and 5a on the astroglial expression of prototypical genes related to astroglial activation showed that both Wnt ligands increased the astroglial expression of interleukin 1β depending on the experimental context, while did not modulate tumor necrosis factor α, interleukin 6, transforming growth factor β1 and glial fibrillary acidic protein expression. These results strongly suggest that the Wnt family of proteins is involved in how astrocytes modulate and respond to the physiological and pathological CNS.

Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf

SummaryKremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.

Abstract 311: Opposing Roles TCF7/LEF1 and TCF7L2 in Wnt/β-Catenin and BMP Signaling During Late Heart Development

T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors are important nuclear mediators of Wnt/β-catenin signaling. Their roles in cardiac development and diseases remain to be determined. We found that these TCF/LEF family members, including LEF1, TCF7, TCF7L1, and TCF7L2 had unique expression pattern during mouse heart development. At embryonic day (E) 13.5, LEF1 was one of the most abundant TCF/LEF members at mRNA level in the murine heart. By immunohistochemical staining, mesenchymal cells in valvular regions had much stronger intensity of LEF1 than cardiomyocytes in ventricles. Cardiomyocytes with weak to moderate nuclear staining for LEF1 were scattered in compact and trabecular layers. At E17.5, LEF1 mRNA levels dropped dramatically and its protein was no longer detected in cardiomyocytes. TCF7L1 mRNA levels also decreased from E13.5 to E17.5. Weak cytoplasmic, but not nuclear TCF7L1 signal was detected in cardiomyocytes at E13.5 by immunochemistry. TCF7 mRNA was the lowest among all four TCF/LEF members at E13.5 and it further decreased at E17.5. TCF7 protein was only detected by immunohistochemistry in mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. TCF7L2 mRNA became the most abundant TCF/LEFs at E17.5. Immunolocalization revealed that TCF7L2 formed an intensity gradient with the highest levels at the inner trabecular layer, opposite to the distribution of β-catenin. Wnt signaling activation by cardiac deletion of adenomatosis polyposis coli ( Apc ), a negative Wnt regulator, led to a dramatic increase in cyclin D2, Bmp4, LEF1, and TCF7. BMP mediators, SMAD1/5/8, were increasingly phosphorylated. Chromatin immunoprecipitation revealed that TCF7L2 was removed from and replaced by LEF1 and TCF7 in the promoter of cyclin D2 and Bmp4 to promote β-catenin recruitment. Furthermore, there was co-occupancy of TCF/LEF and SMAD in the adjacent TCF/LEF and SMAD binding sites of Wnt target genes. Finally, we proved that TCF7L2 was a transcriptional suppressor of cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments. Our results indicate that TCF7L2 is transcriptional repressor of Wnt/β-Catenin pathway in the heart.

Clinical factors and the role of Wnt regulators in racial disparity of metastatic colorectal cancer survival.

6551Background: African American (AA) colorectal cancer (CRC) patients experience lower survival compared to Caucasian Americans (CA). Aberrant Wnt/beta catenin (bcat) signaling is the hallmark of ...

Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis.

Wnt/β-catenin signaling enables developmental transitions during valvulogenesis.

Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector β-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/β-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/β-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/β-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects.

Abstract C21: Generation of Wnt- and mitogenic receptor binding bispecific antibodies to target cancer stem cells

Abstract Background: In colorectal cancer (CRC) and other solid tumors, cancer stem cells (CSC) contribute to tumor progression and resistance to standard chemotherapies. The continuous regeneration of the colon is dependent on strict control of developmental (e.g. Wnt) and mitogenic (e.g. EGF) pathway signaling; dysregulation results in uncontrolled proliferation forming the basis of aggressive tumors with metastatic potential. Here we describe the generation of novel bispecific antibodies designed to target CSC through Wnt signaling receptors and block growth factor signaling. The Wnt targets LGR5, LGR4, ZNRF3 and RNF43 were selected since their expression is modulated in CSC populations. The GPCR family members LGR4/LGR5 are positive Wnt regulators and the transmembrane E3 ligases ZNRF3/RNF43 are negative Wnt regulators. The growth factor receptor EGFR is frequently (>70%) overexpressed in CRC and its blockade has demonstrated clinical benefit in a subgroup of patients. More recently, HER3 pathway activation has been implicated in resistance to EGFR-targeted therapies. Experimental procedures and results: Two parallel strategies were applied to generate panels of common light chain (cLC) Fab against LGR4, LGR5, ZNRF3 and RNF43. Humanized cLC mice (MeMo®) were immunized with recombinant protein or DNA, and materials harvested from these mice used to generate Fab regions against these antigens. The second approach utilized large and diverse synthetic cLC Fab-phagemid libraries. Combined, these methods resulted in ∼1500 unique antigen-specific Fab from which ∼300 were selected for further testing. Bispecific antibodies were produced in a human cLC IgG1 format using substitutions in the IgG Fc regions for coexpression of two different heavy chains resulting in the generation of large panels of pure and stable bispecific IgG suitable for screening. The Wnt target specific Fab were combined with a Tetanus toxoid-specific control Fab arm allowing for stringent ranking of these Wnt-specific panels in a monovalent format for specificity, affinity, stability, and ligand (R-Spondin3) blocking potency. Based on this characterization the 54 most promising Wnt targeting arms were combined with a panel of previously characterized EGFR and HER-3 specific Fab arms resulting in ∼ 500 different cLC bispecific IgG for functional testing. All bispecific IgG were screened for potency of growth inhibition of CSC using novel 3D high content imaging readouts on patient-derived CRC organoids. The organoids are cultured using growth factors that allow for the maintenance and proliferation of healthy and diseased stem cells and their offspring. Functional analysis revealed several bispecific antibodies that inhibited CRC organoid growth much more potently than comparator drugs such as cetuximab or erlotinib. Conclusion: Bispecific antibodies present a biological modality that result in unexpected functional activities by mechanisms possible unique to the architecture of these molecules. Identifying these unique properties requires the rapid generation and screening of large panels of bispecific IgG directly in the therapeutic format in relevant functional assays. Initial screening results of the bispecific antibodies targeting Wnt and HER family members supports the concept of CSC targeting and several leads are currently undergoing more extensive characterization. Citation Format: Berina Eppink, Rob Roovers, Bram Herpers, Wim de Lau, Carina Clements, Vanessa Zondag van der Zande, Abdul Basmeleh, Willem Bartelink, Marc van de Wetering, Robert Vries, Leo Price, John De Kruif, Mark Throsby. Generation of Wnt- and mitogenic receptor binding bispecific antibodies to target cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C21.