Increased Wnt Activity Research Articles

An investigation on the alterations in Wnt signaling in ADHD across developmental stages

The canonical Wnt signaling pathway plays a vital role in the developmental processes of the Central Nervous System throughout both prenatal and postnatal stages, as well as in maintaining homeostasis during adulthood. Its complex intracellular cascade involves the participation of key proteins (i.e., GSK3β and β-catenin) to activate the transcription of Wnt target genes. These genes subsequently control processes like cell proliferation, maturation, and the determination of cell fate. Previous studies suggest that this pathway can also be associated with Attention-Deficit Hyperactivity Disorder (ADHD), a neurodevelopmental disorder with multifactorial etiology. This study aimed to clarify if and at what developmental stage the Wnt pathway is altered in ADHD. Accordingly, we carried out proteomic and functional assessments of the Wnt pathway using Western Blot and reporter assays, respectively. These assessments were performed at the induced pluripotent stem cell (iPSC), neural stem cell (NSC), and neuronal phases. IPSCs were generated from somatic cells retrieved from 5 controls and 5 patients diagnosed with ADHD. As opposed to the developmental stage of iPSCs, ADHD NSCs showed alterations in the protein expression of both GSK3β and β-catenin, suggesting increased Wnt activity in the ADHD group. Moreover, Wnt reporter assays confirmed higher Wnt activity in ADHD NSCs. Our molecular findings in NSCs correlated with genetic predisposition to ADHD and clinical traits displayed by their respective donors. In conclusion, these results suggest that a crucial cellular pathway is disrupted in patient-specific NSCs, potentially explaining the developmental deficits clinically exhibited by ADHD patients.

Normal Skin Cells Increase Aggressiveness of Cutaneous Melanoma by Promoting Epithelial-to-Mesenchymal Transition via Nodal and Wnt Activity.

Melanoma is a lethal form of skin cancer triggered by genetic and environmental factors. Excision of early-stage, poorly aggressive melanoma often leads to a successful outcome; however, left undiagnosed these lesions can progress to metastatic disease. This research investigates whether the exposure of poorly aggressive melanoma to certain normal skin cells can explain how non-metastatic melanoma becomes more aggressive while still confined to the skin. To this end, we used a serial co-culture approach to sequentially expose cells from two different, poorly aggressive human melanoma cell lines against normal cells of the skin beginning with normal melanocytes, then epidermal keratinocytes, and finally dermal fibroblasts. Protein extraction of melanoma cells occurred at each step of the co-culture sequence for western blot (WB) analysis. In addition, morphological and functional changes were assessed to detect differences between the serially co-cultured melanoma cells and non-co-cultured cells. Results show that the co-cultured melanoma cells assumed a more mesenchymal morphology and displayed a significant increase in proliferation and invasiveness compared to control or reference cells. WB analysis of protein from the co-cultured melanoma cells showed increased expression of Snail and decreased levels of E-cadherin suggesting that epithelial-to-mesenchymal transition (EMT) is occurring in these co-cultured cells. Additional WB analysis showed increased levels of Nodal protein and signaling and signs of increased Wnt activity in the co-cultured melanoma cells compared to reference cells. These data suggest that interaction between poorly aggressive melanoma cells with normal cells of the skin may regulate the transition from localized, poorly aggressive melanoma to invasive, metastatic disease via Nodal and/or Wnt induced EMT.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

BackgroundThe Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor.ResultsHydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model.ConclusionsWe show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Abstract B77: PORCN inhibition prolongs survival, decreases tumor burden, and alters the immune microenvironment in ovarian cancer

Abstract Background: Wnt/β-catenin pathway upregulation has been correlated with immune evasion in multiple cancers, including epithelial ovarian cancer (EOC). Porcupine (PORCN) is an enzyme necessary for cells to produce WNT ligand, which is necessary for the activation of the pathway. Our hypothesis was that the administration of a PORCN inhibitor (CGX1321) would decrease Wnt signaling and thereby lead to a decrease in immune evasion, creating a “hot” tumor microenvironment (TME), which would result in increased survival and a decrease tumor burden. Methods: RNA sequencing was performed in a cohort of primary ovarian cancer samples. CGX1321 was administered to C57Bl6 mice injected intraperitoneally with ID8 or ID8p53−/− cells. Tumor growth was measured by bioluminescence and omental weight. H&E slides were analyzed for amount of tumor in the omentum. Amount of tumor-infiltrating lymphocytes (TILs), Tregs, dendritic cells (DCs), macrophages, and monocytes in the TME were quantified via flow cytometry. CD11c-cre x β-catenin-flox C57Bl6 mice, a mouse model with no DC-intrinsic β-catenin, and the administration of anti-CD8β antibody were used to evaluate tumor burden with and without CGX1321. Results: Increased Wnt activity was correlated with a decreased T-cell signature in our human ovarian cancer samples. Treatment with CGX1321 prolonged survival (P=0.0001) and decreased omentum weight (P=0.0056) in mice injected with ID8 cells. H&E slides revealed decreased amount of tumor in the omentum with treatment. There was an increase of DCs (P=0.0159), macrophages (P=0.0079), and monocytes (P value=0.0079) with treatment, which was only significant for mice injected with ID8 cells, not ID8p53−/− mice. Tumor burden was decreased in CD11c-cre x β-catenin-flox mice (P=0.1014) without treatment, and with CGX 1321 treatment (P=0.0450). Tumor burden was not decreased with CGX1321 treatment in the absence of CD8+ T-cells (P=0.3175). Conclusions: Consistent with EOC TCGA data, we identified a correlation between increased Wnt signaling and decreased T-cell signature (i.e., “cold” tumor). ID8 cells have a higher level of total β-catenin and expression of WNT related genes compared with ID8p53−/−, which may explain the prolonged survival, decreased tumor burden, and more profound increase of DCs, macrophages, and monocytes in the TME. This may suggest a reliance on these cells for tumor recognition with PORCN inhibition. Furthermore, tumor burden was decreased in a DC-intrinsic β-catenin absent model, suggesting the presence of β-catenin in this antigen-presenting cell contributes to immune evasion in the TME. The lack of effect of CGX1321 on tumor burden in the absence of CD8+ T-cells indicates a partial reliance on CD8+ T-cells for tumor recognition. These data warrant further investigation of Wnt inhibition in both preclinical models and clinical trials in EOC. Citation Format: Whitney N. Goldsberry, Selene Meza-Perez, Ashwini A. Katre, Angelina Londoño, Bryan T. Mott, Jaclyn M. Arquiette, Troy D. Randall, Sara J. Cooper, Lyse A. Norian, Rebecca C. Arend. PORCN inhibition prolongs survival, decreases tumor burden, and alters the immune microenvironment in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B77.

Zebrafish Wtx is a negative regulator of Wnt signaling but is dispensable for embryonic development and organ homeostasis.

The X-chromosomally linked gene WTX is a human disease gene and a member of the AMER family. Mutations in WTX are found in Wilms tumor, a form of pediatric kidney cancer and in patients suffering from OSCS (Osteopathia striata with cranial sclerosis), a sclerosing bone disorder. Functional data suggest WTX to be an inhibitor of the Wnt/β-catenin signaling pathway. Deletion of Wtx in mouse leads to perinatal death, impeding the analysis of its physiological role. To gain insights into the function of Wtx in development and homeostasis we have used zebrafish as a model and performed both knockdown and knockout studies using morpholinos and transcription activator-like effector nucleases (TALENs), respectively. Wtx knockdown led to increased Wnt activity and embryonic dorsalization. Also, wtx mutants showed a transient upregulation of Wnt target genes in the context of caudal fin regeneration. Surprisingly, however, wtx as well as wtx/amer2/amer3 triple mutants developed normally, were fertile and did not show any anomalies in organ maintenance. Our data show that members of the zebrafish wtx/amer gene family, while sharing a partially overlapping expression pattern do not compensate for each other. This observation demonstrates a remarkable robustness during development and regeneration in zebrafish.

MEK inhibitors activate Wnt signalling and induce stem cell plasticity in colorectal cancer

In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/β-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.

Enabling precision medicine by unravelling disease pathophysiology: quantifying signal transduction pathway activity across cell and tissue types

Signal transduction pathways are important in physiology and pathophysiology. Targeted drugs aim at modifying pathogenic pathway activity, e.g., in cancer. Optimal treatment choice requires assays to measure pathway activity in individual patient tissue or cell samples. We developed a method enabling quantitative measurement of functional pathway activity based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the pathway-associated transcription factor. Oestrogen receptor, Wnt, and PI3K-FOXO pathway assays have been described previously. Here, we report model development for androgen receptor, Hedgehog, TGFβ, and NFκB pathway assays, biological validation on multiple cell types, and analysis of data from published clinical studies (multiple sclerosis, amyotrophic lateral sclerosis, contact dermatitis, Ewing sarcoma, lymphoma, medulloblastoma, ependymoma, skin and prostate cancer). Multiple pathway analysis of clinical prostate cancer (PCa) studies showed increased AR activity in hyperplasia and primary PCa but variable AR activity in castrate resistant (CR) PCa, loss of TGFβ activity in PCa, increased Wnt activity in TMPRSS2:ERG fusion protein-positive PCa, active PI3K pathway in advanced PCa, and active PI3K and NFκB as potential hormonal resistance pathways. Potential value for future clinical practice includes disease subtyping and prediction and targeted therapy response prediction and monitoring.

Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer

Nearly all patients with small cell lung cancer (SCLC) eventually relapse with chemoresistant disease. The molecular mechanisms driving chemoresistance in SCLC remain un-characterized. Here, we describe whole-exome sequencing of paired SCLC tumor samples procured at diagnosis and relapse from 12 patients, and unpaired relapse samples from 18 additional patients. Multiple somatic copy number alterations, including gains in ABCC1 and deletions in MYCL, MSH2, and MSH6, are identifiable in relapsed samples. Relapse samples also exhibit recurrent mutations and loss of heterozygosity in regulators of WNT signaling, including CHD8 and APC. Analysis of RNA-sequencing data shows enrichment for an ASCL1-low expression subtype and WNT activation in relapse samples. Activation of WNT signaling in chemosensitive human SCLC cell lines through APC knockdown induces chemoresistance. Additionally, in vitro-derived chemoresistant cell lines demonstrate increased WNT activity. Overall, our results suggest WNT signaling activation as a mechanism of chemoresistance in relapsed SCLC.

Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting ROR\u03b2

BackgroundColorectal cancer remains one of the most common malignant tumors worldwide. Colorectal cancer initiating cells (CCICs) are a small subpopulation responsible for malignant behaviors of colorectal cancer. Aberrant activation of the Wnt pathways regulates the self-renewal of CCIC. However, the underlying mechanism(s) remain poorly understood.MethodsVia retroviral library screening, we identified Nuclear Receptor-Interacting Protein 2 (NRIP2) as a novel interactor of the Wnt pathway from enriched colorectal cancer colosphere cells. The expression levels of NRIP2 and retinoic acid-related orphan receptor β (RORβ) were further examined by FISH, qRT-PCR, IHC and Western blot. NRIP2 overexpressed and knockdown colorectal cancer cells were produced to study the role of NRIP2 in Wnt pathway. We also verified the binding between NRIP2 and RORβ and investigated the effect of RORβ on CCICs both in vitro and in vivo. Genechip-scanning speculated downstream target HBP1. Western blot, ChIP and luciferase reporter were carried to investigate the interaction between NRIP2, RORβ, and HBP1.ResultsNRIP2 was significantly up-regulated in CCICs from both cell lines and primary colorectal cancer tissues. Reinforced expression of NRIP2 increased Wnt activity, while silencing of NRIP2 attenuated Wnt activity. The transcription factor RORβ was a key target through which NRIP2 regulated Wnt pathway activity. RORβ was a transcriptional enhancer of inhibitor HBP1 of the Wnt pathway. NRIP2 prevented RORβ to bind with downstream HBP1 promoter regions and reduced the transcription of HBP1. This, in turn, attenuated the HBP1-dependent inhibition of TCF4-mediated transcription.ConclusionsNRIP2 is a novel interactor of the Wnt pathway in colorectal cancer initiating cells. interactions between NRIP2, RORβ, and HBP1 mediate a new mechanism for CCIC self-renewal via the Wnt activity.

Dentin Dysplasia in Notum Knockout Mice.

Secreted WNT proteins control cell differentiation and proliferation in many tissues, and NOTUM is a secreted enzyme that modulates WNT morphogens by removing a palmitoleoylate moiety that is essential for their activity. To better understand the role this enzyme in development, the authors produced NOTUM-deficient mice by targeted insertional disruption of the Notum gene. The authors discovered a critical role for NOTUM in dentin morphogenesis suggesting that increased WNT activity can disrupt odontoblast differentiation and orientation in both incisor and molar teeth. Although molars in Notum(-/-) mice had normal-shaped crowns and normal mantle dentin, the defective crown dentin resulted in enamel prone to fracture during mastication and made teeth more susceptible to endodontal inflammation and necrosis. The dentin dysplasia and short roots contributed to tooth hypermobility and to the spread of periodontal inflammation, which often progressed to periapical abscess formation. The additional incidental finding of renal agenesis in some Notum (-/-) mice indicated that NOTUM also has a role in kidney development, with undiagnosed bilateral renal agenesis most likely responsible for the observed decreased perinatal viability of Notum(-/-) mice. The findings support a significant role for NOTUM in modulating WNT signaling pathways that have pleiotropic effects on tooth and kidney development.

Abstract P4-08-03: DEAD-box RNA helicase DP103 as a novel regulator of Wnt/β-catenin signaling pathway and promotes cancer stem cell-like behavior in triple negative breast cancers

Abstract Despite recent advances in breast cancer therapeutics, mortality of metastatic triple negative breast cancer (TNBC) subtype remains high; due to their lack of hormone receptors expression for targeted therapy. Aberrant activation of Wnt/β-catenin signaling has been associated with breast cancers; where 40% of total breast cancers have elevated β-catenin levels with increased Wnt activity. Recently, we identified DEAD-box RNA helicase DP103 as a novel prognostic biomarker and metastasis-driving oncogene; highly expressed in TNBC subtype. Interestingly, we found high DP103 expression to be positively correlated with high β-catenin expression in clinical specimens (n=400). This led us to hypothesize a possible role of DP103 in modulating the Wnt/β-catenin pathway in TNBCs. Depletion of DP103 in metastatic TNBC cells decreases Wnt/β-catenin activity and expression of downstream Wnt target genes, while overexpression of DP103 increases Wnt activity. Depletion of DP103 also decreases phosphorylation of LRP6 and several important Wnt modulators required for downstream Wnt activation. Moreover, induction of Wnt/β-catenin signaling in Wnt responsive TNBC cells also significantly increased DP103 expression, indicating a possible positive feedback loop. Both canonical and non-canonical Wnt signaling is known to independently promote stem cell growth in mammospheres. Herein, we will also provide evidence on the role of DP103 in promoting breast cancer stem cell-like properties. Collectively, our data show a novel regulatory role of DP103 in the Wnt/β-catenin signaling pathway and in promoting breast cancer stem cell-like behavior, presenting itself as a potential drug target in TNBC patients. Citation Format: Cai W, Cheong JK, Edison E, Banerjee A, Tan TZ, Gaboury L, Yousef EM, Thiery JP, Lobie PE, Virshup DM, Yap CT, Kumar AP. DEAD-box RNA helicase DP103 as a novel regulator of Wnt/β-catenin signaling pathway and promotes cancer stem cell-like behavior in triple negative breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-03.

OVOL2, an Inhibitor of WNT Signaling, Reduces Invasive Activities of Human and Mouse Cancer Cells and Is Down-regulated in Human Colorectal Tumors

Activation of WNT signaling promotes the invasive activities of several types of cancer cells, but it is not clear if it regulates the same processes in colorectal cancer (CRC) cells, or what mechanisms are involved. We studied the expression and function of OVOL2, a member of the Ovo family of conserved zinc-finger transcription factors regulated by the WNT signaling pathway, in intestinal tumors of mice and human beings. We analyzed the expression of OVOL2 protein and messenger RNA in CRC cell lines and tissue arrays, as well as CRC samples from patients who underwent surgery at Xiamen University in China from 2009 to 2012; clinical information also was collected. CRC cell lines (SW620) were infected with lentivirus expressing OVOL2, analyzed in migration and invasion assays, and injected into nude mice to assess tumor growth and metastasis. Tandem affinity purification was used to purify the OVOL2-containing complex from CRC cells; the complex was analyzed by liquid chromatography, tandem mass spectrometry, and immunoprecipitation experiments. Gene promoter activities were measured in luciferase reporter assays. We analyzed mice with an intestine-specific disruption of Ovol2 (Ovol2(flox/+) transgenic mice), as well as Apc(min/+) mice; these mice were crossed and analyzed. Analysis of data from patients indicated that the levels of OVOL2 messenger RNA were significantly lower in colon carcinomas than adenomas, and decreased significantly as carcinomas progressed from grades 2 to 4. Immunohistochemical analysis of a tissue array of 275 CRC samples showed a negative association between tumor stage and OVOL2 level. Overexpression of OVOL2 in SW620 cells decreased their migration and invasion, reduced markers of the epithelial-to-mesenchymal transition, and suppressed their metastasis as xenograft tumors in nude mice; knockdown of OVOL2 caused LS174T cells to transition from epithelial to mesenchymal phenotypes. OVOL2 bound T-cell factor (TCF)4 and β-catenin, facilitating recruitment of histone deacetylase 1 to the TCF4-β-catenin complex; this inhibited expression of epithelial-to-mesenchymal transition-related genes regulated by WNT, such as SLUG, in CRC cell lines. OVOL2 was a downstream target of WNT signaling in LS174T and SW480 cells. The OVOL2 promoter was hypermethylated in late-stage CRC specimens from patients and in SW620 cells; hypermethylation resulted in OVOL2 down-regulation and an inability to inhibit WNT signaling. Disruption of Ovol2 in Apc(min/+) mice increased WNT activity in intestinal tissues and the formation of invasive intestinal tumors. OVOL2 is a colorectal tumor suppressor that blocks WNT signaling by facilitating the recruitment of histone deacetylase 1 to the TCF4-β-catenin complex. Strategies to increase levels of OVOL2 might be developed to reduce colorectal tumor progression and metastasis.

High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression.

BackgroundElevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.MethodsTwelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.ResultsHigh basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.ConclusionsHigh basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.

Pigment Epithelium Derived Factor Suppresses Expression of Sost/Sclerostin by Osteocytes: Implication for Its Role in Bone Matrix Mineralization

Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. Mechanisms by which PEDF regulates matrix mineralization remain unknown. We examined effect of exogenous PEDF on expression of osteoblastic and osteocytic related genes and proteins in mineralizing osteoblast culture. Mineralizing human osteoblasts supplemented with exogenous PEDF for 14 days deposited 47% more mineral than cells cultured without PEDF. Analysis of selected gene expression by cells in mineralizing cultures supplemented with exogenous PEDF showed reduction in expression of Sclerostin (Sost) by 70%, matrix extracellular phosphoglycoprotein (MEPE) by 75% and dentin matrix protein (DMP-1) by 20% at day 14 of culture. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) expression was not affected. Western blotting and immunoprecipitation showed that sclerostin and MEPE synthesis by osteocytes were reduced by 50% and 60% respectively in mineralizing osteoblasts containing exogenous PEDF. Primary osteocytes exposed to PEDF also reduced synthesis of Sost/sclerostin by 50% within 24 h. For osteoblastic genes, Bone sialoprotein (BSP) was expressed at 75% higher by day 7 in cultures containing exogenous PEDF while Col1A1 expression remained high at all-time points. Total beta-catenin was increased in mineralizing osteoblastic cells suggesting increased Wnt activity. Taken together, the data indicate that PEDF suppressed expression of factors that inhibit mineralization while enhancing those that promote mineralization. The findings also suggest that PEDF may regulate Sost expression by osteocytes leading to enhanced osteoblastic differentiation and increased matrix mineralization.

Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists.

BackgroundThere is emerging evidence that Wnt pathway activity may increase during the progression from colorectal adenoma to carcinoma and that this increase is potentially an important step towards the invasive stage. Here, we investigated whether epigenetic silencing of Wnt antagonists is the biological driver for this increased Wnt activity in human tissues and how these methylation changes correlate with MSI (Microsatelite Instability) and CIMP (CpG Island Methylator Phenotype) statuses as well as known mutations in genes driving colorectal neoplasia.MethodsWe conducted a systematic analysis by pyrosequencing, to determine the promoter methylation of CpG islands associated with 17 Wnt signaling component genes. Methylation levels were correlated with MSI and CIMP statuses and known mutations within the APC, BRAF and KRAS genes in 264 matched samples representing the progression from normal to pre-invasive adenoma to colorectal carcinoma.ResultsWe discovered widespread hypermethylation of the Wnt antagonists SFRP1, SFRP2, SFRP5, DKK2, WIF1 and SOX17 in the transition from normal to adenoma with only the Wnt antagonists SFRP1, SFRP2, DKK2 and WIF1 showing further significant increase in methylation from adenoma to carcinoma. We show this to be accompanied by loss of expression of these Wnt antagonists, and by an increase in nuclear Wnt pathway activity. Mixed effects models revealed that mutations in APC, BRAF and KRAS occur at the transition from normal to adenoma stages whilst the hypermethylation of the Wnt antagonists continued to accumulate during the transitions from adenoma to carcinoma stages.ConclusionOur study provides strong evidence for a correlation between progressive hypermethylation and silencing of several Wnt antagonists with stepping-up in Wnt pathway activity beyond the APC loss associated tumour-initiating Wnt signalling levels.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-891) contains supplementary material, which is available to authorized users.

Abstract 1794: Cross-regulation of polycomb group protein BMI1 and WNT inhibitors.

Abstract Polycomb group (PcG) proteins are evolutionarily conserved gene silencers, which determine cell fate decisions during development. These proteins are often aberrantly expressed in cancer cells. In particular, BMI1 and EZH2 are known to be overexpressed in a number of human malignancies including breast and prostate cancers. In vitro models of cancer development strongly support oncogenic role of overexpressed BMI1 in cancer and metastasis. BMI1 is known to be required for self-renewal of neural, hematopoietic, intestinal and mammary stem cells. In addition, BMI1 is downregulated in senescent cells, and this downregulation may contribute to age-related pathologies. BMI1 is also suspected to play a role in cancer stem cell development and increase stem cell-ness of tumors to promote drug resistance. Despite its role in cancer, stem cell phenotype, development and aging, very little is known about the signaling pathways that regulate the expression of BMI1. Here we report that BMI1 activates WNT pathway and upregulates its target genes such as Cyclin D1 and c-Myc. Using promoter-reporter assays, quantitative RT-PCR and Western blot analyses, we show that BMI1 auto-activates its expression via an E-box present in its promoter. Because auto-activation of BMI1 depends on E-box sequences present in its promoter, we hypothesized that BMI1 may regulate expression of c-Myc directly or indirectly via activation of WNT pathway. Indeed, our data suggest that BMI1 upregulated expression of c-Myc via activation of WNT pathway. Further dissection of this regulation revealed that BMI1 represses expression of WNT inhibitors, in particular DKK family of proteins such as DKK1, DKK2 and DKK3. Interestingly, we also found that DKK1 inhibits expression of BMI1, which also depends on E-box present in BMI1 promoter. Thus BMI1 and WNT inhibitors such as DKK1 cross-regulate each other's expression. We speculate that such regulation might favor BMI1 overexpression in cancer cells via increased WNT activity and c-Myc expression. On the other hand, increased expression of WNT inhibitors such as DKK1 might favor BMI1 downregulation in senescent and aged tissues. Thus, a fine tuned positive feedback loop controls BMI1 expression via WNT signaling pathway, and this feedback loop may be relevant to the role of BMI1 in cancer, stem cells and senescence. Citation Format: Joon-Ho Cho, Manjari Dimri, Goberdhan P. Dimri. Cross-regulation of polycomb group protein BMI1 and WNT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1794. doi:10.1158/1538-7445.AM2013-1794

Receptors on Neurons Help Shape Worm Development

Animal development relies on signals that trigger or inhibit growth and differentiation in precise places and at precise times, so that a new organ, say, forms “right here” instead of “over there.” The outline of that signaling process has been known for some time, with diffusible molecules released from various sites establishing a set of gradients that form an ever-more specific map of the developing body, beginning with the anterior-posterior axis and leading to location of limbs and organs. In C. elegans, two mid-body neurons (CANL, CANR), whose axons span the anterior-posterior axis, use their posterior axons (green arrow) to help direct head to tail patterning by a Wnt that is secreted in the posterior body (orange arrow). A challenge for such a system is how to maintain spatial precision in the face of the increasing distances and physical changes of the growing form. In this issue of PLOS Biology, Katarzyna Modzelewska, Nadeem Moghal, and colleagues show that in the roundworm, neurons help meet that challenge and play a central role in specifying the location of one crucial organ, the vulva. In the worm, the vulva arises in the middle of the body from three progenitor cells, denoted P5.p, P6.p, and P7.p. Among the many genes that are involved with proper vulval development, the authors were intrigued by vab-8, about which little was known except that mutations in it led to aberrant placement of the vulva along the anterior-posterior axis, and aberrant migration and axon outgrowth of a few neurons. They found that lack of the VAB-8 protein caused a variety of vulval abnormalities, including an increased frequency of developing anterior vulval progenitors, an alteration in the symmetry of the normal mid-body vulva, and formation of vulval tissue from more posterior progenitors than normal. The vab-8 gene was expressed in a total of ten neurons, including the two “canal associated neurons,” or CANs, which stretch the length of the head-tail (or anterior-posterior) axis. Vab-8 gene mutation displaced the CAN cell bodies toward the head, and shortened the posterior CAN axons. When a functional vab-8 transgene was directed only to the CAN neurons in vab-8 mutants, it rescued the vulval phenotypes, confirming that these neurons, and not others in which vab-8 normally is expressed, play a central role in vulval development. Several crucial events in the development of the vulva are controlled by growth factors in the Wnt family, such as EGL-20 and CWN-1, released from cells toward the tail end. Using a marker for Wnt signaling, the authors found that, in vab-8 mutants, there was increased Wnt activity in multiple progenitor cells. This suggested that CANs normally inhibited Wnt signaling, and that the anterior displacement and shortened axon of the mutant neurons prevented this inhibition. Suspicion about the identity of that inhibitor fell on Ror/CAM-1, a membrane-bound tyrosine kinase that binds Wnts, including EGL-20 and CWN-1. Mutation of the cam-1 gene induced a vulval phenotype much like that of vab-8, but only when the mutation affected the Wnt-binding domain, and not when it affected the interior tyrosine kinase domain. This raised the possibility that the normal function of the receptor on CANs is to sequester Wnts to inhibit signaling. Labeling of EGL-20 and CAM-1 showed similar patterns of distribution along the CAN axon, with axonal colocalization of EGL-20 being dependent on CAM-1. Thus, EGL-20 is likely enriched at CAM-1 sites, strengthening the argument that the receptor sequesters EGL-20 to reduce the total Wnt signaling reaching the vulval progenitors. Taken together, these results paint a picture of worm development in which the two CANs play a critical role in positioning the vulva, through mopping up excess Wnt signals emanating from the posterior, reducing the local concentration sufficiently so that vulval progenitors arise and differentiate “here” and not “there.” A salient feature of this system is that it does not seem to involve signaling from the neuron itself; it is the neuron's position, not its behavior, that appears to be critical. It will now be interesting to see if the same phenomenon occurs not only elsewhere in the worm, but also elsewhere in the animal kingdom, and if so, whether neuronal signaling may also play a role in sculpting development. Modzelewska K, Lauritzen A, Hasenoeder S, Brown L, Georgiou J, et al. (2013) Neurons Refine the Caenorhabditis elegans Body Plan by Directing Axial Patterning by Wnts. doi:10.1371/journal.pbio.1001465

SSc—fibrosis takes flight with Wingless inhibition

Recent studies have highlighted a potentially important role for Wnts as profibrotic mediators, and implicated increased Wnt activity in systemic sclerosis and other fibrotic diseases. Strikingly, new data indicates that Wnts have a central role in the profibrotic activity of TGF-β.

Chemical and genetic evidence for the involvement of Wnt antagonist Dickkopf2 in regulation of glucose metabolism

Mutations in Wnt receptor LRP5/6 and polymorphism in Wnt-regulated transcription factor TCF7L2 are associated with dysregulation of glucose metabolism. However, it is not clear whether Wnt antagonist Dickkopf (Dkk) has a significant role in the regulation of glucose metabolism. Here, we identified small-molecule inhibitors of Wnt antagonist Dkk through molecular modeling, computation-based virtual screens, and biological assays. One of the Dkk inhibitors reduced basal blood-glucose concentrations and improved glucose tolerance in mice. This Dkk inhibitor appeared to act through DKK2 because the inhibitor exerted no additional effects on glucose metabolism in the Dkk2(-/-) mice. Our study of Dkk2(-/-) mice showed that DKK2 deficiency was associated with increased hepatic glycogen accumulation and decreased hepatic glucose output. DKK2 deficiency did not cause in increase in insulin production but resulted in increased Wnt activity and GLP1 production in the intestines. Given that the Dkk inhibitor improved glucose tolerance in a murine model of type 2 diabetes (db/db), we suggest that DKK2 may be a potential therapeutic target for treating type 2 diabetes.

Abstract 2249: Mutations to the GSK3β regulatory region of CTNNB1 increase TCF transcription activity and are resistant to degradation by GSK3β

Abstract Hepatocellular carcinoma (HCC) is currently the third most common cause of cancer related mortality worldwide. As with many cancers, aberrant activation of Wnt/β-catenin signaling plays a critical pathogenic role in the disease. Somatic mutations in genes of the Wnt pathway are widely observed in tumor tissue, where it has been estimated that 15-20% of tumors possess activating mutations in beta-catenin (CTNNB1). The turnover of CTNNB1 protein is tightly regulated in cells by the degradation complex which includes the CK1α and GSK3β kinases in a complex with Axin and APC. Mutations within the CTNNB1 gene at the CK1α phosphorylation site (Ser45) and GSK3β phosphorylation sites (Ser33, Ser37, Thr41) are observed clinically and result in CTNNB1 protein accumulation and enhanced transcription of Wnt target genes. The goal of this study is to characterize additional clinically observed CTNNB1 mutations in HCC. It is hypothesized that mutations within the phosphorylation domain in exon 3 will impact the phosphorylation of these key regulatory sites of CTNNB1. To assess the impact of three observed CTNNB1 mutations (H36P, I35S and S23R) on Wnt signaling activity, mutant CTNNB1 constructs were first created and their impact on Wnt signaling was measured using a luciferase-based Wnt reporter assay in transfected HEK293T cells. Compared to the wild type CTNNB1 control, Wnt transcription activity in the presence of H36P, I35S, and S23R mutants were increased by 6.55 ± 2.08, 6.76 ± 2.44, and 2.36 ± 1.01 fold, respectively. This compared to a 5.91 ± 1.38 fold increase observed with the phosphorylation mutant S45P. To evaluate if these mutations had a measurable impact on GSK3β regulation of CTNNB1 levels, each mutant was coexpressed with a GSK3β expression vector and evaluated using the same Wnt reporter system. As anticipated, GSK3β coexpression reduced Wnt reporter activity of the CTNNB1 control by 3.6 fold ± .33 where the S45P mutant was not affected. Coexpression of GSK3β did not attenuate increased Wnt activity observed with the mutants H36P and I35S, however it did decrease activity observed with the S23R by a similar magnitude of the wild type CTNNB1 control (2.27 fold ± .93). These results describe the activating nature of H36P, S45P, and I35S which likely mediate their effect via impaired CTNNB1 phosphorylation. To a lesser extent, S23R activated Wnt activity, however, is subject to attenuation by GSK3β phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2249. doi:1538-7445.AM2012-2249