Activation Of Wnt Signaling Research Articles

Invitro atlas of dorsal spinal interneurons reveals Wnt signaling as a critical regulator of progenitor expansion.

SUMMARYRestoring sensation after injury or disease requires a reproducible method for generating large quantities of bona fide somatosensory interneurons. Toward this goal, we assess the mechanisms by which dorsal spinal interneurons (dIs; dI1–dI6) can be derived from mouse embryonic stem cells (mESCs). Using two developmentally relevant growth factors, retinoic acid (RA) and bone morphogenetic protein (BMP) 4, we recapitulate the complete in vivo program of dI differentiation through a neuromesodermal intermediate. Transcriptional profiling reveals that mESC-derived dIs strikingly resemble endogenous dIs, with the correct molecular and functional signatures. We further demonstrate that RA specifies dI4–dI6 fates through a default multipotential state, while the addition of BMP4 induces dI1–dI3 fates and activates Wnt signaling to enhance progenitor proliferation. Constitutively activating Wnt signaling permits the dramatic expansion of neural progenitor cultures. These cultures retain the capacity to differentiate into diverse populations of dIs, thereby providing a method of increasing neuronal yield.

Metalloprotease ADAM9 cleaves ephrin-B ligands and differentially regulates Wnt and mTOR signaling downstream of Akt kinase in colorectal cancer cells

Ephrin-B signaling has been implicated in many normal and pathological processes, including neural crest development and tumor metastasis. We showed previously that proteolysis of ephrin-B ligands by the disintegrin metalloprotease ADAM13 is necessary for canonical Wnt signal activation and neural crest induction in Xenopus, but it was unclear if these mechanisms are conserved in mammals. Here, we report that mammalian ADAM9 cleaves ephrin-B1 and ephrin-B2 and can substitute for Xenopus ADAM13 to induce the neural crest. We found that ADAM9 expression is elevated in human colorectal cancer (CRC) tissues and that knockdown (KD) of ADAM9 inhibits the migration and invasion of SW620 and HCT116 CRC cells by reducing the activity of Akt kinase, which is antagonized by ephrin-Bs. Akt is a signaling node that activates multiple downstream pathways, including the Wnt and mTOR pathways, both of which can promote CRC cell migration/invasion. Surprisingly, we also found that KD of ADAM9 downregulates Wnt signaling but has negligible effects on mTOR signaling in SW620 cells; in contrast, mTOR activity is suppressed while Wnt signaling remains unaffected by ADAM9 KD in HCT116 cells. These results suggest that mammalian ADAM9 cleaves ephrin-Bs to derepress Akt and promote CRC migration and invasion; however, the signaling pathways downstream of Akt are differentially regulated by ADAM9 in different CRC cell lines, reflecting the heterogeneity of CRC cells in responding to manipulations of upstream Akt regulators.

Small Molecules Promote Selective Denaturation and Degradation of Tubulin Heterodimers through a Low-Barrier Hydrogen Bond.

Here, we report a novel mechanism to selectively degrade target proteins. 3-(3-Phenoxybenzyl)amino-β-carboline (PAC), a tubulin inhibitor, promotes selective degradation of αβ-tubulin heterodimers. Biochemical studies have revealed that PAC specifically denatures tubulin, making it prone to aggregation that predisposes it to ubiquitinylation and then degradation. The degradation is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and βGlu198, which is identified as a low-barrier hydrogen bond (LBHB). In contrast, another two tubulin inhibitors that only form normal hydrogen bonds with βGlu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. We then screened for compounds capable of forming an LBHB with βGlu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimer degradation through the LBHB. Our study provided a unique example of LBHB function and identified a novel approach to obtain tubulin degraders.

Upregulation of β-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis.

SummaryAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2–5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator β-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS.

Abstract 6246: WNT signaling-mediated DKC1 expression drives stemness associated with colorectal cancer

Abstract The emergence of subclonal mutations in high grade colorectal (CRC) tumors driving resistance against EGFR targeting is a major challenge, thus necessitating the discovery of alternative therapeutic strategies. Targeting aberrantly active WNT signaling provides an alternate route for CRC treatment. Here we show that the high DKC1 expression in CRC patients associates with advanced-stage disease regardless of RAS/RAF mutation status, indicating the potential of DKC1 as a pan-CRC therapeutic target. We show a direct regulation of DKC1 downstream of canonical WNT signaling and confirm it as one of the WNT targets. Mechanistically, DKC1 regulates several critical cellular processes such as telomere maintenance, pseudouridylation of ribosomal RNAs, ribosomal biogenesis, etc. However, its plausible association with cancer stemness remains largely unexplored. We generated stable DKC1 knockdown (DKC1-KD) cell lines, namely WiDr and Colo320HSR to investigate its role in cancer stemness and CRC progression. DKC1-KD cells show a significant reduction in the tumor sphere forming ability along with diminished cell surface expression of stemness markers. Using multiple publicly available gene expression datasets for CRC patients, we performed Gene Set Variation Analysis to characterize the association of DKC1 with upregulated gene signature of consensus molecular subtype 2 (CMS2) of CRC, which has been associated with the highest incidence rate and active WNT signaling. Further, Database for Annotation Visualization and Integrated Discovery tool-based analysis across these CRC patient samples striated based on DKC1 expression shows positive enrichment of biological processes related to cell cycle regulation. Consistently, we observed a G2/M phase cell cycle arrest upon silencing DKC1 in CRC cells. We also show that silencing DKC1 results in enhanced chemosensitivity with a remarkable reduction in lipid accumulation. Moreover, upon DKC1 silencing, an increase in epithelial marker E-cadherin and a decrease in mesenchymal marker N-cadherin expression was observed, suggesting its role in epithelial-to-mesenchymal transition (EMT) in CRC. Our findings demonstrate the role of DKC1 in regulating stemness, drug resistance, and EMT to drive CRC oncogenicity. Taken together, we highlight the role of DKC1 as the most suitable pan-CRC drug target by employing the potential of pharmacological inhibitors against WNT signaling. Citation Format: Shivansh Nigam, Ayush Praveen, Bushra Ateeq. WNT signaling-mediated DKC1 expression drives stemness associated with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6246.

Abstract 5843: Truncated APC & AXIN2 expression contribute to maintaining β-catenin/WNT signaling at the “Just Right” level for CRC cell proliferation

Abstract Adenomatous polyposis coli (APC) mutation leads to increased WNT signaling and stem cell overpopulation, which drives development of colorectal cancer (CRC). APC mutations often encode a truncated protein that has loss of APC’s domains that mediate APC interaction with AXIN2. A substantial body of scientific evidence indicates that truncated APC is essential for CRC cell proliferation by maintaining beta-catenin levels and WNT signaling at the “just right” level (known as the “Just Right” Hypothesis). Thus, AXIN2 is of particular interest because both wild-type APC and AXIN2 are part of the beta-catenin destruction complex, which is essential for tumor-suppressing ability. Hypothesis: the degree of AXIN2 expression, in the context of truncated APC, is critical to maintain WNT-signaling activity at the “just right” level which is optimal for CRC cell proliferation. Accordingly, we evaluated the effect of AXIN2 levels in CRC cells that contain APC protein-truncating mutations on WNT signaling (Top Flash assay), expression of WNT-targeted genes, and CRC cell proliferation and differentiation. We found that modulating AXIN2 expression altered WNT signaling, expression of WNT-targeted genes, and CRC cell proliferation in CRC cell lines with different APC-truncation causing mutations. Indeed, western blot results showed that knock down of AXIN2 gene expression increased truncated-APC and AXIN1 protein expression, and decreased expression of several WNT-target genes (e.g. LGR5, CD44, c-Jun, c-Myc, Cyclin D1), particularly genes that have reported roles in cell cycle regulation. These findings were consistent with our independent bioinformatics analyses of NCI TCGA RNA-seq data on CRC patients. Overall, our study suggests that i) The beta-catenin destruction complex is still functional at a certain level despite APC-truncating mutations; ii) Perturbing AXIN2 expression leads to changes in WNT-signaling activity that deviate from a level that is “just right” for malignant cell proliferation, likely by affecting the function of the destruction complex; iii) Deviation from “just right” WNT signaling levels alters WNT-target gene expression and diminishes the ability of APC-mutant CRC cells to maintain uncontrolled proliferation. Citation Format: Chi Zhang, Caroline O. Facey, Katherine Funk, Lynn M. Opdenaker, Bruce M. Boman. Truncated APC & AXIN2 expression contribute to maintaining β-catenin/WNT signaling at the “Just Right” level for CRC cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5843.

Abstract 3164: Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells

Abstract Multiple myeloma (MM) is the second most common hematological malignancy characterized by a malignant expansion of abnormal antibody-producing cells. Despite available treatments, relapsed disease is a common occurrence with patients receiving survival predictions of less than one year. Relapsed MM is often refractory to multiple lines of therapy and occurs due to the uncontrolled regeneration of a population of malignant cells displaying progenitor-cell-like phenotypes. Selectively targeting this cancer stem cell (CSC) population provides an effective way to limit incidence of relapsed disease, improving the prognosis for high-risk patients. In MM, the defining characteristics of CSCs remain poorly understood, though signaling pathways promoting ‘stemness’ that are often deregulated in CSCs, such as interleukin-6 (IL-6) signaling, as well as canonical and non-canonical Wnt signaling are also aberrantly activated in MM. In this context, the central objective of this project was to explore the functionality of two proteins which have been shown to be downstream targets of IL-6/JAK2/STAT3 signaling and potentiate Wnt signaling, the embryonic G-protein coupled receptor LGR4 and the fetal-oncogene tyrosine kinase like orphan receptor-1 (ROR1), as CSC biomarker candidates, and explore the feasibility of CSC targeting therapeutics for MM. Receptor expression was assessed by quantitative PCR and flow cytometry in primary plasma cell dyscrasia patient samples across the spectrum of disease, as well as in human myeloma cell lines (HMCLs). Whole transcriptome sequencing analyses were carried out in HMCLs induced to overexpress LGR4, which enriched genes associated with ion transport and embryonic development. Cell adhesion and extracellular matrix genes were downregulated, along with genes associated with metabolic and mitochondrial pathways. Results of ROR1 gene expression and flow cytometry studies suggest that a subset of MM patient samples harbor a low-frequency ROR1-positive cell population, and expression is enriched in an in vitro model of acquired drug resistance. Overexpression of interferon regulatory factor-4 (IRF4) - a myeloma cell-enriched transcription factor that we recently showed promotes myeloma regeneration - also contributed to increased human ROR1 protein levels. HMCLs with high endogenous ROR1 expression were sensitive to in vitro killing by anti-ROR1 CAR T-cell treatment, which employs an scFv generated from the fully humanized ROR1-specific monoclonal antibody cirmtuzumab, at effector:target cell ratios (ET) as low as 0.33:1. Together, these results suggest a central role for Wnt signaling in MM stem cell phenotypes and activation of embryonic transcriptional pathways, while also paving the way towards development of clinical therapies that target a CSC population in MM. Citation Format: Lara C. Avsharian, Christopher S. Oh, Ashni A. Vora, Jayde Fernandez, Caitlin Shin, Navyaa Sinha, Charles Prussak, Leslie Crews. Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3164.

A p300/GATA6 axis determines differentiation and Wnt dependency in pancreatic cancer models.

Wnt signaling regulates the balance between stemness and differentiation in multiple tissues and in cancer. RNF43-mutant pancreatic cancers are dependent on Wnt production, and pharmacologic blockade of the pathway, e.g., by PORCN inhibitors, leads to tumor differentiation. However, primary resistance to these inhibitors has been observed. To elucidate potential mechanisms, we performed in vivo CRISPR screens in PORCN inhibitor–sensitive RNF43-mutant pancreatic cancer xenografts. As expected, genes in the Wnt pathway whose loss conferred drug resistance were identified, including APC, AXIN1, and CTNNBIP1. Unexpectedly, the screen also identified the histone acetyltransferase EP300 (p300), but not its paralog, CREBBP (CBP). We found that EP300 is silenced due to genetic alterations in all the existing RNF43-mutant pancreatic cancer cell lines that are resistant to PORCN inhibitors. Mechanistically, loss of EP300 directly downregulated GATA6 expression, thereby silencing the GATA6-regulated differentiation program and leading to a phenotypic transition from the classical subtype to the dedifferentiated basal-like/squamous subtype of pancreatic cancer. EP300 mutation and loss of GATA6 function bypassed the antidifferentiation activity of Wnt signaling, rendering these cancer cells resistant to Wnt inhibition.

Abstract 776: Expression of SOX15 and Wnt pathway genes in papillary thyroid carcinoma

Abstract Although thyroid cancer (TC) is a rare cancer with a rate of 2.1%, it is the most common cancer type among endocrine tumors. The molecular mechanisms of thyroid cancer, which develops as a result of environmental and genetic factors, are still unclear. SOX family genes are transcription factors that are expressed in a tissue-specific manner and play a role in the developmental processes. SRY-box transcription factor 15 (SOX15) is an important member of the SOX family and is involved in carcinogenesis. Its downregulation has been associated with the development and progression of different human malignancies. Some SOX family members have been reported to control cell proliferation by acting as negative regulators of the Wnt/β-catenin signaling pathway. MYC, CCND1 and CTNNB1 genes are important components of the Wnt signaling pathway. Recently, we have shown that epigenetic silencing of the SOX15 gene is associated with the pathogenesis of papillary thyroid carcinoma,In the present study, to further the understanding of the molecular mechanisms of SOX15 in papillary thyroid carcinoma (PTC) we investigated SOX15 expression in association with the transcriptional targets of the Wnt/β-catenin pathway.Primary thyroid tumors and adjacent nonmalignant tissue samples were collected from 52 patients, prior to any treatment. Total RNA was isolated from tissue samples and reverse transcription was performed. Expression levels of the SOX15, CTNNB1, c-MYC and CCND1 genes were analyzed by qRT-PCR using the SYBR green and Light-Cycler 480 system (Roche Diagnostics, Germany). β2M was used as the reference to normalize the mRNA levels. Statistical analysis was performed using the SPSS 21.0 (IBM® SPSS® Statistics, IBM Corporation Somers, NY, USA) program.SOX15 gene expression was significantly downregulated in 64.6% of PTC tissues compared to their normal counterparts. In contrast, CCND1, CTNNB1 and c-MYC gene expressions were significantly upregulated in 70.2%, 60% and 57.8% of the patients. Among the group of patients with downregulated SOX15 expression CCND1, CTNNB1 and c-MYC expression were upregulated in 46.8%, 40% and 40% of the subjects, respectively. Our data suggest that SOX15 downregulation may contribute to activation of Wnt signaling and provides further evidence indicating involvement of SOX15 in modulating the Wnt/β-catenin pathway in thyroid carcinogenesis. Citation Format: Ayse Nur Buyru, Ayşegul Soysal, Ahmet Ozaydın, Betul Seyhan, Soykan Arıkan, Nejat Dalay. Expression of SOX15 and Wnt pathway genes in papillary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 776.

Discovery of GSK3β Inhibitors through In Silico Prediction-and-Experiment Cycling Strategy, and Biological Evaluation.

Direct inhibitors of glycogen synthase kinase 3β (GSK3β) have been investigated and reported for the past 20 years. In the search for novel scaffold inhibitors, 3000 compounds were selected through structure-based virtual screening (SBVS), and then high-throughput enzyme screening was performed. Among the active hit compounds, pyrazolo [1,5-a]pyrimidin-7-amine derivatives showed strong inhibitory potencies on the GSK3β enzyme and markedly activated Wnt signaling. The result of the molecular dynamics (MD) simulation, enhanced by the upper-wall restraint, was used as an advanced structural query for the SBVS. In this study, strong inhibitors designed to inhibit the GSK3β enzyme were discovered through SBVS. Our study provides structural insights into the binding mode of the inhibitors for further lead optimization.

Non-canonical WNT signalling in cardiovascular disease: mechanisms and therapeutic implications.

WNT signalling comprises a diverse spectrum of receptor-mediated pathways activated by a large family of WNT ligands and influencing fundamental biological processes. WNT signalling includes the β-catenin canonical pathway and the non-canonical pathways, namely the planar cell polarity and the calcium-dependent pathways. Advances over the past decade have linked non-canonical WNT signalling with key mechanisms of atherosclerosis, including oxidative stress, endothelial dysfunction, macrophage activation and vascular smooth muscle cell phenotype regulation. In addition, non-canonical WNT signalling is involved in crucial aspects of myocardial biology, from fibrosis to hypertrophy and oxidative stress. Importantly, non-canonical WNT signalling activation has complex effects in adipose tissue in the context of obesity, thereby potentially linking metabolic and vascular diseases. Tissue-specific targeting of non-canonical WNT signalling might be associated with substantial risks of off-target tumorigenesis, challenging its therapeutic potential. However, novel technologies, such as monoclonal antibodies, recombinant decoy receptors, tissue-specific gene silencing with small interfering RNAs and gene editing with CRISPR–Cas9, might enable more efficient therapeutic targeting of WNT signalling in the cardiovascular system. In this Review, we summarize the components of non-canonical WNT signalling, their links with the main mechanisms of atherosclerosis, heart failure and arrhythmias, and the rationale for targeting individual components of non-canonical WNT signalling for the treatment of cardiovascular disease.

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.

RARE-29. Transcriptome characterization of pediatric adamantinomatous craniopharyngioma at the cellular level

Abstract BACKGROUND: Adamantinomatous Craniopharyngioma (ACP) is a neurologically devastating brain tumor that affects children and adults. It is histologically heterogeneous with epithelial populations that are characterized by the nuclear accumulation of mutated β-catenin, and activated Wnt signaling. Current models suggest that ACP growth is driven through paracine mechanisms characterized by the senescence-associated secretory phenotype (SASP). However, detailed pathogenic mechanisms remain unknown. Improved definition of the various cellular phenotypes that compose ACP will inform and advance our understanding of this disease. METHODS: Single cell RNA-sequencing (scRNA-seq) and multiplex ELISA were performed on pediatric ACP tissue and cyst fluid, respectively. Reference scRNA-seq data was obtained from PanglaoDB. Preprocessing and standard analyses were conducted using Seurat software. Cellular phenotypes were annotated using the Human Primary Cell atlas. Differential expression and functional enrichment analyses were utilized to identify Wnt-signaling activation and epithelial subpopulations. Paracrine signaling was inferred via CellChatDB. SASP Atlas was utilized to query marker gene lists. Pseudotemporal ordering was performed using monocle3. RESULTS: ACP tissue is heterogenous and contains multiple distinct immune signatures. ACP tissue contains 2 unique epithelial subpopulations, which demonstrate canonical Wnt-signaling and SASP, respectively. Pseudotemporal ordering suggests the initial oncogenic event to be of epidermal character, with subsequent aggressive behavior from a separate epithelial cell population. CONCLUSIONS: Based on gene expression, cell populations that correspond to the histologically identifiable epithelial whorls and palisading epithelium can be identified. These subpopulations display unique functional signatures. Simultaneous and synergistic therapeutic targeting of these separate epithelial populations may lead to improved patient care.

MEDB-12. Severe developmental abnormalities and proliferative cerebellar lesions induced by combined activity of Wnt signalling and loss of SMARCA4

Almost all medulloblastomas (MB) of the WNT subgroup are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations as well as the tumor origin in the dorsal brain stem. Around 20 % of WNT MB additionally carry SMARCA4mutations, but the functional role of these alterations is mostly unknown. We therefore amended previously described Blbp-cre::Ctnnb1(ex3)Fl/+mice by the introduction of a floxed Smarca4 allele. In contrast to existing literature, Blbp-cre::Ctnnb1(ex3)Fl/+ mice had a maximum life span of only 17 days, even after breeding into two different genetic backgrounds (C57BL/6J and 129S2/Sv). The mice displayed a severe developmental phenotype including a thinned cerebral cortex, hydrocephalus, missing cerebellar foliation and layering as well as non-proliferative cell accumulations in brain stem and cerebellum. An additional homozygous loss of SMARCA4 even resulted in prenatal death for most mice and caused big proliferative lesions in the cerebellum at embryonal day 14.5. These lesions appear to originate from SOX2-positive progenitor cells in the cerebellar ventricular zone. In a next experiment, cells isolated from this region will be characterized in vitro and will be transplanted orthotopically to evaluate their neoplastic potential in vivo. Altogether, we hope to elucidate how a loss of SMARCA4 and mutations of Ctnnb1 cooperate during hindbrain development and tumor formation within this region.

MEDB-81. Combined inhibition of CDK11 and EZH2 results in regression of MYC-amplified medulloblastoma

We explored an shRNA library screen on 20 cyclin-dependent kinases to establish cyclin-dependent kinase 11 (CDK11) as a critical mediator in MYC-driven medulloblastoma. The effect and molecular mechanism of CDK11 in the proliferation and growth of medulloblastoma were investigated in vitro. Pharmaceutic inhibitors and genetic depletion of CDK11 resulted in cessation of tumor growth in xenograft mouse models. Through combination chemical screening, we identified that 5-FU enhanced the apoptosis which induced by inhibition of CDK11 in medulloblastoma cells. In addition, we found CDK11 is a significant candidate kinase participating in the negative control of Wnt/b-catenin signaling. Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes through activation of transmembrane Frizzled (FZD) receptors which is suppressed by H3K27Me3. RNASeq and cut&run revealed that Cdk11 and mediator associated Cdk8 kinase regulate a common set of genes. Lack of Cdk8 and Cdk11 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 tri-methylation. We concluded that combined EZH2 and CDK8/CDK11 inhibitors treatment concurrently activated Wnt signaling may be an effective treatment for Group 3 medulloblastoma.

Molecular correlates of Delta-like-ligand 3 (DLL3) expression in neuroendocrine neoplasms (NENs).

4127 Background: NENs can occur in many locations but have limited precision therapy options. DLL3 is a cell surface protein that is emerging as a promising therapeutic target in NENs including neuroendocrine prostate cancer (NEPC) and small cell lung cancer (SCLC). Our recent study indicated that ̃77% of NEPCs expressed DLL3, with expression in circulating tumor cells being highly concordant with matched biopsies. While there are ongoing clinical trials of drugs targeting DLL3, the repertoire of clinical and genomic features shared across other DLL3-expressing NEN cancers is ill-defined. Methods: We analyzed WES and WTS data from NENs identified across 29 different sites of origin using the Caris Life Sciences platform, excluding SCLC and including neuroendocrine carcinomas and neuroendocrine tumors. We used values above or below median DLL3 expression of all NEN samples to define DDL3-High/Low (H/L). Significance of molecular alterations in DLL3-H vs L was determined using Fisher’s-Exact/Mann Whitney/X2 test with Benjamini-Hochberg correction. Results: DLL3 expression across all 2672 NEN samples was observed in 27 of 29 NENs after excluding SCLC. NENs of anus, prostate, lung, bladder, and bile duct exhibited the greatest median DLL3 expression, whereas adrenal gland, small bowel, and nervous system displayed the lowest. Certain tissues of origin displayed more robust DLL3 expression, with 71% (50/66) of NEPC, 75% (87/122) of lung, and 77.3% (51/66) of bladder being DLL3-H compared to 14.4% (13/90) of adrenal and 7.9% (12/151) of small bowel NENs. DLL3-H NENs were associated with TMB-high status ( > 10 muts/Mb; 12.1% vs 4.5%, OR 2.7, q < 0.001) and more genomic alterations in several driver genes, including tumor suppressors TP53 (51% vs 23%, OR 2.3, q < 0.001) and RB1 (42% vs 10%, OR 4.2, q < 0.001), and oncogenes KRAS (14% vs 5.4%, OR 2.5, q < 0.001), MYC (5.7% vs 0.9%, OR 6.3, q < 0.001) and CCNE1 (5.3% vs 1.3%, OR 4.0, q = 0.001). Conversely, DLL3-L NENs exhibited more alterations in CTNNB1 (2.2% vs 5.2%, OR 0.42, q = 0.04), MEN1 (3.3% vs 11%, OR 0.30, q < 0.001), and BCOR (1.3% vs 4.1%, OR 0.32, q = 0.02). DLL3-H NENs also had significantly more alterations in PIK3CA (6.4% vs 3.0%, OR 2.1, q = 0.04), chromatin remodeling genes KMT2D (6.7% vs 2.6% OR 2.6, q = 0.005) and CREBBP (3.2% vs 0.9%, OR 3.6, q = 0.03), and WNT signaling gene APC (9.7% vs 5.2%, OR 1.9, q = 0.02). Conclusions: We confirmed DLL3 expression in NENs across different tissues of origin, with highest expression in poorly differentiated NENs. DLL3-H expression was associated with genomic features considered “undruggable” based on current precision therapy approaches. Therefore, DLL3-targeted therapies may serve as a promising strategy for NEN patients with functional loss of tumor suppressors TP53 and RB1, as well as increased activity of KRAS, WNT and MYC signaling.

Sodium Selenite Promotes Osteoblast Differentiation via The WNT/ß-Catenin Signaling Pathway.

ObjectivesOsteoporosis is regarded as a silent disorder affecting bone slowly, leading to an increased risk of fractures. Lately, selenium has been found to be associated with the acquisition and maintenance of bone health by affecting the bone remodeling process. However, the mechanism of action of selenium on bone is poorly understood. Here, the objective of this study is to examine the protective effects and mechanism of sodium selenite on the differentiation process of osteoblasts as well as under oxidative stress-induced conditions by evaluating the expression of osteoblast differentiation markers in the sodium selenite and/or hydrogen peroxide (H2O2)-treated MC3T3-E1 cell line.Materials and Methods In this experimental study, we confirmed the inducible osteogenic effect of sodium selenite on MC3T3-E1 cells. Moreover, we investigated the recovery of expression levels of osteogenic markers of sodium selenite in H2O2-treated MC3T3-E1 cells. Results It was observed that sodium selenite could promote alkaline phosphatase (ALP) activity and collagen synthesis in pre-osteoblasts. Also, sodium selenite enhanced the mRNA expression levels of osteogenic transcriptional factors, like osterix (OSX) and runt-related transcription factor 2 (Runx2). In addition, the terminal differentiation markers, such as osteocalcin (OCN) and collagen 1α (Col1α) were also increased after the treatment of sodium selenite. Also treatment of sodium selenite recused the H2O2-induced inhibition of osteoblastic differentiation of pre-osteoblasts cells via the WNT signaling pathway, implicating its antioxidant activity. Furthermore, sodium selenite restored the H2O2 repressed β-catenin stability and axin-2 reporter activity in MC3T3-E1 cells. ConclusionIt may be concluded that sodium selenite can stimulate bone formation and rescue the oxidative repression of osteogenesis by activating WNT signaling pathways. Further detailed studies on the role of selenium and its ability to stimulate bone formation via the WNT signaling pathway may project it as a potential therapeutic intervention for osteoporosis.

Cadmium impairs zebrafish swim bladder development via ROS mediated inhibition of the Wnt / Hedgehog pathway.

The posterior swim bladder is an important organ in teleost fishes, that primarily maintains buoyancy and motility for swimming and survival. In this study, we examined the molecular mechanisms of the toxicity of cadmium (Cd) on the early development of the swim bladder in zebrafish. Embryonic Cd exposure resulted in the non-inflation of the swim bladder when the ambient Cd concentration was greater than or equal to 0.25mg/L. Cd disturbed surfactant lipid distribution and inhibited the formation of all three tissue layers in the swim bladder. Additionally, excessive Cd down-regulated Wnt (fzd3, nkd1, fzd7 and axin2) and Hedgehog (ihh, shh, ptc1 and ptc2) signaling pathways. Conversely, Wnt signaling activation partially neutralized Cd-induced swim bladder developmental defects. Moreover, ROS scavenger reduced Glutathione (GSH) effectively recovered Cd induced defects in swim bladder and Wnt/Hedgehog signaling. Taken together, our results first revealed that Cd caused swim bladder developmental defects via ROS-mediated inhibition of the Wnt and Hedgehog pathways. These results herein provide important data for future toxicological studies and risk assessments of Cd.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in axolotl limb development and regeneration.

The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.