Upregulation Of Wnt Signaling Research Articles

Saroglitazar Enhances Memory Functions and Adult Neurogenesis via Up-Regulation of Wnt/β Catenin Signaling in the Rat Model of Dementia.

Peroxisome proliferator-activated receptors (PPARs) have emerged as a promising target for the treatment of various neurodegenerative disorders. Studies have shown that both PPAR α & γ individually modulate various pathophysiological events like neuroinflammation and insulin resistance, which are known to variedly affect neurogenesis. Our study aimed to evaluate the effect of saroglitazar (SGZR), a dual PPAR agonist, on adult neurogenesis and spatial learning and memory, in intracerebroventricular streptozotocin (ICV STZ)-induced dementia in rats. We have found that SGZR at the dose of 4 mg/kg per oral showed significant improvement in learning and memory compared to ICV STZ-treated rats. A substantial increase in neurogenesis was observed in the subventricular zone (SVZ) and the dentate gyrus (DG), as indicated by an increase in the number of 5-bromo-2'-deoxyuridine (BrdU)+ cells, BrdU+ nestin+ cells, and doublecortin (DCX)+cells. Treatment with SGZR also decreased the active form of glycogen synthase kinase 3β (GSK3β) and hence enhanced the nuclear translocation of the β-catenin. Enhanced expression of Wnt transcription factors and target genes indicates that the up-regulation of Wnt signaling might be involved in the observed increase in neurogenesis. Hence, it can be concluded that the SGZR enhances memory functions and adult neurogenesis via the upregulation of Wnt β-catenin signaling in ICV STZ-treated rats.

Role of U11/U12 minor spliceosome gene ZCRB1 in Ciliogenesis and WNT Signaling.

Despite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally, zcrb1 CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.

The concept of onychodermis containing onychofibroblasts has histological (microanatomical), immunohistochemical as well as molecular basis.

The terms "onychofibroblast" (nail-specific fibroblast) and onychodermis (nail-specific dermis) were first introduced in 2006 and 2012, respectively, based on distinctive histologic and immunohistochemical features from the dermis of the surrounding skin and have been demonstrated in multiple studies. Recently, based on molecular research, the definition of onychodermis containing onychofibroblasts has been expanded to encompass the area located between the nail matrix and bed epithelium and periosteum. Single-cell RNA sequencing and in situ hybridization demonstrated that onychofibroblasts within the onychodermis express the genes including RSPO4, MSX1, WIF-1, and BMP5, which are implicated in nail formation and/or in disorders with nail phenotype. A mutation in RSPO4, a component of the Wnt signaling pathway, causes anonychia congenita. Nail matrix onychodermis and nail bed onychodermis share many similar characteristics which differ from the surrounding normal dermis of the skin. Comparative spatial transcriptomic and single-cell analyses of human nail units and hair follicles suggest that onychodermis is the counterpart of follicular dermal papilla, which plays a key role in hair follicle growth and morphogenesis. Onychomatricoma, as a nail-specific tumor, has been demonstrated to be a mesenchymal tumor that originates from onychofibroblasts and is associated with the upregulation of Wnt signaling. Collectively, the onychodermis and onychofibroblasts play crucial roles in nail development and these specialized nail mesenchymal elements are key components in the pathogenesis of onychomatricoma. The concept of onychodermis containing onychofibroblasts is very important for nail biology and pathology.

Fluoxetine Rescues Excessive Myelin Formation and Psychological Behaviors in a Murine PTSD Model.

Posttraumatic stress disorder (PTSD) is a complex mental disorder notable for traumatic experience memory. Although current first-line treatments are linked with clinically important symptom reduction, a large proportion of patients retained to experience considerable residual symptoms, indicating pathogenic mechanism should be illustrated further. Recent studies reported that newly formed myelin could shape neural circuit function and be implicated in fear memory preservation. However, its role in PTSD remains to be elucidated. In this study, we adopted a restraint stress-induced PTSD mouse model and found that PTSD-related neuropsychiatric symptoms were accompanied by increased myelination in the posterior parietal cortex and hippocampus. Fluoxetine, but not risperidone or sertraline, has a more profound rescue effect on neuropsychological behaviors and myelin abnormalities. Further mechanistic experiments revealed that fluoxetine could directly interfere with oligodendroglial differentiation by upregulating Wnt signaling. Our data demonstrated the correlation between PTSD and abnormal myelination, suggesting that the oligodendroglial lineage could be a target for PTSD treatment.

Combinatorial interpretation of BMP and WNT controls the decision between primitive streak and extraembryonic fates

BMP signaling is essential for mammalian gastrulation, as it initiates a cascade of signals that control self-organized patterning. As development is highly dynamic, it is crucial to understand how time-dependent combinatorial signaling affects cellular differentiation. Here, we show that BMP signaling duration is a crucial control parameter that determines cell fates upon the exit from pluripotency through its interplay with the induced secondary signal WNT. BMP signaling directly converts cells from pluripotent to extraembryonic fates while simultaneously upregulating Wnt signaling, which promotes primitive streak and mesodermal specification. Using live-cell imaging of signaling and cell fate reporters together with a simple mathematical model, we show that this circuit produces a temporal morphogen effect where, once BMP signal duration is above a threshold for differentiation, intermediate and long pulses of BMP signaling produce specification of mesoderm and extraembryonic fates, respectively. Our results provide a systems-level picture of how these signaling pathways control the landscape of early human development.

Abstract 3807: CXCL9:SPP1 macrophage polarity as a prognostic biomarker for localized colorectal cancer after surgical resection

Abstract Background: There is an unmet need for additive predictive biomarkers for the recurrence of stage II/III colorectal cancer (CRC) after surgery. As recent studies showing the potential prognostic role of macrophage polarity defined by CXCL9 and SPP1, instead of conventional M1 and M2, we tried to evaluate the prognostic role of this biomarker in real-world practice setting, delineating biologic mechanisms. Method: The RNA sequencing data of 273 tumors derived from patietns with stage II/III colorectal cancer who underwent surgical resection in 5 Korean cancer-centers were available for analysis. We initially divided two subgroups based on the ratio of expression level of CXCL9 per SPP1 expressions (CS ratio) and evaluated prognostic outcomes and compared immune cell proportions by deconvolutional method between subgroups. Result: The CS ratio was higher in consensus molecular subtype (CMS)2 or CMS3 than others, and no significant differences were observed according to sidedness, stage, microsatellite instability status and medical centers. Total 69 (25,3%) patients were classified as low level of CS ratio group, and there were no significant differences in baseline characteristics, including age, sex, CMS, sidedness, stage, microsatellite instability status and medical centers. With a median follow-up of 58.2 months, the low CS ratio group showed poor 5-year recurrence-free survival (50.6 vs. 71.1%, HR=2.27, P = 0.002) and 5-year overall survival (69.7% vs. 85.9%, HR=1.75, P = 0.033). These trends were consistent across the subgroups. In the multivariate analyses including the potential prognostic factors (P < 0.1 in the univariate analyses), low CS ratio was significantly associated with poorer for RFS (adjusted HR = 1.96, P = 0.011) and OS (adjusted HR = 1.81, P = 0.002). These trends were consistent in the TCGA dataset (5 years OS: 69.7% vs. 85.9%, HR=1.98, P = 0.011). The low CS ratio group showed upregulated WNT signaling, and extracellular matrix organization pathway. Immune cell deconvolutional method exhibited the low CS ratio group showed more proportion of neutrophils, monocytes, whereas CD8+T cells, activated memory CD4+T cells were decreased, compared to high CS ratio group. Conclusions: These results suggest that CS ratio could be an applicable prognostic biomarker in locally advanced colorectal cancer after surgical resection. Based on this classification, more advanced treatment strategy may be required for low CS ratio group to improve surgical outcomes. Citation Format: Yeong Hak Bang, Ji Hye Choi, Hyunsu Kim, Kyunghee Park, Dae Hee Pyo, Yong Beom Cho, Woong-Yang Park. CXCL9:SPP1 macrophage polarity as a prognostic biomarker for localized colorectal cancer after surgical resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3807.

Pinoresinol diglucoside mitigates dexamethasone-induced osteoporosis and chondrodysplasia in zebrafish

BackgroundThe global increase in the aging population has led to a higher incidence of osteoporosis among the elderly. ObjectiveThis study aimed to evaluate the protective properties of pinoresinol diglucoside (PDG), an active constituent of Eucommia ulmoides, against dexamethasone-induced osteoporosis and chondrodysplasia. MethodsA zebrafish model of osteoporosis was established by exposing larval zebrafish to dexamethasone. The impact of PDG on bone mineralization was assessed through alizarin red and calcein staining. Alkaline phosphatase activity was quantified to evaluate osteoblast function. The influence of PDG on chondrogenesis was estimated using alcian blue staining. Fluorescence imaging and motor behavior analysis were employed to assess the protective effect of PDG on the structure and function of dexamethasone-induced skeletal teratogenesis. qPCR determined the expression of osteogenesis and Wnt signaling-related genes. Molecular docking was used to assess the potential interactions between PDG and Wnt receptors. ResultsPDG significantly increased bone mineralization and corrected spinal curvature and cartilage malformations in the zebrafish model. Furthermore, PDG enhanced swimming abilities compared to the model group. PDG mitigated dexamethasone-induced skeletal abnormalities in zebrafish by upregulating Wnt signaling, showing potential interaction with Wnt receptors FZD2 and FZD5. ConclusionPDG mitigates dexamethasone-induced osteoporosis and chondrodysplasia by promoting bone formation and activating Wnt signaling.

SMARCA4 Loss and Mutated β-Catenin Induce Proliferative Lesions in the Murine Embryonic Cerebellum.

Almost all medulloblastomas (MB) of the Wingless/Int-1 (WNT) type are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations. Additional alterations in SMARCA4 are detected in ∼20% of WNT MB, but their functional role is mostly unknown. We, therefore, amended previously described brain lipid binding protein (Blbp)-cre::Ctnnb1(ex3)fl/wt mice by the introduction of floxed Smarca4 alleles. Unexpectedly, mutated and thereby stabilized β-catenin on its own induced severe developmental phenotypes in male and female Blbp-cre::Ctnnb1(ex3)fl/wt mice in our hands, including a thinned cerebral cortex, hydrocephalus, missing cerebellar layering, and cell accumulations in the brainstem and cerebellum. An additional loss of SMARCA4 even resulted in prenatal death for most mice. Respective Blbp-cre::Ctnnb1(ex3)fl/wt::Smarca4fl/rec mutants (male and female) developed large proliferative lesions in the cerebellum evolving from E13.5 to E16.5. Histological and molecular analysis of these lesions by DNA methylation profiling and single-cell RNA sequencing suggested an origin in early undifferentiated SOX2-positive cerebellar progenitors. Furthermore, upregulated WNT signaling, altered actin/cytoskeleton organization, and reduced neuronal differentiation were evident in mutant cells. In vitro, cells harboring alterations in both Ctnnb1 and Smarca4 were negatively selected and did not show tumorigenic potential after transplantation in adult female recipient mice. However, in cerebellar explant cultures, mutant cells displayed significantly increased proliferation, suggesting an important role of the embryonic microenvironment in the development of lesions. Altogether, these results represent an important first step toward the unraveling of tumorigenic mechanisms induced by aberrant WNT signaling and SMARCA4 deficiency.

Ex vivo modeling of acquired drug resistance in BRAF - mutated pancreatic cancer organoids uncovers individual therapeutic vulnerabilities

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis due to late detection and limited treatment options. Some PDAC patients harbor alterations that qualify for targeted treatment strategies but develop acquired resistance, leading to treatment failure. We here report the ex vivo modeling of acquired drug resistance by creating a PDAC patient-derived tumor organoid (PDTO) model harboring a rare BRAF R506_K507ins VLR mutation resulting in a resistance to trametinib, a MEK inhibitor. Genomic and transcriptomic analyses revealed upregulated WNT signaling in resistant PDTO clones compared to treatment-naïve parental control cells. By combining genomic and transcriptomic analysis with a functional drug testing approach, we uncovered a de novo upregulation and circumventive reliance on WNT signaling in resistant PDTO clones. Ex vivo models such as PDTOs represent valuable tools for resistance modelling and offer the discovery of novel therapeutic approaches for patients in need where clinical diagnostic tools are currently at the limit.

Abstract B087: CTNNB1 mutations can mediate resistance to EGFR targeted therapies in Non-Small Cell Lung Cancer

Abstract Resistance to targeted therapy remains an ongoing challenge in lung cancer. β-Catenin (CTNNB1) mutations are observed in lung cancer, often co-occurring with known oncogenic alterations such as EGFR/KRAS/ALK. Oncogenic CTNNB1 mutations disrupt the β-catenin destruction complex upregulating Wnt signaling that may cause resistance to therapies targeting oncogenic drivers. An example of this was identified in post-mortem donated NSCLC tissue of a patient treated with a combination of carboplatin, pemetrexed, and bevacizumab that harbored both an acquired CTNNB1-T41A mutation and an EGFR exon 20 insertion. In addition, the CTNNB1 mutation spectrum was analyzed in lung cancer patients at Moffitt Cancer Center to identify prevalent CTNNB1 mutations for functional analyses. T41A, along with the most frequent Exon 3 CTNNB1 mutations were selected and transduced into HCC827 mutant cell lines to determine their ability to drive resistance to targeted therapies. Subsequently, western blotting and RNA sequencing were performed to assess differences between cells expressing wild-type and the most-resistant CTNNB1 mutant. A viability screen was also performed in which these cells were treated with a panel of drugs with known targets (COCKTAIL), in combination with EGFR TKI erlotinib, to identify new vulnerabilities of this CTNNB1 mutant erlotinib-resistant cells. Our data indicate that some CTNNB1 mutations, T41A, S37F, S45C and D32H, can cause resistance to EGFR inhibitor therapy, while others, D32Y, S33C, S33Y, S45P, and S37C, do not. Western blot analysis shows corresponding stabilization and higher accumulation of active β-catenin in cells expressing CTNNB1 mutants that are resistant to erlotinib. Of the mutations tested, cells expressing CTNNB1 T41A demonstrated the strongest resistance to erlotinib. Further analysis reveals higher levels of β-catenin regulated proteins, like axin2 and survivin, as well as EMT markers, such as vimentin and N-cadherin, in HCC827 CTNNB1 T41A cells when compared to the HCC827 CTNNB1 WT. Pathway analysis of genes differentially expressed between the WT and T41A mutant cells reveal WNT pathway to be the top upregulated pathway in the mutant cells. HCC827 T41A also showed sustained ERK, AKT and GSK3β signaling, and lower PARP cleavage compared to wild type, which may explain the resistance to EGFR TKI seen in these cells. COCKTAIL screening, comparing drug sensitivities of the WT and T41A mutant HCC827 cells to erlotinib in combination with various other targeted agents, identified PI3K, mTOR and MEK inhibitors that can reverse erlotinib resistance in the HCC827 CTNNB1 T41A cells. Our data suggests that CTNNB1 may be an important co-occurring alteration in developing resistance to targeted EGFR therapy. However, we show that different CTNNB1 mutations have differing abilities to drive resistance to EGFR TKI which correlates with the level of stabilization and activation of CTNNB1 and its downstream targets by these mutations. Citation Format: Anurima Majumder, Liznair Bridenstine, Benjamin Meyer, Fumi Kinose, J. Kevin Hicks, Theresa A Boyle, Eric B Haura. CTNNB1 mutations can mediate resistance to EGFR targeted therapies in Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B087.

Ketamine Promoted Breast Cancer Invasion and Metastasis Through Up-regulating Wnt, BMP, and EGFR Signaling.

In this study, we used an orthotropic breast cancer model combined with ketamine addiction and next-generation sequencing (NGS) to comprehensively investigate molecular alterations in ketamine-mediated metastasis. Ketamine is widely used in anesthesia and drug abuse. Our previous study revealed that ketamine promotes the growth of breast cancer cells; however, the detailed molecular mechanism remains unknown. An orthotropic breast cancer model was established by injecting EO771 breast cancer cells into the mammary fat pad of mice intraperitoneally administered ketamine (30 mg/kg, daily) for 68 days. Tumors collected at day 38 were frozen for future analysis, and their metastasis state was checked at day 68. Tumors were grouped and subjected to NGS analysis, followed by differential gene expression analysis (DEseq) and pathway identification. DEseq analysis showed that ketamine up-regulated metastasis-related signaling, and the key genes were BMP5, FZD6, MMP1B, EGFR, WNT5A, BMP7, and DCN. Ketamine addiction up-regulates the expression of genes involved in the Wnt, EGFR, and BMP signaling cascades, which may be associated with breast cancer progression and metastasis.

Region-specific Wnt signaling responses promote gastric polyp formation in patients with familial adenomatous polyposis.

Germline adenomatous polyposis coli (APC) mutation in patients with familial adenomatous polyposis (FAP) promotes gastrointestinal polyposis, including the formation of frequent gastric fundic gland polyps (FGPs). In this study, we investigated how dysregulated Wnt signaling promotes FGPs and why they localize to the corpus region of the stomach. We developed a biobank of FGP and surrounding nonpolyp corpus biopsies and organoids from patients with FAP for comparative studies. Polyp biopsies and polyp-derived organoids exhibited enhanced Wnt target gene expression. Polyp-derived organoids with intrinsically upregulated Wnt signaling showed poor tolerance to further induction, suggesting that high Wnt restricts growth. Targeted genomic sequencing revealed that most gastric polyps did not arise via APC loss of heterozygosity. Studies in genetic mouse models demonstrated that heterozygous Apc loss increased epithelial cell proliferation in the corpus but not the antrum, while homozygous Apc loss was not maintained in the corpus yet induced hyperproliferation in the antrum. Our findings suggest that heterozygous APC mutation in patients with FAP may be sufficient to drive polyp formation in the corpus region while subsequent loss of heterozygosity to further enhance Wnt signaling is not tolerated. This finding contextualizes the abundant yet benign nature of gastric polyps in FAP patient corpus compared with the rare, yet adenomatous polyps in the antrum.

Benzophenone induces cardiac developmental toxicity in zebrafish embryos by upregulating Wnt signaling

Benzophenone (BP) is found in many popular consumer products, such as cosmetics. BP potential toxicity to humans and aquatic organisms has emerged as an increased concern. In current study, we utilized a zebrafish model to assess BP-induced developmental cardiotoxicity. Following BP exposure, zebrafish embryos exhibited developmental toxicity, including increased mortality, reduced hatchability, delayed yolk sac absorption, and shortened body length. Besides, BP exposure induced cardiac defects in zebrafish embryos, comprising pericardial edema, reduced myocardial contractility and rhythm disturbances, and altered expression levels of cardiac developmental marker genes. Mechanistically, BP exposure disturbed the redox state and increased the level of apoptosis in zebrafish cardiomyocytes. Transcriptional expression levels of Wnt signaling genes, involving lef1, axin2, and β-catenin, were upregulated after BP treatment. Inhibition of Wnt signaling with IWR-1 could rescue the BP-induced cardiotoxicity in zebrafish. In summary, BP exposure causes cardiotoxicity via upregulation of the Wnt signaling pathway in zebrafish embryos.

Wnt signaling pathway and sclerostin in the development of atherosclerosis and vascular calcification.

Atherosclerosis is a complex process involving endothelial dysfunction, vascular inflammation, vascular smooth muscle cell (VSMC) proliferation, angiogenesis, and calcification. One of the pathomechanisms of atherosclerosis is the upregulation of Wnt signaling. This study aimed to summarize the current knowledge regarding the role of Wnt signaling and sclerostin in atherosclerosis, vascular calcification, aneurysms, and mortality based on the PubMed database. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendation and identified 160 papers that were included in this systematic review. The published data highlight that the upregulation of Wnt components facilitates the initiation and progression of atherosclerosis, arterial remodeling, VSMCs proliferation and phenotypic transition to the osteoblastic lineage in the arterial wall. This results in protein secretion, cell migration, calcification, fibrosis and aneurysm formation. The transformation of VSMCs into osteoblast-like cells that is observed in atherosclerosis results in sclerostin expression inhibiting the Wnt pathway. Furthermore, it was shown that sclerostin, expressed in atherosclerotic plaques, inhibits aneurysm formation in a mouse model. However, in humans, while the antisclerostin antibody romosozumab inhibits bone resorption, biochemical parameters of endothelial activation and inflammation are not affected, and the incidence of aneurysms is not increased. It was suggested that detecting sclerostin in the calcified aortic atherosclerotic plaques reflects a defense mechanism against Wnt activation and inhibition of atherosclerosis, although this has only been shown in animal models. Moreover, an increased number of vascular cells converted to osteogenic phenotypes results in increased plasma sclerostin concentrations. Therefore, plasma sclerostin derived from bone limits its importance as a global marker of vascular calcification.

Loss of endothelial glucocorticoid receptor accelerates organ fibrosis in db/db mice.

Endothelial cells play a key role in maintaining homeostasis and are deranged in many disease processes, including fibrotic conditions. Absence of the endothelial glucocorticoid receptor (GR) has been shown to accelerate diabetic kidney fibrosis in part through upregulation of Wnt signaling. The db/db mouse model is a model of spontaneous type 2 diabetes that has been noted to develop fibrosis in multiple organs over time, including the kidneys. This study aimed to determine the effect of loss of endothelial GR on organ fibrosis in the db/db model. db/db mice lacking endothelial GR showed more severe fibrosis in multiple organs compared with endothelial GR-replete db/db mice. Organ fibrosis could be substantially improved either through administration of a Wnt inhibitor or metformin. IL-6 is a key cytokine driving the fibrosis phenotype and is mechanistically linked to Wnt signaling. The db/db model is an important tool to study the mechanisms of fibrosis and its phenotype in the absence of endothelial GR highlights the synergistic effects of Wnt signaling and inflammation in the pathogenesis or organ fibrosis.NEW & NOTEWORTHY The major finding of this work is that endothelial glucocorticoid receptor-mediated upregulation of Wnt signaling and concurrent hyperinflammation work synergistically to exacerbate organ fibrosis in a genetic mouse model of diabetes. This study adds to our understanding of diabetic renal fibrosis and has important implications for the use of metformin in this condition.

Inhibition of Mertk Signaling Enhances Bone Healing after Tooth Extraction

Regeneration of alveolar bone is an essential step in restoring healthy function following tooth extraction. Growth of new bone in the healing extraction socket can be variable and often unpredictable when systemic comorbidities are present, leading to the need for additional therapeutic targets to accelerate the regenerative process. One such target is the TAM family (Tyro3, Axl, Mertk) of receptor tyrosine kinases. These proteins have been shown to help resolve inflammation and maintain bone homeostasis and thus may have therapeutic benefits in bone regeneration following extraction. Treatment of mice with a pan-TAM inhibitor (RXDX-106) led to accelerated alveolar bone fill following first molar extraction in a mouse model without changing immune infiltrate. Treatment of human alveolar bone mesenchymal stem cells with RXDX-106 upregulated Wnt signaling and primed the cells for osteogenic differentiation. Differentiation of human alveolar bone mesenchymal stem cells with osteogenic media and TAM-targeted inhibitor RXDX-106 (pan-TAM), ASP-2215 (Axl specific), or MRX-2843 (Mertk specific) showed enhanced mineralization with pan-TAM or Mertk-specific inhibitors and no change with Axl-specific inhibitor. First molar extractions in Mertk–/– mice had increased alveolar bone regeneration in the extraction socket relative to wild type controls 7 d postextraction. Flow cytometry of 7-d extraction sockets showed no difference in immune cell numbers between Mertk –/– and wild type mice. RNAseq of day 7 extraction sockets showed increased innate immune-related pathways and genes associated with bone differentiation in Mertk –/– mice. Together, these results indicate that TAM receptor signaling, specifically through Mertk, can be targeted to enhance bone regeneration after injury.

The association of Wnt-signalling and EMT markers with clinical characteristics in women with endometrial cancer.

Endometrial cancer is the most common gynecologic malignancy in the developed world. Risk stratification and treatment approaches are changing due to better understanding of tumor biology. Upregulated Wnt signaling plays an important role in cancer initiation and progression with promising potential for development of specific Wnt inhibitor therapy. One of the ways in which Wnt signaling contributes to progression of cancer, is by activating epithelial-to-mesenchymal transition (EMT) in tumor cells, causing the expression of mesenchymal markers, and enabling tumor cells to dissociate and migrate. This study analyzed the expression of Wnt signaling and EMT markers in endometrial cancer. Wnt signaling and EMT markers were significantly correlated with hormone receptors status in EC, but not with other clinico-pathological characteristics. Expression of Wnt antagonist, Dkk1 was significantly different between the ESGO-ESTRO-ESP patient risk assessment categories using integrated molecular risk assessment.

EVADR ceRNA transcript variants upregulate WNT and PI3K signaling pathways in SW480 and HCT116 cells by sponging miR-7 and miR-29b.

Long noncoding RNAs are cancer regulators and EVADR-lncRNA is highly upregulated in colorectal cancer (CRC). Accordingly, we aimed to functionally characterize the EVADR in CRC-originated cells. Firstly, during the amplification of EVADR full-length cDNA (named EVADR-v1), a novel/shorter variant (EVADR-v2) was discovered. Then, RT-qPCR analysis confirmed that EVADR is upregulated in tumors, consistent with RNA-seq analysis. Interestingly, bioinformatics analysis and dual-luciferase assay verified that EVADR sponges miR-7 and miR-29b. When both EVADR-v1/-v2 variants were overexpressed in SW480/HCT116 cells, miR-7 and miR-29b target genes (involved in the WNT/PI3K signaling) were upregulated. Furthermore, EVADR-v1/-v2 overexpression resulted in elevated PI3K activity (verified by western blotting and RT-qPCR) and upregulation of WNT signaling (confirmed by western blotting, TopFlash assay, and RT-qPCR). Consistently, overexpression of EVADR-v1/-v2 variants was followed by increased cell cycle progression, viability and migration aswell as reduced early/late apoptotic rate, and Bax/Bcl2 ratio of the CRC cells, detected by the cell cycle analysis, MTT,wound-healing, Annexin-V/PI, and RT-qPCR methods, respectively. Overall, we introduced two oncogenic transcript variants for EVADR that by sponging miR-7/miR-29b, upregulate WNT and PI3K signaling. Given the crucial role of these pathways in CRC, EVADR may present potential therapy use.

Proteogenomic insights into the biology and treatment of pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor prognosis. Proteogenomic characterization and integrative proteomic analysis provide a functional context to annotate genomic abnormalities with prognostic value.MethodsWe performed an integrated multi-omics analysis, including whole-exome sequencing, RNA-seq, proteomic, and phosphoproteomic analysis of 217 PDAC tumors with paired non-tumor adjacent tissues. In vivo functional experiments were performed to further illustrate the biological events related to PDAC tumorigenesis and progression.ResultsA comprehensive proteogenomic landscape revealed that TP53 mutations upregulated the CDK4-mediated cell proliferation process and led to poor prognosis in younger patients. Integrative multi-omics analysis illustrated the proteomic and phosphoproteomic alteration led by genomic alterations such as KRAS mutations and ADAM9 amplification of PDAC tumorigenesis. Proteogenomic analysis combined with in vivo experiments revealed that the higher amplification frequency of ADAM9 (8p11.22) could drive PDAC metastasis, though downregulating adhesion junction and upregulating WNT signaling pathway. Proteome-based stratification of PDAC revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Immune clustering defined a metabolic tumor subset that harbored FH amplicons led to better prognosis. Functional experiments revealed the role of FH in altering tumor glycolysis and in impacting PDAC tumor microenvironments. Experiments utilizing both in vivo and in vitro assay proved that loss of HOGA1 promoted the tumor growth via activating LARP7-CDK1 pathway.ConclusionsThis proteogenomic dataset provided a valuable resource for researchers and clinicians seeking for better understanding and treatment of PDAC.

Retinoic acid effects on in vitro palatal fusion and WNT signaling.

Retinoic acid is the main active vitamin A derivate and a key regulator of embryonic development. Excess of retinoic acid can disturb palate development in mice leading to cleft palate. WNT signaling is one of the main pathways in palate development. We evaluated the effects of retinoic acid on palate fusion and WNT signaling in in vitro explant cultures. Unfused palates from E13.5 mouse embryos were cultured for 4 days with 0.5μM, 2μM or without retinoic acid. Apoptosis, proliferation, WNT signaling and bone formation were analyzed by histology and quantitative PCR. Retinoic acid treatment with 0.5 and 2.0μM reduced palate fusion from 84% (SD 6.8%) in the controls to 56% (SD 26%) and 16% (SD 19%), respectively. Additionally, 2μM retinoic acid treatment increased Axin2 expression. Retinoic acid also increased the proliferation marker Pcna as well as the number of Ki-67-positive cells in the palate epithelium. At the same time, the WNT inhibitors Dkk1, Dkk3, Wif1 and Sfrp1 were downregulated at least two-fold. Retinoic acid also down-regulated Alpl and Col1a2 gene expression. Alkaline phosphatase (ALP) activity was notably reduced in the osteogenic areas of the retinoic acid- treated palates. Our data suggest that retinoic acid impairs palate fusion and bone formation by upregulation of WNT signaling.