Wnt Signaling Cascade Research Articles

Therapeutic Potential of Traditional Chinese Medicine Against Osteoarthritis: Targeting the Wnt Signaling Pathway.

Osteoarthritis (OA) is a chronic degenerative articular disease that leads to physical disability and reduced quality of life. The key pathological events in OA are cartilage degradation and synovial inflammation. Conventional therapies often lead to adverse effects that some patients are unwilling to endure. Traditional Chinese Medicines (TCMs) have long been known for their efficacy in treating OA with minimal side effects. The wingless-type (Wnt) signaling pathway is believed to play a role in OA progression, but there is still a lack of comprehensive understanding on how TCM may treat OA via the Wnt signaling pathway. This study aims to fill this gap by reviewing relevant research on the association between the Wnt signaling pathway and cartilage degradation and synovial inflammation in OA. Meanwhile, we also summarized and categorized TCMs and their active components, such as alkaloids, polysaccharides, flavonoids, sesquiterpene lactones, etc., which have shown varying efficacy in treating OA through modulation of the Wnt/[Formula: see text]-catenin signaling pathway. This work underscores the pivotal role of the Wnt signaling pathway in OA pathogenesis and progression, suggesting that targeting this pathway holds promise as a prospective therapeutic strategy for OA management in the future. TCMs and their active components have the potential to alleviate OA by modulating the Wnt signaling cascade. Harnessing TCMs and their active components to regulate the Wnt signaling pathway presents an encouraging avenue for delivering substantial therapeutic benefits to individuals with OA.

Ulk1 regulates T Cell Development and Response to Listeria monocytogenes Stimulation.

T cells are crucial for the normal functioning of the immune system. The development and response of these cells to foreign antigens involve many complex stages and interactions between various types of cells. However, many details of these processes are still unclear. Our research revealed a key role for a protein called ULK1, a serine/threonine protein kinase, in regulating T-cell development and function. During T-cell maturation, the absence of Ulk1 (as in Ulk1-/- mice) leads to an increase in a cell type called DN3 in the thymus. We also found a reduction in the number of T cells in peripheral immune organs, such as the spleen, in Ulk1-/- mice. In response to Listeria infection, Ulk1-/- mice have a weaker ability to clear this bacterium, and their T cells also have defects in producing cytokines. However, the absence of Ulk1 did not affect the activation or apoptosis of naïve CD4+ T cells in vitro. In a bone marrow chimeric mouse model, T cells from Ulk1-/- mice did not differ developmentally from those from control mice. Furthermore, RNA-seq revealed that Ulk1 deficiency affects the metabolic function of splenocytes and T-cell function in mice, potentially through the canonical Wnt signaling cascade and the ERK1/ERK2 signaling cascades. Overall, these results suggest that Ulk1 is essential for T-cell maturation in the thymus, the balance of peripheral T cells, and the functional response of T cells to antigens.

STAU1-mediated CNBP mRNA degradation by LINC00665 alters stem cell characteristics in ovarian cancer

BackgroundTo investigate the role of lncRNA LINC00665 in modulating ovarian cancer stemness and its influence on treatment resistance and cancer development.MethodsWe isolated ovarian cancer stem cells (OCSCs) from the COC1 cell line using a combination of chemotherapeutic agents and growth factors, and verified their stemness through western blotting and immunofluorescence for stem cell markers. Employing bioinformatics, we identified lncRNAs associated with ovarian cancer, with a focus on LINC00665 and its interaction with the CNBP mRNA. In situ hybridization, immunohistochemistry, and qPCR were utilized to examine their expression and localization, alongside functional assays to determine the effects of LINC00665 on CNBP.ResultsLINC00665 employs its Alu elements to interact with the 3’-UTR of CNBP mRNA, targeting it for degradation. This molecular crosstalk enhances stemness by promoting the STAU1-mediated decay of CNBP mRNA, thereby modulating the Wnt and Notch signaling cascades that are pivotal for maintaining CSC characteristics and driving tumor progression. These mechanistic insights were corroborated by a series of in vitro assays and validated in vivo using tumor xenograft models. Furthermore, we established a positive correlation between elevated CNBP levels and increased disease-free survival in patients with ovarian cancer, underscoring the prognostic value of CNBP in this context.ConclusionslncRNA LINC00665 enhances stemness in ovarian cancer by mediating the degradation of CNBP mRNA, thereby identifying LINC00665 as a potential therapeutic target to counteract drug resistance and tumor recurrence associated with CSCs.

Wnt-Ror-Dvl signalling and the dystrophin complex organize planar-polarized membrane compartments in C. elegans muscles

Cell polarity mechanisms allow the formation of specialized membrane domains with unique protein compositions, signalling properties, and functional characteristics. By analyzing the localization of potassium channels and proteins belonging to the dystrophin-associated protein complex, we reveal the existence of distinct planar-polarized membrane compartments at the surface of C. elegans muscle cells. We find that muscle polarity is controlled by a non-canonical Wnt signalling cascade involving the ligand EGL-20/Wnt, the receptor CAM-1/Ror, and the intracellular effector DSH-1/Dishevelled. Interestingly, classical planar cell polarity proteins are not required for this process. Using time-resolved protein degradation, we demonstrate that –while it is essentially in place by the end of embryogenesis– muscle polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the unsuspected complexity of the C. elegans muscle membrane and establish a genetically tractable model system to study cellular polarity and membrane compartmentalization in vivo.

WNT Signaling Cascade Proteins and LRP6 in the Formation of Various Types of Coronary Lesions in Patients With Coronary Artery Disease.

Assessment of WNT1, WNT3a, and LRP6 concentrations in patients with ischemic heart disease (IHD) and obstructive and non-obstructive coronary artery (CA) disease. This cross-sectional observational study included 50 IHD patients (verified by coronary angiography, CAG), of which 25 (50%) were men, mean age 64.9±8.1 years; 20 patients had non-obstructive CA disease (stenosis <50%), and 30 patients had hemodynamically significant stenosis. Concentrations of WNT1, WNT3a and LRP6 were measured in all patients. The concentrations of WNT1 and WNT3a proteins were significantly higher in patients with IHD and obstructive CA disease (p < 0.001), while the concentration of LRP6 was higher in the group with non-obstructive CA disease (p = 0.016). Data analysis of the group with obstructive CA disease showed a moderate correlation between WNT1 and LRP6 (ρ=0.374; p=0.042). Correlation analysis of all groups of patients with CA disease revealed a moderate association between the concentrations of WNT1 and uric acid (ρ=0.416; p=0.007). Regression analysis showed that risk factors for the development of IHD, such as increased body mass index, age, smoking, dyslipidemia, and hypertension, did not significantly influence the type of CA disease in IHD patients. According to ROC analysis, the obstructive form of IHD was predicted by a WNT3a concentration higher than 0.155 ng/ml and a LRP6 concentration lower than 12.94 ng/ml. IHD patients with non-obstructive CA disease had the greatest increase in LRP6, while patients with obstructive CA disease had significantly higher concentrations of the canonical WNT cascade proteins, WNT1 and WNT3a. According to the ROC analysis, a WNT3a concentration >0.155 ng/ml can serve as a predictor for the presence of hemodynamically significant CA stenosis in IHD patients (sensitivity 96.7%; specificity 70%), whereas a LRP6 concentration >12.94 ng/ml can predict the development of non-obstructive CA disease (sensitivity 76.7%; specificity 65%).

Sizzled (Frzb3) physically interacts with noncanonical Wnt ligands to inhibit gastrulation cell movement

The coordinated movement of germ layer progenitor cells reaches its peak at the dorsal side, where the Bmp signaling gradient is low, and minimum at the ventral side, where the Bmp gradient is high. This dynamic cell movement is regulated by the interplay of various signaling pathways. The non-canonical Wnt signaling cascade serves as a pivotal regulator of convergent and extension cellular movement, facilitated by the activation of small GTPases such as Rho, Rab, and Rac. However, the underlying cause of limited cell movement at the ventral side remains elusive. To explore the functional role of a key regulator in constraining gastrulation cell movement at the ventral side, we investigated the Bmp4-direct target gene, sizzled, to assess its potential role in inhibiting non-canonical Wnt signaling. In our current study, we demonstrated that ectopic expression of sizzled led to gastrulation defects in a dose-dependent manner, without altering cell fate specification. Overexpression of sizzled resulted in decreased elongation of Activin-treated animal cap and Keller explants. Furthermore, our immunoprecipitation assay unveiled the physical interaction of Sizzled with non-canonical Wnt ligand proteins (Wnt5 and Wnt11). Additionally, the activation of small GTPases involved in Wnt signaling mediation (RhoA and Rac1) was diminished upon sizzled overexpression. In summary, our findings suggest that Bmp4 signaling negatively modulates cell movement from the ventral side of the embryo by inducing sizzled expression during early Xenopus gastrulation.

Frizzled receptors (FZDs) in Wnt signaling: potential therapeutic targets for human cancers.

Frizzled receptors (FZDs) are key contributors intrinsic to the Wnt signaling pathway, activation of FZDs triggering the Wnt signaling cascade is frequently observed in human tumors and intimately associated with an aggressive carcinoma phenotype. It has been shown that the abnormal expression of FZD receptors contributes to the manifestation of malignant characteristics in human tumors such as enhanced cell proliferation, metastasis, chemotherapy resistance as well as the acquisition of cancer stemness. Given the essential roles of FZD receptors in the Wnt signaling in human tumors, this review aims to consolidate the prevailing knowledge on the specific status of FZD receptors (FZD1-10) and elucidate their respective functions in tumor progression. Furthermore, we delineate the structural basis for binding of FZD and its co-receptors to Wnt, and provide a better theoretical foundation for subsequent studies on related mechanisms. Finally, we describe the existing biological classes of small molecule-based FZD inhibitors in detail in the hope that they can provide useful assistance for design and development of novel drug candidates targeted FZDs.

Analysing the Anticancer Properties of Pterostilbene Through Absorption, Distribution, Metabolism, and Excretion (ADME) and Molecular Docking Studies.

Aim The aim of this study is to examine the possible therapeutic effect of pterostilbene (PTS), a chemical present in grapes and blueberries, in the treatment of liver cancer by analysing its interactions with important proteins linked to the wingless/integrated (Wnt) signaling system. Objective Using computational techniques like molecular docking and absorption, distribution, metabolism, and excretion (ADME) studies, this research focuses on examining the pharmacokinetics and molecular interactions of PTS with proteins such as vimentin (Vim), glycogen synthase kinase 3 beta (GSK3-β), epithelial cadherin (E-cadherin), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), c-Jun N-terminal kinase (JNK), and Wnt, all of which are connected to the Wnt signaling pathway in liver cancer. Methods The study includes the synthesis of proteins and ligands, ADME investigations for PTS, and AutoDock Vina molecular docking simulations to evaluate binding affinities and interactions. PTS is obtained from PubChem, while protein structures are obtained from the Protein Data Bank. Results Strong binding affinities between PTS and essential proteins in the Wnt signaling cascade are shown by molecular docking, which also highlights noteworthy hydrogen bonds, hydrophobic interactions, and electrostatic contacts. According to an ADME study, PTS has advantageous pharmacokinetic properties, such as moderate solubility, membrane permeability, and a minimal chance of drug interactions. Conclusion The extensive study highlights PTS's potential as a viable treatment option for liver cancer. The study promotes its investigation in cutting-edge liver cancer therapy approaches and urges more investigation into the molecular mechanisms, underpinning its anticancer properties. This paper sheds important light on the role of natural chemicals in cancer therapy and emphasizes the need for computational methods in drug discovery.

Pectolinarigenin ameliorates osteoporosis via enhancing Wnt signaling cascade in PPARβ-dependent manner

BackgroundOsteoporosis is a prevalent metabolic bone disease in older adults. Peroxisome proliferator-activated receptor β (PPARβ), the most abundant PPAR isotype expressed in bone tissues, plays a critical role in regulating the energy metabolism of osteoblasts. However, the botanical compounds targeting PPARβ for the treatment of osteoporosis remain largely unexplored. PurposeTo discover a potent PPARβ agonist from botanical compounds, as well as to investigate the anti-osteoporosis effects and to elucidate the underlying mechanisms of the newly identified PPARβ agonist. MethodsThe PPARβ agonist effects of botanical compounds were screened by an in vitro luciferase reporter gene assay. The PPARβ agonist effects of pectolinarigenin (PEC) in bone marrow mesenchymal stromal cells (BMSCs) were validated by Western blotting. RNA-seq transcriptome analyses were conducted to reveal the underlying osteoporosis mechanisms of PEC in BMSCs. The PPARβ antagonist (GSK0660) and Wnt signaling inhibitor (XAV969) were used to explore the role of the PPARβ and Wnt signaling cascade in the anti-osteoporosis effects of PEC. PEC or the PEG-PLGA nanoparticles of PEC (PEC–NP) were intraperitoneally administrated in both wild-type mice and ovariectomy-induced osteoporosis mice to examine its anti-osteoporotic effects in vivo. ResultsPEC, a newly identified naturally occurring PPARβ agonist, significantly promotes osteogenic differentiation and up-regulates the osteogenic differentiation-related genes (Runx2, Osterix, and Bmp2) in BMSCs. RNA sequencing and functional gene enrichment analysis suggested that PEC could activate osteogenic-related signaling pathways, including Wnt and PPAR signaling pathways. Further investigations suggested that PEC could enhance Wnt/β-catenin signaling in a PPARβ-dependent manner in BMSCs. Animal tests showed that PEC–NP promoted bone mass and density, increased the bone cell matrix protein, and accelerated bone formation in wild-type mice, while PEC–NP also played a preventive role in ovariectomy-induced osteoporosis mice via maintaining the expression level of bone cell matrix protein, balancing the rate of bone formation, and slowing down bone loss. Additionally, PEC–NP did not cause any organ injury and body weight loss after long-term use (11 weeks). ConclusionPEC significantly promotes bone formation and reduces bone loss in both BMSCs and ovariectomy-induced osteoporosis mice via enhancing the Wnt signaling cascade in a PPARβ-dependent manner, providing a new alternative therapy for preventing estrogen deficiency-induced osteoporotic diseases.

Molecular signaling directing neural plate border formation.

During embryonic development, the vertebrate embryonic epiblast is divided into two parts including neural and superficial ectoderm. The neural plate border (NPB) is a narrow transitional area which locates between these parts and contains multipotent progenitor cells. Despite its small size, the cellular heterogeneity in this region produces specific differentiated cells. Signaling pathways, transcription factors, and the expression/repression of certain genes are directly involved in these differentiation processes. Different factors such as the Wnt signaling cascade, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) signaling, and Notch, which are involved in various stages of the growth, proliferation, and differentiation of embryonic cells, are also involved in the determination and differentiation of neural plate border stem cells. Therefore, it is essential to consider the interactions and temporospatial coordination related to cells, tissues, and adjacent structures. This review examines our present knowledge of the formation of the neural plate border and emphasizes the requirement for interaction between different signaling pathways, including the BMP and Wnt cascades, the expression of its special target genes and their regulations, and the precise tissue crosstalk which defines the neural crest fate in the ectoderm at the early human embryonic stages.

Gestational Fisetin Exerts Neuroprotection by Regulating Mitochondria-Directed Canonical Wnt Signaling, BBB Integrity, and Apoptosis in Prenatal VPA-Induced Rodent Model of Autism.

Embryonic valproic acid (VPA) has been considered a potential risk factor for autism. Majority of studies indicated that targeting autism-associated alterations in VPA-induced autistic model could be promising in defining and designing therapeutics for autism. Numerous investigations in this field investigated the role of canonical Wnt signaling cascade in regulating the pathophysiology of autism. The impaired blood-brain barrier (BBB) permeability and mitochondrial dysfunction are some key implied features of the autistic brain. So, the current study was conducted to target canonical Wnt signaling pathway with a natural polyphenolic modulator cum antioxidant namely fisetin. A single dose of intraperitoneal VPA sodium salt (400 mg/kg) at gestational day 12.5 induced developmental delays, social behaviour impairments (tube dominance test), and anxiety-like behaviour (sucrose preference test) similar to autism. VPA induced mitochondrial damage and over-activated the canonical Wnt signaling which further increased the blood-brain barrier (BBB) disruption, apoptosis, and neuronal damage. Our findings revealed that oral administration of 10 mg/kg gestational fisetin (GD 13-till parturition) improved social and anxiety-like behaviour by modulating the ROS-regulated mitochondrial-canonical Wnt signaling. Moreover, fisetin controls BBB permeability, apoptosis, and neuronal damage in autism model proving its neuroprotective efficacy. Collectively, our findings revealed that fisetin-evoked modulation of the Wnt signaling cascade successfully relieved the associated symptoms of autism along with developmental delays in the model and indicates its potential as a bioceutical against autism.

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.

Wnt10b protects cardiomyocytes against doxorubicin-induced cell death via MAPK modulation.

Doxorubicin, an anthracycline chemotherapeutic known to incur heart damage, decreases heart function in up to 11% of patients. Recent investigations have implicated the Wnt signaling cascade as a key modulator of cardiac tissue repair after myocardial infarction. Wnt upregulation in murine models resulted in stimulation of angiogenesis and suppression of fibrosis after ischemic insult. However, the molecular mechanisms of Wnt in mitigating doxorubicin-induced cardiac insult require further investigation. Identifying cardioprotective mechanisms of Wnt is imperative to reducing debilitating cardiovascular adverse events in oncologic patients undergoing treatment. Exposing human cardiomyocyte AC16 cells to varying concentrations of Wnt10b and DOX, we observed key metrics of cell viability. To assess the viability and apoptotic rates, we utilized MTT and TUNEL assays. We quantified cell and mitochondrial membrane stability via LDH release and JC-1 staining. To investigate how Wnt10b mitigates doxorubicin-induced apoptosis, we introduced pharmacologic inhibitors of key enzymes involved in apoptosis: FR180204 and SB203580, ERK1/2 and p38 inhibitors. Further, we quantified apoptotic executor enzymes, caspase 3/7, via immunofluorescence. AC16 cells exposed solely to doxorubicin were shrunken with distorted morphology. Cardioprotective effects of Wnt10b were demonstrated via a reduction in apoptosis, from 70.1% to 50.1%. LDH release was also reduced between doxorubicin and combination groups from 2.27-fold to 1.56-fold relative to the healthy AC16 control group. Mitochondrial membrane stability was increased from 0.67-fold in the doxorubicin group to 5.73 in co-treated groups relative to control. Apoptotic protein expression was stifled by Wnt10b, with caspase3/7 expression reduced from 2.4- to 1.3-fold, and both a 20% decrease in p38 and 40% increase in ERK1/2 activity. Our data with the AC16 cell model demonstrates that Wnt10b provides defense mechanisms against doxorubicin-induced cardiotoxicity and apoptosis. Further, we explain a mechanism of this beneficial effect involving the mitochondria through simultaneous suppression of pro-apoptotic p38 and anti-apoptotic ERK1/2 activities.

Anti-proliferative activity, molecular genetics, docking analysis, and computational calculations of uracil cellulosic aldehyde derivatives

In this study, the oxidation of microcrystalline cellulose using NaIO4 to yield the corresponding cellulose aldehyde utilized microwave irradiation as a green tool, the obtained cellulosic aldehyde was confirmed through spectral analysis and it has an active site to react with the synthesized uracil acetamide to afford the corresponding arylidene cellulosic MDAU(4), the latter compound which can easily due to presence of active CH=group behind a cyano group react with nitrogen nucleophile’s and cyclized with hydrazine hydrate to give pyrazole cellulosic MDPA(5). The spectral analysis of the obtained cellulosic derivatives was confirmed with FT-IR, NMR, and SEM. Additionally, a neutral red uptake analysis has been used to investigate the cytotoxic activity of the cellulosic compounds MDAC(2), MDAU(4), and MDAP(5) against the cancer cells A549 and Caco2. After 48 h, Compound MDAU(4) had a stronger inhibitory effect on the growth of A549 and Caco2, compared to control cells. Then, using QRT-PCR, the expression levels of the genes β-Catenin, c-Myc, Cyclin D1, and MMP7 in A549 cells were examined. By reducing the expression levels of the Wnt signaling cascade genes (β-Catenin, c-Myc, Cyclin D1, and MMP7) when administered to A549 cells, compound MDAU(4) was shown in this investigation to be a viable candidate compared to lung cancer. Additionally, docking simulation was used to explore the uracil cellulosic heterocycles attached to different proteins, and computational investigations of these compounds looked at how well their physical characteristics matched the outcomes of their experiments.

Different expression of DACT1, DACT2, and CYCLIN D1 genes in human colorectal cancer tissues and its association with clinicopathological characteristics

Aberrant activation of Wnt pathway is linked to dysregulation of several genes. DACT1 and DACT2 are members of the DACT family that participate in antagonizing of the Wnt signaling cascade. Thus in this study, we assessed the mRNA levels of DACT1, DACT2, and CYCLIN D1 in 70 pairs of CRC tissues compared to the adjacent tissues. Determination of the mRNA levels of DACT1, DACT2, and CYCLIN D1 was done by Quantitative Real-Time PCR (qRT-PCR). The correlation between DACT1, DACT2, and CYCLIN D1 genes was also examined. Receiver operating characteristic (ROC) curves was plotted to assess the diagnostic power. The association between histopathological parameters and the DACT1, DACT2, and CYCLIN D1 genes was investigated. The expression levels of DACT1 and CYCLIN D1 were remarkably higher in CRC tissues compared to the adjacent tissues (p < 0.0001). However, the expression of DACT2 was decreased (p < 0.001). Our results showed a significant correlation between the expression of DACT1 and CYCLIN D1 (p < 0.0001). DACT1 (AUC = 0.74, p < 0.0001), DACT2 (AUC = 0.69, p < 0.0003), and CYCLIN D1 (AUC = 0.75, p < 0.0001) had good effectiveness in separation between CRC samples and adjacent tissues. We found a significant association between DACT1 expression with tumor site (p < 0.01). Also, a significant association was detected between DACT2 and CYCLIN D1 with tumor stage (p < 0.005 and p < 0.038, respectively). The findings suggested that DACT1 could function as an oncogene, whereas DACT2 was downregulated and can be considered as a tumor suppressor in CRC.

Unwinding the role of Wnt signaling cascade and molecular triggers of motor neuron degeneration in amyotrophic lateral sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative condition, triggered by various factors causing the degeneration of upper and lower motor neurons, resulting in progressive muscle wasting, paralysis, and death. Multiple in vivo and in vitro models have been established to unravel the molecular events leading to the deterioration of motor neurons in ALS. The canonical and non-canonical Wnt signaling pathway has been implicated to play a crucial role in the progression of neurodegenerative disorders. This review discusses the role of Wnt signaling in the reported causes of ALS such as oxidative stress, mitochondrial dysfunction, autophagy, and apoptosis. Mutations in ALS-associated genes such as SOD1, C9orf72, TDP43, FUS, and OPTN cause an imbalance in neuronal integrity and homeostasis leading to motor neuron demise. Wnt signaling is also observed to play a crucial role in the muscle sparing of oculomotor neurons. The non-canonical Wnt/Ca2+ pathway which regulates intrinsic electrophysiological properties and mobilizes calcium ions to maintain neuronal integrity has been found to be altered in the stem cell-derived ALS model. Thus, the interplay of dysregulated canonical and non-canonical Wnt pathways in multiple motor neuron disease models has shown that Wnt contributes to disease progression indicating it to be utilized as a potential target for ALS.

Rational construction of a practical enzyme-activatable fluorogenic substrate for hNotum and its applications in functional imaging and inhibitor screening

Notum, a negative regulator of the Wnt signaling cascade via removing the essential palmitoleate moieties on the Wnt proteins, has been validated as a promising target for treating various Wnt/β-catenin related diseases. Herein, a rational substrate design strategy was used to construct a highly specific and high-affinity fluorogenic substrate for real-time sensing and functional imaging of hNotum in complex biological systems. Utilizing computer-aided substrate design and phenotyping assays, 3-O-methyl-6-O-octanoylfluorescein (MOF) was selected as an ideal enzyme-activatable fluorogenic substrate for hNotum from a set of ester derivatives of the fluorophores bearing an optically adjustable phenolic group. Under physiological conditions, MOF could be readily hydrolyzed by hNotum to release a brightly fluorescent product 3-O-methylfluorescein (3-MF), which could be easily captured by the devices equipped with fluorescence detector(s). Further studies showed that MOF displayed good performance for sensing hNotum in complex biological samples with rapid response, ultrahigh sensitivity, excellent specificity, and high binding affinity (Km = 101.4 nM). MOF also exhibited excellent performance for in-situ functional imaging of hNotum in living cells, cancerous tissues, and tumor-xenograft mice. MOF was then used to construct a fluorescence-based biochemical assay for high-throughput screening (HTS) of hNotum inhibitors, while isoalantolactone (IAL) was identified as a potent hNotum inhibitor and an efficacious activator of Wnt signaling cascade from an in-house compound library. Collectively, this study devised the first-generation fluorogenic substrate for sensing and imaging hNotum activities in various biological systems, which offered a practical tool for deciphering the biological roles of hNotum and its relevance to human diseases.

A New Wave of Targeting 'Undruggable' Wnt Signaling for Cancer Therapy: Challenges and Opportunities.

Aberrant Wnt signaling activation is frequently observed in many cancers. The mutation acquisition of Wnt signaling leads to tumorigenesis, whereas the inhibition of Wnt signaling robustly suppresses tumor development in various in vivo models. Based on the excellent preclinical effect of targeting Wnt signaling, over the past 40 years, numerous Wnt-targeted therapies have been investigated for cancer treatment. However, Wnt signaling-targeting drugs are still not clinically available. A major obstacle to Wnt targeting is the concomitant side effects during treatment due to the pleiotropic role of Wnt signaling in development, tissue homeostasis, and stem cells. Additionally, the complexity of the Wnt signaling cascades across different cancer contexts hinders the development of optimized targeted therapies. Although the therapeutic targeting of Wnt signaling remains challenging, alternative strategies have been continuously developed alongside technological advances. In this review, we give an overview of current Wnt targeting strategies and discuss recent promising trials that have the potential to be clinically realized based on their mechanism of action. Furthermore, we highlight new waves of Wnt targeting that combine recently developed technologies such as PROTAC/molecular glue, antibody-drug conjugates (ADC), and anti-sense oligonucleotides (ASO), which may provide us with new opportunities to target 'undruggable' Wnt signaling.

Identification of Rare Variants Involved in High Myopia Unraveled by Whole Genome Sequencing

Myopia (nearsightedness) is a condition in which a refractive error (RE) affects vision. Although common variants explain part of the genetic predisposition (18%), most of the estimated 70% heritability is missing. Here, we investigate the contribution of rare genetic variation because this might explain more of the missing heritability in the more severe forms of myopia. In particular, high myopia can lead to blindness and has a tremendous impact on a patient and at the societal level. The exact molecular mechanisms behind this condition are not yet completely unraveled, but whole genome sequencing (WGS) studies have the potential to identify novel (rare) disease genes, explaining the high heritability. Cross-sectional study performed in the Netherlands. We investigated 159 European patients with high myopia (RE > -10 diopters). We performed WGS using a stepwise filtering approach and burden analysis. The contribution of common variants was calculated as a genetic risk score (GRS). Rare variant burden, GRS. In 25% (n= 40) of these patients, there was a high (> 75th percentile) contribution of common predisposing variants; that is, these participants had higher GRSs. In 7 of the remaining 119 patients (6%), deleterious variants in genes associated with known (ocular) disorders, such as retinal dystrophy disease (prominin 1 [PROM1]) or ocular development (ATP binding cassette subfamily B member 6 [ABCB6], TGFB induced factor homeobox 1 [TGIF1]), were identified. Furthermore, without using a gene panel, we identified a high burden of rare variants in 8 novel genes associated with myopia. The genes heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) (proportion in study population vs. the Genome Aggregation Database (GnomAD) 0.14 vs. 0.03, P= 4.22E-17), RNA binding motif protein 20 (RBM20) (0.15 vs. 0.06, P= 4.98E-05), and MAP7 domain containing 1 (MAP7D1) (0.19 vs. 0.06, P= 1.16E-10) were involved in the Wnt signaling cascade, melatonin degradation, and ocular development and showed most biologically plausible associations. We found different contributions of common and rare variants in low and high grade myopia. Using WGS, we identified some interesting candidate genes that could explain the high myopia phenotype in some patients. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Abstract 1077: Porcupine inhibition enhances enzalutamide efficacy in drug resistant prostate cancer

Abstract Androgen receptor (AR) signaling continues to participate as a vital component of castration-resistant prostate cancer (CRPC). Subsequently, this has led to the development of Androgen Signaling Inhibitors (ASI), specifically Enzalutamide (ENZ), which is a direct inhibitor of AR, to clinically manage CRPC. Inevitably, ENZ treatment only provides improvement for approximately two months before advancing to an incurable form, ENZ-resistant CRPC. With PCa ranking as the second leading cause of cancer-related deaths in USA males, there is an urgency and necessity for the discovery and development of novel therapeutic approaches for CRPC. Wnt signaling has been extensively documented in its involvement in PCa and the tumor microenvironment (TME), however the mechanism of how the Wnt signaling cascades contribute to ENZ resistance is still ambiguous. Recently we have published that the activation of the canonical Wnt pathway contributes to the progression of ENZ resistance in CRPC and using a combination of β-catenin inhibitor with ENZ resulted in the synergistic inhibition of patient derived xenograft (PDX) tumor growth. Regarding the non-canonical Wnt pathway, we confirmed its contribution to invasion and migration which leads to metastasis in ENZ-resistant CRPC, and when the downstream effector ROCK is depleted or depleted ROCK cells are treated with ENZ, there is a significant hindering of cell migration and invasion. Also, utilizing a combination therapy of ROCK inhibitor with ENZ synergistically inhibited the growth of PDX tumors. Hence the reasoning that by simultaneously inhibiting both the canonical and non-canonical Wnt signaling cascade will result in the inhibition of cell proliferation, migration, and invasion. The goal of this study was to define the mechanism of PORCN in ENZ-resistant CRPC and develop a therapeutic approach to combat this disease. My research has determined that Porcupine (PORCN) is associated with CRPC progression to ENZ-resistance, and that an inhibition or loss of PORCN has resulted in the regain of ENZ sensitivity in ENZ-resistant models. This model has also demonstrated that PORCN and Wnt signaling engage in a paramount role contributing to AR activation, promoting CRPC progression, and the development of ENZ resistance. Citation Format: Katelyn Makayla Jones, Xiaoqi Liu. Porcupine inhibition enhances enzalutamide efficacy in drug resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1077.