Wnt Signaling Pathway Research Articles

LOXL1 promotes gastric cancer progression by β-catenin-cyclinD mediated proliferation.

Although much progress has been made in chemotherapy or target therapy for advanced gastric cancer, the prognosis is still poor. It is necessary to screen biomarkers for early diagnosis and prognosis prediction. However, the prognostic value of LOX family in gastric cancer and the underlying molecular mechanisms for promoting the progression of gastric cancer remains unclear. Among five members of LOX family, LOXL1 was the unique independent prognostic risk factor. The nomogram established based on the expression of LOXL1 and other clinical parameters could predict the overall survival rate of gastric cancer. Knockdown (KD) of LOXL1 decreased cell proliferation and led to G1 phase arrest in gastric cells. According to GSEA analysis that LOXL1 was positively correlated with the WNT signaling pathway, in vitro experiment proved that LOXL1-KD reduced the phosphorylation level of β-catenin and the expression of the downstream G1 phase checkpoint CCND1.In Conclusion, LOXL1 has been identified as a potential risk prognostic biomarker for gastric cancer by promoting gastric cancer proliferation via WNT/β-catenin/cyclinD1 pathway.

The Wnt/Pyruvate kinase, Muscle axis plays an essential role in the differentiation of mouse neuroblastoma cells

Neuronal differentiation and neurite growth are essential processes in nervous system development and are regulated by several factors. Although all-trans retinoic acid (ATRA) has been shown to mediate the differentiation of mouse neuroblastoma cells via the activation of several pathways, including Wnt/β-catenin signaling, the mechanism remains unclear. The pyruvate kinase, muscle (PKM) plays an important role in the glycolysis of neuroblastoma cells and regulates the Wnt signaling pathway in various cancer cells. In this study, we hypothesized that the Wnt/PKM axis regulates the differentiation of neuroblastoma cells (Neuro-2a and N1E-115). To test this hypothesis, we used inhibitors and activators of the Wnt/β-catenin and glycolytic pathways in ATRA-induced differentiated Neuro-2a and N1E-115 cells and established cell lines with silenced or a mutant replacement of Pkm. Western blot and qPCR showed that ATRA treatment activated the Wnt signaling pathway and inhibited PKM-mediated glycolysis. The oxygen consumption rate (indicating oxidative phosphorylation) significantly increased, whereas the extracellular acidification rate (indicating glycolysis) significantly decreased during differentiation; these effects were reversed upon PKM inhibition. The Wnt inhibitor ICG-001 and PKM activator ML-265 inhibited ATRA-induced Neuro-2a and N1E-115 differentiation, whereas RNA interference-mediated Pkm silencing promoted Neuro-2a and N1E-115 differentiation, which was reversed by PKM overexpression. Treatment with the Wnt activator kenpaullone promoted Neuro-2a and N1E-115 differentiation, which was reversed by ML-265 administration. These results indicate that Wnt/β-catenin signaling promotes Neuro-2a and N1E-115 differentiation by inhibiting PKM-mediated glycolysis during ATRA-induced differentiation. These findings may provide a new theoretical basis for the role of glycolysis in nerve differentiation.

CK2B is a Prognostic Biomarker and a Potential Drug Target for Hepatocellular Carcinoma.

Although casein kinase II subunit beta (CK2B) was previously reported to be involved in human cancers, such as hepatocellular carcinoma (HCC), there has been no systematic assessment of CK2B in HCC. To assess the potential function of CK2B as a prognostic biomarker and possible druggable target in HCC. The Cancer Genome Atlas database was accessed to investigate the potential oncogenic and prognostic roles of CK2B in HCC. Diverse analytical methods were used to obtain a fuller understanding of CK2B, including CIBERSORT, The Tumor Immune Estimation Resource (TIMER), gene set enrichment analyses (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene ontology (GO). Furthermore, the Comparative Toxicogenomic Database (CTD) was used to identify potential drugs to treat CK2B-overexpressing HCC. Patents for these drugs were reviewed using Patentscope® and Worldwide Espacenet®. Upregulated CK2B expression was markedly associated with more aggressive pathological features, including G3, G4 (vs. G1, G2), and T2, T3 (vs. T1). Kaplan-Meier survival curves indicated that patients with HCC with higher expression of CK2B had worse overall survival (P = 0.005), progression-free interval (P = 0.001), and disease-specific survival (P = 0.011). GO and KEGG analysis revealed that CK2B dysregulation affects mitotic chromosome condensation, protein stabilization and binding, regulation of signal transduction of p53 class mediator, and cancer-related pathways. GSEA identified six well-known pathways, including MAPK, WNT, Hedgehog, and TGFβ signaling pathways. Finally, CTD identified six compounds that might represent targeted drugs to treat HCC with CK2B overexpression. A review of patents indicated these compounds showed promising anticancer results; however, whether CK2B interacts with these drugs and improves drug outcomes for patients with HCC was not confirmed. CK2B is a biomarker for HCC prognosis and could be a potential new drug target. Moreover, the association between infiltrating immune cells and CK2B in the HCC tumor microenvironment might provide a solid basis for further investigation and a potent strategy for immunotherapy of HCC.

Parental arsenic exposure and tissue-specific DNA methylation in Bangladeshi infants with spina bifida.

An emerging hypothesis linking arsenic toxicity involves altered epigenetic mechanisms, such as DNA methylation. In this study, we examined the relationship between parents' arsenic exposure and DNA methylation in tissues obtained from 28 infants with spina bifida from Bangladesh. We analyzed arsenic in parents' toenails using inductively coupled plasma mass spectrometry (ICP-MS). DNA methylation was measured in infants' dural tissue, buccal swabs, and whole blood using the Illumina Infinium MethylationEPIC BeadChip. We performed epigenome-wide association analyses (EWAS) and tested differentially methylated regions (DMRs). In EWAS, DNA methylation at cg24039697 in dural tissue was positively associated (β = 0.59, p = 7.6 × 10-9) with father's toenail arsenic concentrations, adjusting for covariates. We did not identify any CpG sites related to father's arsenic exposure in the other tissues, or any CpG sites related to mother's arsenic exposure. Gene ontology analysis identified many biological pathways of interest, including the Wnt signaling pathways. We identified several DMRs across the tissues related to arsenic exposure that included probes mapping to genes that have previously been identified in studies of neural tube defects. This study emphasizes the potential impact of arsenic exposure in fathers, often understudied in epidemiological studies, on DNA methylation in a unique neurological tissue specific to spina bifida.

A redox-related lncRNA signature in bladder cancer.

The redox status is intricately linked to the development and progression of cancer, a process that can be modulated by long non-coding RNAs (lncRNAs). Previous studies have demonstrated that redox regulation can be considered a potential therapeutic approach for cancer. However, the redox-related lncRNA predictive signature specific to bladder cancer (BCa) has yet to be fully elucidated. The purpose of our study is to establish a redox-related lncRNA signature to improve the prognostic prediction for BCa patients. To achieve this, we downloaded transcriptome and clinical data from the Cancer Genome Atlas (TCGA) database. Prognostic redox-related lncRNAs were identified through univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression analysis, resulting in the establishment of two risk groups. A comprehensive analysis corresponding to clinical features between high-risk and low-risk groups was conducted. Eight redox-related lncRNAs (AC018653.3, AC090229.1, AL357033.4, AL662844.4, AP003352.1, LINC00649, LINC01138, and MAFG-DT) were selected to construct the risk model. The overall survival (OS) in the high-risk group was worse than that in the low-risk group (p < 0.001). The redox-related lncRNA signature exhibits superior predictive accuracy compared to traditional clinicopathological characteristics. Gene Set Enrichment Analysis (GSEA) showed that the MAPK signaling pathway and Wnt signaling pathway were enriched in the high-risk group. Compared with the low-risk group, patients in the high-risk group demonstrated increased sensitivity to cisplatin, docetaxel, and paclitaxel. Furthermore, IGF2BP2, a potential target gene of MAFG-DT, was found to be overexpressed in tumor tissues and correlated with overall survival (OS). Our study demonstrated that the predictive signature based on eight redox-related lncRNAs can independently and accurately predict the prognosis of BCa patients.

Molecular Interactions of the Plant Steroid Hormone Epibrassinolide on Human Drug-Sensitive and Drug-Resistant Small-Cell Lung Carcinoma Cells

Background: Small-cell lung cancer (SCLC) has a poor prognosis because it is often diagnosed after it has spread and develops multi-drug resistance. Epibrassinolide (EB) is a plant steroid hormone with widespread distribution and physiological effects. In plants, EB-activated gene expression occurs via a GSK-mediated signaling pathway, similar to Wnt-β-catenin signaling in animal cells that is elevated in cancer cells. Methods: This mechanistic parallel prompted investigations of the molecular interactions of EB on drug-sensitive (H69) and multi-drug-resistant (VPA) SCLC cells. Cellular and molecular investigations were performed. Results: Pharmacologic interactions between EB and the Wnt signaling inhibitors IGC-011 and PRI-724 were determined by the combination index method and showed antagonism, indicating that EB acts on the same pathway as these inhibitors. Following incubation of drug-sensitive and drug-resistant SCLC cells with EB, there was a reduction in β-catenin (e.g., 3.8 to 0.7 pg/µg protein), accompanied by a reduction in β-catenin promoter activity, measured by firefly luciferase-coupled promoter element transfection. Cellular β-catenin concentration is regulated by the active form of GSK3β. In Wnt signaling, active GSK3β is converted to inactive pGSK3β, thereby increasing the concentration of β-catenin. After incubation of SCLC cells with EB, there was a reduction in the inactive form (pGSK3β) and a relative increase in the active form (GSK3β). In vitro enzyme assays showed that EB did not inhibit purified GSK3β, but there was non-competitive inhibition when SCLC cell extracts were used as the source of enzyme. This indirect inhibition by EB indicates that it may act on the Wnt pathway by blocking the phosphorylation of GSK3β. The protein levels of three SCLC tumor markers, namely, NSE, CAV1, and MYCL1, were elevated in drug-resistant SCLC cells. EB incubation led to a significant reduction in the levels of the three markers. Two major effects of EB on SCLC cells are the promotion of apoptosis and the reversal of drug resistance. Transcriptional analyses showed that after exposure of SCLC cells to EB, there were increases in the expression of genes encoding apoptotic inducers (e.g., BAX and FAS) and effectors (e.g., CASP3) and reductions in the expression of genes encoding apoptosis inhibitors (e.g., survivin). PGP1 and MRP1, two membrane efflux pumps expressed in SCLC cells, were elevated in drug-resistant cells, but EB incubation did not affect these protein levels. Cellular assays of drug efflux by PGP1 showed an increase in drug-resistant cells, but EB did not alter efflux activity. Following exposure to human liver microsomes, EB was metabolized by NADPH-dependent oxidation and UDPG-dependent glucuronidation, as evidenced by the elimination of EB cytotoxicity against SCLC cells. Conclusions: Taken together, these data indicate that EB, a steroid hormone in plants consumed in the human diet, is pharmacologically active in drug-sensitive and drug-resistant SCLC cells in the Wnt signaling pathway, alters apoptotic gene expression, and is a substrate for microsomal modifications.

Construction and Analysis of miRNA–mRNA Interaction Network in Ovarian Tissue of Wanxi White Geese Across Different Breeding Stages

Ovarian development significantly influences the laying performance of geese. In this study, the transcriptome analysis was conducted on the ovarian tissues of Wanxi White Geese during the pre-laying (KL), laying (CL), and ceased-laying period (XL). Short Time-series Expression Miner (STEM) analysis and miRNA–mRNA regulatory network construction were performed to identify the key genes and miRNAs regulating laying traits. Comparative analysis of KL vs. CL, CL vs. XL, and XL vs. KL groups resulted in the identification of 337, 136, and 525 differentially expressed genes (DEGs), and 258, 1131, and 909 differentially expressed miRNAs (DEMs), respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (p &lt; 0.05) revealed that the main enrichment pathways of DEGs and DEMs at different breeding periods were Neuroactive ligand–receptor interaction, GnRH signaling pathway and Wnt signaling pathway, all associated with ovarian development. According to the three groups of common pathways, four DEGs were screened out, including INHBB, BMP5, PRL, and CGA, along with five DEMs, including let-7-x, miR-124-y, miR-1-y, and miR-10926-z, all of them may affect ovarian development. A miRNA–mRNA regulatory network was constructed through integrated analysis of DEGs and DEMs, revealing nine miRNAs highly associated with ovarian development: miR-101-y, let-7-x, miR-1-x, miR-17-y, miR-103-z, miR-204-x, miR-101-x, miR-301-y, and miR-151-x. The dual-luciferase reporter gene verified the target relationship between WIF1 and miR-204-x, suggesting that these miRNAs may influence ovarian development in Wanxi White Goose by regulating the expression levels of their target genes within ovarian tissue. This study provides a theoretical foundation for analyzing the mechanisms of ovarian development across different breeding periods and accelerating the cultivation of new breeds through post-transcriptional regulation levels.

Anti-inflammatory role of low-intensity pulsed ultrasound in inhibiting lipopolysaccharide-induced M1 polarization of RAW264.7 cells via Wnt2b/AXIN/β-catenin

Background Low-intensity pulsed ultrasound (LIPUS) is a special type of low-intensity ultrasound. In periodontal disease, LIPUS is applied as an adjuvant and non-invasive treatment. It has been reported that LIPUS significantly shifts the macrophage phenotype from M1 to M2, but the specific mechanism behind this shift is still unknown. Methods RAW264.7 cells were induced to M1/M2 polarization with lipopolysaccharide (LPS)/interleukin-4 (IL4). LIPUS was performed for 25 min two times, 24 h apart, at an intensity of 45 mW/cm2 to stimulate RAW264.7 cells. PolyA mRNA sequencing was conducted of both the LPS-induced RAW264.7 cells and the LPS-induced RAW264.7 cells with LIPUS treatment. The expression of Wnt2b in RAW264.7 cells was downregulated by siRNA. The macrophage surface markers and downstream inflammatory cytokines were detected using flow cytometry. The relative expression of proteins in the Wnt2b/AXIN/β-catenin pathway was assessed using reverse transcription real-time polymerase chain reaction (RT-qPCR) and Western blot. Results LIPUS reversed the M1 polarization of RAW264.7 cells, with decreased expression of CD80 and CD86. In addition, LIPUS enhanced the M2 polarization of RAW264.7 cells, with upregulated expression of CD163 and CD206. The polyA mRNA sequencing results indicated that the Wnt signaling pathway participated in the M1 polarization of LIPUS-treated RAW264.7. The results of the RT-qPCR showed a higher expression of Wnt2b in LIPUS-treated and M1- or M2-polarized RAW264.7 cells. Knocking down Wnt2b was shown to reverse the inhibitory effect of LIPUS on M1 polarization and increase the expression of CD80 and CD86. Wnt2b knockdown also regulated downstream AXIN, β-catenin, and inflammatory factors such as tumor necrosis factor alpha (TNFα) and interleukin-6 (IL6). Conclusions LIPUS plays an anti-inflammatory role by inhibiting LPS-induced M1 polarization of RAW264.7 cells in a Wnt2b/AXIN/β-catenin-dependent way. LIPUS may play a therapeutic role in periodontal diseases by inhibiting inflammation through the regulation of macrophage differentiation.

Primary Osteoporosis Induced by Androgen and Estrogen Deficiency: The Molecular and Cellular Perspective on Pathophysiological Mechanisms and Treatments

Primary osteoporosis is closely linked to hormone deficiency, which disrupts the balance of bone remodeling. It affects postmenopausal women but also significantly impacts older men. Estrogen can promote the production of osteoprotegerin, a decoy receptor for RANKL, thereby preventing RANKL from activating osteoclasts. Furthermore, estrogen promotes osteoblast survival and function via activation of the Wnt signaling pathway. Likewise, androgens play a critical role in bone metabolism, primarily through their conversion to estrogen in men. Estrogen deficiency accelerates bone resorption through a rise in pro-inflammatory cytokines (IL-1, IL-6, TNF-α) and RANKL, which promote osteoclastogenesis. In the classic genomic pathway, estrogen binds to estrogen receptors in the cytoplasm, forming a complex that migrates to the nucleus and binds to estrogen response elements on DNA, regulating gene transcription. Androgens can be defined as high-affinity ligands for the androgen receptor; their combination can serve as a ligand-inducible transcription factor. Hormone replacement therapy has shown promise but comes with associated risks and side effects. In contrast, the non-genomic pathway involves rapid signaling cascades initiated at the cell membrane, influencing cellular functions without directly altering gene expression. Therefore, the ligand-independent actions and rapid signaling pathways of estrogen and androgen receptors can be harnessed to develop new drugs that provide bone protection without the side effects of traditional hormone therapies. To manage primary osteoporosis, other pharmacological treatments (bisphosphonates, teriparatide, RANKL inhibitors, sclerostin inhibitors, SERMs, and calcitonin salmon) can ameliorate osteoporosis and improve BMD via actions on different pathways. Non-pharmacological treatments include nutritional support and exercise, as well as the dietary intake of antioxidants and natural products. The current study reviews the processes of bone remodeling, hormone actions, hormone receptor status, and therapeutic targets of primary osteoporosis. However, many detailed cellular and molecular mechanisms underlying primary osteoporosis seem complicated and unexplored and warrant further investigation.

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation.

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

Exploring the spatial distribution of interstitial cells in kidney tissue.

Interstitial cells are crucial to the development of kidney structure and function, although the mechanism underlying their role in it remains unclear to date. Our previous study identified cell clusters in human fetal kidney tissue, and we further analyzed the interstitial cell cluster within this context. We extracted the barcoded cDNA from tissue samples and prepared spatial transcriptome libraries. Sequencing data was quality-checked, normalized, and clusters were identified using Seurat. Single-cell and spatial data were integrated using MIA and cell types were deconvoluted. DEGs in interstitial cells were identified and functionally annotated using DAVID. CellPhoneDB was used to predict ligand-receptor interactions between cell types. The results of the present study revealed that this cluster of interstitial cells appeared to be scattered in the junction between the cortical and medullary regions. The subsequent KEGG pathway analysis revealed that the differentially expressed genes (DEGs) in this cluster of interstitial cells were involved in the WNT signaling pathway. The Gene Ontology (GO) analysis revealed that these DEGs were involved in multiple pathways associated with kidney development, with six of the genes (NKD2, TCF21, WNT5A, WNT4, MDK, and SFRP1) associated with kidney development exhibiting significant upregulation. Accordingly, it was inferred that these interstitial cells might be involved in regulating epithelial cell differentiation, ureteral bud development, and morphogenesis. The subsequent cell-cell communication analysis revealed that the cellular crosstalk was primarily regulated mainly by ligand-receptor pairs. Additionally, 17 genes reported to be associated with kidney disease were focused on, and these genes were found to be predominantly expressed in a single cell type. In summary, the present study revealed the characteristics of a previously identified cluster of interstitial cells in the kidney tissue, thereby providing fresh insights into the process of kidney development.

Transcriptome-wide methylated RNA immunoprecipitation sequencing profiling reveals m6A modification involved in response to heat stress in Apostichopus japonicus.

Global warming-induced environmental stresses have diverse effects on gene expression and regulation in the life processes of various aquatic organisms. N6 adenylate methylation (m6A) modifications are known to influence mRNA transcription, localization, translation, stability, splicing, and nuclear export, which are pivotal in mediating stress responses. Apostichopus japonicus is a significant species in aquaculture and a representative of benthic organisms in ecosystems, thus there is a growing need for research on its heat stress mechanism. In this study, m6A-modified whole transcriptome profiles of the respiratory tree tissues of A. japonicus in the control (T18) and high-temperature stress (T32) groups were obtained using MeRIP-seq technology. The results showed that 7,211 common m6A peaks, and 9,459 genes containing common m6A were identified in three replicates T18 and T32 groups. The m6A peaks were found to be highly enriched in the 3' untranslated region, and the common sequence of the m6A peak was also enriched, which was shown as RRACH (R = G or A; H = A, C, or U). A total of 1,200 peaks were identified as significantly differentially enriched in the T32 group compared with the T18 group. Among them, 245 peaks were upregulated and 955 were downregulated, which indicated that high temperature stress significantly altered the methylation pattern of m6A, and there were more demethylation sites in the T32 group. Conjoint analysis of the m6A methylation modification and the transcript expression level (the MeRIP-seq and RNA-seq data) showed co-differentiated 395 genes were identified, which were subsequently divided into four groups with a predominant pattern that more genes with decreased m6A modification and up-regulated expression, including HSP70IV, EIF2AK1, etc. GO enrichment and KEGG analyses of differential m6A peak related genes and co-differentiated genes showed the genes were significantly associated with transcription process and pathways such as protein processing in the endoplasmic reticulum, Wnt signaling pathway, and mTOR signaling pathway, etc. CONCLUSION: The comparisons of m6A modification patterns and conjoint analyses of m6A modification and gene expression profiles suggest that m6A modification was involved in the regulation of heat stress-responsive genes and important functional pathways in A. japonicus in response to high-temperature stress. The study will contribute to elucidate the regulatory mechanism of m6A modification in the response of A. japonicus to environmental stress, as well as the conservation and utilization of sea cucumber resources in the context of environmental changes.

R-Spondin 1 Suppresses Inflammatory Cytokine Production in Human Cortical Astrocytes

Background/Objectives: Wnt signaling pathways are essential in various biological processes, including embryonic development and tissue homeostasis, and are implicated in many diseases. The R-Spondin (RSpo) family, particularly RSpo1, plays a significant role in modulating Wnt signaling. This study aims to explore how RSpo1 binding to astrocytic LGR6 receptors influences central nervous system (CNS) homeostasis, particularly in the context of inflammation. Methods: Human-induced pluripotent stem cell-derived astrocytes were treated with RSpo1 to assess its impact on inflammatory cytokine release. A proteomic analysis was conducted using a Human Cytokine Array Kit to measure differential protein expression. Pathway enrichment analysis was performed to identify affected signaling pathways. Results: RSpo1 treatment led to a suppression of inflammatory cytokines such as IL-10, IFN-γ, and IL-23 in astrocytes, while TNF-α and CXCL12 levels were increased. Pathway analysis revealed significant alterations in key signaling pathways, including cytokine–cytokine receptor interaction, chemokine signaling, and TNF signaling pathways, suggesting RSpo1’s role in modulating immune responses within the CNS. Conclusions: RSpo1 significantly influences inflammatory responses in astrocytes by modulating cytokine release and altering key signaling pathways. These findings enhance our understanding of the interaction between cell-specific Wnt signaling and CNS inflammation, suggesting potential therapeutic applications of RSpo1 in neuroinflammatory and neurodegenerative diseases.

TMIC-46. TRANSCRIPTIONAL HETEROGENEITY AND MECHANISTIC PATHWAYS OF RECURRENT GLIOBLASTOMA: INSIGHTS FROM A PRE-CLINICAL RECURRENT TUMOR MODEL

Abstract Despite aggressive therapy combining surgical resection, radiation and chemotherapy, glioblastoma (GBM) almost always recurs. Currently, there is no standard treatment for recurrent GBM, and patient inclusion in clinical trials is based on the genetic and molecular profile of the primary tumor even though the effects of chemoradiation on the transcriptional subtype in matched primary and recurrent GBM remain largely understudied. Paralleling the GLASS consortium, we sought to perform a longitudinal assessment of GBM recurrence after therapy based on molecular subtype with the hypothesis that recurrent tumors exhibit a distinct transcriptional landscape different from treatment-naive tumors. To address this, we utilized primary tumor-derived glioma stem cells (GSCs) representing classical and proneural subtypes. We then undertook in vivo intracranial implantation studies and performed standard concomitant treatment that mimics the Stupp protocol. Upon bulk RNAseq data analyses of untreated (n=21) and recurrent tumors (n=17) we identified that while the molecular subtype of recurrent tumors does not change post therapy, their transcriptional profile is more heterogenous compared to untreated tumors for both subtypes. Hierarchical cluster analyses revealed distinct sub-clusters of recurrent tumors. Common mechanisms of recurrence include upregulation of angiogenesis pathways and PI3k/Akt/mTOR signaling pathways. Interestingly, recurrent tumors exhibit upregulation of genes involved in cellular metabolism and membrane transport/ potential. The source of heterogeneity in recurrent tumors in both subtypes appears to be due to the diversity in expression of gene sets involved in hallmark of cancer pathways such as cell cycle, Notch and Wnt signaling pathways and heterogeneity in oncogenic signatures and cell states resembling fetal or neural cells. Further research into the recurrent tumor microenvironment by spatial multi-omics approaches using this pre-clinical recurrent GBM model will elucidate the linkage between cellular as well as molecular heterogeneity and enable identification of targets that can help drive therapeutic decisions for recurrent GBM patients.

Integrated Profiling Identifies Long-Term Molecular Consequences of Prenatal Dexamethasone Treatment in the Rat Brain-Potential Triggers of Depressive Phenotype and Cognitive Impairment.

Prenatal excess of glucocorticoids (GCs) is considered to be one of the highly impacting factors contributing to depression development. Although GCs are crucial for normal fetal development and their administration (mainly dexamethasone, DEX) is a life-saving procedure for those at risk of preterm delivery, exposure to excess levels of GCs during pregnancy can yield detrimental consequences. Therefore, we aimed to systematically investigate the brain molecular alterations triggered by prenatal DEX administration. We used a rat model of depression based on prenatal exposure to DEX and performed integrative multi-level methylomic, transcriptomic, and proteomic analyses of adult rats' brains (i.e., frontal cortex (FCx) and hippocampus (Hp)) to identify the outcomes of DEX action. Each of the investigated levels was significantly affected by DEX in the long-term manner. Particularly, we found 200 CpG islands to be differentially methylated in the FCx and 200 in the Hp of prenatally DEX-treated rats. Global transcriptomic analysis uncovered differential expression of transcripts mostly in FCx (271) and 1 in Hp, while proteomic study identified 146 differentially expressed proteins in FCx and 123 in Hp. Among the identified enriched molecular networks, we found altered pathways involved in synaptic plasticity (i.e., cAMP, calcium, and Wnt signaling pathways or tight junctions and adhesion molecules), which may contribute to cognitive impairment, observed in DEX-treated animals. Moreover, in the FCx, DEX administration in the prenatal period downregulates the expression of ribosome protein genes associated both with large and small ribosomal subunit assembly which can lead to a global decrease in translation and protein synthesis processes and, indirectly, alterations in the neurotransmission process.

Single-cell landscape identified SERPINB9 as a key player contributing to stemness and metastasis in non-seminomas.

Embryonal carcinoma (EC), characterized by a high degree of stemness similar to that of embryonic stem cells, is the most malignant subtype within non-seminomatous testicular germ cell tumors (TGCTs). However, the mechanisms underlying its malignancy remain unknown. In this study, we employed single-cell RNA sequencing to analyze four non-seminoma samples. Our differential expression analysis revealed high expression of SERPINB9 in metastatic EC cells. We conducted in vitro experiments to further investigate SERPINB9's role in the progression of EC. Functionally, the knockdown of SERPINB9 in NCCIT and NTERA-2 leads to a diminished migratory capability and decreased cis-platin resistance, as demonstrated by Transwell migration assay and drug sensitivity assay. Moreover, embryoid bodies showed reduced size and lower OCT4 expression, alongside heightened expression of differentiation markers AFP, ACTA2, and CD57 in shSERPINB9 cells. In vivo, the role of SERPINB9 in maintaining cancer stemness was validated by the limiting dilution assay. Mechanistically, Bulk RNA-seq further showed downregulation of ERK1/2 signaling and WNT signaling pathways with concomitant upregulation of differentiation pathways subsequent to SERPINB9 knockdown. Additionally, the analysis indicated increased levels of cytokines linked to tertiary lymphoid structures (TLS), such as IL6, IL11, IL15, CCL2, CCL5, and CXCL13 in shSERPINB9 cells, which were further validated by ELISA. Our research indicates that SERPINB9 plays a key role in driving tumor progression by enhancing tumor stemness and suppressing TLS. This study stands as the first to elucidate the molecular signature of non-seminomas at a single-cell level, presenting a wealth of promising targets with substantial potential for informing the development of future therapeutic interventions.

Mechanism of vitamin C alleviating the immunotoxicity of 17α-methyltestosterone in Carassius auratus.

In recent years, the use of endocrine-disrupting chemicals (EDCs) has become increasingly common, leading to severe environmental pollution and harm to aquatic organisms. 17α-Methyltestosterone (MT) is a synthetic androgen that can cause immunotoxicity in aquaculture, affecting fish health. To address this issue, this study aimed to investigate the effect of Vitamin C (VC) on MT-induced immunotoxicity and determine the optimal VC supplementation. Carassius auratus was exposed to 50 ng/L MT and treated with 25, 50, and 150mg/kg VC for 7, 14, and 21 d. Morphological indicators, histological characteristics, hepatic antioxidant capacity, and immune-related gene expression were analyzed. Additionally, RNA-seq was performed on the liver tissues of the control, MT, and MT + 25mg/kg VC groups after 21 d. Results showed that, MT treatment significantly increased liver malondialdehyde content and inhibited immune-related gene expression (TNF-α, IL-8, INF-γ, IL-10, Caspase-9, and IGF-I), causing oxidative stress and immunotoxicity, leading to hepatic steatosis. However, supplementation with 25-50mg/kg VC effectively alleviated the MT-induced damage to the hepatic structure and immune system. RNA-seq revealed significant enrichment of differentially expressed genes in multiple signaling pathways, including the mTOR, MAPK, and Wnt pathways. In summary, 25-50mg/kg VC alleviated inhibitory effect of MT on immune-related genes in C. auratus liver, reducing MT-induced tissue damage. VC not only alleviated inflammation, oxidative stress, and immunotoxicity induced by MT through the regulation of the mTOR, MAPK, and Wnt signaling pathways, but also indirectly enhanced cellular antioxidant defense mechanisms by regulating the NRF2 pathway. This provides a theoretical basis for VC application in aquaculture, improving fish health and increasing efficiency.

Structural and Functional Insights into Dishevelled-Mediated Wnt Signaling

Dishevelled (DVL) proteins precisely control Wnt signaling pathways with many effectors. While substantial research has advanced our understanding of DVL’s role in Wnt pathways, key questions regarding its regulatory mechanisms and interactions remain unresolved. Herein, we present the recent advances and perspectives on how DVL regulates signaling. The experimentally determined conserved domain structures of DVL in conjunction with AlphaFold-predicted structures are used to understand the DVL’s role in Wnt signaling regulation. We also summarize the role of DVL in various diseases and provide insights into further directions for research on the DVL-mediated signaling mechanisms. These findings underscore the importance of DVL as a pharmaceutical target or biological marker in diseases, offering exciting potential for future biomedical applications.

Emerging roles and biomarker potential of WNT6 in human cancers.

The WNT6 ligand is a well-known activator of the WNT signaling pathway, considered a vital player in several important physiologic processes during embryonic development and maintaining homeostasis throughout life, regulating the proliferation and differentiation of multiple stem/progenitor cell types. More recently, as it is the case for many key molecular regulators of embryonic development, dysregulation of WNT6 has been implicated in cancer development and progression in multiple studies. In this review, we overview the most significant recent findings regarding WNT6 in the context of human malignancies, exploring its influence on multiple dimensions of tumor pathophysiology and highlighting the putative underlying WNT6-associated molecular mechanisms. We also discuss the potential clinical implications of WNT6 as a prognostic and therapeutic biomarker. This critical review highlights the emerging relevance of WNT6 in multiple human cancers, and its potential as a clinically-useful biomarker, addressing key unanswered questions that could lead to new opportunities in patient diagnosis, stratification, and the development of rationally-designed precision therapies.