Dickkopf WNT Signaling Pathway Inhibitor Research Articles

Analysis of the types and functions of CD34+ cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing

Objective: To analyze the types and functions of CD34+ cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing. Methods: This study was an experimental study. The CD34+ cell lineage tracing mouse was produced, and the visualization of CD34+ cells under the fluorescent condition was realized. Six male CD34+ cell lineage tracing mice aged 7-8 weeks (designated as diabetic group) were intraperitoneally injected with streptozotocin to establish a diabetic model, and full-thickness skin defect wounds were prepared on their backs when they reached 13 weeks old. Another 6 male CD34+ cell lineage tracing mice aged 13 weeks (designated as control group) were also subjected to full-thickness skin defect wounds on their backs. On post-injury day (PID) 4, wound tissue was collected from 3 mice in control group and 2 mice in diabetic group, and digested to prepare single-cell suspensions. CD34+ cells were screened using fluorescence-activated cell sorting, followed by single-cell RNA sequencing. The Seurat 4.0.2 program in the R programming language was utilized for dimensionality reduction, visualization, and cell clustering analysis of CD34+ cell types, and to screen and annotate the marker genes for each CD34+ cell subpopulation. Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis was performed to analyze the differentially expressed genes (DEGs) of CD34+ fibroblasts (Fbs), smooth muscle cells (SMCs), keratinocytes (KCs), and chondrocyte-like cells (CLCs) in the wound tissue of two groups of mice for exploring cellular functions. Results: On PID 4, CD34+ cells in the wound tissue of both groups of mice were consisted of 7 cell types, specifically endothelial cells, Fbs, KCs, macrophages, T cells, SMCs, and CLCs. Among these, Fbs were further classified into 5 subpopulations. Compared with those in control group, the proportions of CD34+ endothelial cells, Fbs subpopulation 1, Fbs subpopulation 4, KCs, and CLCs in the wound tissue of mice were increased in diabetic group, while the proportions of CD34+ Fbs subpopulation 2, Fbs subpopulation 3, and SMCs were decreased. The marker genes for annotating CD34+ CLCs, endothelial cells, Fbs subpopulation 1, Fbs subpopulation 2, Fbs subpopulation 3, Fbs subpopulation 4, Fbs subpopulation 5, KCs, macrophages, SMCs, and T cells were respectively metastasis-associated lung adenocarcinoma transcript 1, fatty acid binding protein 4, Gremlin 1, complement component 4B, H19 imprinted maternally expressed transcript, Dickkopf Wnt signaling pathway inhibitor 2, fibromodulin, keratin 5, CD74 molecule, regulator of G protein signaling 5, and inducible T-cell co-stimulator molecule. KEGG and GO enrichment analysis revealed that, compared with those in control group, DEGs with significant differential expression (SDE) in CD34+ Fbs from the wound tissue of mice in diabetic group on PID 4 were significantly enriched in terms related to inflammatory response, extracellular matrix (ECM) organization, regulation of cell proliferation, and aging (with Pvalues all <0.05), DEGs with SDE in CD34+ SMCs were significantly enriched in terms related to cell migration, apoptotic process, positive regulation of transcription, and phagosome (with P values all <0.05), DEGs with SDE in CD34+ KCs were significantly enriched in terms related to mitochondrial function, transcription, and neurodegenerative diseases (with P values all <0.05), and DEGs with SDE in CD34+ CLCs were significantly enriched in terms related to rhythm regulation, ECM, and viral infection (with P values all <0.05). Conclusions: CD34+ cells display high heterogeneity in the healing process of full-thickness skin defect wounds in both normal mice and diabetic mice. The significantly enriched functions of DEGs with SDE in CD34+ cell subpopulations in the wound tissue of the two mouse groups are closely related to the wound healing process.

Integrated analysis strategy of genome-wide functional gene mining reveals DKK2 gene underlying meat quality in Shaziling synthesized pigs

BackgroundShaziling pig is a well-known indigenous breed in China who has superior meat quality traits. However, the genetic mechanism and genomic evidence underlying meat quality characteristics of Shaziling pigs are still unclear. To explore and investigate the germplasm characteristics of Shaziling pigs, we totally analyzed 67 individual’s whole genome sequencing data for the first time (20 Shaziling pigs [S], 20 Dabasha pigs [DBS], 11 Yorkshire pigs [Y], 10 Berkshire pigs [BKX], 5 Basha pigs [BS] and 1 Warthog).ResultsA total of 2,538,577 SNPs with high quality were detected and 9 candidate genes which was specifically selected in S and shared in S to DBS were precisely mined and screened using an integrated analysis strategy of identity-by-descent (IBD) and selective sweep. Of them, dickkopf WNT signaling pathway inhibitor 2 (DKK2), the antagonist of Wnt signaling pathway, was the most promising candidate gene which was not only identified an association of palmitic acid and palmitoleic acid quantitative trait locus in PigQTLdb, but also specifically selected in S compared to other 48 Chinese local pigs of 12 populations and 39 foreign pigs of 4 populations. Subsequently, a mutation at 12,726-bp of DKK2 intron 1 (g.114874954 A > C) was identified associated with intramuscular fat content using method of PCR–RFLP in 21 different pig populations. We observed DKK2 specifically expressed in adipose tissues. Overexpression of DKK2 decreased the content of triglyceride, fatty acid synthase and expression of relevant genes of adipogenic and Wnt signaling pathway, while interference of DKK2 got contrary effect during adipogenesis differentiation of porcine preadipocytes and 3T3-L1 cells.ConclusionsOur findings provide an analysis strategy for mining functional genes of important economic traits and provide fundamental data and molecular evidence for improving pig meat quality traits and molecular breeding.

Role of prognostic gene DKK1 in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common squamous cell carcinomas of the head and neck. The morbidity and mortality rates of OSCC have increased in recent years. However, the pathogenesis of this disease remains unknown. The present study aimed to identify predictive biomarkers and therapeutic targets for OSCC. Bioinformatics screening of differentially expressed genes in OSCC was performed based on data from The Cancer Genome Atlas and Gene Expression Omnibus databases. Dickkopf Wnt signaling pathway inhibitor 1 (DKK1) was identified to be associated with survival, tumor immunity and DNA repair in OSCC. Furthermore, the effects of DKK1 were evaluated by the knockdown of DKK1 in two OSCC cell lines. The proliferation, clonogenicity, migration and invasion of the cells were assessed in vitro using Cell Counting Kit-8, colony formation, wound healing and Transwell assays, respectively, and were found to be inhibited by DKK1 knockdown. The present study suggests that DKK1 may be used in the prognosis of patients with OSCC and that targeting DKK1 is a potential strategy for OSCC therapy.

Single Cell Multi-Omic Analysis of Neoplastic Plasma Cells across the Disease Spectrum Identifies Novel Pathobiologic Mediators and Potential Therapeutic Targets in Multiple Myeloma (MM)

Background: A challenge to developing a full understanding of MM pathobiology using bulk profiling techniques is the large degree of intra-tumoral heterogeneity from minor subclones that, despite their low representation, nonetheless contribute to therapy resistance and disease progression. Moreover, our current therapies tend to be targeted against pathways common to both neoplastic and normal plasma cells (PCs), indicating a need to define novel tumor biology modifiers that could potentially serve to improve risk stratification and as therapeutic targets. Methods: To overcome these limitations, we simultaneously evaluated individual PC single-cell transcriptome and B-cell receptor variable (VDJ) sequences from freshly enriched bone marrow biopsies. These included 33 patients with precursor conditions, 17 each with newly diagnosed symptomatic or relapsed/refractory disease (RRMM), and 3 controls using the 10x Genomics platform. A total of ~320,000 PCs were analyzed, yielding median per sample cell counts of 4,626 and ~300 million total reads. Identical immunoglobulin VDJ sequences defined monoclonal PCs, while diverse VDJ sequences at low frequencies defined polyclonal cells, allowing each patient to serve as their own control. Results: The percentage of monoclonal PCs in active MM was &amp;gt;84%, while precursor stages were more variable (40-100% in smoldering MM; 0.3-82% in monoclonal gammopathy of undetermined significance (MGUS)). Though we could not identify monoclonal PCs in two MGUS cases, we identified them by scRNA-seq in four MGUS patients whose bone marrow showed no clonal cells by standard clinical multiparametric flow cytometry. In differential gene expression analysis where samples were grouped based on the diagnosis, 501 dysregulated genes were identified in monoclonal PCs from symptomatic MM compared to polyclonal PCs. These included genes previously identified as disease modifiers, including Hepatocyte growth factor, Lactate dehydrogenase A, Dickkopf WNT signaling pathway inhibitor 1, CD27, and Lysosomal associated membrane protein family member 5, providing validation for our approach. Next, we identified 279 genes that were commonly dysregulated in monoclonal PCs from symptomatic vs. asymptomatic MM. We also performed unsupervised hierarchical clustering of the top 1000 most variable genes and defined three diagnostic categories. Significant ontology terms characterizing these categories broadly involved antigen processing and recognition in Group 1 (enriched in precursor stages); transcription and translation control in Group 2 (enriched in active MM); and cell division and DNA metabolism in Group 3 (enriched with RRMM) ( A). We focused on dysregulated targets that: showed differential expression in monoclonal vs. polyclonal PCs; expression in most patients within a diagnostic category; a correlation with outcomes in the CoMMpassstudy; and novelty in MM. Among these were Mitotic arrest deficient 2 like 1 (MAD2L1), a component of the mitotic spindle assembly checkpoint, and Methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of S-adenosylmethionine, a key methyl donor in multiple cellular processes, from methionine and ATP. Notably, MAD2L1 inhibition with M2I1, and MAT2A targeting with the allosteric inhibitor AG-270, both produced a dose- and time-dependent inhibition in cell proliferation and reduction in myeloma cell line viability. Importantly, the effects of AG-270 ( B) were associated with induction of markers of apoptosis, including Caspase cleavage, and a reduction of Histone H3 dimethylation. Moreover, they occurred at clinically relevant concentrations based on the known pharmacokinetics of this drug in Phase I testing. Conclusions: Our combined single-cell transcriptomics and VDJ sequencing allowed a greater understanding of heterogeneity among neoplastic PCs compared to each patient's own polyclonal PCs. These analyses can identify putative novel mediators of disease pathobiology that impact prognosis, and that can serve as potential new therapeutic targets using either novel agents, or drug repurposed from other therapeutic areas, setting the stage for their translation to the clinic.

Dormant tumors circumvent tumor-specific adaptive immunity by establishing a Treg-dominated niche via DKK3.

Approximately 30% of breast cancer survivors deemed free of disease will experience locoregional or metastatic recurrence even up to 30 years after initial diagnosis, yet how residual/dormant tumor cells escape immunity elicited by the primary tumor remains unclear. We demonstrate that intrinsically dormant tumor cells are indeed recognized and lysed by antigen-specific T cells in vitro and elicit robust immune responses in vivo. However, despite close proximity to CD8+ killer T cells, dormant tumor cells themselves support early accumulation of protective FoxP3+ T regulatory cells (Tregs), which can be targeted to reduce tumor burden. These intrinsically dormant tumor cells maintain a hybrid epithelial/mesenchymal state that is associated with immune dysfunction, and we find that the tumor-derived, stem cell/basal cell protein Dickkopf WNT signaling pathway inhibitor 3 (DKK3) is critical for Treg inhibition of CD8+ T cells. We also demonstrate that DKK3 promotes immune-mediated progression of proliferative tumors and is significantly associated with poor survival and immunosuppression in human breast cancers. Together, these findings reveal that latent tumors can use fundamental mechanisms of tolerance to alter the T cell microenvironment and subvert immune detection. Thus, targeting these pathways, such as DKK3, may help render dormant tumors susceptible to immunotherapies.

Immune Cell Infiltration and Clinical Significance of Angiogenesis-related Genes in Lung Adenocarcinoma.

Angiogenesis is one of the hallmarks of cancer. However, the role of molecular subtypes of angiogenesis-associated genes (AAGs) in the tumor immune microenvironment (TIME) of lung adenocarcinoma (LUAD) remains unclear. The expression of AAGs in patients with LUAD were studied. Consensus clustering was performed to identify new AAG-associated molecular subgroups. The TIME and immune status of the subgroups were analyzed. Functional enrichment analysis was performed on the differentially expression genes among the clustered subgroups to analyze their relationship with AAGs. Furthermore, a prognostic risk model and clinical nomogram associated with survival time were constructed. Risk scores of drug sensitivity, immune checkpoint molecules, tumor mutational burden, and tumor cell stemness were analyzed. Finally, a series of in vitro experiments were performed to investigate the role of dickkopf WNT signaling pathway inhibitor 1 (DKK1) in LUAD. Two molecular subgroups with significantly different survival rates and TIME were identified. Immune checkpoint scores were higher in the subgroup with a worse prognosis. Moreover, differentially expressed genes were enriched in cell-cycle regulation, protein metabolism, and the immune microenvironment. The risk model and clinical nomogram constructed based on AAGs accurately predicted the prognosis of patients with LUAD. Patients with high-risk scores were less sensitive to chemotherapy but more sensitive to immunotherapy. DKK1 was highly expressed in basal cells and luminal cells. In addition, the knockdown of DKK1 reduced LUAD cell proliferation, invasion, and migration. Models based on AAGs can play an important role in predicting LUAD prognosis and immunotherapy effects. We further characterized the angiogenesis of TIME and studied the AAG DKK1. Our findings provide a theoretical basis for antitumor strategies targeting angiogenesis.

DKK3 expression is correlated with poorer prognosis in head and neck squamous cell carcinoma: A bioinformatics study based on the TCGA database

ObjectiveWe previously reported that dickkopf WNT signaling pathway inhibitor 3 (DKK3) expression is correlated with poorer prognosis in head and neck squamous cell carcinoma (HNSCC). Here we investigated DKK3 expression by using The Cancer Genome Atlas (TCGA) public database and bioinformatic analyses. MethodsWe used the RNA sequence data and divided the tumor samples into “DKK3-high” and “DKK3-low” groups according to median DKK3 expression. The correlations between DKK3 expression and the clinical data were investigated. Differentially expressed genes (DEGs) were detected using DESEq2 and analyzed by ShinyGO 0.77. A gene set enrichment analysis (GSEA) was also performed using GSEA software. The DEGs were also analyzed with TargetMine to establish the protein–protein interaction (PPI) network. ResultsDKK3 expression was significantly increased in cancer samples, and a high DKK3 expression was significantly associated with shorter overall survival. We identified 854 DEGs, including 284 up-regulated and 570 down-regulated. Functional enrichment analyses revealed several Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with extracellular matrix remodeling. The PPI network identified COL8A1, AGTR1, FN1, P4HA3, PDGFRB, and CEP126 as the key genes. ConclusionsThese results suggested the cancer-promoting ability of DKK3, the expression of which is a promising prognostic marker and therapeutic target for HNSCC.

A pilot genome-wide association study meta-analysis of gastroparesis.

Gastroparesis (GP) is characterized by delayed gastric emptying in the absence of mechanical obstruction. Genetic predisposition may play a role; however, investigation at the genome-wide level has not been performed. We carried out a genome-wide association study (GWAS) meta-analysis on (i) 478 GP patients from the National Institute of Diabetes and Digestive and Kidney Diseases Gastroparesis Clinical Research Consortium (GpCRC) compared to 9931 population-based controls from the University of Michigan Health and Retirement Study; and (ii) 402 GP cases compared to 48,340 non-gastroparesis controls from the Michigan Genomics Initiative. Associations for 5,811,784 high-quality SNPs were tested on a total of 880 GP patients and 58,271 controls, using logistic mixed models adjusted for age, sex, and principal components. Gene mapping was obtained based on genomic position and expression quantitative trait loci, and a gene-set network enrichment analysis was performed. Genetic associations with clinical data were tested in GpCRC patients. Protein expression of selected candidate genes was determined in full thickness gastric biopsies from GpCRC patients and controls. While no SNP associations were detected at strict significance (p≤5×10-8 ), nine independent genomic loci were associated at suggestive significance (p≤1×10-5 ), with the strongest signal (rs9273363, odds ratio=1.4, p=1×10-7 ) mapped to the human leukocyte antigen region. Computational annotation of suggestive risk loci identified 14 protein-coding candidate genes. Gene-set network enrichment analysis revealed pathways potentially involved in immune and motor dysregulation (pFDR ≤ 0.05). The GP risk allele rs6984536A (Peroxidasin-Like; PXDNL) was associated with increased abdominal pain severity scores (Beta=0.13, p=0.03). Gastric muscularis expression of PXDNL also positively correlated with abdominal pain in GP patients (r=0.8, p=0.02). Dickkopf WNT Signaling Pathway Inhibitor 1 showed decreased expression in diabetic GP patients (p=0.005 vs. controls). We report preliminary GWAS findings for GP, which highlight candidate genes and pathways related to immune and sensory-motor dysregulation. Larger studies are needed to validate and expand these findings in independent datasets.

Identification of Potential Abnormal Methylation-Modified Genes in Coronary Artery Ectasia.

Coronary artery ectasia (CAE) is an easily recognized abnormality of coronary artery anatomy and morphology. However, its pathogenesis remains unclear. This study aimed to identify abnormal methylation-modified genes in patients with CAE, which could provide a research basis for CAE. Peripheral venous blood samples from patients with CAE were collected for RNA sequencing to identify differentially expressed genes (DEGs), followed by functional enrichment. Then, the DNA methylation profile of CAE was downloaded from GSE87016 (HumanMethylation450 BeadChip data, involving 11 cases and 12 normal controls) to identify differentially methylated genes (DMGs). Finally, after taking interaction genes between DEGs and DMGs, abnormal methylation-modified genes were identified, followed by protein-protein interaction analysis and expression validation using reverse transcriptase polymerase chain reaction. A total of 152 DEGs and 4318 DMGs were obtained from RNA sequencing and the GSE87016 dataset, respectively. After taking interaction genes, 9 down-regulated DEGs due to hypermethylation and 11 up-regulated DEGs due to hypomethylation were identified in CAE. A total of 10 core abnormal methylation-modified genes were identified, including six down-regulated DEGs due to hypermethylation (netrin G1, ADAM metallopeptidase domain 12, immunoglobulin superfamily member 10, sarcoglycan dela, Dickkopf WNT signaling pathway inhibitor 3, and GATA binding protein 6), and four up-regulated DEGs due to hypomethylation (adrenomedullin, ubiquitin specific peptidase 18, lymphocyte antigen 6 family member E, and MX dynamin-like GTPase 1). Some signaling pathways were identified in patients with CAE, including cell adhesion molecule, O-glycan biosynthesis, and the renin-angiotensin system. Abnormal methylation-modified DEGs involved in signaling pathways may be involved in CAE development.

Prediction of prognosis and immunotherapy response in lung adenocarcinoma based on CD79A, DKK1 and VEGFC

BackgroundTumor immune microenvironment (TIME) is crucial for tumor initiation, progression, and metastasis; however, its relationship with lung adenocarcinoma (LUAD) is unknown. Traditional predictive models screen for biomarkers that are too general and infrequently associated with immune genes. MethodsRNA sequencing data of LUAD patients and immune-related gene sets were retrieved from public databases. Using the common genes shared by The Cancer Genome Atlas (TCGA) and Immunology Database and Analysis Portal (ImmPort), differential gene expression analysis, survival analysis, Lasso regression analysis, and univariate and multivariate Cox regression analyses were performed to generate a novel risk score model. LUAD cohort in International Cancer Genome Consortium (ICGC), GSE68465 cohort in Gene Expression Omnibus (GEO) and an immunohistochemical assay were used to validate the key genes constructed risk score. The LUAD-related prognosis, clinical indicators, immune infiltrate characteristics, response to immunotherapy, and response to chemotherapeutic agents in different risk groups were evaluated by CIBERSORT, ImmuCellAI, pRRophetic and other tools. ResultsThe risk score model was constructed using CD79a molecule (CD79A), Dickkopf WNT signaling pathway inhibitor 1 (DKK1), and vascular endothelial growth factor C (VEGFC). High risk score was identified as a negative predictor for overall survival (OS) in subgroup analyses with tumor stage, TNM classification, therapy outcome, and ESTIMATE scores (P < 0.05). Low risk score was positively associated with plasma cells, memory B cells, CD8 T cells, regulatory T cells and γδT cells (P < 0.05). In low-risk group, programmed cell death 1 receptor (PD1), cytotoxic T-lymphocyte associated protein 4 (CTLA4), and lymphocyte activating 3 (LAG3) and indoleamine 2,3-dioxygenase (IDO) were more robustly expressed (P < 0.05). The treatment responses of immune checkpoint blockade (ICB) therapy and chemotherapy were likewise superior in low-risk group (P < 0.05). In immunohistochemical analysis, the tumor group had significantly higher levels of CD79A, DKK1, and VEGFC than the adjacent normal group (P < 0.01). ConclusionsCD79A, DKK1 and VEGFC are important differential genes related to LUAD, risk score could reliably predict prognosis, composition of TIME and immunotherapy responses in LUAD patients. The excellent performance of the risk model shows its strong and broad application potential.

Association of clinical characteristics and biomarkers with recurrent atrial fibrillation after hybrid ablation

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Atrial fibrillation ablation research at our institution was supported by an unrestricted grant from AtriCure. Background Hybrid ablation is an alternative treatment for both persisting and therapy-resistant, symptomatic atrial fibrillation (AF), combining the strengths of endocardial catheter and epicardial surgical ablation. Although results exhibit lower rates of recurrence and higher reduction of AF burden, there is a considerable risk of procedure-related complications. Therefore, the identification of patient groups who benefit most of this procedure can help in pre-procedural selection and tailoring of optimal therapy post-ablation. Purpose This study aimed to identify clinical factors and blood biomarkers associated with recurrence of AF within 1 year post-ablation. Methods A total of 96 consecutive patients undergoing hybrid ablation between January 2015 and December 2018 in a tertiary care center were enrolled. Blood samples were collected pre-ablation. Seven biomarkers [Bone Morphogenetic Protein 10 (BMP10), angiopoietin-2 (ANG2), Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3), Endothelial Cell Specific Molecule 1 (ESM1), Fibroblast Growth Factor 23 (FGF23), Insulin Like Growth Factor Binding Protein 7 (IGFBP7) and total N-terminal pro b-type natriuretic peptide (NT-proBNP)] were assayed at Roche Diagnostics based on their possible association with recurrence of AF. Clinical follow-up visits combined with 12-lead ECG and 24-hour Holter monitoring were scheduled at 3, 6 and 12 months post-procedure. Cox proportional hazards regression models were used to study the association with recurrence of AF. Results Out of the 96 patients (mean age 58±8 years; 18% women), 17 (17.7%) developed recurrence of AF within 1 year post-ablation. Median time to diagnosis was 191 days (range: 92-344). Comparisons between patients with and without recurrence of AF are depicted in Table 1. Multivariable Cox proportional hazard regression, adjusted for sex, showed that older age (HR 1.104, 95% CI 1.003-1.215, p=0.043), type of AF (HR 0.322, 95% CI 0.117-0.886, p=0.028), a history of peripheral vascular disease (HR 13.84, 95% CI 3.13-61.14, p&amp;lt;0.001) and recurrence in the blanking period (HR 14.25, 95% CI 3.99-50.85, p&amp;lt;0.001) were associated with recurrence of AF within 1 year post-ablation, but no blood biomarkers (Figure 1). Conclusion In AF patients undergoing extensive ablation, older age, type of AF, a history of peripheral vascular disease and recurrence in the blanking period were determinants of AF recurrence within 1 year post-ablation.

Upregulation of long intergenic non-coding RNA LINC00326 inhibits non-small cell lung carcinoma progression by blocking Wnt/β-catenin pathway through modulating the miR-657/dickkopf WNT signaling pathway inhibitor 2 axis

BackgroundLong intergenic non-coding RNA 326 (LINC00326) modulates hepatocarcinogenic lipid metabolism. However, the ability of LINC00326 to modulate the highly aggressive non-small cell lung carcinoma (NSCLC) is unknown. Here, LINC00326 in NSCLC was investigated, together with its effects on tumor malignancy and the underlying mechanisms of action.MethodsLINC00326 levels in tumor tissues and cell lines were measured by Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and RNA fluorescence in situ hybridization (FISH). Proliferation and apoptosis were assessed in cell lines by Cell Counting Kit-8 (CCK-8), EdU staining assays and flow cytometry, respectively, and tumor growth was measured in mouse models. Possible microRNA targets of LINC00326 were predicted by bioinformatics and verified by RNA pull-down and immunoprecipitation and luciferase reporter assays. Western blotting was used to evaluate the expression of Wnt/β-catenin-associated proteins.Results LINC00326 was downregulated in tumor tissues and cell lines. Knockdown of LINC00326 stimulated NSCLC cell proliferation and suppressed apoptosis in vitro, as well as enhancing xenograft tumor growth. LINC00326 sponged miR-657, and dickkopf WNT signaling pathway inhibitor 2 (DKK2) was found to be directly targeted by miR-657, with LINC00326 positively regulating its expression through sponging miR-657. The actions of LINC00326 knockdown on proliferation and apoptosis were reversed by stimulation of the miR-657/DKK2 axis. Furthermore, overexpression of miR-657 mitigated DKK2 inhibition on Wnt/β-catenin signaling.ConclusionsLINC00326/miR-657/DKK2 axis signaling blocked tumor-associated functions in NSCLC cells through the targeting Wnt/β-catenin pathway. This suggests that this pathway could be a target for NSCLC treatment.

Abstract PR015: Dormant mammary tumors persist long-term despite adaptive immunity by establishing a Treg-dominated niche via DKK3

Abstract A significant portion of breast cancer survivors eventually experience locoregional or metastatic recurrence. It is clear that the adaptive immune system controls proliferative tumor outgrowth, but how dormant tumor cells escape to eventually form new lesions remains unclear. Previous studies showed that proliferative D2A1 or D2.OR tumors are restricted by adaptive immunity in Balb/c versus SCID mice whereas dormant D2.1 tumors are largely unaffected. Further, D2.1 tumors are highly infiltrated with T cells, particularly CD4+ cells and Tregs. Thus, the aim of the present study was to determine the extent to which dormant D2.1 tumors evade antigen-specific immunity and how these tumors shape the T cell niche. Experiments were performed using Balb/c syngeneic D2 mammary tumor cells – D2A1 cells grow rapidly upon orthotopic injection, D2.OR cells proliferate but to a lesser degree than D2A1, and D2.1 cells remain dormant long term. Cells were also modified to express enhanced green fluorescent protein (eGFP) for use with Just eGFP Death Inducing (JeDI) T cells containing a T cell receptor (TCR) targeted to eGFP200-208. Transfer of JeDI T cells caused complete rejection of D2A1-eGFP tumors in the mammary fat pad (MFP) while no difference was observed in D2.1-eGFP tumors. In contrast, treatment with anti-CTLA4 alone, anti-PD-1 alone, or combined anti-CTLA4/PD-1 reduced D2.1 tumor progression compared to isotype control. Whole JeDI splenocytes were also stimulated in vitro with eGFP200-208 peptide in the presence of D2A1 or D2.1 conditioned medium (CM). Surprisingly, while CD8 cells proliferated in both D2A1 and D2.1 CM, CD4+ T cells also proliferated in D2.1 CM and the Treg fraction of CD4+ cells was significantly elevated in D2.1 CM compared to D2A1 CM or control medium. RNA-sequencing confirmed substantial immune activity in D2.1 tumors compared to D2A1. D2.1 tumors also displayed a hybrid epithelial/mesenchymal (E/M) phenotype and expressed genes associated with both mammary epithelium (Krt8, Krt14, Cdh1) and mesenchymal cells (Itga5, Snai2, Notch1, Zeb1). Moreover, Gene Set Enrichment Analysis (GSEA) showed an enrichment of mammary stem and luminal progenitor cell signatures in D2.1 tumors. Of the top upregulated genes, Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) was chosen for further investigation. Interestingly, increased CD8 proliferation and IFNγ production, but decreased CD4 and Treg abundance, was observed in DKK3 shRNA knockdown (k/d) D2.1 CM compared to shScramble control. In vivo, overexpressing DKK3 in D2A1 cells significantly increased MFP tumor growth in Balb/c but not SCID mice, altered T cell activation locally and systemically, and decreased eGFP-specific antibodies. Similar results were observed in DKK3-overexpressing D2.OR tumors. Conversely, DKK3 k/d in D2.1 cells resulted in ~60% complete tumor rejection. Together, these data indicate that CD4 cells, particularly Tregs, are crucial in establishing a protective niche for long-term tumor dormancy with tumor-derived DKK3 emerging as a mediator of the Treg niche. Citation Format: Timothy N. Trotter, Delila Serra, Carina Dagotto, Tao Wang, Xiao Yang, Junping Wei, Gangjun Lei, H. Kim Lyerly, Zachary C. Hartman. Dormant mammary tumors persist long-term despite adaptive immunity by establishing a Treg-dominated niche via DKK3 [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR015.

Apogossypolone Inhibits Cell Proliferation and Epithelial-Mesenchymal Transition in Cervical Cancer via Activating DKK3.

Apogossypolone (ApoG2), a novel derivative of gossypol lacking of two aldehyde groups, exhibits anti-tumor effects. However, the mechanisms by which ApoG2 regulates cervical cancer (CC) cells remain unclear. In this study, we treated two CC cell lines (CaSki and HeLa) with an increasing concentration of ApoG2 for 24 h. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, flow cytometry and transwell invasion assay were utilized to detect cell proliferation, apoptosis and invasion in vitro. We first observed that ApoG2 inhibited cell proliferation, invasion and epithelial-to-mesenchymal transition (EMT) process in CC cells, along with upregulation of Dickkopf Wnt signaling pathway inhibitor 3 (DKK3) in a dose-dependent manner. The immunohistochemistry confirmed the downregulation of DKK3 in tumor tissues. Moreover, DKK3 was correlated with FIGO stage and lymph node metastasis. Functionally, DKK3 overexpression significantly suppressed cell viability, colony formation and invasion, but promoted apoptosis in CaSki and HeLa cells. Overexpression of DKK3 upregulated the protein levels of cleaved caspase-3 and E-cadherin, but downregulated the protein levels of Bcl-2, N-cadherin and Vimentin. Furthermore, DKK3 knockdown reversed the suppressive effects of ApoG2 on CaSki cell proliferation, invasion and EMT markers, while DKK3 overexpression enhanced these effects. In addition, ApoG2 treatment inhibited CC xenograft tumor growth and upregulated the protein levels of DKK3, cleaved caspase-3 and E-cadherin. In conclusions, these findings suggested that ApoG2 could effectively inhibit the growth and invasion of CC cells at least partly by activating DKK3.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

Abstract 2451: Dormant mammary tumors resist antigen-specific killing and regulate systemic immunity in association with PTN and DKK3

Abstract Little is known regarding why the immune system is ineffective at eliminating certain disseminated/dormant tumor cells before overt metastatic recurrence. Previous findings revealed that the adaptive immune system reduces growth of proliferative D2A1 tumors in Balb/c versus SCID mice while dormant D2.1 tumors are largely unaffected by adaptive immunity, despite being significantly infiltrated by T cells. Therefore, the aim of the present study was to determine the downstream effects and molecular underpinnings of dormancy-mediated alterations to the immune environment. Experiments were performed using two independent clones of a spontaneous mammary tumor from a female Balb/c mouse that display varying degrees of dormancy in vivo. D2A1 cells grow rapidly upon orthotopic or systemic injection while D2.1 cells remain dormant long term in local and metastatic sites. Both cell lines were modified to express enhanced green fluorescent protein (eGFP) for use with Just eGFP Death Inducing (JeDI) T cells which contain a T cell receptor (TCR) targeted to eGFP200-208. Adoptive transfer of JeDI T cells caused complete rejection of D2A1-eGFP tumors in the mammary fat pad (MFP), while no difference in growth was observed in D2.1-eGFP tumors. Interestingly, contralateral MFP implantation of D2.1-eGFP tumors resulted in a partial rescue of D2A1-eGFP rejection suggesting systemic immune suppression by distant, dormant tumor cells. Bulk RNA-sequencing of D2A1 and D2.1 tumors revealed significant upregulation of pathways associated with immune activation and extracellular matrix (ECM) remodeling in dormant tumors. Further, Gene Set Enrichment Analysis (GSEA) showed an enrichment for signatures of mammary stem cells and luminal progenitor cells, as well as exhausted vs memory CD8 cells and T cell anergy, in D2.1 tumors. Of the top upregulated genes in D2.1 tumors, pleiotrophin (PTN) and Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) were chosen for further investigation. Overexpressing PTN or DKK3 in D2A1 cells significantly increased MFP tumor growth in Balb/c but not SCID mice. In addition, PTN or DKK3 overexpression altered both local and systemic T cell profiles, including decreased effector memory (CD62l-, CD44+) and increased naïve (CD62l+, CD44-) CD4 and CD8 cells. Humoral immunity was also affected, as indicated by decreased eGFP-specific antibodies in serum from mice bearing PTN or DKK3 overexpressing tumors. Conversely, knocking down PTN or DKK3 in D2.1 cells had the opposite effect on systemic immunity. Taken together, these data indicate that dormant tumors can resist antigen-specific killing by CD8 T cells despite high T cell infiltration, and possibly suppress antigen-specific immunity in distant tumor sites. Moreover, both PTN and DKK3 emerge as potential mediators of dormancy-induced immune evasion. Citation Format: Timothy N. Trotter, Delila Serra, Carina Dagotto, Tao Wang, Xiao Yang, Junping Wei, Gangjun Lei, H. Kim Lyerly, Zachary C. Hartman. Dormant mammary tumors resist antigen-specific killing and regulate systemic immunity in association with PTN and DKK3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2451.

Chondroitin Sulfates Control Invasiveness of the Basal-Like Breast Cancer Cell Line MDA-MB-231 Through ROR1

Extracellular and cell surface chondroitin sulfates (CSs) regulate cancer cell properties, including proliferation and invasion. Thus, it is necessary to understand the mechanisms underlying their roles in cancer. Although we have shown that CS has an inherent ability to enhance the invasive activity of the human triple-negative breast cancer cell line MDA-MB-231, its molecular mechanism remains elusive. Here, we focused on receptor tyrosine kinase-like orphan receptor 1 (ROR1) and dickkopf WNT signaling pathway inhibitor 1 (DKK1). MDA-MB-231 cells express high levels of ROR1; their invasive potential depends on ROR1 signaling. Although accumulating evidence has demonstrated that ROR1 is associated with aggressive breast-cancer phenotypes, the whole picture of its biological function remains poorly understood. In this study, we examined whether CS controls ROR1 function. Surface plasmon resonance analysis indicated that CSs were bound to ROR1 in the presence of WNT5A. The invasive activity of MDA-MB-231 cells enhanced by CSs was completely suppressed by ROR1 knockdown. In addition, knockdown of the CS biosynthetic enzymes CHST11 and CHST15 inhibited invasive activity, even in the presence of ROR1. These results suggest that CS is required to induce an ROR1-dependent, aggressive MDA-MB-231 phenotype. ROR1 signaling in MDA-MB-231 cells activated c-Jun N-terminal kinase (JNK), leading to increased invasive potential; moreover, exogenous CSs activated JNK. MDA-MB-231 cells express DKK1, a tumor suppressor factor that binds to CS, at high levels. Knockdown of DKK1 enhanced CS-stimulated tumor invasion activity of MDA-MB-231 cells, suggesting that DKK1 sequesters CS to block ROR1/JNK signaling. These results showed that CSs promotes cancer aggressiveness through the ROR1−JNK axis in MDA-MB-231 cells.

Molecular networks of hepatoblastoma predisposition and oncogenesis in Beckwith-Wiedemann syndrome.

Beckwith‐Wiedemann Syndrome (BWS) is the most common human overgrowth disorder caused by structural and epigenetic changes to chromosome 11p15. Patients with BWS are predisposed to developing hepatoblastoma (HB). To better understand the mechanism of HB oncogenesis in this cancer predisposition background, we performed the first multi‐dimensional study of HB samples collected from patients diagnosed with BWS. This multi‐omic investigation of seven BWS HB and five matched nontumor BWS liver samples from 7 unique patients included examination of whole exome sequences, messenger RNA/microRNA expression, and methylation levels to elucidate the genomic, transcriptomic, and epigenomic landscape of BWS‐associated HB. We compared the transcriptional profiles of the BWS samples, both HB and nontumor, to that of control livers. Genes differentially expressed across BWS tissues were identified as BWS HB predisposition factors; this gene group included cell cycle regulators, chromatin organizers, and WNT, mitogen‐activated protein kinase (MAPK), and phosphoinositide 3‐kinase (PI3K)/AKT members. We also compared transcriptional changes associated with non‐syndromic HB carrying BWS‐like 11p15 alterations compared to those without, as well as to BWS HB. Through this analysis, we identified factors specific to 11p15‐altered HB oncogenesis, termed the BWS oncogenesis network. We propose that 11p15 alterations drive HB oncogenesis by initially dysregulating cell‐cycle regulators and chromatin organizers, including histone deacetylase 1 (HDAC1), ATP‐dependent helicase X, and F‐Box and WD repeat domain containing 7. Furthermore, we found oncogenic factors such as dickkopf WNT signaling pathway inhibitor 1 and 4, WNT16, forkhead box O3 (FOXO3), and MAPK10 are differentially expressed in 11p15‐altered HB in both the BWS and non‐syndromic backgrounds. These genes warrant further investigation as diagnostic or therapeutic targets.

Intratumoral heterogeneity of MYC drives medulloblastoma metastasis and angiogenesis.

Intratumoral heterogeneity is crucially involved in metastasis, resistance to therapy, and cancer relapse. Amplifications of the proto-oncogene MYC display notable heterogeneity at the single-cell level and are associated with a particularly dismal prognosis in high-risk medulloblastomas (MBs). The aim of this study was to establish the relevance of interclonal cross-talk between MYC-driven and non-MYC-driven MB cells. We used fluorescence in situ hybridization, single-cell transcriptomics, and immunohistochemistry, in vitro isogenic cell models, non-targeted proteomics, mass spectrometry-based metabolite quantification, HUVECs tube formation assay, and orthotopic in vivo experiments to investigate interclonal cross-talk in MB. We found that the release of lactate dehydrogenase A (LDHA) from MYC-driven cells facilitates metastatic seeding and outgrowth, while secretion of dickkopf WNT signaling pathway inhibitor 3 from non-MYC-driven cells promotes tumor angiogenesis. This tumor-supporting interaction between both subclones was abrogated by targeting the secretome through pharmacological and genetic inhibition of LDHA, which significantly suppressed tumor cell migration. Our study reveals the functional relevance of clonal diversity and highlights the therapeutic potential of targeting the secretome to interrupt interclonal communication and progression in high-risk MB.

Uremic Toxin Lanthionine Induces Endothelial Cell Mineralization In Vitro.

Vascular calcification (VC) is a pathological event caused by the unusual deposition of minerals in the vascular system, representing the leading cause of cardiovascular mortality in chronic kidney disease (CKD). In CKD, the deregulation of calcium and phosphate metabolism, along with the effect of several uremic toxins, act as key processes conveying altered mineralization. In this work, we tested the ability of lanthionine, a novel uremic toxin, to promote calcification in human endothelial cell cultures (Ea.hy926). We evaluated the effects of lanthionine, at a concentration similar to that actually detected in CKD patients, alone and under pro-calcifying culture conditions using calcium and phosphate. In pro-calcific culture conditions, lanthionine increased both the intracellular and extracellular calcium content and induced the expression of Bone Morphogenetic Protein 2 (BMP2) and RUNX Family Transcription Factor 2 (RUNX2). Lanthionine treatment, in pro-calcifying conditions, raised levels of tissue-nonspecific alkaline phosphatase (ALPL), whose expression also overlapped with Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1) gene expression, suggesting a possible role of the latter gene in the activation of ALPL. In addition, treatment with lanthionine alone or in combination with calcium and phosphate reduced Inorganic Pyrophosphate Transport Regulator (ANKH) gene expression, a protective factor toward the mineralizing process. Moreover, lanthionine in a pro-calcifying condition induced the activation of ERK1/2, which is not associated with an increase in DKK1 protein levels. Our data underscored a link between mineral disease and the alterations of sulfur amino acid metabolisms at a cell and molecular level. These results set the basis for the understanding of the link between uremic toxins and mineral-bone disorder during CKD progression.