DKK3 Research Articles

Macrophage-to-osteocyte communication: Impact in a 3D in vitro implant-associated infection model

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1β and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. Statement of significanceBiomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication.

DKK1-SE recruits AP1 to activate the target gene DKK1 thereby promoting pancreatic cancer progression

Super-enhancers are a class of DNA cis-regulatory elements that can regulate cell identity, cell fate, stem cell pluripotency, and even tumorigenesis. Increasing evidence shows that epigenetic modifications play an important role in the pathogenesis of various types of cancer. However, the current research is far from enough to reveal the complex mechanism behind it. This study found a super-enhancer enriched with abnormally active histone modifications in pancreatic ductal adenocarcinoma (PDAC), called DKK1-super-enhancer (DKK1-SE). The major active component of DKK1-SE is component enhancer e1. Mechanistically, AP1 induces chromatin remodeling in component enhancer e1 and activates the transcriptional activity of DKK1. Moreover, DKK1 was closely related to the malignant clinical features of PDAC. Deletion or knockdown of DKK1-SE significantly inhibited the proliferation, colony formation, motility, migration, and invasion of PDAC cells in vitro, and these phenomena were partly mitigated upon rescuing DKK1 expression. In vivo, DKK1-SE deficiency not only inhibited tumor proliferation but also reduced the complexity of the tumor microenvironment. This study identifies that DKK1-SE drives DKK1 expression by recruiting AP1 transcription factors, exerting oncogenic effects in PDAC, and enhancing the complexity of the tumor microenvironment.

RhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals

BackgroundRecombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro.MethodsrhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes.ResultsThe data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo.ConclusionsAltogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

Abstract 1024: Differential gene expression of colorectal cancer biomarkers in Hispanic individuals

Abstract Colorectal cancer (CRC) is a leading cause of cancer death among men and women in Puerto Rico and the United States. About 60-70% of CRC cases are found at advanced stages (III/IV), suggesting limited adherence to available screening methods. Several molecular biomarkers have been identified as discriminatory for CRC (e.g., KRAS, NRAS, MSI), improving CRC management and patient outcomes. This study evaluates the differential expression patterns of DKK3, PKM2, IGFBP2, and SEPT9 genes between matched CRC tumors and adjacent mucosa samples. The secondary outcome was to examine the association between the presence of the selected biomarkers in tissue compared to blood samples. Using a retrospective study design, we examined 112 tissue samples of Hispanic individuals with a pathology-confirmed CRC diagnosis recruited throughout the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR). We quantified expression levels of target genes by RT-qPCR normalized with endogenous control GAPDH, using TaqMan Gene Expression Assay in the QuantStudio 3 (Applied Biosystems). Wilcoxon signed-rank test was used to calculate differences between gene expression from tumor and adjacent mucosal tissue samples. Additionally, 39 paired blood and tissue samples were analyzed to explore the association between biomarkers in these two types of samples. Protein biomarkers were measured using a commercially available ELISA kit according to manufacturer protocols (Thermo Scientific Federick, MD and Biosource San Diego, CA). mSEPT9 was measured using the Epi proColon® 2.0 CE test. The association between biomarker presence in tissue and blood samples was assessed using the Chi-square test of independence, with biomarker concentrations dichotomized at the median value. Statistical analyses were performed using Stata 17 and R version 4.3.1. DKK3 and IGFBP2 expression levels were higher among CRC tumor samples. However, expression levels of PKM2 and SEPT9 genes were slightly lower. DKK3 was the only target gene significantly different (p<0.05) when comparing overall gene expression between the matched samples. Upon comparison between early and advanced CRC stages, DKK3 expression levels were higher in advanced-stage tumor samples when compared to adjacent mucosal tissue samples (p<0.05). Moreover, a significant association between IGFBG2 presence in tissue compared to blood was detected at a 90% confidence level (p-value<0.10). This association was evident in early-stage CRC cases; there is a relationship between IGFBG2 biomarker presence in tissue and IGFBG2 biomarker presence in blood (p<0.05). Our findings indicate DKK3 and IGFBP2 as potential biomarkers for CRC progression and early detection. The study’s insights into the differential expression patterns of these biomarkers can inform targeted screening strategies. Further research is needed to assess their potential to enhance early tumor detection in Hispanics. Citation Format: Elba V. Caraballo-Rivera, Hilmaris Centeno-Girona, Gabriel De Jesus-Astacio, Camille Zenon-Melendez, Brenda Torres-Velasquez, Madeline Martir-Ocasio, Marcia R. Cruz-Correa. Differential gene expression of colorectal cancer biomarkers in Hispanic individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1024.

Transcriptomic Analysis Reveals Intrinsic Abnormalities in Endometrial Polyps.

Endometrial polyps (EPs) are benign overgrowths of the endometrial tissue lining the uterus, often causing abnormal bleeding or infertility. This study analyzed gene expression differences between EPs and adjacent endometrial tissue to elucidate intrinsic abnormalities promoting pathological overgrowth. RNA sequencing of 12 pairs of EPs and the surrounding endometrial tissue from infertile women revealed 322 differentially expressed genes. Protein-protein interaction network analysis revealed significant alterations in specific signaling pathways, notably Wnt signaling and vascular smooth muscle regulation, suggesting these pathways play critical roles in the pathophysiology of EPs. Wnt-related genes DKK1 and DKKL1 were upregulated, while GPC3, GREM1, RSPO3, SFRP5, and WNT10B were downregulated. Relevant genes for vascular smooth muscle contraction were nearly all downregulated in EPs, including ACTA2, ACTG2, KCNMB1, KCNMB2, MYL9, PPP1R12B, and TAGLN. Overall, the results indicate fundamental gene expression changes promote EP formation through unrestrained growth signaling and vascular defects. The intrinsic signaling abnormalities likely contribute to clinical symptoms of abnormal uterine bleeding and infertility common in EP patients. This analysis provides molecular insights into abnormal endometrial overgrowth to guide improved diagnostic and therapeutic approaches for this troublesome women's health condition. Confirmation of expanded cohorts and further investigations into implicated regulatory relationships are warranted.

Investigation of iso-propylchaetominine anticancer activity on apoptosis, cell cycle and Wnt signaling pathway in different cancer models

Dysregulation of the Wnt signaling pathway contributes to the development of many cancer types. Natural compounds produced with biotechnological systems have been the focus of research for being a new drug candidate both with unlimited resources and cost-effective production.In this study, it was aimed to reveal the effects of isopropylchaetominine on cytotoxic, cytostatic, apoptotic and Wnt signaling pathways in brain, pancreatic and prostate cancer.The IC50 values of isopropylchaetominine in U-87 MG, PANC1, PC3 and LNCaP cells were calculated as 91.94 μM, 41.68 μM, 54.54 μM and 7.86 μM in 72nd h, respectively. The metabolite arrests the cell cycle in G0/G1 phase in each cancer cells. Iso-propylchaetominine induced a 4.3-fold and 1.9-fold increase in apoptosis in PC3 and PANC1 cells, respectively. The toxicity of isopropylchaetominine in healthy fibroblast cells was assessed using the annexin V method, and no significant apoptotic activity was observed between the groups treated with the active substance and untreated. In U-87 MG, PANC1, PC3, and LNCaP cells under treatment with isopropylchaetominin, the expression levels of DKK3, TLE1, AES, DKK1, FRZB, DAB2, AXIN1/2, PPARD, SFRP4, APC and SOX17 tumor suppressor genes increased significantly. Decreases in expression of Wnt1, Wnt2, Wnt3, Wnt4, Wnt5, Wnt6, Wnt10, Wnt11, FRZ2, FRZ3, FRZ7, TCF7L1, BCL9, PYGO, CCND2, c-MYC, WISP1 and CTNNB1 oncogenic genes were detected.All these result shows that isopropylchaetominine can present promising new treatment strategy in different cancer types.

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.

Expression of DKK1 in Endometrial Endometrioid Carcinoma and Its Correlation with Wnt/β-catenin Signalling Pathway.

Endometrial cancer is the most prevalent form of cancer affecting female reproductive organs. The most common histologic type, endometrioid carcinoma, accounts for 75-80% of all endometrial cancer cases. Studies on DKK1 expression profiles and their inhibitory role in the Wnt signalling pathway in the genesis and development of endometrial carcinoma are scarce. This study aimed to investigate DKK1 expression in endometrial carcinoma and its correlation with the Wnt/β-catenin pathway. A total of 160 formalin-fixed paraffin-embedded samples were included in this study (50 cases of endometrial atypical hyperplasia, 50 cases of endometrioid endometrial carcinoma, 30 cases of proliferative endometrium and 30 cases of secretory endometrium). The expression patterns of DKK1, E-cadherin, β-catenin and c-Myc in endometrial atypical hyperplasia and carcinoma were investigated and compared with that of proliferative and secretory endometrium. Immunohistochemistry and analysis were performed from July 2018 to June 2020. A decreasing pattern of immunopositivity for DKK1, E-cadherin and β-catenin from proliferative/secretory endometrium to endometrial atypical hyperplasia and endometrioid carcinoma was found. Increasing c-Myc immunopositivity was noted from proliferative/secretory endometrium to endometrial atypical hyperplasia and endometrioid carcinoma. Moreover, decreasing DKK1 immunopositivity was well correlated with E-cadherin, β-catenin and c-Myc immunopositivity. Decreasing DKK1 positivity from benign endometrium to endometrioid carcinoma suggests a negative regulatory function of DKK1 in endometrioid carcinoma. DKK1 is downregulated in the Wnt signalling pathway in endometrioid endometrial carcinoma. Therefore, DKK1 is promising as a biomarker for screening endometrioid carcinoma. Future studies should examine the reactivation of the DKK1 gene, which may be a valuable strategy for antagonising the Wnt signalling pathway.

Mir-4699 promotes the osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are drawing considerable attention in the field of regenerative medicine due to their differentiation capabilities. The miRNAs are among the most important epigenetic regulators of MSC differentiation. Our previous study identified miR-4699 as a direct suppressor of the DKK1 and TNSF11 gene expression. However, the precise osteogenic-related phenotype or mechanism caused by miR-4699 change has yet to be dealt with in depth. In the present study, miR-4699 mimics were transfected into human Adipose tissue-derived mesenchymal stem cells (hAd-MSCs) and osteoblast marker gene expression (RUNX2, ALP, and OCN), was analyzed to investigate whether miR-4699 promotes osteoblast differentiation of hAd-MSCs through targeting the DKK-1 and TNFSF11. We further examined and compared the effects of recombinant human BMP2 with miR-4699 on cell differentiation. In addition to quantitative PCR, analysis of alkaline phosphatase activity, calcium content assay, and Alizarin red staining were used to explore osteogenic differentiation. To evaluate the effect of miR-4699 on its target gene (on protein level) we utilized the western blotting technique. The overexpression of miR-4699 in hAd-MSCs resulted in the stimulation of alkaline phosphatase activity, osteoblast mineralization, and the expression of RUNX2, ALP, and OCN osteoblast marker genes. Our findings indicated that miR-4699 supported and synergized the BMP2-induced osteoblast differentiation of mesenchymal stem cells. We suggest, thereof, the utilization of hsa-miR-4699 for further in vivo experimental investigation to reveal the potential therapeutic impact of regenerative medicine for different types of bone defects.

Upregulation of the Oct3/4 Network in Basal Breast Cancer Is Associated with Its Metastatic Potential and Shows Tissue Dependent Variability.

Adaptive plasticity of Breast Cancer stem cells (BCSCs) is strongly correlated with cancer progression and resistance, leading to a poor prognosis. In this study, we report the expression profile of several pioneer transcription factors of the Oct3/4 network associated with tumor initiation and metastasis. In the triple negative breast cancer cell line (MDA-MB-231) stably transfected with human Oct3/4-GFP, differentially expressed genes (DEGs) were identified using qPCR and microarray, and the resistance to paclitaxel was assessed using an MTS assay. The tumor-seeding potential in immunocompromised (NOD-SCID) mice and DEGs in the tumors were also assessed along with the intra-tumor (CD44+/CD24-) expression using flow cytometry. Unlike 2-D cultures, the Oct3/4-GFP expression was homogenous and stable in 3-D mammospheres developed from BCSCs. A total of 25 DEGs including Gata6, FoxA2, Sall4, Zic2, H2afJ, Stc1 and Bmi1 were identified in Oct3/4 activated cells coupled with a significantly increased resistance to paclitaxel. In mice, the higher Oct3/4 expression in tumors correlated with enhanced tumorigenic potential and aggressive growth, with metastatic lesions showing a >5-fold upregulation of DEGs compared to orthotopic tumors and variability in different tissues with the highest modulation in the brain. Serially re-implanting tumors in mice as a model of recurrence and metastasis highlighted the sustained upregulation of Sall4, c-Myc, Mmp1, Mmp9 and Dkk1 genes in metastatic lesions with a 2-fold higher expression of stem cell markers (CD44+/CD24-). Thus, Oct3/4 transcriptome may drive the differentiation and maintenance of BCSCs, promoting their tumorigenic potential, metastasis and resistance to drugs such as paclitaxel with tissue-specific heterogeneity.

Role of Dickkopf-3 in Blood Pressure Regulation in Mice and Hypertensive Rats.

Dkk3 (Dickkopf-3) is a secreted glycoprotein known for its proapoptotic and angiogenic activity. The role of Dkk3 in cardiovascular homeostasis is largely unknown. Remarkably, the Dkk3 gene maps within a chromosome segment linked to the hypertensive phenotype in spontaneously hypertensive rats (SHR). We used Dkk3-/- mice or stroke-resistant (sr) and stroke-prone (sp) SHR to examine the role of Dkk3 in the central and peripheral regulation of blood pressure (BP). We used lentiviral expression vector to rescue Dkk3 in knockout mice or to induce Dkk3 overexpression or silencing in SHR. Genetic deletion of Dkk3 in mice enhanced BP and impaired endothelium-dependent acetylcholine-induced relaxation of resistance arteries. These alterations were rescued by restoring Dkk3 expression either in the periphery or in the central nervous system (CNS). Dkk3 was required for the constitutive expression of VEGF (vascular endothelium growth factor), and the action of Dkk3 on BP and endothelium-dependent vasorelaxation was mediated by VEGF-stimulated phosphatidylinositol-3-kinase pathway, leading to eNOS (endothelial NO synthase) activation both in resistance arteries and the CNS. The regulatory function of Dkk3 on BP was confirmed in SHR stroke-resistant and SHR stroke-prone in which was blunted in both resistance arteries and brainstem. In SHR stroke-resistant, lentiviral expression vector-induced Dkk3 expression in the CNS largely reduced BP, whereas Dkk3 knock-down further enhanced BP. In SHR stroke-prone challenged with a hypersodic diet, lentiviral expression vector-induced Dkk3 expression in the CNS displayed a substantial antihypertensive effect and delayed the occurrence of stroke. These findings demonstrate that Dkk3 acts as peripheral and central regulator of BP by promoting VEGF expression and activating a VEGF/Akt (protein kinase B)/eNOS hypotensive axis.

A systematic and comprehensive analysis of T cell exhaustion related to therapy in lung adenocarcinoma tumor microenvironment.

Background: T cell exhaustion (TEX) is an important immune escape mechanism, and an in-depth understanding of it can help improve cancer immunotherapy. However, the prognostic role of TEX in malignant lung adenocarcinoma (LUAD) remains unclear. Methods: Through TCGA and GEO datasets, we enrolled a total of 498 LUAD patients. The patients in TCGA-LUAD were unsupervised clustered into four clusters according to TEX signaling pathway. WGCNA analysis, survival random forest analysis and lasso regression analysis were used to select five differentially expressed genes among different clusters to construct a TEX risk model. The risk model was subsequently validated with GEO31210. By analyzing signaling pathways, immune cells and immune checkpoints using GSEA, GSVA and Cibersortx, the relationship between TEX risk score and these variables was evaluated. In addition, we further analyzed the expression of CCL20 at the level of single-cell RNA-seq and verified it in cell experiments. Results: According to TEX signaling pathway, people with better prognosis can be distinguished. The risk model constructed by CD109, CCL20, DKK1, TNS4, and TRIM29 genes could further accurately identify the population with poor prognosis. Subsequently, it was found that dendritic cells, CD44 and risk score were closely related. The final single-cell sequencing suggested that CCL2O is a potential therapeutic target of TEX, and the interaction between TEX and CD8 + T is closely related. Conclusion: The classification of T cell depletion plays a crucial role in the clinical decision-making of lung adenocarcinoma and needs to be further deepened.

Reserve of Wnt/β-catenin Signaling Alleviates Mycoplasma pneumoniae P1-C-induced Inflammation in airway epithelial cells and lungs of mice

Mycoplasma pneumoniae (M. pneumoniae) is the most common pathogen of respiratory tract infections in both children and adults. M. pneumoniae P1 adhesin plays an important role in the pathogenesis of M. pneumoniae infection by mediating the attachment of pathogen to host cells. The inoculation of C-terminal residuals of P1 (P1-C) showed a protective role from M. pneumoniae infection. Accumulated evidence suggests that the Wnt/β-Catenin signaling is implicated in regulation of inflammatory responses to bacterial infections. However, mechanisms underlying the regulatory roles of Wnt signaling in host cells in response to M. pneumoniae infections are incompletely understood. In the present study, the impact and molecular mechanism of Wnt/β-catenin signaling in immune responses induced by M. pneumoniae P1-C were investigated. The results demonstrated that the P1-C could activate Wnt/β-catenin and Toll-like receptor (TLR) signaling in primary mouse airway epithelial cells cultured in an air-liquid interface (ALI) state. Interestingly, the inhibition of Wnt/β-catenin signaling by an adenovirus-mediated Wnt inhibitor Dickkopf-1 (Dkk1) gene transduction alleviated the P1-C induced inflammation fibrosis in mouse lung, accompanied by the reduced expression of epithelial mesenchymal transition (EMT) markers. Mechanistical analysis further demonstrated that the Dkk1 could suppress the expression of JAK2/STAT1-STAT3 and Caspase3, 8/Bax signaling in mouse lung tissues. In vitro study further revealed that XAV939, a small molecule of Wnt/β-catenin inhibitor, inhibited the P1-C-activated TLR4/MyD88 signaling and cytokine productions in primary mouse airway ALI epithelial cells. This study thus provides an insight into the function of Wnt/β-catenin signaling in regulation of the pathogenesis of M. pneumoniae infection, suggesting that targeting Wnt/β-catenin signaling by gene transduction of Dkk1, or pharmacological molecules of inhibitor may be a promised approach that worthy of further investigation in the treatment of M. pneumoniae pneumonia.

SMARCC2 mediates the regulation of DKK1 by the transcription factor EGR1 through chromatin remodeling to reduce the proliferative capacity of glioblastoma

Switch/sucrose-nonfermenting (SWI/SNF) complexes play a key role in chromatin remodeling. Recent studies have found that SMARCC2, as the core subunit of the fundamental module of the complex, plays a key role in its early assembly. In this study, we found a unique function of SMARCC2 in inhibiting the progression of glioblastoma by targeting the DKK1 signaling axis. Low expression of SMARCC2 is found in malignant glioblastoma (GBM) compared with low-grade gliomas. SMARCC2 knockout promoted the proliferation of glioblastoma cells, while its overexpression showed the opposite effect. Mechanistically, SMARCC2 negatively regulates transcription by dynamically regulating the chromatin structure and closing the promoter region of the target gene DKK1, which can be bound by the transcription factor EGR1. DKK1 knockdown significantly reduced the proliferation of glioblastoma cell lines by inhibiting the PI3K–AKT pathway. We also studied the functions of the SWIRM and SANT domains of SMARCC2 and found that the SWIRM domain plays a more important role in the complete chromatin remodeling function of SMARCC2. In addition, in vivo studies confirmed that overexpression of SMARCC2 could significantly inhibit the size of intracranial gliomas in situ in nude mice. Overall, this study shows that SMARCC2, as a tumor suppressor, inhibits the proliferation of glioblastoma by targeting the transcription of the oncogene DKK1 through chromatin remodeling, indicating that SMARCC2 is a potentially attractive therapeutic target in glioblastoma.

Establishment of anti-DKK3 peptide for the cancer control in head and neck squamous cell carcinoma (HNSCC)

BackgroundHead and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck. We identified cancer-specific genes in HNSCC and focused on DKK3 expression. DKK3 gene codes two isoforms of proteins (secreted and non-secreted) with two distinct cysteine rich domains (CRDs). It is reported that DKK3 functions as a negative regulator of oncogenic Wnt signaling and, is therefore, considered to be a tumor suppressor gene. However, our series of studies have demonstrated that DKK3 expression is specifically high in HNSCC tissues and cells, and that DKK3 might determine the malignant potentials of HNSCC cells via the activation of Akt. Further analyses strongly suggested that both secreted DKK3 and non-secreted DKK3 could activate Akt signaling in discrete ways, and consequently exert tumor promoting effects. We hypothesized that DKK3 might be a specific druggable target, and it is necessary to establish a DKK3 inhibitor that can inhibit both secreted and non-secreted isoforms of DKK3.MethodsUsing inverse polymerase chain reaction, we generated mutant expression plasmids that express DKK3 without CRD1, CRD2, or both CRD1 and CRD2 (DKK3ΔC1, DKK3ΔC2, and DKK3ΔC1ΔC2, respectively). These plasmids were then transfected into HNSCC-derived cells to determine the domain responsible for DKK3-mediated Akt activation. We designed antisense peptides using the MIMETEC program, targeting DKK3-specific amino acid sequences within CRD1 and CRD2. The structural models for peptides and DKK3 were generated using Raptor X, and then a docking simulation was performed using CluPro2. Afterward, the best set of the peptides was applied into HNSCC-derived cells, and the effects on Akt phosphorylation, cellular proliferation, invasion, and migration were assessed. We also investigated the therapeutic effects of the peptides in the xenograft models.ResultsTransfection of mutant expression plasmids and subsequent functional analyses revealed that it is necessary to delete both CRD1 and CRD2 to inhibit Akt activation and inhibition of proliferation, migration, and invasion. The inhibitory peptides for CRD1 and CRD2 of DKK3 significantly reduced the phosphorylation of Akt, and consequently suppressed cellular proliferation, migration, invasion and in vivo tumor growth at very low doses.ConclusionsThis inhibitory peptide represents a promising new therapeutic strategy for HNSCC treatment.

The RNA-binding protein PCBP1 represses lung adenocarcinoma progression by stabilizing DKK1 mRNA and subsequently downregulating β-catenin

BackgroundPolyC-RNA-binding protein 1 (PCBP1) functions as a tumour suppressor and RNA regulator that is downregulated in human cancers. Here, we aimed to reveal the biological function of PCBP1 in lung adenocarcinoma (LUAD).MethodsFirst, PCBP1 was identified as an important biomarker that maintains LUAD through The Cancer Genome Atlas (TCGA) project screening and confirmed by immunohistochemistry and qPCR. Via colony formation, CCK8, IncuCyte cell proliferation, wound healing and Transwell assays, we confirmed that PCBP1 was closely related to the proliferation and migration of LUAD cells. The downstream gene DKK1 was discovered by RNA sequencing of PCBP1 knockdown cells. The underlying mechanisms were further investigated using western blot, qPCR, RIP, RNA pulldown and mRNA stability assays.ResultsWe demonstrate that PCBP1 is downregulated in LUAD tumour tissues. The reduction in PCBP1 promotes the proliferation, migration and invasion of LUAD in vitro and in vivo. Mechanistically, the RNA-binding protein PCBP1 represses LUAD by stabilizing DKK1 mRNA. Subsequently, decreased expression of the DKK1 protein relieves the inhibitory effect on the Wnt/β-catenin signalling pathway. Taken together, these results show that PCBP1 acts as a tumour suppressor gene, inhibiting the tumorigenesis of LUAD.ConclusionsWe found that PCBP1 inhibits LUAD development by upregulating DKK1 to inactivate the Wnt/β-catenin pathway. Our findings highlight the potential of PCBP1 as a promising therapeutic target.

Comparing the Effects of Regular Aerobic Training, Hyaluronic Acid, and Mesenchymal Stem Cells on Wnt/β-Catenin Signaling of Cardiac Tissue in Rats with the Experimental Model of Knee Osteoarthritis

Background: The present study aimed to compare the effects of three therapeutic methods, including regular aerobic training, hyaluronic acid (HA), and mesenchymal stem cells, on Wnt/β-catenin signaling of cardiac tissue in rats with the experimental model of knee osteoarthritis. Methods: Sixty-three male rats were divided into nine groups (seven in each group): (1) healthy control, (2) patient control, (3) sham, (4) saline, (5) exercise (EXT), (6) mesenchymal stem cells (MSCs), (7) hyaluronic acid, (8) EXT + MSCs, and (9) EXT + HA. After inducing the osteoarthritis (OA) model, we conducted 5 days of running on the treadmill for five weeks for the EXT group. Also, HA was injected intra-articularly. After 12 to 14 hours of fasting and 72 hours following the last training session, we conducted cardiac tissue sampling for β-catenin, GSK-3β, Wnt, Fz, TCF, and DKK1 analysis. We used RT-PCR to analyze the expression of the β-catenin, GSK-3β, Wnt, Fz, TCF, and DKK1 genes in cardiac tissue. The data were analyzed using one-way ANOVA and Tukey's post hoc test (P < 0.05). Results: Osteoarthritis induction significantly decreased the expression of the GSK-3β and DKK1 genes but significantly increased the expression of the β-catenin, Wnt, Fz, and TCF genes of cardiac tissue compared to the control group. However, the EXT, HA, MSC, and combination methods increased the expression of the GSK-3β and DKK1 genes but decreased the expression of the β-catenin, Wnt, Fz, and TCF genes of cardiac tissue, which were significant in the combination group. Conclusions: Regular exercise, along with HA and MSCs, may have protective effects for the following reasons: (1) reducing the expression of the β-catenin, Wnt, Fz, and TCF genes; (2) increasing the expression of the GSK-3β and DKK1 genes in cardiac tissue; and (3) inhibiting Wnt signaling in the heart.

Multi-biomarker approach and IBR index to evaluate the effects of bisphenol A on embryonic stages of zebrafish (Danio rerio)

This study assessed the effects of Bisphenol A in embryonic stages of zebrafish, applying an IBR multi-biomarker approach that included alterations in growth and oxidative status and relates it with the expression of Nrf1, Nrf2, Wnt3a, Wnt8a, COX-2, Qdpra, and DKK1 genes. For this purpose, we exposed zebrafish embryos to eight environmentally relevant concentrations of BPA (220, 380, 540, 700, 860, 1180, 1340, and 1500 ng L−1) until 96 h post-fertilization. Our results show that BPA induces several malformations in embryos (developmental delay, hypopigmentation, tail malformations, and on), leading to their death. The LC50, EC50 of malformations, and teratogenic index (TI) were 1234.60 ng L−1, 987.77 ng L−1, and 1.25, respectively; thus, this emerging contaminant is teratogenic. Regarding oxidative stress and gene expression, we demonstrated BPA altered oxidative status and the gene expression in embryos of Danio rerio.

Fibroblast morphology, growth rate and gene expression in facial melasma

BackgroundIn addition to melanocytic hyperfunction, changes are observed in the upper dermis of melasma, and fibroblasts play a central role in collagen synthesis and pigmentation induction. ObjectiveTo explore the morphology, growth rate, and gene expression profile of fibroblasts from the skin with melasma in comparison to fibroblasts from the adjacent healthy skin. MethodsTen women with facial melasma were biopsied (lesion and adjacent healthy skin), and the fragments were processed for fibroblast culture. Samples from five participants were seeded to evaluate growth (days 2, 5 and 8) and senescence (SA-β-gal) curves. The samples from the other participants were submitted to real-time PCR to comparatively evaluation of the expression of 39 genes. ResultsCultured fibroblasts from melasma skin were morphologically less fusiform in appearance and on average a 34% (95% CI 4%‒63%) greater proportion of cells labeled with SA-β-gal than the fibroblasts from the adjacent skin. The cell growth rate was lower for the melasma samples after eight days (p < 0.01). TheWNT3A, EDN3, ESR2, PTG2, MMP1, and SOD2 genes were up-regulated, whereas the COL4A1, CSF2, DKK3, COL7A1, TIMP4, CCL2, and CDH11 genes were down-regulated in melasma skin fibroblasts when compared to the ones from adjacent healthy skin. Study limitationsSmall sample size; absence of functional tests. ConclusionsFibroblasts from the skin with melasma showed a lower growth rate, less fusiform morphology and greater accumulation of SA-β-gal than those from adjacent photo exposed skin. Moreover, their gene expression profile comprised factors that may contribute to upper dermis damage and sustained melanogenesis.

DKK-1 Is Underexpressed in Mesenchymal Stem Cells from Patients with Ankylosing Spondylitis and Further Downregulated by IL-17

Dickkopf-1 (Dkk-1) is a key regulator of bone remodeling in spondyloarthropathies. Nevertheless, data regarding its expression in cells of pathophysiologic relevance, such as mesenchymal stem cells (MSCs), are lacking. Herein, we aimed to address DKK1 gene expression and Wnt pathway activation in MSCs from patients with ankylosing spondylitis (AS) and explore the effect of IL-17 on MSCs with respect to DKK-1 expression and Wnt pathway activation. Primary MSCs were isolated from the bone marrow of the femoral head of two patients with AS and two healthy controls undergoing orthopedic surgery. MSCs were cultured for 7 days in expansion medium and for 21 days in osteogenic medium in the presence or absence of IL-17A. Gene expression of DKK-1 and osteoblastic markers was determined by RT-PCR. Alkaline phosphatase activity, alizarin red and Van Kossa staining were used to assess osteoblastic function and mineralization capacity. DKK-1 was significantly downregulated in MSCs and osteoblasts from patients with AS compared to controls. Moreover, MSCs and osteoblasts from AS patients displayed increased Wnt pathway activation and enhanced osteoblastic activity, as indicated by increased expression of osteoblast marker genes and alkaline phosphatase activity. IL-17 downregulated DKK-1 expression and increased osteoblastic activity and mineralization capacity. DKK-1 is underexpressed in MSCs from AS patients compared to controls, whereas IL-17 has an inhibitory effect on DKK-1 expression and stimulates osteoblastic function. These data may have pathogenetic and clinical implications in AS.