DACT2 modulates atrial fibrillation through TGF/ß and Wnt signaling pathways.

Atrial fibrillation (AF) is a common cardiac arrhythmia that seriously affects the quality of life of patients. Effective treatment and prevention are important to control the morbidity and mortality of AF. It has been found that cardiac fibrosis promotes the onset and progression of AF. It is now known that transforming growth factor ß (TGF-ß), an important fibrotic cytokine, plays an important role in cardiac fibrosis by inducing myofibroblast activation via the activation of classical (SMAD-based) and non-classical (non-SMAD-based) signaling pathways. In addition, specific activation of the Wnt/ß-catenin pathway has been shown to promote the transformation of fibroblasts into myofibroblasts. In recent years, a new family of proteins, namely Disheveled-associated antagonist of beta-catenin (DACT) 2, can affect the Wnt/ß-catenin and TGF-ß signaling pathways by regulating the phosphorylation levels of these target proteins, which in turn affects the progression of fibrosis. The present study focuses on the effect of DACT2-guided ß-catenin on atrial fibrosis. It is expected that the summarized information can be helpful in the treatment of AF.

[Lycium barbarum miR2911-loaded exosomes promote spermatogenic function recovery in rats with non-obstructive azoospermia by regulating Wnt/ß-catenin signaling pathways].

OBJECTIVE: To investigate the effect of exosomes loaded with Lycium barbarum miRNA (Lb-miR2911) on spermatogenic function recovery in non-obstructive azoospermia (NOA) rats through cross-regulation of the Wnt/ß-catenin signaling pathways. METHODS: We established an NOA model in 30 four-week-old male SD rats by intraperitoneal injection of busulfan. At 5 weeks after modeling, we equally randomized the rats into a model control group (MC，untreated), an Lb-miR2911EXO group (Lb-miR2911EXO ，treated by intratesticular injection of Lb-miR2911-loaded exosomes), and a sham group (Shame，treated by intratesticular injection of exosomes-empty drug), with another 10 male SD rats taken as normal controls（NC）. We observed the uptake and metabolic changes of Lb-miR2911 in the testis tissue of the rats by RNA FISH at 2 and 6 weeks after treatment, detected cell proliferation, spermatogenesis and gene expressions of the Wnt/ß-catenin signaling pathways in the testis tissue by Transcriptome sequencing analysis combined with Western blot and RT-PCR at 12 weeks, evaluated the recovery of the spermatogenic function based on the testis tissue morphology and sperm quality, and assessed the organ toxicity of Lb-miR2911 in the tissue and organs of the rats based on histomorphological analysis and the levels of serum TNF-α, IL-1ß, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and other relevant indicators. RESULTS: After 12 weeks of treatment, histomorphological analysis showed regular arrangement of spermatogenic cells at all levels in the testis tissue, with a large number of mature sperm in the tubular lumen, and with significantly higher Johnsen scores, testis weight, testicular index, sperm concentration and sperm motility in the Lb-miR2911EXO than in the sham group (all P< 0.05). Compared with the model controls, the Lb-miR2911EXO group exhibited remarkably down-regulated gene expression of DACT3 (P< 0.05), up-regulated expressions of DVL2 and ß-catenin (P< 0.05), elevated levels of p-DVL2 and ß-catenin (nucleus) proteins (P< 0.05), increased expressions of cell proliferation-related genes CCND1, CCNE1 and CCNE2 (P< 0.05) and spermatogenesis-related genes DMC1, CCR6, JAM2 and KLC3 (P< 0.05). No pathological changes were observed in the lung, liver and kidney tissues of the rats, or in the levels of serum TNF-α, IL-1ß, AST, ALT, creatinine and urea nitrogen in the rats treated with Lb-miR2911EXO compared with the normal controls (P > 0.05). CONCLUSION: Lb-miR2911-loaded exosomes promote spermatogenic function recovery in NOA rats through cross-regulation of the DACT3, Wnt and ß-catenin signaling pathways.

Dishevelled-associated antagonist of ß-catenin homolog 3 (DACT3) suppresses glioma progression though Notch1 signaling pathway in ß-catenin-dependent manner.

The disheveled-associated antagonist of ß-catenin homolog 3 (DACT3) has been recognized as a tumor suppressor in various cancers. However, the function of DACT3 on glioma malignant progression along with potential molecular mechanisms is poorly clarified. This research aimed to investigate how DACT3 contributes to suppressing the progression of glioma. In our investigation, a pronounced decrease in DACT3 expression was observed in glioma tissues. Through the overexpression of DACT3, we noted a significant suppression in the proliferation, invasion, and migration of glioma cells, while concurrently observing an increase in cell adhesion. Our exploration into the molecular mechanisms revealed that DACT3 executes its tumor-suppressive role by impeding the expression of notch 1 intracellular domain (NICD) and translocating into the nucleus by downregulating the expression of ß-catenin. Consequently, this process leads to the suppression of Notch1 signaling. To summarize, our findings reveal the function of DACT3 to inhibit glioma progression via the Notch1 signaling pathway in ß-catenin dependent manner. This study stands as the pioneer in examining the role of DACT3 in glioma progression and comprehensively elucidating its molecular mechanisms in glioma development. Therefore, our results suggest that DACT3 holds promise as both a prognostic factor and a potential biomarker for guiding treatment strategies in glioma patients (Graphical Abstract).

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

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

Loss of Dact2 alleviates cisplatin-induced nephrotoxicity through regulation of the Igfl-MAPK pathway axis.

Wnt signaling is a principal pathway regulating the essential activities of cell proliferation. Here, we investigated the effect of Wnt/ß-catenin signaling on in vivo drug-induced renal injury through the deletion of Dact2, a Wnt antagonist, and deciphered the underlying mechanism. Wild-type (WT) and Dact2 knockout (KO) mice were administered a single intraperitoneal injection of cisplatin to induce renal injury. The injury was alleviated in Dact2 KO mice, which showed lower levels of blood urea nitrogen and creatinine. RNA sequencing revealed 194 differentially expressed genes (DEGs) between WT and Dact2 KO mouse kidney before cisplatin treatment. Among them, higher levels of Igf1, one of the Wnt target genes responsible for "Positive regulation of cell proliferation" in KO mice, were confirmed along with the induction of Ki67 expression. In RNA-seq analysis comparing WT and Dact2 KO mice after cisplatin treatment, genes related to "Apoptosis" and "Activation of mitogen-activated protein kinase (MAPK) activity" were among the downregulated DEGs in KO mice. These results were corroborated in western blotting of proteins related to apoptosis and proapoptotic MAPK pathway; the expression of which was found to be lower in cisplatin-treated KO mice. Importantly, ß-catenin was found to directly bind to and regulate the transcription of Igf1, leading to the alleviation of cisplatin-induced cytotoxicity by the Wnt agonist, CHIR-99021. In addition, Igf1 knockdown accelerated cisplatin-induced cytotoxicity, accompanied by the MAPK upregulation. Our findings suggest that Dact2 knockout could protect cisplatin-induced nephrotoxicity by inhibiting apoptosis, possibly through the regulation of the Igf1-MAPK axis associated with Wnt/ß-catenin signaling.

Loss of cancer-associated fibroblast-derived exosomal DACT3-AS1 promotes malignant transformation and ferroptosis-mediated oxaliplatin resistance in gastric cancer.

AIMS: Long non-coding RNAs (lncRNAs), as one of the components of exosomes derived from cancer-associated fibroblasts (CAFs), exhibit a crucial role in the pathogenesis and chemoresistance of gastric cancer (GC). Herein, we investigated the role and mechanism of a novel lncRNA disheveled binding antagonist of beta catenin3 antisense1 (DACT3-AS1) and its involvement in GC. METHODS: DACT3-AS1 was identified by RNA-sequencing and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The functional role of DACT3-AS1 in GC was evaluated using in vitro and in vivo experiments including Transwell assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay, immunoblotting, and xenograft tumor mouse model. Dual-luciferase reporter assay was performed to assess the association between genes. RESULTS: DACT3-AS1 was downregulated and involved in poor prognosis of patients with GC. The results from both in vitro and in vivo experiments showed that DACT3-AS1 suppressed cell proliferation, migration, and invasion through targeting miR-181a-5p/sirtuin 1 (SIRT1) axis. Additionally, DACT3-AS1 was transmitted from CAFs to GC cells mainly via exosomes. Exosomal DACT3-AS1 alleviated xenograft tumor growth. DACT3-AS1 conferred sensitivity of cancer cells to oxaliplatin through SIRT1-mediated ferroptosis both in vitro and in vivo. CONCLUSIONS: CAFs-derived exosomal DACT3-AS1 is a suppressive regulator in malignant transformation and oxaliplatin resistance. DACT3-AS1 could be used for diagnosis and treatment of GC.

Mitochondrial dysfunction due to in vitro exposure to atrazine and its metabolite in striatum.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) has been described as a potential toxic for dopaminergic metabolism both in vivo and in vitro. Its main metabolite diamino-chloro triazine (DACT) has been shown to achieve higher levels in brain tissue than atrazine. The aim of this study was to evaluate the in vitro effects of atrazine and DACT on striatal mitochondrial function, active oxygen species generation, and nitric oxide (NO) content. Incubation of mitochondria with atrazine (10 µM) was not able to modify oxygen consumption. However, a 50% increase in malate-glutamate state 4 respiratory rates was observed after DACT treatment (100 µM) without changes in respiratory state 3. Atrazine was able to inhibit complex I-III activity by 30% and DACT induced a tendency to decrease by 17% in the striatum. Regarding reactive oxygen species (ROS), DACT increased H2 O2 production by 43%. Also, superoxide anion levels were higher (14%) after atrazine exposure than in control mitochondria. Incubation of striatal mitochondria with atrazine and DACT induced membrane depolarization by 15% and 19%, respectively. Also, atrazine increased NO content by 10% but no significant changes were observed after exposure of mitochondria to DACT. Glutathione peroxidase activity was inhibited (56%) by DACT and atrazine inhibited superoxide dismutase activity by 60%. Also, cardiolipin oxidation (15%) was observed after atrazine treatment. Summing up, the obtained results suggest that in vitro atrazine and DACT induce ROS production affecting striatal mitochondrial function. The atrazine effects would be attributed to a direct effect on the mitochondrial respiratory chain and superoxide dismutase activity while DACT appears to disturb glutathione-related enzyme system.

CircDUSP1 regulates tumor growth, metastasis, and paclitaxel sensitivity in triple-negative breast cancer by targeting miR-761/DACT2 signaling axis.

Triple-negative breast cancer TNBC) is a malignant tumor with high incidence and high mortality that threaten the health of women worldwide. Circular RNAs (circRNAs) are a new class of noncoding RNAs that participate in the biological processes of various tumors, but the regulatory roles of circRNAs in TNBC have not been fully elucidated. In this study, the expression and characterization of circDUSP1 was detected via quantitative real-time PCR, nuclear-cytoplasmic fractionation assay, and fluorescence in situ hybridization. Then, in vitro and in vivo functional experiments were performed to evaluate the effects of circDUSP1 in TNBC. The interaction among circDUSP1, miR-761, DACT2 were confirmed by dual luciferase reporter assay, RNA pull-down, and RNA immunoprecipitation experiments. We identified the circRNA named circDUSP1 that was inversely correlated with tumorigenesis and progression in TNBC. Overexpression of circDUSP1 significantly attenuated cell proliferation, migration, invasion, and epithelial-mesenchymal transition, while increased the sensitivity of TNBC cells to paclitaxel. In-depth mechanism analysis indicated that circDUSP1 acts as an endogenous sponge of miR-761 to reduce its suppression on target gene DACT2 expression in TNBC. Upregulation of miR-761 or downregulation of DACT2 partially reversed the biological process of TNBC and the prognosis of paclitaxel affected by circDUSP1. Taken together, our findings revealed a role for the regulation of the miR-761/DACT2 axis by circDUSP1 in the biological process of TNBC. These results provided new insights into the biological mechanism and targeted therapy of TNBC.

DACT2 protects against pulmonary fibrosis via suppressing glycolysis in lung myofibroblasts.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrotic lung disease with poor prognosis and few treatment options. Dapper homolog 2 (DACT2), a member of the DACT gene family, plays crucial roles in tissue development and injury. However, its functions and molecular mechanisms in IPF remain largely unknown. We aimed to investigate the role of DACT2 in the development of pulmonary fibrosis and the therapeutic potential of targeting DACT2 related signaling pathways. METHODS: In our study, adeno-associated virus serotype 6 (AAV6)-mediated DACT2 overexpression was assessed in several mice models of experimental pulmonary fibrosis in vivo. The role of DACT2 in lung myofibroblast differentiation was determined by DACT2 overexpression in vitro. The glucose uptake, extracellular acidification rate, intracellular adenosine-triphosphate (ATP) level and lactate levels of myofibroblasts were detected after DACT2 overexpression. The LDHA degradation rate and colocalization with lysosomes were monitored as well. RESULTS: Intratracheal administration of AAV6-mediated DACT2 overexpression apparently attenuated pulmonary fibrosis in experimental pulmonary fibrosis models. In vitro experiments revealed that DACT2 inhibited TGF-ß-induced myofibroblast differentiation by promoting lysosome-mediated LDHA degradation and thus suppressing glycolysis in myofibroblasts. CONCLUSION: In conclusion, our findings support for DACT2 as a novel pharmacological target for pulmonary fibrosis treatments.

The Role of DACT Family Members in Tumorigenesis and Tumor Progression.

Disheveled-associated antagonist of ß-catenin (DACT), which ubiquitously expressed in human tissue, is critical for regulating cell proliferation and several developmental processes in different cellular contexts. In addition, DACT is essential for some other cellular processes, such as cell apoptosis, migration and differentiation. Given the importance of DACT in these cellular processes, many scientists are gradually interested in studying the role of DACT in tumorigenesis and cancer progression. This review article focuses on the latest research regarding the essential functions and potential DACT mechanisms in the occurrence and progression of tumors. Our study indicates that DACT may act as a tumor biomarker for cancer diagnosis and prognosis, as well as a promising therapeutic target in cancers.

CKD-581 Downregulates Wnt/ß-Catenin Pathway by DACT3 Induction in Hematologic Malignancy.

The present study evaluated the anti-cancer activity of histone deacetylase (HDAC)-inhibiting CKD-581 in multiple myeloma (MM) and its pharmacological mechanisms. CKD-581 potently inhibited a broad spectrum of HDAC isozymes. It concentration-dependently inhibited proliferation of hematologic cancer cells including MM (MM.1S and RPMI8226) and T cell lymphoma (HH and MJ). It increased the expression of the dishevelled binding antagonist of ß-catenin 3 (DACT3) in T cell lymphoma and MM cells, and decreased the expression of c-Myc and ß-catenin in MM cells. Additionally, it enhanced phosphorylated p53, p21, cleaved caspase-3 and the subG1 population, and reversely, downregulated cyclin D1, CDK4 and the anti-apoptotic BCL-2 family. Finally, administration of CKD-581 exerted a significant anti-cancer activity in MM.1S-implanted xenografts. Overall, CKD-581 shows anticancer activity via inhibition of the Wnt/ß-catenin signaling pathway in hematologic malignancies. This finding is evidence of the therapeutic potential and rationale of CKD-581 for treatment of MM.

LncRNA CRNDE hinders the progression of osteoarthritis by epigenetic regulation of DACT1.

Osteoarthritis (OA) is mainly characterized by articular cartilage degeneration, synovial fibrosis, and inflammation. LncRNA CRNDE (colorectal neoplasia differentially expressed) has been reported to be down-regulated in age-related OA, but its role in injury-induced OA needs to be further explored. In this study, an OA rat model was established using anterior cruciate ligament transection, and the adenovirus-mediated CRNDE overexpression (Ad-CRNDE) or DACT1 (dapper antagonist of catenin-1) interference (sh-DACT1) vectors were administered by intraarticular injection. Moreover, chondrocyte­like ATDC5 cells were treated with IL-1ß (10 ng/mL) to simulate OA conditions in vitro. We found that overexpression of CRNDE alleviated cartilage damage and synovitis in OA rats, and suppressed IL-1ß-induced apoptosis, inflammation, and extracellular matrix (ECM) degradation in chondrocyte­like ATDC5 cells, while silencing DACT1 effectively antagonized the protective effect of CRNDE both in vivo and in vitro. Mechanism studies revealed that DACT1 could act as a downstream target of CRNDE. By recruiting p300, CRNDE promoted the enrichment of H3K27ac in the DACT1 promoter, thus promoting DACT1 transcription. In addition, CRNDE hindered the activation of the Wnt/ß-catenin pathway in IL-1ß-stimulated cells by inducing DACT1 expression. In conclusion, CRNDE promoted DACT1 expression through epigenetic modification and restrained the activation of Wnt/ß-catenin signaling to impede the progression of OA.

Differential expression of regulators of the canonical Wnt pathway during the compensatory beta-cell hyperplasia in prediabetic mice.

We previously reported that the canonical Wnt signaling pathway is activated during compensatory islet hyperplasia in prediabetic mice. Here, we aimed to expand our knowledge concerning the Wnt signaling partners and modulators involved in this process. We report here that Axin1, Axin2, and DACT1, inhibitors of the canonical Wnt signaling pathway, displayed no change in their expression, while GSK-3ß, a multi-functional kinase that acts as a negative regulator of this pathway as well as affects insulin secretion/action, was up-regulated in hyperplastic islets of prediabetic mice. We also observed that COUP-TFII, a protein that acts positively on Wnt-target genes related to cell proliferation, displays a significant increase in gene expression and protein content and is highly immunolabeled in islet cell nuclei of prediabetic mice compared to control islets. These findings suggest that GSK-3ß and COUP-TFII may play a role in beta-cell dysfunction and hyperplasia during type 2 prediabetes.

Disheveled binding antagonist of ß-catenin 1 interacted with ß-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes.

Previously, we demonstrated that the disheveled binding antagonist of ß-catenin 1 (DACT1) was involved in atrial fibrillation by regulating the reorganization of connexin 43 and ß-catenin in cardiomyocytes. Little is known, however, about DACT1 in human normal myocardial cells. Therefore, we used cardiomyocytes (CMs) derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate the role of DACT1 and its connection with ß-catenin and connexin 43. While the ESC-CMs and iPSC-CMs were differentiated using commercial differentiation kits, the cardiac-specific markers were detected by immunofluorescence. The expression level of DACT1 was detected using western blotting, whereas the interaction of DACT1 and connexin 43 or ß-catenin was detected by immunofluorescence and co-immunoprecipitation (co-IP) assays. Both H1-CMs and SF-CMs were immunostained for cardiac-specific markers, including Troponin I, Troponin T, α-actinin, NKX2.5, and GATA6. While DACT1 was not expressed in both H1 ESCs and SF-iPSCs, it was, however, highly expressed in differentiated CMs, being also localized in the cytoplasm and the nucleus of differentiated CMs. Interestingly, the DACT1 expression in different nuclei was different in the same multinucleated cell. Moreover, DACT1 colocalized with ß-catenin in both the cytoplasm and nucleus of differentiated CMs, and it also colocalized with connexin 43 in the perinuclear region and the gap junctions of differentiated CMs. Co-IP results showed that DACT1 could directly bind to ß-catenin and connexin 43. Taken together, DACT1 interacted with ß-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes.

Targeting miR-9 in Glioma Stem Cell-Derived Extracellular Vesicles: A Novel Diagnostic and Therapeutic Biomarker.

BACKGROUND: Glioblastoma (GBM) is a lethal brain tumor with no effective strategies in early diagnosis and treatment. This study was aimed to assess the miRNA expression profiles in EVs from CSF and tissue of glioblastoma patients to identify significantly upregulated miRNAs and investigate the underlying neoplastic mechanisms. METHODS: EVs were measured by TEM and NTA assays. Differentially regulated miRNAs were measured using RNA sequencing in GBM CSF EVs and in GBM tissues compared with controls. RT-qPCR was employed to analyze miRNA and gene expression. Luciferase report assay was used to investigate gene target of miR-9. The proliferation ability was detected by EdU and CCK-8 experiment while cell migration was measured by transwell and wound healing assay. RESULTS: The expression level of miR-9 was significantly higher in GBM CSF EVs and tissues than controls (p = 0.038). The area under curve for CSF EV miR-9 was 0.800 (95% CI: 0.583-1.000, p = 0.033). The expression of miR-9 was significantly higher in Glioma stem cells (GSCs) and GSC-derived EVs than in glioblastoma cells. GSC-derives EVs could promote GBM growth and migration Moreover, inhibition of miR-9 in GSCs showed the reverse anti-tumor effects through secreted EVs. MiR-9 could bind to the 3'UTR region of DACT3 and suppress its expression. The miR-9/DACT3 axis might attribute to GBM malignant phenotype. CONCLUSION: MiR-9 in CSF EVs may act as a novel diagnostic biomarker for GBM and targeting miR-9 by GSC-derived EVs may be a specific and efficient strategy for GBM biotherapy.

Methylation analysis of APC, AXIN2, DACT1, RASSF1A and MGMT gene promoters in non-small cell lung cancer.

Silencing of tumour-suppressor genes through promoter methylation is frequently observed in carcinogenesis. In this study, we determined the methylation status of RASSF1A, MGMT, APC, AXIN2 and DACT1 genes in 73 cases of non-small cell lung cancer. Methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyse the promoter methylation, which was further validated with Bisulfite pyrosequencing or Sanger sequencing. Promoter methylation of RASSF1A and APC was frequently found (56% and 49% of cases, respectively), while methylation of MGMT, AXIN2, DACT1 was observed in 30%, 19% and 16%, respectively. Concurrent gene methylation of at least two genes was observed in 55% of the examined cases, with a total of 89% of samples displaying methylation in one or more of the investigated genes. Further analysis of concurrent methylation revealed a positive correlation between AXIN2-DACT1 and an inverse correlation of APC-MGMT. Associations of methylated genes and clinicopathological features were emerged. In more detail, APC promoter methylation was correlated with smoking status (p= 0.020) and non-metastatic cases (p= 0.003). Moreover, MGMT methylation was preferentially found in TTF1-negative cases (p= 0.049). Interestingly, correlation occurred between AXIN2/DACT1 methylation and smoking status (p= 0.009) as well as tumour grade (p= 0.013), as none of these genes was methylated in the majority of smokers and one of the genes was methylated in high-grade tumours. We conclude that aberrant promoter methylation was observed in our cohort while concurrent methylation patterns were also determined. APC, MGMT and AXIN2/DACT1 methylation are potentially of clinical importance regarding prognosis and histological subtyping of NSCLC.

In vitro evaluation of the pogostone effects on the expression of PTEN and DACT1 tumor suppressor genes, cell cycle, and apoptosis in ovarian cancer cell line.

Background and purpose: Ovarian cancer is one of the most dangerous cancers among women. Pogostone has anticancer effects and is rich in polyphenol compounds. In the present study, we investigated the effects of pogostone on ovarian cancer cell lines (OVCAR-3). Experimental approach: OVCAR-3 cells were treated with pogostone at IC50(90 µg/mL) for 24 and 48 h. Cell viability and apoptotic rate in the cells were measured using MTT assay and flow cytometry. Real-time PCR was used to determine the expression of genes involved in the cell cycle and apoptosis. The expression of caspase-3 (CASP3) protein was evaluated by the CASP3 assay. Findings/Results: Treatment of OVCAR-3 cells with pogostone increased the expression levels of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and Dapper antagonist of catenin-1 (DACT1) tumor suppressor genes, as well as the apoptotic genes CASPs3, 8, and 9. Moreover, the ratio of the expressed BCL2 associated X (BAX)/BCl2 genes, as pro- and anti-apoptotic genes, was increased. The expression levels of the genes related to the cell cycle progression including cyclin D1 (CCND1) and cyclin- dependent kinase 4 (CDK4) were inhibited. The data obtained from flow cytometry indicated that pogostone induced cell apoptosis in 24 and 48 pogostone groups. The CASP3 colorimetric assay revealed that pogostone increased the expression of CASP3 protein in the treated groups. Conclusion and implication: Pogostone, by inducing the expression of PTEN and DACT1 tumor suppressor genes and regulation of downstream genes may decrease cell proliferation and increase the rate of apoptosis in OVCAR-3.

DACT3 has a tumor-inhibiting role in acute myeloid leukemia via the suppression of Wnt/ß-catenin signaling by DVL2.

Dapper antagonist of catenin-3 (DACT3) is a new tumor-related protein associated with a diverse set of tumors. However, whether DACT3 plays a role in acute myeloid leukemia (AML) is not fully understood. Our findings showed low DACT3 level in AML tissue, which was corrected with shorter survival rates. Upregulation of DACT3 effectively repressed cellular proliferation, and promoted cell cycle arrest and apoptosis of AML cells. Upregulation of DACT3 decreased levels of Dishevelled2 (DVL2), phospho-glycogen synthase kinase-3ß (GSK-3ß), and active ß-catenin, which collectively suppressed Wnt/ß-catenin-mediated transcriptional activity. Overexpression of DVL2 reversed DACT3-mediated suppression of Wnt/ß-catenin pathway. Reactivation of Wnt/ß-catenin abrogated DACT3-upregulation-evoked tumor-suppression in AML cells. Overexpression of DACT3 impeded the formation and growth of AML-derived xenograft tumor. Collectively, our work reveals a tumor-suppressive role of DACT3, a protein that negatively adjusts Wnt/ß-catenin pathway via downregulation of DVL2 in AML.

PCSK9 regulates the NODAL signaling pathway and cellular proliferation in hiPSCs.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein (LDL) cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in hepatocytes. Here, we show that PCSK9 is highly expressed in undifferentiated human induced pluripotent stem cells (hiPSCs). PCSK9 inhibition in hiPSCs with the use of short hairpin RNA (shRNA), CRISPR/cas9-mediated knockout, or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. In fact, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in transforming growth factor (TGF) ß-R1 lysosomal degradation. Using different PCSK9 constructs, we show that PCSK9 interacts with DACT2 through its Cys-His-rich domain (CHRD) domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development.

Intratumor Epigenetic Heterogeneity-A Panel Gene Methylation Study in Thyroid Cancer.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy, and the incidence is increasing very fast. Surgical resection and radioactive iodine ablation are major therapeutic methods, however, around 10% of differentiated thyroid cancer and all anaplastic thyroid carcinoma (ATC) are failed. Comprehensive understanding the molecular mechanisms may provide new therapeutic strategies for thyroid cancer. Even though genetic heterogeneity is rigorously studied in various cancers, epigenetic heterogeneity in human cancer remains unclear. METHODS: A total of 405 surgical resected thyroid cancer samples were employed (three spatially isolated specimens were obtained from different regions of the same tumor). Twenty-four genes were selected for methylation screening, and frequently methylated genes in thyroid cancer were used for further validation. Methylation specific PCR (MSP) approach was employed to detect the gene promoter region methylation. RESULTS: Five genes (AP2, CDH1, DACT2, HIN1, and RASSF1A) are found frequently methylated (>30%) in thyroid cancer. The five genes panel is used for further epigenetic heterogeneity analysis. AP2 methylation is associated with gender (P < 0.05), DACT2 methylation is associated with age, gender and tumor size (all P < 0.05), HIN1 methylation is associated to tumor size (P < 0.05) and extra-thyroidal extension (P < 0.01). RASSF1A methylation is associated with lymph node metastasis (P < 0.01). For heterogeneity analysis, AP2 methylation heterogeneity is associated with tumor size (P < 0.01), CDH1 methylation heterogeneity is associated with lymph node metastasis (P < 0.05), DACT2 methylation heterogeneity is associated with tumor size (P < 0.01), HIN1 methylation heterogeneity is associated with tumor size and extra-thyroidal extension (all P < 0.01). The multivariable analysis suggested that the risk of lymph node metastasis is 2.5 times in CDH1 heterogeneous methylation group (OR = 2.512, 95% CI 1.135, 5.557, P = 0.023). The risk of extra-thyroidal extension is almost 3 times in HIN1 heterogeneous methylation group (OR = 2.607, 95% CI 1.138, 5.971, P = 0.023). CONCLUSION: Five of twenty-four genes were found frequently methylated in human thyroid cancer. Based on 5 genes panel analysis, epigenetic heterogeneity is an universal event. Epigenetic heterogeneity is associated with cancer development and progression.