Dickkopf-1 Gene Research Articles

7881 Using Selective Estrogen Receptor Modulators to Reveal Therapeutic Vulnerabilities in ESR1 Mutant Breast Cancer

Abstract Disclosure: S.W. Fanning: Grant Recipient; Self; Olema Oncology. Hotspot activating somatic mutations to ESR1, the gene for estrogen receptor alpha (ER), enable hormone therapy resistance and drive breast cancer metastasis. The Y537S missense mutation within the ER ligand binding domain is the most activating, therapy resistant, and increases metastatic potential. Next generation antiestrogens including lasofoxifene (laso) and elacestrant (RAD1901) show improved progression-free survival over fulvestrant (fulv), but further progress is needed to achieve durable response. To fully address Y537S ESR1, it is critically important to understand how the mutation boosts metastasis. We have developed a small molecule antiestrogen, T6I-29, that uniquely impacts transcriptional pathways related to cell-cell adhesions and morphology. Two allele-specific phenotypes identified by the Oesterreich Laboratory enhance the metastasis of Y537S ESR1 breast cancer cells. T6I-29-specific pathway effects were driven by a significant downregulation of dickkopf-1 (DKK1), which was also observed but to a lesser extent with fulv, laso, and RAD1901. DKK1 is an immunosuppressive, tumor-secreted glycoprotein and its overexpression correlates with poor outcomes in multiple cancers. However, DKK1 acts as a tumor-suppressor in colorectal cancer, which does not rely on ER. Our preliminary studies suggest that DKK1 plays a role in ER+ breast cancer progression. We measured significantly elevated DKK1 protein levels in the blood plasma of 108 diverse ER+ breast cancer patients compared to 100 matched healthy female donors and levels increase coincident with tumor stage. Unfortunately, we lack ESR1 mutation status and other critical details to make further insights on these patients. DKK1 gene expression and protein secretion is stimulated in WT cells by the pathogenic hormone 17β-estradiol (E2), while it is constitutively expressed in Y537S ESR1 cells. DKK1 is characterized as a WNT antagonist but can also induce PI3K/Akt signaling. Interestingly, DKK1 is elevated and ERα responsive in WT not mutant PI3KCA cell lines. DKK1 knockdown also reduced proliferation of Y537S ESR1 breast cancer cells where elevated DKK1 was observed. Presentation: 6/3/2024

Advanced Glycation End Products and Bone Metabolism in Patients with Chronic Kidney Disease.

Advanced glycation end products (AGEs) accumulation may be involved in the progression of CKD-bone disorders. We sought to determine the relationship between AGEs measured in the blood, skin, and bone with histomorphometry parameters, bone protein, gene expression, and serum biomarkers ofbone metabolism in patients with CKD stages 3 to 5D patients. Serum levels of AGEs were estimated by pentosidine, glycated hemoglobin (A1c), and N-carboxymethyl lysine (CML). The accumulation of AGEs in the skin was estimated from skin autofluorescence (SAF). Bone AGEs accumulation and multiligand receptor for AGEs (RAGEs) expression were evaluated by immunohistochemistry; bone samples were used to evaluate protein and gene expression and histomorphometric analysis. Data are from 86 patients (age: 51 ± 13 years; 60 [70%] on dialysis). Median serum levels of pentosidine, CML, A1c, and SAF were 71.6pmol/mL, 15.2ng/mL, 5.4%, and 3.05 arbitrary units, respectively. AGEs covered 3.92% of trabecular bone and 5.42% of the cortical bone surface, whereas RAGEs were expressed in 0.7% and 0.83% of trabecular and cortical bone surfaces, respectively. AGEs accumulation in bone was inversely related to serum receptor activator of NF-κB ligand/parathyroid hormone (PTH) ratio (R=-0.25; p=0.03), and RAGE expression was negatively related to serum tartrate-resistant acid phosphatase-5b/PTH (R=-0.31; p=0.01). Patients with higher AGEs accumulation presented decreased bone protein expression (sclerostin [1.96 (0.11-40.3) vs. 89.3 (2.88-401) ng/mg; p=0.004]; Dickkopf-related protein 1 [0.064 (0.03-0.46) vs. 1.36 (0.39-5.87) ng/mg; p=0.0001]; FGF-23 [1.07 (0.4-32.6) vs. 44.1 (6-162) ng/mg; p=0.01]; and osteoprotegerin [0.16 (0.08-2.4) vs. 6.5 (1.1-23.7) ng/mg; p=0.001]), upregulation of the p53 gene, and downregulation of Dickkopf-1 gene expression. Patients with high serum A1c levels presented greater cortical porosity and Mlt and reduced osteoblast surface/bone surface, eroded surface/bone surface, osteoclast surface/bone surface, mineral apposition rate, and adjusted area. Cortical thickness was negatively correlated with serum A1c (R=-0.28; p=0.02) and pentosidine levels (R=-0.27; p=0.02). AGEs accumulation in the bone of CKD patients was related to decreased bone protein expression, gene expression changes, and increased skeletal resistance to PTH; A1c and pentosidine levels were related to decreased cortical thickness; and A1c levels were related to increased cortical porosity and Mlt. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

MO415: Kidney Expression of Canonical WNT Inhibitors and Β-Catenin in Experimental Models of Arterial Hypertension and Nephrectomy Induced CKD

Abstract BACKGROUND AND AIMS The canonical Wnt (cWnt) is known to be involved in tubular recovery after kidney injury and fibrosis, while arterial hypertension (AH) promotes cWnt activity. The aim of the study was to investigate the kidney expression of β-catenin and cWnt inhibitors in AH and chronic kidney disease (CKD) models. METHOD AH-CKD was performed by 3/4 nephrectomy (NE) in spontaneously hypertensive rats (SHR) with 2 or 6 months of experimental exposure (Table). Sham-operated (SO) SHRs and Wistar Kyoto rats (WKY) served as controls, and 6 mo exposure SO-SHRs were used as AH-induced CKD. Serum creatinine (Cr), renal sclerostin (Sost) and dickkopf-1 (Dkk1) gene expression were measured. The following parameters of kidney morphology were analyzed and calculated quantitatively: the areas of tubular dystrophy (TD) (PAS), renal fibrosis (RF) (Masson's trichrome), β-catenin (BC), sclerostin (SOST) and dickkopf-1 (DKK1) IHC-staining. RESULTS The experimental models were corresponded to early (SO6, NE2) and mild (NE6) CKD (Table). In all groups, BC membrane IHC staining in epithelial cells and nuclear expression in podocytes were determined, SOST and DKK1 cytoplasmic IHC staining in tubular epithelium were detected (Figure). The tubular and interstitial lesions in NE (Table) were accompanied by cytoplasmic BC localization in epithelial and interstitial cells (Figure) with a decrease in dickkopf-1 gene (Table) and protein (Figure) expression, increase in Sost gene expression and SOST IHC staining of tubular intracellular vesicles (Figure). Long-term exposure to hypertension (SO6) resulted in the renal function decline and RF comparable to NE2 (Table), while TD and levels of Wnt inhibitors and BC IHC staining area did not differ significantly vs SO2, WKY (Table). In the pooled analysis, the RF directly correlated with the BC renal expression (r = 0.76, P = 0.005) and negatively with Dkk1 expression (r = −0.45, P = 0.028); the TD negatively correlated with Sost expression (r = −0.80, P = 0.002). CONCLUSION Tubular and interstitial damage in NE and AH-induced CKD is associated with BC up-regulation. The decrease in dickkopf1 and BC cytoplasmic accumulation may be required for renal fibrosis. The sclerostin expression is likely to be essential for the pathogenesis of TD.

The Dickkopf1 and FOXM1 positive feedback loop promotes tumor growth in pancreatic and esophageal cancers

Dickkopf1 (DKK1) is overexpressed in various cancers and promotes cancer cell proliferation by binding to cytoskeleton-associated protein 4 (CKAP4). However, the mechanisms underlying DKK1 expression are poorly understood. RNA sequence analysis revealed that expression of the transcription factor forkhead box M1 (FOXM1) and its target genes concordantly fluctuated with expression of DKK1 in pancreatic ductal adenocarcinoma (PDAC) cells. DKK1 knockdown decreased FOXM1 expression and vice versa in PDAC and esophageal squamous cell carcinoma (ESCC) cells. Inhibition of either the DKK1-CKAP4-AKT pathway or the ERK pathway suppressed FOXM1 expression, and simultaneous inhibition of both pathways showed synergistic effects. A FOXM1 binding site was identified in the 5ʹ-untranslated region of the DKK1 gene, and its depletion decreased DKK1 expression and cancer cell proliferation. Clinicopathological and database analysis revealed that PDAC and ESCC patients who simultaneously express DKK1 and FOXM1 have a poorer prognosis. Multivariate analysis demonstrated that expression of both DKK1 and FOXM1 is the independent prognostic factor in ESCC patients. Although it has been reported that FOXM1 enhances Wnt signaling, FOXM1 induced DKK1 expression independently of Wnt signaling in PDAC and ESCC cells. These results suggest that DKK1 and FOXM1 create a positive feedback loop to promote cancer cell proliferation.

Dickkopf-1: A Promising Target for Cancer Immunotherapy.

Clinical studies in a range of cancers have detected elevated levels of the Wnt antagonist Dickkopf-1 (DKK1) in the serum or tumors of patients, and this was frequently associated with a poor prognosis. Our analysis of DKK1 gene profile using data from TCGA also proves the high expression of DKK1 in 14 types of cancers. Numerous preclinical studies have demonstrated the cancer-promoting effects of DKK1 in both in vitro cell models and in vivo animal models. Furthermore, DKK1 showed the ability to modulate immune cell activities as well as the immunosuppressive cancer microenvironment. Expression level of DKK1 is positively correlated with infiltrating levels of myeloid-derived suppressor cells (MDSCs) in 20 types of cancers, while negatively associated with CD8+ T cells in 4 of these 20 cancer types. Emerging experimental evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. This review introduces the rapidly growing body of literature revealing the cancer-promoting and immune regulatory activities of DKK1. In addition, this review also predicts that by understanding the interaction between different domains of DKK1 through computational modeling and functional studies, the underlying functional mechanism of DKK1 could be further elucidated, thus facilitating the development of anti-DKK1 drugs with more promising efficacy in cancer immunotherapy.

The expression and biological function of DKK1 in oral squamous cell carcinomas by bioinformatics analysis

Objective: To clear the expression of transcription factor Dickkopf-1 (DKK1) in Oral Squamous Cell Carcinoma (OSCC) using the method of bioinformatics analysis. And to clarify the relationship between the expression of DKK1 and the clinicopathological characteristics of OSCC using the method of molecular biology and cytobiology, in order to determine the early diagnosis and significance of OSCC according to the marker of DKK1. Methods: In this study, the expression level of DKK1 in OSCC tissues was analyzed using GEPIA and TCGA databases, and then verified in vitro by qRT-PCR and Western-blot analysis. The correlation between DKK1 gene expression and the clinical pathological parameters of OSCC, and also the impact of DKK1 on prognosis were determined using the Linked Omics database. In addition, DKK1 was knocked down by RNA interference in SCC-4 and SCC-25 OSCC cell lines and the proliferation ability of OSCC cells was assessed by MTT assay. Results: High expression of DKK1 in OSCC is positively correlated with the pathological grade and T stage of OSCC. According to the TCGA results, DKK1 mRNA was highly expressed and it is related to the overall survival rate of OSCC. In addition, the expression level of both DKK1mRNA and protein were significantly raised in the cell line SCC25 and SCC-4. Furthermore, MTT analysis showed that DKK1 knockdown resulted in reduced proliferation of OSCC cells. Conclusions: TCGA database analysis showed that DKK1 was highly expressed in OSCC, and it is closely correlated to the pathological parameters of OSCC, which will provide important theoretical guidance for the subsequent study of oral squamous cell carcinoma.

The Expression and Biological Function of DKK1 in Oral Squamous Cell Carcinomas by Bioinformatics Analysis

Objective: To clear the expression of transcription factor Dickkopf-1 (DKK1) in Oral Squamous Cell Carcinoma (OSCC) using the method of bioinformatics analysis. And to clarify the relationship between the expression of DKK1 and the clinicopathological characteristics of OSCC using the method of molecular biology and cytobiology, in order to determine the early diagnosis and significance of OSCC according to the marker of DKK1. Methods: In this study, the expression level of DKK1 in OSCC tissues was analyzed using GEPIA and TCGA databases, and then verified in vitro by qRTPCR and Western-blot analysis. The correlation between DKK1 gene expression and the clinical pathological parameters of OSCC, and also the impact of DKK1 on prognosis were determined using the LinkedOmics database. In addition, DKK1 was knocked down by RNA interference in SCC-4 and SCC-25 OSCC cell lines and the proliferation ability of OSCC cells was assessed by MTT assay. Results: High expression of DKK1 in OSCC is positively correlated with the pathological grade and T stage of OSCC. According to the TCGA results, DKK1 mRNA was highly expressed and it is related to the overall survival rate of OSCC. In addition, the expression level of both DKK1mRNA and protein were significantly raised in the cell line SCC-25 and SCC-4. Furthermore, MTT analysis showed that DKK1 knockdown resulted in reduced proliferation of OSCC cells. Conclusions: TCGA database analysis showed that DKK1 was highly expressed in OSCC, and it is closely correlated to the pathological parameters of OSCC, which will provide important theoretical guidance for the subsequent study of oral squamous cell carcinoma.

Expression and methylation of Dickkopf-1 in the pathogenesis and malignant transformation of oral submucous fibrosis.

Dickkopf-1 is an inhibitor of the Wnt/β-catenin pathway, but the role of Dickkopf-1 in oral submucous fibrosis remains unclear. We evaluated the protein expression and gene methylation levels of dickkopf-1 to determine the mechanism underlying abnormal Wnt/β-catenin pathway activation. Healthy mucosa, oral submucous fibrosis, oral squamous cell carcinoma, and cancer-adjacent tissues were collected. The expression and promoter methylation levels of dickkopf-1 were analyzed. The expression levels of dickkopf-1 in oral submucous fibrosis and oral squamous cell carcinoma tissues were lower than those in healthy and cancer-adjacent tissues. The methylation levels of the dickkopf-1 gene in oral submucous fibrosis and oral squamous cell carcinoma tissues were higher than those in healthy and cancer-adjacent tissues. Dickkopf-1 expression was negatively correlated with dickkopf-1 gene methylation. High dickkopf-1 methylation levels in oral submucous fibrosis and oral squamous cell carcinoma tissues may decrease dickkopf-1 expression, which may induce an abnormal activation of the Wnt/β-catenin pathway and oral submucous fibrosis pathogenesis.

Dickkopf-3 (DKK3) Signaling in IL-1α-Challenged Chondrocytes: Involvement of the NF-κB Pathway.

Osteoarthritis (OA) is an age-related biomechanical and low-grade inflammometabolic disease of the joints and one of the costliest and disabling forms of arthritis. Studies on matrix-degrading enzymes such as metalloproteases, which are implicated in the increased catabolism of extracellular matrix, are of paramount relevance. DKK3 is a member of DKK family and is best known for its role in cancer. Although there is some information about the participation of DKK3 in cartilage pathophysiology and on metalloproteases regulation, in particular, little is known about DKK3 signaling mechanisms. Thus, the aim of this study is to explore how DKK3 regulates matrix metalloproteinase-13 (MMP-13) expression. Gene, protein expression and protein phosphorylation in primary human chondrocytes and ATDC5 mouse cells were assessed by RT-qPCR and Western blot analysis. Further studies on DKK3 activity were performed by targeting DKK3 gene with a specific siRNA. DKK3 expression was found to be higher in OA human chondrocytes than healthy cells, being its expression decreased in interleukin-1α (IL-1α)-stimulated cells. DKK3 knockdown increased the induction of MMP-13 elicited by IL-1α in human and mouse chondrocytes and after the analysis of different signalling pathways, we observed that NF-κB pathway was involved in the regulation of MMP-13 expression by DKK3. Herein we have demonstrated, for the first time, that DKK3 gene silencing exacerbated NF-κB activation, resulting in an increased IL-1α-driven induction of MMP-13. Our results further confirm that DKK3 may play a protective role in OA by attenuating NF-κB activation and the subsequent production of metalloproteases.

Intracavernous delivery of Dickkopf3 gene or peptide rescues erectile function through enhanced cavernous angiogenesis in the diabetic mouse.

Severe peripheral angiopathy in patients with diabetes is a major contributing factor for low response rate to phosphodiesterase-5 inhibitors. To examine whether and how Dickkopf3 (DKK3), a secreted modulator of the Wnt pathway that known to be involved in endothelial cell repair and vascular progenitor cell migration, restores erectile function in diabetic mice. Eight-week-old C57BL/6 mice received intraperitoneal injections of streptozotocin (50mg/kg for 5days). Eight weeks after the diabetes was induced, the efficacy of DKK3 was determined by three independent experiments: experiment 1 (DKK3 peptide [5μg in 20μL PBS]); experiment 2 (DKK3 plasmid DNA with electroporation [10, 40, or 100μg in 20μL PBS, respectively]); and experiment 3 (DKK3 adenovirus [1×107 , 1×108 , 1×109 virus particles per 20μL, respectively]). Erectile function was measured by electrical stimulation of the cavernous nerve one week (for peptide) or two weeks (for genes) after treatment. The angiogenic activity of DKK3 was determined in diabetic penis in vivo and in primary cultured mouse cavernous endothelial cells (MCECs) in vitro. The cavernous expression of DKK3 protein was significantly lower in the diabetic mice than in controls. DKK3 peptide or adenovirus significantly improved erectile function in diabetic mice (70% of the control values). DKK3 adenovirus profoundly restored cavernous endothelial cell and pericyte contents and increased endothelial junction proteins in diabetic mice in vivo. DKK3 peptide induced upregulation of angiogenic factors (angiopoietin-1, vascular endothelial growth factor, and basic fibroblast growth factor) and accelerated tube formation in MCECs cultivated under the high-glucose condition in vitro. DKK3 restored cavernous vascular integrity and improved erectile function in diabetic mice. Therapeutic cavernous angiogenesis by the use of DKK3 will be a promising therapeutic strategy to treat diabetic erectile dysfunction.

A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells

Background: Wnt signaling plays an essential role in tumor cell growth, including the development of malignant mesothelioma (MM). Epigenetic silencing of negative Wnt regulators leading to constitutive Wnt signaling has been observed in various cancers and warrants further attention. We have reported that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic effects in MM cells. Thus, in this study, we investigated whether the anti-MM effect of T3E could be mediated via the epigenetic alteration of the Wnt antagonist gene, Dickkopf-1 (DKK1). Methods: WST-1 and cell analyzers were employed to analyze the effects of T3E on cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR and Western blot were performed to evaluate the expression at mRNA and protein levels. Methylation status and epigenetic modifications of DKK1’s promoter regions after T3E treatment in MM cells were studied using methylation-specific PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown ­(siRNA), and specific inhibitors, were used to validate DKK1 as a target of T3E. Results: T3E markedly impaired MM cell viability, increased the expression of phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, which promotes cell proliferation as a Wnt-independent DKK1 receptor was inhibited by T3E. Silencing CKAP4 by ­siRNA did not appear to directly affect MM cell viability, thereby indicating that expression of both DKK1 and CKAP4 is required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases (DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells leads to increased DKK1 expression, thereby promoting tumor growth inhibition. MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells following T3E treatment is positively correlated with the methylation status of its promoter; T3E decreased DNA methylation and increased histone acetylation. Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation activity, whereas no effects were observed on histone H3K9 and H3K27. Conclusions: Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.

Structural Characterization and Association of Ovine Dickkopf-1 Gene with Wool Production and Quality Traits in Chinese Merino

Dickkopf-1 (DKK1) is an inhibitor of canonical Wnt signaling pathway and regulates hair follicle morphogenesis and cycling. To investigate the potential involvement of DKK1 in wool production and quality traits, we characterized the genomic structure of ovine DKK1, performed polymorphism detection and association analysis of ovine DKK1 with wool production and quality traits in Chinese Merino. Our results showed that ovine DKK1 consists of four exons and three introns, which encodes a protein of 262 amino acids. The coding sequence of ovine DKK1 and its deduced amino acid sequence were highly conserved in mammals. Eleven single nucleotide polymorphisms (SNPs) were identified within the ovine DKK1 genomic region. Gene-wide association analysis showed that SNP5 was significantly associated with mean fiber diameter (MFD) in the B (selected for long wool fiber and high-quality wool), PW (selected for high reproductive capacity, high clean wool yield and high-quality wool) and U (selected for long wool fiber with good uniformity, high wool yield and lower fiber diameter) strains (p < 4.55 × 10−3 = 0.05/11). Single Nucleotide Polymorphisms wide association analysis showed that SNP8 was significantly associated with MFD in A strain and fleece weight in A (selected for large body size), PM (selected for large body size, high reproductive capacity and high meat yield) and SF (selected for mean fiber diameter less than 18 μm and wool fiber length between 5 and 9 cm) strains (p < 0.05), SNP9 was significantly associated with curvature in B and U strains (p < 0.05) and SNP10 was significantly associated with coefficient of variation of fiber diameter in A, PW and PM strains and standard deviation of fiber diameter in A and PM strains (p < 0.05). The haplotypes derived from these 11 identified SNPs were significantly associated with MFD (p < 0.05). In conclusion, our results suggest that DKK1 may be a major gene controlling wool production and quality traits, also the identified SNPs (SNPs5, 8, 9 and 10) might be used as potential molecular markers for improving sheep wool production and quality in sheep breeding.

Dickkopf1 destabilizes atherosclerotic plaques and promotes plaque formation by inducing apoptosis of endothelial cells through activation of ER stress

Several clinical studies reported that Dickkopf1 (DKK1) plasma levels are correlated with atherosclerosis. However, the impact of DKK1 on the formation and vulnerability of atherosclerotic plaques remains elusive. This study investigated DKK1’s effects on enlargement and destabilization of plaques by targeting endothelial cells and assessing the possible cellular mechanisms involved. The effects of DKK1 on atherogenesis and plaque stability were evaluated in ApoE−/− mice using lentivirus injections to knockdown and knock-in the DKK1 gene. The presence of DKK1 resulted in enlarged and destabilized atherosclerotic lesions and increased apoptosis, while silencing of DKK1 alleviated plaque formation and vulnerability in the whole progression of atherosclerosis. DKK1 expression was upregulated in response to ox-LDL treatment in a time- and concentration-dependent manner on human umbilical vein endothelial cell (HUVEC). The interference of DKK1 reversed ox-LDL-induced apoptosis in HUVECs. The mechanism underlying this effect was DKK1’s activation of the JNK signal transduction pathway and inhibition of canonical Wnt signaling, following by activation of the IRE1α and eif2α/CHOP pathways. In conclusion, DKK1 promotes plaque formation and vulnerability partly by inducing apoptosis in endothelial cells, which partly through inducing the JNK-endoplasmic reticulum stress pathway and inhibiting canonical Wnt signaling.

Dysregulation of the DKK1 gene in pediatric B-cell acute lymphoblastic leukemia

The canonical Wingless-type (WNT) pathway is involved in normal hematopoietic cell development and deregulated WNT signaling is implicated in the development of hematological malignancies. Dickkopf 1 (DKK1) acts as a modulator of the β-catenin regulated canonical pathway. Activation of DKK1 leads to apoptosis and growth suppression, whereas silencing by promoter hypermethylation results in abnormal WNT activation. The secreted inhibitor Dickkopf can antagonize WNT signaling by competitively binding to low density lipoprotein receptors (LRPs) 5 and 6. We studied DKK1 gene promoter methylation and investigated DKK1, β-catenin, LRP5, and LRP6 mRNA levels in B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 90). Methylation-specific PCR and bisulfite sequencing were used for methylation profiling and quantitative real-time PCR was used for mRNA detection. The DKK1 gene was examined for its promoter methylation and only 33% of patients were found methylated. On the other hand, B-ALL cases showed high expression of DKK1 (P = 0.01), LRP5 (P = 0.04), and LRP6 (P = 0.02) compared to normal bone marrow cells. DKK1 methylation exists in some of cases but is not sufficient for WNT pathway activation alone in pediatric B-ALL.

Study of Dickkopf-1 (DKK-1) Gene Expression in Hepatocellular Carcinoma Patients.

Hepatocellular Carcinoma (HCC) is the sixth most common cancer in the world. Dickkopf -1 (DKK-1) protein is a new biomarker used in conjunction with Alpha Fetoprotein (AFP) to differentiate HCC from "non-malignant" liver disease. DKK-1 is an inhibitor of Wnt/β-catenin signaling pathway which is involved in embryogenesis and has been implicated in tumorigenesis in many tissues. To investigate the level of DKK-1 gene expression in the peripheral blood of patients with HCC who had a history of Hepatitis C Virus (HCV) and schistosomal infections. This "cross-sectional" study was carried out in the Tropical Medicine Department of Tanta University Hospital on 50 patients with HCC and 10 healthy volunteers served as control. All patients were tested for HCV antibodies and "anti-schistosomal" antibodies. All groups were tested for DKK-1 gene expression which was measured with quantitative real-time PCR. DKK-1 gene was over-expressed in HCC patients than in the control group with mean 3.269±4.762 versus 1.00 in controls (p< 0.005). "Over- expression" of DKK-1 was found in: 8/20 of patients with negative serology for both infections (40%; p<0.001), 7/18 of patients with positive anti-HCV antibodies (38.89%; p<0.001) and 11/12 of patients with positive anti-schistosomal antibodies (91.66%; p<0.001). There was no statistically significant correlation between DKK-1 expression and HCV infection (p=0.139) but there was significant correlation between the gene expression and schistosomal infection (p<0.001). These data suggest the role of DKK-1 over-expression in HCC development in patients with combined HCV and schistosomal infections and that induction of the Wnt pathway or using DKK-1 antagonist may represent a key advance in the area of genetic prevention of HCC in these "high-risk" patients.

Effect of Decitabine on DKK1 Gene Demethylation in Leukemia Cells

To explore the effect of decitabine on Dickkopf-1 (DKK1) gene expression level and its downstream Wnt signaling pathway in acute myeloid leukemia (AML) cell line HL-60. Flow cytometry and DNA ladder analysis were performed to detect apoptosis in HL-60 cell treated with different concentration of decitabine. Methylation-specific polymerase chain reaction (MS-PCR) was used to examine the methylation status of DKK1 gene. The expressions of mRNA and protein were determined by qRT-PCR and Western blot, respectively. Flow cytometric detection showed that after treating HL-60 cell line with decitabine of different concentrations for 48 h, the early apoptosis of HL-60 cells increased significantly as compared with control group (P < 0.05). DNA ladder analysis showed that the DNA ladder and demethylation of DKK1 gene appeared. RT-PCR and Western blot showed that the expressions of mRNA and protein increased. The protein expressions of β-catenin and C-MYC decreased. The decitabine can promote the apoptosis of HL-60 cells throngh demethylation of DDK1 gene and inhibition of Wnt signalling pathway.

Research Progress on DKK1 Gene in Leukemia

A number of studies have demonstrated that the methylation of Dickkopf-1 (DKK1) gene promoter is related with the occurrence and development of many neoplastic diseases. By means of binding with corresponding receptors, DKK1 blocks the transduction pathway of Wnt/β-catenin/TCF and inhibits the proliferation and invasion of tumor cells, inducing apoptosis. Leukemia is a hyperplastic disease of hematopoietic stem cell malignant clone. Its pathogenesis has been confirmed to be closely related with the aberrant activation of Wnt signaling pathway. This pathway is associated with the self-renewal and proliferation of the hematopoietic stem cells, which can regulate growth, differentiation, migration of the cells, angiogenesis and embryonic development. Its expression is regulated by some suppressor genes like Dickkopf 1 (DKK1). Leukemia often accompanied by methylation modification of the DKK1 gene, so as to leads to silencing itself and activation of the Wnt signaling pathway, which cause the occurrence of leukemia. Some therapeutic methods on leukemia aiming at DKK1 gene have been reported, among which DKK1 gene was demethylated. The intensive study on the expression and function of DKK1 should be important for the early diagnosis, treatment and prognosis. This article reviews the current progress in this field.

Differential expression of Dickkopf-1 among non-small cell lung cancer cells.

Dickkopf-1 (DKK1) is a negative regulator of the Wnt/β-catenin signaling pathway, which is expressed in various human cancers. It was hypothesized that DKK1 was oncogenic and involved in invasive growth in non-small cell lung cancer (NSCLC) cells. The present study aimed to investigate whether DKK1 gene expression levels differ among various NSCLC cells. The DKK1 expression pattern was analyzed in various human NSCLC cell lines and tissues. The DKK1 protein and gene expression levels were quantified using immunoblotting, polymerase chain reaction analysis and immunohistochemistry. The majority of the lung cancer cell lines analyzed revealed increased expression levels of DKK1. Furthermore, DKK1 expression was highly transactivated in the majority of these cancer cell lines. Clinical samples were obtained from 98 NSCLC patients for immunohistochemical analysis. Of the 98 samples analyzed, 62 (63.3%) demonstrated positive staining for DKK1, whereas the remaining 36 (37%) exhibited negative staining. However, no immunohistopathological staining was detected in normal tissues. The relative effects of DKK1 were assessed in a high-expression cell line (LTEP-a-2) and a low-expression cell line (95D). The differential expression of genes involved in cell cycle, apoptosis, signaling pathway, invasion and metastasis were evaluated, relative to DKK1 levels. In conclusion, the results of the present study indicated that DKK1 functioned as a key regulator in the progression of NSCLC. The results confirmed the differential expression of DKK1 in NSCLC cells, which may present a potential therapeutic target for cancer prevention.

DKK1 eukaryotic expression plasmid and expression product identification.

We constructed the human dickkopf 1 (DKK1) eukaryotic expression plasmid and expressed, purified, and identified its expression product. We extracted cancer cells from cervical cancer tissue, followed by extraction of mRNA. Reverse transcription-polymerase chain reaction was conducted to obtain DKK1 gene fragments. Using these fragments, we prepared the recombinant plasmid pCMV-HA2/DKK1. The recombinant plasmid was restriction enzyme-digested and sequenced, and using liposome vectors, was transiently transfected into Free-Style 293-F cells (serum-free medium). DKK1 protein was detected by western blotting. The amplification product showed the expected size. Restriction enzyme digestion and sequence analysis showed that the recombinant plasmid was PCMV-HA2/DKK1. The expression product was verified properly by western blotting using an anti-DKKI antibody. The successful cloning of the DKKI gene and expression of DKKI protein will be useful for studying the biological activity of tumorigenesis.

Heritability and genetics of serum dickkopf 1 levels in African ancestry families.

The aim of the study was to determine the heritability of serum dickkopf-1 (DKK1) and its association with DKK1 polymorphisms in African ancestry subjects. Serum DKK1 was measured in 422 Afro-Caribbean men and women aged 18+ from 7 large, multi-generational families (mean family size: 60; 3,215 relative pairs). Twenty-four common single nucleotide polymorphisms (SNPs) were genotyped within an 80 kilobase-pair region encompassing the DKK1 gene. Heritability was estimated and SNPs were tested for association with serum DKK1 using variance components analysis. DKK1 mRNA expression was tested in peripheral blood of 16 individuals from each of the rs7069912 genotypes. Mean serum DKK1 was 1724.1 pg/mL and was significantly lower in women than men (P = 0.043). Residual genetic heritability of serum DKK1 was 0.4460 (P < 0.0001). Six SNPs reached nominal significance with DKK1, with rs7069912 being significant after adjustment for multiple comparisons. Two of these six SNPs represented independent association signals (rs7069912 and rs16928725), which accounted for 4.6% of the phenotypic variation in DKK1. Additionally, carriers of the rs7069912 variant had significantly greater DKK1 expression than non-carriers (P = 0.036). Serum DKK1 levels are highly heritable in the African ancestry families. Two SNPs within the DKK1 region accounted for nearly 5% of the variation in serum DKK1.