Dickkopf-1 mRNA Research Articles

The prognostic value of Dickkopf-3 (Dkk3), TGFB1 and ECM-1 in prostate cancer

Prostate cancer (PCa) is considered one of the most common cancers worldwide. Despite advances in patient diagnosis, management, and risk stratification, 10%–20% of patients progress to castration-resistant disease. Our previous report highlighted a protective role of Dickkopf-3 (DKK3) in PCa stroma. This role was proposed to be mediated through opposing extracellular matrix protein 1 (ECM-1) and TGF-β signalling activity. However, a detailed analysis of the prognostic value of DKK3, ECM-1 and members of the TGF-β signalling pathway in PCa was not thoroughly investigated. In this study, we explored the prognostic value of DKK3, ECM-1 and TGFB1 using a bioinformatical approach through analysis of large publicly available datasets from The Cancer Genome Atlas Program (TGCA) and Pan-Cancer Atlas databases. Our results showed a significant gradual loss of DKK3 expression with PCa progression (p < 0.0001) associated with increased DNA methylation in its promoter region (p < 1.63E-12). In contrast, patients with metastatic lesions showed significantly higher levels of TGFB1 expression compared to primary tumours (p < 0.00001). Our results also showed a marginal association between more advanced tumour stage presented as positive lymph node involvement and low DKK3 mRNA expression (p = 0.082). However, while ECM1 showed no association with tumour stage (p = 0.773), high TGFB1 expression showed a significant association with more advanced stage presented as advanced T3 stage compared to patients with low TGFB1 mRNA expression (p < 0.001). Interestingly, while ECM1 showed no significant association with patient outcome, patients with high DKK3 mRNA expression showed a significant association with favourable outcomes presented as prolonged disease-specific (p = 0.0266), progression-free survival (p = 0.047) and disease-free (p = 0.05). In contrast, high TGFB1 mRNA expression showed a significant association with poor patient outcomes presented as shortened progression-free (p = 0.00032) and disease-free survival (p = 0.0433). Moreover, DKK3, TGFB1 and ECM1 have acted as immune-associated genes in the PCa tumour microenvironment. In conclusion, our findings showed a distinct prognostic value for this three-gene signature in PCa. While both DKK3 and TGFB1 showed a potential role as a clinical marker for PCa stratification, ECM1 showed no significant association with the majority of clinicopathological parameters, which reduce its clinical significance as a reliable prognostic marker.

Dickkopf-1 is an immune infiltration-related prognostic biomarker of head and neck squamous cell carcinoma.

Immunotherapy is currently one of the most viable therapies for head and neck squamous cell carcinoma (HNSCC), characterized by high immune cell infiltration. The Wnt-signaling inhibitor and immune activation mediator, Dickkopf-1 (DKK1), has a strong correlation with tumor growth, tumor microenvironment, and, consequently, disease prognosis. Nevertheless, it is still unclear how DKK1 expression, HNSCC prognosis, and tumor-infiltrating lymphocytes are related. To better understand these associations, we examined how DKK1 expression varies across different tumor and normal tissues. In our study, we investigated the association between DKK1 mRNA expression and clinical outcomes. Next, we assessed the link between DKK1 expression and tumor immune cell infiltration. Additionally, using immunohistochemistry, we evaluated the expression of DKK1 in 15 healthy head and neck tissue samples, and the expression of CD3, CD4, and DKK1 in 27 HNSCC samples. We also explored aberrant DKK1 expression during tumorigenesis. DKK1 expression was remarkably higher in HNSCC tissues than in healthy tissues, and was shown to be associated with tumor stage, grade, lymph node metastasis, histology, and a dismal clinical prognosis in HNSCC. DKK1 expression in HNSCC tissues was inversely correlated with CD3+ (P < 0.0001) and CD4+ (P < 0.0001) immune cell infiltration, while that in immune cells was inversely associated with HNSCC prognosis. These findings offer a bioinformatics perspective on the function of DKK1 in HNSCC immunotherapy and provide justification for clinical research on DKK1-targeted HNSCC treatments. DKK1 is a central target for improving the efficacy of HNSCC immunotherapy.

Abstract 376: DKK1 inhibition can overcome sorafenib resistance in hepatocellular carcinoma cells through beta-catenin suppression

Abstract As part of our previous studies on the anti-tumor mechanisms of sorafenib, one of the primary treatments for advanced hepatocellular carcinoma (HCC), we found that suppression of cyclin E1 in HCC cells correlates with sensitivity to sorafenib (Chiun Hsu et al. Clin Cancer Res. 2016:2555). Furthermore, the combination of sorafenib and a cyclin-dependent kinase inhibitor was found to have synergistic anti-tumor effects. Specifically, we found that inhibiting DKK1 (dickkopf1), an essential regulator of Wnt signaling, had a synergistic effect. In sorafenib-sensitive HCC cells, DKK1 mRNA and protein expressions were suppressed following treatment with sorafenib, but not in sorafenib-resistant cells. HCC cells resistant to sorafenib were induced to apoptosis with knockdown of DKK1 expression. The apoptosis-inducing effects of sorafenib were enhanced by DKK1 inhibitors, and beta-catenin suppression was found to be crucial to this effect. In addition, mouse tumors secrete DKK1 into the blood and its presence correlates positively with tumor size and survival during sorafenib treatment. Suppression of DKK1 in hepatocellular carcinoma cells may serve as a pharmacodynamic biomarker for predicting sorafenib or CDK inhibitor efficacy. Sorafenib combined with a DKK1 inhibitor may improve sorafenib's efficacy in hepatocellular carcinoma. (Supported by MOST 106-2314-B-002-229-MY3, MOST 109-2634-F-002-043, MOST 109-2314-B-002-229 -MY3, MOST 110-2634-F-002-044 from Ministry of Science and Technology, Taiwan) Citation Format: Lin Li, Chia-Lang Hsu, Liang-In Lin, Bin-Shyun Lee, Ping-Yun Ou, Ann-Lii Cheng, Chiun Hsu, Da-Liang Ou. DKK1 inhibition can overcome sorafenib resistance in hepatocellular carcinoma cells through beta-catenin suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 376.

Correlation of NNMT and DKK1 Protein Expression With Clinicopathological Characteristics and Prognosis of Breast Cancer.

Nicotinamide N-methyltransferase (NNMT) and Dickkopf-1 (DKK1) play an important role in the development of breast cancer, and the purpose of this study was designed to examine the clinical and prognostic significance of NNMT and DKK1 in breast cancer. The GEPIA2 database was used to evaluate the expression and survival of NNMT mRNA and DKK1 mRNA of breast cancer. Then an immunohistochemical study was carried out on 374 cases of breast tissue to identify the protein expression and significance of NNMT and DKK1. Next, the prognostic significance of DKK1 in breast cancer was explored by COX and Kaplan-Meier models. Protein NNMT expression was correlated with lymph node metastasis and histological grade (P < .05) while protein DKK1 expression was related to tumor size, pT stage, histological grade, and Ki-67 (P < .05). Protein DKK1 was related to disease-specific survival (DSS), and low DKK1 expression indicated a poor prognosis of breast cancer patients (P < .05). Combined expression of protein NNMT and protein DKK1 predicted different prognosis of DSS (P < .05). Nicotinamide N-methyltransferase and DKK1 were linked to breast cancer malignancy and invasion. Breast cancer patients with low DKK1 expression had a worse prognosis. Oncotypes of NNMT and DKK1 expression predicted patient outcomes.

Abstract 5009: DKK1 is a potential target for enhancing the efficiency of radioimmunotherapy in head and neck squamous carcinoma

Abstract Immune checkpoint inhibitors (ICIs) have been approved to treat various types of tumors, including head and neck squamous carcinoma (HNSC). However, most HNSC patients do not respond to ICIs. Radioimmunotherapy has been proposed to enhance the immune response rate in HNSC. Herein, we aimed to explore the effect of DKK1 on radiotherapy and immunotherapy in HNSC. We collected the gene expression profile and clinical information of HNSC patients from TCGA, GEO, and ICGC. The immune cell infiltration, level of related pathways and biological processes, signature scores of gene sets of interest were assessed using CIBERSORT, GSEA or GSVA respectively. One of the genes, Dickkopf-1 (DKK1), a secreted protein which plays important roles in the Wnt signaling pathways, was identified because of its significantly higher expression in HNSC. Then, we confirmed the DKK1 expression at protein level by immunohistochemistry of HNSC tissue microarray. CCK8, apoptosis detection and survival curves were performed to investigate the response of DKK1-knockdown HSC-3 cell line to radiation. We found the mRNA and protein expression level of DKK1 was significantly higher in HNSC tissue. The DKK1high group had a shorter survival time in both disease-free survival and overall survival than the DKK1low group. The DKK1high group showed a more immunosuppressive microenvironment with lower infiltration of T cells and higher infiltration of MDSCs. Furthermore, the DKK1high group was enriched in radioresistance related to wound healing, angiogenesis, and autophagy. Patients in the DKK1high group who received radiotherapy had much poorer disease-free survival and overall survival than those in the DKK1low group. HSC-3 cell line was more sensitive to radiation after knockdown of DKK1 by siRNA. Finally, we established a risk score system based on the 9 genes from 41 DKK1-associated immunomodulators and created a nomogram model to predict the prognosis of the patients. Our data show that DKK1 can affect both radiotherapy and immunotherapy in HNSC, suggesting that DKK1 can be a potential target for radioimmunology in HNSC. Citation Format: Xinyu YE, JIAN Zhang, Yi Lu, Yingxuan Gong, Xinyu Jiang, Yueming Li, Yinghao Wang, Xiuzhi Li, Jingwen Liu, Rencui Quan. DKK1 is a potential target for enhancing the efficiency of radioimmunotherapy in head and neck squamous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5009.

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.

TGFβ1 Suppressed Matrix Mineralization of Osteoblasts Differentiation by Regulating SMURF1–C/EBPβ–DKK1 Axis

Transforming growth factor β1 (TGFβ1) is a major mediator in the modulation of osteoblast differentiation. However, the underlying molecular mechanism is still not fully understood. Here, we show that TGFβ1 has a dual stage-dependent role in osteoblast differentiation; TGFβ1 induced matrix maturation but inhibited matrix mineralization. We discovered the underlying mechanism of the TGFβ1 inhibitory role in mineralization using human osteoprogenitors. In particular, the matrix mineralization-related genes of osteoblasts such as osteocalcin (OCN), Dickkopf 1 (DKK1), and CCAAT/enhancer-binding protein beta (C/EBPβ) were dramatically suppressed by TGFβ1 treatment. The suppressive effects of TGFβ1 were reversed with anti-TGFβ1 treatment. Mechanically, TGFβ1 decreased protein levels of C/EBPβ without changing mRNA levels and reduced both mRNA and protein levels of DKK1. The degradation of the C/EBPβ protein by TGFβ1 was dependent on the ubiquitin–proteasome pathway. TGFβ1 degraded the C/EBPβ protein by inducing the expression of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (SMURF1) at the transcript level, thereby reducing the C/EBPβ-DKK1 regulatory mechanism. Collectively, our findings suggest that TGFβ1 suppressed the matrix mineralization of osteoblast differentiation by regulating the SMURF1-C/EBPβ-DKK1 axis.

A neutralizing antibody against DKK1 does not reduce plaque formation in classical murine models of atherosclerosis: Is the therapeutic potential lost in translation?

Background and aimsClinical interventions targeting nonlipid risk factors are needed given the high residual risk of atherothrombotic events despite effective control of dyslipidemia. Dickkopf-1 (DKK1) plays a lipid-independent role in vascular pathophysiology but its involvement in atherosclerosis development and its therapeutic attractiveness remain to be established. MethodsPatient data, in vitro studies and pharmacological intervention in murine models of atherosclerosis were utilized. ResultsIn patients' material (n = 127 late stage plaque specimens and n = 10 control vessels), DKK1 mRNA was found to be higher in atherosclerotic plaques versus control arteries. DKK1 protein was detected in the luminal intimal area and in the necrotic core of plaques. DKK1 was released from isolated primary human platelets (~12 - 21-fold) and endothelial cells (~1.4–2.5-fold) upon stimulation with different pathophysiological stimuli. In ApoE−/− and Ldlr−/− mice, plasma DKK1 concentrations were similar to those observed in humans, whereas DKK1 expression in different atheroprone arterial segments was very low/absent. Chronic treatment with a neutralizing DKK1 antibody effectively reduced plasma concentrations, however, plaque lesion area was not reduced in ApoE−/− and Ldlr−/− mice fed a western diet for 14 and 16 weeks. Anti-DKK1 treatment increased bone volume and bone mineral content. ConclusionsFunctional inhibition of DKK1 with an antibody does not alter atherosclerosis progression in classical murine models. This may reflect the absence of DKK1 expression in plaques and more advanced animal disease models could be needed to evaluate the role and therapeutic attractiveness of DKK1 in late stage complications such as plaque destabilization, calcification, rupture and thrombosis.

Interaction between Stromal Cell-Derived Factor 1 Alpha and Dickkopf-1 Involves in Occurence of Myeloma Bone Disease

To explore the role of interaction between osteoclast stimulator stromal derived factor 1 alpha (SDF-1α) and osteoblast inhibitor dickkopf-1 (DKK-1) in the development of multiple myeloma (MM) bone disease. The serum samples of 51 patients with newly diagnosed MM, 30 age-matched healthy controls, and 35 non-Hodgkin lymphoma patients from June 2011 to May 2014 in Peking Union Medical College Hospital were collected. The serum SDF-1α and DKK-1 were detected by ELISA. Primary myeloma cells and human MM cell line RPMI 8226 were treated with SDF-1α, then DKK-1 mRNA expression was detected by real time PCR. Primary bone marrow stromal cells (BMSCs) were treated with Wnt-3a and/or DKK-1, and the transc-ription level of SDF-1α mRNA was assayed. Serum SDF-1α in MM patients was significantly higher than that in control group (3231.0±1269.5 pg/ml vs 2817.5±419.6 pg/ml)(P=0.036), so was serum DKK-1 (3057.4±1874.7 pg/ml vs 1867.7±1148.4 pg/ml)(P=0.01). There was a positive correlation between serum SDF-1α and DKK-1 in MM patients (r=0.301, P=0.032), but there was no correlation between control group (r=0.15, P=0.428) and non-Hodgkin lymphoma patients (r=0.227, P=0.095). After treated with SDF-1α (20 ng/ml) for 8 and 36 h, the DKK-1 mRNA transcription level in RPMI 8226 increased by 1.92 and 4.19-folds respectively(P=0.365, P=0.099). Moreover, the high transcription level of DKK-1 mRNA was observed in 5 out of 9 MM patients. The detection showed that after treatment with SDF-1α, the transcription level was up-regulated(P=0.043), the Wnt-3a (200 ng/ml) could decrease the expression of SDF-1α mRNA in primary BMSC to 29% of baseline(P=0.028), the adding DKK-1 could reverse the down-regulation effect. The serum SDF-1α and DKK-1 level in MM patients is high than normal leve, moreover shows the positive correlation between them. The SDF-1α and DKK-1 can interreact, therefore accerate the formation of MM bone disease.

Dickkopf-1 Can Lead to Immune Evasion in Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).

Ectodysplasin-A2 induces dickkopf 1 expression in human balding dermal papilla cells overexpressing the ectodysplasin A2 receptor

Androgenetic alopecia (AGA) is a common genetic disorder, and a X-chromosomal locus that contains the androgen receptor (AR) and ectodysplasin A2 receptor (EDA2R) genes represents a major susceptibility locus for AGA. In our previous study, we reported that ectodysplasin-A2 (EDA-A2) induces apoptosis in cultured human hair follicle (HF) cells and promotes the regression of HFs in mice. However, the role of the EDA-A2/EDA2R in AGA remains unknown, as the causative gene in this pathway has not yet been identified and potential functional connections between EDA-A2 signaling and the androgen pathway remain unclear. In this study, we investigated the expression of EDA2R in balding HFs and matched with non-balding HFs. The EDA2R level was upregulated in the balding dermal papilla (DP) cells compared with non-balding DP cells derived from patients with AGA. However, EDA2R was strongly expressed in both balding and non-balding outer root sheath (ORS) cells. We screened EDA-A2-regulated genes in balding DP cells and identified dickkopf 1 (DKK-1) as catagen inducer during the hair cycle. The mRNA and protein expression levels of DKK-1 were both upregulated by EDA-A2. In addition, DKK-1 expression was induced by EDA-A2 both in cultured human HFs and in mouse HFs. Moreover, the EDA-A2-induced apoptosis of DP and ORS cells was reversed by the antibody-mediated neutralization of DKK-1.Collectively, our data strongly suggest that EDA-A2 induces DKK-1 secretion and causes apoptosis in HFs by binding EDA2R, which is overexpressed in the bald scalp. EDA-A2/EDA2R signaling could inhibit hair growth through DKK-1 induction, and an inhibitor of EDA-A2/EDA2R signaling may be a promising agent for the treatment and prevention of AGA.

P0096RENAL EXPRESSION AND CIRCULATING LEVELS OF CANONICAL WNT PATHWAY INHIBITORS IN THE EXPERIMENTAL MODEL OF EARLY CHRONIC KIDNEY DISEASE

Abstract Background and Aims Molecular mechanisms implicated in the initial stages of inorganic phosphate (Pi) imbalance in chronic kidney disease (CKD) remain poorly understood. The aim of the study was to evaluate whether canonical Wnt pathway inhibitors (iWnt) involved in early response to Pi retention in CKD. Method Mild CKD was induced by 3/4 nephrectomy (NE, n=10) in spontaneously hypertensive rats (SHR) fed rat chow diet containing 0.8 % phosphate. Controls were sham operated SHR (SO, n=9). Duration of experimental exposure (NE or SO) was 4 months. Creatinine (Cr), albumin/ creatinine ratio (Alb/Cr), inorganic phosphate (Pi), fractional (FEPi) and daily Pi excretion (uPi24), intact parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho protein, sclerostin (SOST) and Dickkopf-1 (DKK1), SOST and DKK1 mRNA expression in kidney, renal interstitial fibrosis (RF) were analyzed. Results Levels of Alb/Cr, serum Cr, RF and indices of Pi exchange in the experimental model corresponded to mild CKD with Pi retention (Table). No differences were observed in serum levels of Klotho, FGF23 and PTH in experimental group vs controls (Table). Serum SOST and DKK1 concentrations were significantly higher in NE group while the kidney expression of SOST and DKK1 mRNAs decreased compared to SO (Table). Conclusion Early stages of experimental CKD are characterized by increased serum concentrations of sclerostin and Dickkopf-1 in the absence of Klotho, FGF23 or PTH levels alterations. The increase of circulating iWnt levels seems not to be associated with their renal biosynthesis and likely have a bone origin.

Effect of electroacupuncture on neurological function and Wnt signaling pathway in ischemic brain tissue of cerebral infarction rats

To explore the mechanism of electroacupuncture (EA) underlying improvement of cerebral infarction (CI) by investigating its influence on expression of cerebral Wnt7a, lymphoid enhancer factor-1 (LEF1), glycogen synthase kinase 3β(GSK-3β) and Dickkopf-1(DKK1) mRNA and proteins in CI rats. A total of 280 male Wistar rats were randomly divided into blank control (n＝10), sham-operation, model and EA groups，and 90 rats of the last 3 groups were further divided into 1, 3, 6, 9, 12 and 24 h, and 3, 7 and 12 d subgroups with 10 rats in each subgroup. The CI model was established by occlusion of the middle cerebral artery (MCAO). The sham-operation group received the same surgical operation but without thread embolus insertion. EA (2 Hz/15 Hz, 2 mA) was applied to "Shuigou" (GV26) for 20 min, once a day for 1, 3, 7 and 12 d, respectively. The neurological deficit was evaluated by using Neurological Severity Scores (NSS). The expression levels of Wnt7a，LEF1, GSK-3β and DKK1 mRNAs and proteins in the right ischemic brain tissues were detected by Quantative real-time PCR and Western blot, respectively. After MCAO, the NSS score was significantly increased in the model and EA groups relevant to the blank control and sham-operation groups (P<0.01) and gradually decreased with the prolongation of ischemia time. After EA, the NSS scores were notably decreased on day 3, 7 and 12 in the EA group compared with the model group (P<0.05, P<0.01). After modeling, the expression levels of Wnt7a and LEF1 mRNAs from 3 h to 12 d, Wnt7a and LEF1 proteins from 6 h to 12 d were considerably increased (P<0.01, P<0.05), while those of GSK-3β mRNA at 9, 12 and 24 h, GSK-3β protein at 24 h and 3 d, and DKK1 mRNA at 24 h and 3 d and DKK1 protein at 3 d were obviously decreased in the model group relevant to the sham-operation group (P<0.05, P<0.01). After the intervention, the expression levels of Wnt7a mRNA at 12 h to 3 d, Wnt7α protein from 24 h to 12 d, LEF1 mRNA from 24 h to 12 d, and LEF1 protein from 3 d to 12 d were further apparently up-regulated (P<0.01, P<0.05), while those of GSK-3β mRNA at 9 h, 3，7 and 12 d, and GSK-3β protein at 12 h, 7 d and 12 d, and DKK1 mRNA at 12 h, 24 h and 3 d, and DKK1 protein at 24 h to 12 d were obviously down-regulated in the EA group relevant to the model group (P<0.01, P<0.05). No significant difference was found between the blank and sham-operation groups in the NSS scores and expression levels of Wnt7a, LEF1, GSK-3β and DKK1 mRNAs and proteins at all the time points (P>0.05). EA of GV26 can significantly improve the neurological deficit symptoms in MCAO rats, which may be associated with its effects in up-regulating the expression of Wnt7a and LEF1 mRNAs and proteins, and in down-regulating the expression of GSK-3β and DKK1 mRNAs and proteins.

DKN-01 in combination with pembrolizumab in patients with advanced gastroesophageal adenocarcinoma (GEA): Tumoral DKK1 expression as a predictor of response and survival.

357 Background: Dickkopf-1 (DKK1) modulates Wnt signaling and contributes to an immune suppressive tumor microenvironment. DKN-01 (D), a neutralizing DKK1 antibody + pembrolizumab (P) has demonstrated safety and clinical activity in advanced GEA. We report response and survival outcomes in anti-PD-1/anti-PD-L1 naïve GEA patients by high/low tumoral DKK1 expression. Methods: We enrolled advanced anti-PD-1/PD-L1 naïve GEA patients (pts) in a Phase 1b/2a study of D + P (NCT02013154). Tumoral DKK1 mRNA expression was assessed by an in situ hybridization RNAscope assay. Objective response rate (ORR), disease control rate (DCR), progression free survival (PFS) and overall survival (OS) were compared between DKK1 high and low groups. Kaplan-Meier method and Cox-PH model was used for survival analysis and logistic regression was used for clinical benefit/response outcome. Results: 34 GEA pts were enrolled to receive 300 mg D + P. 31 GEA pts had DKK1 expression available (25 response-evaluable/RE) and 27 had both DKK1 and PD-L1 expression available (22 RE). In RE pts, DKK1 high (H-score ≥ upper-tertile [≥35]) had an ORR of 50% (5 PR/10), DCR of 80% (8/10) while DKK1 low ( &lt; upper-tertile) had an ORR of 0% (0/15) and DCR of 20% (3/15); DKK1 high (vs. low) had an OR of 16 (95%CI: 2.2, 118.3; n = 25) and adjusted (for PD-L1 CPS ≥10 vs. &lt; 10) OR of 17.6 (95%CI:1.6, 194.4; n = 22) for clinical benefit/response (PR/SD vs. PD). Median PFS was 22.1 weeks in DKK1 high (n = 11) vs. 5.9 weeks in DKK1 low (n = 20); HR of 0.23 (95%CI: 0.082, 0.66; n = 31). Adjusted (for PD-L1 expression) HR for DKK1 high was 0.20 (95%CI: 0.061,0.68; n = 27) for PFS. DKK1 high (n = 11) had a median OS of 31.6 weeks vs. 17.4 weeks in DKK1 low (n = 20); HR of 0.45 (95%CI: 0.16, 1.3; n = 31) and adjusted HR of 0.62 (95%CI: 0.2,1.9; n = 27). Conclusions: High levels of tumoral DKK1 expression identify advanced anti-PD-1/PD-L1 naïve GEA pts with the greatest benefit from D + P. Improvements in response/clinical benefit and PFS were observed independent of PD-L1 expression. Tumoral DKK1 as a potential predictive biomarker for DKN-01 treated GEA pts will be evaluated in future studies. Overall survival follow-up is ongoing. Clinical trial information: NCT02013154.

Correlation of radiomics of metastatic lesions in gastroesophageal adenocarcinoma (GEA) with tumoral DKK1 mRNA expression and other immune biomarkers in patients (pts) treated with DKN-01.

429 Background: Dickkopf-1 (DKK1) modulates Wnt and PI3K/AKT signaling pathways and contributes to an immune suppressive tumor microenvironment. Recently, anti-DKK1 antibody (DKN-01) [D] plus pembrolizumab (P) in GEA tumors has demonstrated clinical activity. Radiomic Quantitative Texture Analysis (QTA) is a non-invasive method for imaging biomarker discovery that can predict molecular drivers of cancer from CT scans obtained during clinical trials. QTA may be a useful tool for predicting DKK1 expression and other immune biomarkers in metastatic lesions from GEA pts treated with DKN-01. Methods: 13 pts with GEA (12 M; 1F; age 37-81) enrolled in D+P Phase I/II trial with high vs low DKK1 RNA Scope H-Scores (H Score &gt; 35; n = 6 vs H-Score &lt; 35; n = 7) were identified. Metastatic Target lesions (TLs) from baseline CTs with IV contrast underwent additional QTA analysis (TexRAD, CE mark, Essex, UK) after RECIST analysis. ROIs were placed on the TLs and histogram frequency curves (HFC) of the metastatic tumor pixels were generated. First order tumor HFC statistical results (i.e. mean, Standard Deviation-SD, Skewness, Kurtosis, MPP) were correlated with tumoral DKK1 mRNA expression, baseline plasma DKK1 levels and other biomarkers (PD-L1 CPS and MDSCs) along with tumor shrinkage using Pearson’s r correlation (significance p &lt; 0.05). Results: Tumor derived QTA parameter (QTA SD) of the largest metastatic TL from each subject was positively correlated with DKK1 RNAscope H-score (r = 0.661, p value = .014) meaning larger SD texture scores had greater DKK1 expression. Also, tumor derived QTA parameter (QTA MPP) was correlated with baseline plasma DKK1 (r = -0.461, p = 0.005). Finally, QTA SD was correlated with PD-L1 expression (r = 0.630, p = 0.028) and TL shrinkage (r = -0.590, p = 0.034). Conclusions: Radiomics of GEA tumor metastasis using QTA showed significant correlations between tumor texture, tumor DKK1 mRNA expression and baseline plasma DKK1 suggesting that tumor textures may provide a non-invasive tool for assessing DKK1. Although promising as a imaging biomarker, further studies are required to validate these findings. Clinical trial information: NCT02013154.

Dorsomorphin: A novel inhibitor of Dickkopf-1 in breast cancer

Advanced stages of breast cancer are frequently complicated by bone metastases which cause substantial cancer-related morbidity and mortality. The Wnt-signaling antagonist Dickkopf-1 (DKK-1) has emerged as a crucial factor in the development and progression of osteolytic bone metastases. Although several signaling pathways have been implicated in promoting DKK-1 production in breast cancer cells, pharmacological interventions that interfere with tumor DKK-1 synthesis still remain scarce.In the current study, using an unbiased approach, we identified the small molecule Dorsomorphin as a potent suppressor of DKK-1 in several breast cancer cell lines (MDA-MB-231, MDA-Bone, MDA-MET and MCF7, respectively). Here, Dorsomorphin suppressed DKK-1 mRNA and protein production by 70 and 90%, respectively (p <0.001). Whereas bone morphogenic protein (BMP)- and AMP activated protein kinase (AMPK)-signaling are two well-established targets of Dorsomorphin, we show that neither pathway is essentially involved in facilitating its inhibitory effects on DKK-1.In summary, we identified Dorsomorphin as a potent pharmacological inhibitor of DKK-1 production in breast cancer cells. Whether Dorsomorphin reflects a valuable therapeutic agent in breast cancer warrants further investigations.

Inhibitors of canonical Wnt signaling pathway and inorganic phosphate imbalance in experimental chronic kidney disease

BACKGROUND. The molecular mechanisms of the initial stages of inorganic phosphate (Pi) metabolic disorders in chronic kidney disease (CKD) remain poorly understood.THE AIM. To test the hypothesis about changes in canonical Wnt signaling pathway inhibitors biosynthesis and a concomitant decrease in bone turnover as one of early mechanisms of Pi imbalance in CKD.MATERIAL AND METHODS. Creatinine (Cr), inorganic phosphate (Pi), serum parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), osteoprotegerin (OPG), sclerostin (SOST) and Dickkopf-1 (DKK), renal SOST and DKK mRNA expression, albuminuria (Alb), proteinuria (uTP) levels, fractional (FEPi) and daily (uPi24) Pi excretion were analyzed in SHR rats (N = 52) with 3/4 nephrectomy (NE) or sham operation (SO) and observation periods of 2, 4, and 6 months.RESULTS. Experimental model was comparable with 1-2 stages of CKD. In groups NE4 and NE6, the concentration of sPi and renal Pi excretion (FEPi and uPi24) were significantly higher vs corresponding controls SO4 (p = 0.006, p &lt;0.010) and SO6 (p = 0.002, p = 0.028). Serum concentrations of FGF23 and PTH in NE and SO animals did not change significantly. In NE4 and NE6 groups, serum SOST and DKK concentrations were significantly higher vs controls (p &lt;0.049, p &lt;0.043), while the kidney expression SOST and DKK mRNA in NE rats did not change significantly or decreased (p = 0.002, p &lt;0.011). The serum concentration of OPG was higher in the NE6 vs SO6 control (p = 0.028).CONCLUSION. The initial stages of experimental CKD are characterized by an increase in serum concentrations of Dikkopf-1, sclerostin and osteoprotegerin. The obtained data suggest the possible role of canonical Wnt signaling inhibition and reduction of bone turnover in the pathogenesis of Pi metabolic disorders in early stages of CKD.

Correlation of Dickkopf-1 with Inflammation in Crohn Disease

To explore the potential roles of Dickkopf-1 (DKK-1) and b-catenin in Crohn disease, and to evaluate the effects of a tumor necrosis factor (TNF)-a inhibitor on Wnt signaling in patients with the disease. We enrolled 21 patients who received infliximab treatment for one year and achieved clinical remission during the treatment period. Disease activity was graded according to the Pediatric Crohn's Disease Activity Index (PCDAI). Peripheral blood and colonic mucosal specimens were collected from all patients with Crohn disease and from 14 healthy controls. DKK-1 levels in serum were detected by enzyme-linked immunosorbent assay (ELISA). Total RNA for DKK-1 and b-catenin from the frozen colonic tissue were obtained via real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Serum C-reactive protein (CRP) levels, erythrocyte sedimentation rates (ESR), and albumin were also measured in patients with Crohn disease before and after infliximab therapy. The serum levels of DKK-1 were significantly higher in patients with Crohn disease than in healthy controls (P=0.003) and were decreased in those treated with infliximab (P=0.026). Serum DKK-1 level was correlated with levels of ESR (r=0.527, P=0.025), CRP (r=0.502, P=0.034), albumin (r=0.363, P=0.021) and PCDAI (r =0.462, P=0.054) in Crohn disease. DKK-1 mRNA expression in the colonic mucosa was higher in patients than in controls and decreased after infliximab treatment. b-catenin expression in the colonic mucosa was lower in patients than in controls and increased after infliximab treatment. However, the differences were not significant (P<0.05). DKK-1 might be an important mediator of the pathogenesis of Crohn disease, and changes in DKK-1 levels may serve as biomarkers of inflammation in these patients.

Expression level of Dickkopf-1 protein in bone mesenchymal stem cells in Type 2 diabetic rats and its relationship with osteogenic activity

To examine the growth activity and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in rats with Type 2 diabetes mellitus (T2DM) as well as the expression level of Dickkopf-1 (DKK-1) in bone marrow, and to explore the relationship between the osteogenic activity of BMSCs and the expression of DKK-1. Methods: The BMSCs were isolated from T2DM rats and were cultured in vitro. The BMSCs were divided into a T2DM group and a control group. The proliferation of BMSCs was detected by cell counting kit-8 (CCK8). Apoptosis rate was detected by annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining. In the osteogenic induction phase, the expression level of alkaline phosphatase (ALP) in BMSCs was detected by ALP staining and ALP activity assay kit. The osteogenic differentiation of BMSCs was analyzed by alizarin red staining and mineralized nodule quantification. In addition, the expression of Runx2 and DKK-1 in BMSCs was detected by qRT-PCR. Results: Compared with the control group, the proliferation of BMSCs was decreased and the apoptosis was increased in the T2DM group (both P<0.01). In the osteogenic induction process of BMSCs, the expression of ALP significantly decreased, the formation of calcium nodules reduced, and the expression of osteoblast transcription factor Runx2 was down-regulated in the T2DM group compared with those in the control group (all P<0.01). The levels of DKK-1 protein and mRNA were up-regulated in the T2DM group, which were higher than those in the control group (both P<0.01). The levels of DKK-1 protein and mRNA were related to the increase of Runx2 (both P<0.01). Conclusion: The growth activity of BMSCs and the potential of osteogenic differentiation are attenuated in the T2DM rats, which may be related to the increase of DKK-1 expression in BMSCs.