Dickkopf-1 Expression Research Articles

Converging on DKK1 to drive metastasis

Cancer Hepatocellular carcinoma (HCC) is a common form of liver cancer. The Wnt signaling protein Dickkopf-1 (DKK1) and epidermal growth factor receptor (EGFR) are both associated with metastatic progression and poor prognosis in HCC patients. Niu et al. found that these molecular markers are linked. The activation of EGFR in HCC cells induced DKK1 expression through parallel pathways that promoted nuclear translocation of the kinase PKM2 and activation of the acetyltransferase p300. These pathways converged on modifying histone H3 at the DKK1 promoter to activate DKK1 transcription. Sci. Signal. 13 , eabb5727 (2020).

EGF promotes DKK1 transcription in hepatocellular carcinoma by enhancing the phosphorylation and acetylation of histone H3.

The protein Dickkopf-1 (DKK1) is frequently overexpressed at the transcript level in hepatocellular carcinoma (HCC) and promotes metastatic progression through the induction of β-catenin, a Wnt signaling effector. We investigated how DKK1 expression is induced in HCC and found that activation of the epidermal growth factor receptor (EGFR) promoted parallel MEK-ERK and PI3K-Akt pathway signaling that converged to epigenetically stimulate DKK1 transcription. In HCC cell lines stimulated with EGF, EGFR-activated ERK phosphorylated the kinase PKM2 at Ser37, which promoted its nuclear translocation. Also in these cells, EGFR-activated Akt phosphorylated the acetyltransferase p300 at Ser1834 Subsequently, PKM2 and p300 mediated the phosphorylation and acetylation, respectively, of histone H3 at the DKK1 promoter, which synergistically enhanced DKK1 transcription. The mechanism was supported with mutational analyses in cells and in a chemically induced HCC model in rats. The findings suggest that dual inhibition of the MEK and PI3K pathways might suppress the expression of DKK1 and, consequently, tumor metastasis in patients with HCC.

Balanced Wnt/Dickkopf-1 signaling by mesenchymal vascular progenitor cells in the microvascular niche maintains distal lung structure and function.

The well-described Wnt inhibitor Dickkopf-1 (DKK1) plays a role in angiogenesis as well as in regulation of growth factor signaling cascades in pulmonary remodeling associated with chronic lung diseases (CLDs) including emphysema and fibrosis. However, the specific mechanisms by which DKK1 influences mesenchymal vascular progenitor cells (MVPCs), microvascular endothelial cells (MVECs), and smooth muscle cells (SMCs) within the microvascular niche have not been elucidated. In this study, we show that knockdown of DKK1 in Abcg2pos lung mouse adult tissue resident MVPCs alters lung stiffness, parenchymal collagen deposition, microvessel muscularization and density as well as loss of tissue structure in response to hypoxia exposure. To complement the in vivo mouse modeling, we also identified cell- or disease-specific responses to DKK1, in primary lung chronic obstructive pulmonary disease (COPD) MVPCs, COPD MVECs, and SMCs, supporting a paradoxical disease-specific response of cells to well-characterized factors. Cell responses to DKK1 were dose dependent and correlated with varying expressions of the DKK1 receptor, CKAP4. These data demonstrate that DKK1 expression is necessary to maintain the microvascular niche whereas its effects are context specific. They also highlight DKK1 as a regulatory candidate to understand the role of Wnt and DKK1 signaling between cells of the microvascular niche during tissue homeostasis and during the development of chronic lung diseases.

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.

Dickkopf-1 Expression as a Predicting Factor for Bone Metastasis in Breast Cancer

Dickkopf-1 Expression as a Predicting Factor for Bone Metastasis in Breast Cancer

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).

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-1 Overexpression in vitro Nominates Candidate Blood Biomarkers Relating to Alzheimer's Disease Pathology.

Background:Previous studies suggest that Dickkopf-1 (DKK1), an inhibitor of Wnt signaling, plays a role in amyloid-induced toxicity and hence Alzheimer’s disease (AD). However, the effect of DKK1 expression on protein expression, and whether such proteins are altered in disease, is unknown.Objective:We aim to test whether DKK1 induced protein signature obtained in vitro were associated with markers of AD pathology as used in the amyloid/tau/neurodegeneration (ATN) framework as well as with clinical outcomes.Methods:We first overexpressed DKK1 in HEK293A cells and quantified 1,128 proteins in cell lysates using aptamer capture arrays (SomaScan) to obtain a protein signature induced by DKK1. We then used the same assay to measure the DKK1-signature proteins in human plasma in two large cohorts, EMIF (n = 785) and ANM (n = 677).Results:We identified a 100-protein signature induced by DKK1 in vitro. Subsets of proteins, along with age and apolipoprotein E ɛ4 genotype distinguished amyloid pathology (A + T–N–, A+T+N–, A+T–N+, and A+T+N+) from no AD pathology (A–T–N–) with an area under the curve of 0.72, 0.81, 0.88, and 0.85, respectively. Furthermore, we found that some signature proteins (e.g., Complement C3 and albumin) were associated with cognitive score and AD diagnosis in both cohorts.Conclusions:Our results add further evidence for a role of DKK regulation of Wnt signaling in AD and suggest that DKK1 induced signature proteins obtained in vitro could reflect theATNframework as well as predict disease severity and progression in vivo.

Artesunate promotes osteoblast differentiation through miR-34a/DKK1 axis

BackgroundOsteoporosis is characterised by impairment of microarchitecture and bone mass. Therapeutic strategy promoting osteoblast differentiation is considered as a promising direction for the treatment of osteoporosis. Artesunate (ART) is related to osteoporosis, but the relationship between ART and osteogenic differentiation is still unknown. MethodsCells proliferation were measured by MTT, ALP activity assay and Alizarin Red S staining were used to assess osteogenic differentiation of hBMSCs. Western blotting and qRT-PCR were applied for measuring expression of protein and mRNA, respectively. The relationship between miR-34a and Dickkopf-1 (DKK1) was detected by dual luciferase reporter assay. ResultsThe expression of osteoblasts differentiation related proteins (Runx2, OCN, and OPN) were significantly increased by ART. And ART accelerates the osteoblasts differentiation of hBMSCs through promoting Wnt signaling pathway by DKK1 inhibition. Significant higher expression of miR-34a and lower expression of DKK1 could be induced by ART. We firstly proved that miR-34a could bind DKK1 specifically. ConclusionART could promote osteoblast differentiation through miR-34a/DKK1/Wnt pathway. Therefore, our findings may provide a new thought for the treatment of osteoporosis by ART through osteoblast differentiation promotion.

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.

Comparison of the diagnostic value of CEA combined with OPN or DKK1 in non-small cell lung cancer.

Carcinoma embryonic antigen (CEA), osteopontin (OPN), and Dickkopf-1 (DKK1) expressed in serum are associated with hypoxia in tumor progression. However, the role of these proteins in the plasma of patients with non-small cell lung cancer (NSCLC) is poorly understood. The diagnostic values of CEA combined with OPN or DKK1 were compared in non-small cell lung cancer. This study investigated the diagnostic value of CEA combined with OPN and DKK1, respectively, in NSCLC. Eighty patients with NSCLC (NSCLC group) and 60 patients with benign lung diseases (benign lung disease group) admitted to Shandong Provincial Third Hospital from May 2014 to January 2015 were selected as the study subjects. In addition, 60 healthy subjects undergoing normal physical examination were selected as healthy control group. The OPN and DKK1 in serum of the two groups were detected by enzyme linked immunosorbent assay (ELISA), and the CEA expression was measured by Electrochemical Photometric method. The diagnostic value of CEA combined with OPN and DKK1, respectively, in NSCLC was analyzed. The expression of CEA, OPN, and DKK1 in serum of NSCLC group was significantly higher than that of healthy control group and benign lung disease group (P<0.05). The expression of CEA, OPN and DKK1 in serum of NSCLC patients was correlated with tumor diameter, lymph node metastasis, degree of pathological differentiation and clinical stage (P<0.05). ROC curve for diagnosis of NSCLC was drawn by further combination of serum CEA and OPN. The AUC of combined diagnosis of CEA and OPN for NSCLC was 0.920 (95% CI, 0.875-0.964), and the diagnostic sensitivity and specificity were 87.50 and 86.67%, respectively; the AUC of combined diagnosis of CEA and DKK1 for NSCLC was 0.912 (95% CI, 0.866-0.958), and the diagnostic sensitivity and specificity were 92.50 and 76.67%, respectively. CEA, OPN and DKK1 may be involved in the occurrence and progression of NSCLC and have good sensitivity and specificity in the diagnosis of NSCLC and may be new biomarkers for the diagnosis of NSCLC.

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.

Prognostic role of dickkopf-1 in patients with cancer.

As an antagonist for the WNT signal passway, dickkopf-1(DKK1) have a great important role in the occurence and development of various type cancer. The present paper performed a meta-analysis to evaluate the predictive significance of DKK1 in cancer.To assess the relationship between the expression of DKK1 and prognostic role in human cancers, a total of 16 articals were screened from the multiple online databases (Pubmed, EMBASE, CNKI, Web of Science and Google Scholar) in our study. By using the STATA soft,pooled hazard ratio and 95% confidence intervals of overall survival (OS), progression-free survival, disease-free survival and time to recurrence were used to evaluate the strength of this relationship.The meta-analysis showed that higher expression of DKK1 was significantly associated with shorter OS in cancer patients. In stratified analyzes, the higher expression of DKK1 could reduced the OS in patients with breast cancer,digestive system cancer and urogenital system cancer, but not patients with the lung cancer. It also showed that higher expression of DKK1 was significantly associated with shorter progression-free survival, disease-free survival and time to recurrence in cancer patients.The present study indicate that higher expression of DKK1 predict an unfavorable clinical outcome in patients with breast cancer, digestive system cancer and urogenital system cancer.

Exercise Attenuates Brain Aging by Rescuing Down-Regulated Wnt/β-Catenin Signaling in Aged Rats.

Down-regulated Wnt signaling is involved in brain aging with declined cognitive capacity due to its modulation on neuronal function and synaptic plasticity. However, the molecular mechanisms are still unclear. In the present study, the naturally aged rat model was established by feeding rats from 6 months old to 21 months old. The cognitive capacity of aged rats was compared with young rats as the controls and the aged rats upon 12-week exercise interventions including treadmill running, resistance exercise, and alternating exercise with resistance exercise and treadmill running. Wnt signaling was examined in hippocampal tissues of the rats from different groups. Results indicated that the expression of Dickkopf-1 (DKK-1) as an antagonist of Wnt signal pathway, the activation of GSK-3β, and the hyperphosphorylated Tau were markedly increased as the extension of age. Meanwhile, higher p-β-cateninSer33, 37, Thr41 promoted neuronal degradation of aged rats. In contrast, three kinds of exercise interventions rescued the abnormal expression of DKK-1 and synaptophysin such as PSD-93 and PSD-95 in hippocampal tissues of the aged rats; especially 12-week treadmill running suppressed DKK-1 up-regulation, GSK-3β activation, β-catenin phosphorylation, and hyperphosphorylated Tau. In addition, the down-regulated PI3K/AKT and Wnt signal pathways were observed in aged rats, but could be reversed by resistance exercise and treadmill running. Moreover, the increased Bax and reduced Bcl-2 levels in hippocampal tissues of aged rats were also reversed upon treadmill running intervention. Taken together, down-regulated Wnt signaling suppressed PI3K/Akt signal pathway, aggravated synaptotoxicity, induced neuron apoptosis, and accelerated cognitive impairment of aged rats. However, exercise interventions, especially treadmill running, can attenuate their brain aging process via restoring Wnt signaling and corresponding targets.

Analyzing the prognostic value of DKK1 expression in human cancers based on bioinformatics.

BackgroundThe Dickkopf1 (DKK1) gene encodes a protein that belongs to the Dickkopf family. The protein can inhibit the Wnt signaling pathway which plays a key role in the carcinogenesis and progression of various types of cancers. Based on this, we hypothesized that the differential expression of DKK1 may figure significantly in cancers by regulating Wnt signaling pathway transduction. In this study, we conducted bioinformatics analysis to evaluate the prognostic and therapeutic value of DKK1 expression level in human cancers.MethodsThe expression level was analyzed by using the Oncomine database and Gene Expression Profiling Interactive Analysis tool. The analysis of prognosis was conducted by using the UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA), and DriverDBv3 databases. We also investigated using DKK1 promoter methylation to define cancer types through the UALCAN database. Meanwhile, the related functional networks of DKK1 were analyzed by using the GeneMANIA interactive tool and Cytoscape software. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted using the Metascape online website, and we used the cBioPotartal database to explored DKK1 expression, aberrant information, and the co-expression genes in the subgroups of lung cancer. Finally, we performed the overall survival (OS) meta-analysis of the DKK1 expression in lung squamous cell carcinoma (LUSC) via the Lung Cancer Explorer (LCE).ResultsDKK1 was differentially expressed in different types of human cancers. DKK1 was overexpressed in human cancers including head and neck squamous cell carcinoma (HNSC), LUSC, and pancreatic adenocarcinoma (PAAD). Overexpression of DKK1 indicated adverse OS in bladder urothelial carcinoma (BLCA), HNSC, and PADD, but no difference in OS was found between the LUSC and healthy groups. The high expression of DKK1 was also associated with shorter disease-free survival (DFS) in HNSC, LUSC, and PAAD. Gene regulation network analysis indicated that DKK1 was mainly involved in Wnt signaling pathways and several other signaling pathways.ConclusionsOur findings showed that DKK1 is significantly expressed in various cancers and could be a biomarker for targeted therapy and a predictor for prognosis of these specific cancers. The bioinformatics analysis revealed a significant overexpression of DKK1 in HNSC, LUSC, and PAAD, with DKK1 overexpression being associated with adverse outcome in these patients, but how DKK1 expression levels relate to hematological malignancies and prognosis is still unclear. These new insights into the function of DKK1 may provide a basis for new targeted drug therapy and an avenue for further investigation into the mechanisms underlying carcinogenesis of DKK1 in different cancer types.

Overexpression of microRNA-141 inhibits osteoporosis in the jawbones of ovariectomized rats by regulating the Wnt/β-catenin pathway

ObjectiveThis work was aimed to investigate the effect of microRNA-141 (miR-141) overexpression in the jawbones of ovariectomized-induced osteoporosis rats and investigate the role of miR-141 in the Wnt/β-catenin pathway. MethodsTwenty-four female rats were randomly divided into the sham group, ovariectomized osteoporosis group (OP), miR-141 agonist group (miR-141), and miR-141 scramble group (Scramble). Bone mineral density (BMD) and pathological changes of the jaw were detected. Serum receptor activator of nuclear factor-B ligand (RANKL), osteoprotegerin, tartrate-resistant acid phosphatase (TRAP), and bone gla protein (BGP) levels were tested by ELISA. The expression of Runt-related transcription factor 2 (Runx2), and Osterix measured by immunohistochemistry and the expression of Wnt, β-catenin, and Dickkopf1 (DKK1) proteins was measured by Western blot. Furhter, the Wnt agonist DKK2-C2, Wnt inhibitor Endostar were used to verify the effect of miR-141 overexpression on the Wnt/β-catenin pathway. ResultCompared with the OP group, the content of osteoprotegerin increased while the levels of RANKL, BGP, TRAP decreased in the miR-141 and DKK2-C2 groups (p < 0.05). The levels of Runx2 and Osterix increased significantly in the miR-141 and DKK2-C2 groups when compared to the OP group (p < 0.05). Interestingly, the protein expression of Wnt and β-catenin increased while DKK1 was remarkably down-regulated in the miR-141 and DKK2-C2 groups when compared to the OP group (p < 0.05). In contrast to the miR-141 group, the above results were reversed after treatment with the Endostar (p < 0.05). ConclusionOverexpression of miR-141 could inhibit the osteoporosis of jawbones in ovariectomized rats by activating the Wnt/β-catenin pathway.

Activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting siRNA.

BackgroundFibroblast-like synoviocytes (FLSs), resident mesenchymal cells of synovial joints, play an important role in the pathogenesis of rheumatoid arthritis (RA). Dickkopf-1 (DKK-1) has been proposed to be a master regulator of bone remodeling in inflammatory arthritis. Here, potential impairation on the activity of FLSs derived from RA to small interfering RNAs (siRNAs) targeting DKK-1 was investigated.MethodssiRNAs targeting DKK-1 were transfected into FLSs of patients with RA. Interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP) 2, MMP3, MMP9, transforming growth factor (TGF)-β1, TGF-β2 and monocyte chemoattractant protein (MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Invasion assay and 3H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation, respectively. Western blotting was performed to analyze the expression of nuclear factor (NF)-κB, interleukin-1 receptor-associated kinase (IRAK)1, extracellular regulated protein kinases (ERK)1, Jun N-terminal kinase (JNK) and β-catenin in FLSs.ResultsDKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs (P < 0.01). siRNAs induced a significant reduction of the levels of IL-6, IL-8, MMP2, MMP3 and MMP9 in FLSs compared to the control group (P < 0.05). DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs (P < 0.05). Important molecules of pro-inflammatory signaling in FLSs, including IRAK1 and ERK1, were decreased by the inhibition of DKK-1 in FLSs. In contrast, β-catenin, a pivotal downstream molecule of the Wnt signaling pathway was increased.ConclusionsBy inhibiting DKK-1, we were able to inhibit the proliferation, invasion and pro-inflammatory cytokine secretion of FLSs derived from RA, which was mediated by the ERK or the IRAK-1 signaling pathway. These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

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.

Angiotensin II inhibits osteogenic differentiation of isolated synoviocytes by increasing DKK-1 expression.

The renin-angiotensin system contributes to the pathogenesis of rheumatoid arthritis, but that the mechanism is unclear. This study aims to investigate the effect of angiotensin II (Ang II) on osteogenic differentiation of synoviocytes and the underlying mechanism. Ang II was showed to inhibite osteogenic differentiation of synoviocytes, which was mitigated by a Dickkopf-1 (DKK-1) inhibitor. DKK-1 was upregulated by Ang II, which was weakened by the Ang II type 1 receptor (AT1R) blocker, reactive oxygen species (ROS) scavenger, and p38 inhibitor. Ang II increased the levels of AT1R, ROS, and NADPH oxidase (NOX), and the upregulations were mitigated by the AT1R blocker or NOX inhibitor. Furthermore, Ang II activated the p38 pathway, which was blocked by the AT1R blocker, ROS scavenger, or siRNA-MKK3. In brief, these results indicate that Ang II upregulates NOX expression and ROS production via AT1R, activates the MKK3/p38 signaling, and in turn upregulates DKK-1 expression, participating in the inhibition of osteogenic differentiation of synoviocytes.