Dickkopf-1 Is Oncogenic and Involved in Invasive Growth in Non Small Cell Lung Cancer

Dickkopf-1 (DKK1) is an inhibitor of the Wnt/β-catenin signaling pathway. However, the role of DKK1 in the progression of non small cell lung cancer (NSCLC) is not fully understood. In this study, RT-PCR and Western blot were used to examine the expression of DKK1 in a panel of ten human NSCLC cell lines and NSCLC tissues. DKK1 expression was highly transactivated in the great majority of these cancer lines. The expression of DKK1 was upregulated on both mRNA and protein levels in NSCLC tissues compared with the adjacent normal lung tissues. Immunohistochemistry and immunofluoresence revealed that DKK1 was mainly distributed in the cytoplasm in both carcinoma tissues and cell lines. DKK1 protein expression was also evaluated in paraffin sections from 102 patients with NSCLC by immunohistochemistry, and 65(63.73%)tumors were DKK1 positive. Relative analysis showed a significant relationship between DKK1 positive expression and lymph node metastasis(P<0.05). Patients with DKK1-positive tumors had poorer DFS than those with negative ESCC (5-year DFS; 15.4% versus 27%, P = 0.007). To further explore the biological effects of DKK1 in NSCLC cells, we over-expressed DKK1 in NSCLC 95C cell using eukaryotic expression vector pCMV-Tab-2b and performed a knockdown of DKK1 in LTEP-a-2 cell using a short hairpin RNA expression vector pSilencer 5.1. DKK1 did not have any effect on proliferation, but seemed to play a role in migration and invasion capability. Overexpression of DKK1 promotes migratory and invasive activity of 95C, while DKK1 knockdown resulted in the suppression of migration and invasion potentials of LTEP-a-2 cell. Taken together, these results indicate that DKK1 may be a crucial regulator in the progression of NSCLC. DKK1 might be a potential therapeutic target in NSCLC.     

Relationship of Dickkopf1 (DKK1) with Cardiovascular Disease and Bone Metabolism in Caucasian Type 2 Diabetes Mellitus

ObjectivesDickkopf-1 (DKK1) is a potent inhibitor of Wnt signalling, which exerts anabolic effects on bone and also takes part in the regulation of vascular cells. Our aims were to evaluate serum DKK1 in type 2 diabetes (T2DM) patients and to analyze its relationships with cardiovascular disease (CVD). We also evaluated the relationship between DKK1 and bone metabolism.DesignWe conducted a cross-sectional study in which we measured serum DKK1 (ELISA, Biomedica) in 126 subjects: 72 patients with T2DM and 54 non-diabetic subjects. We analysed its relationship with clinical CVD, preclinical CVD expressed as carotid intima media thickness (IMT), and bone metabolism.ResultsT2DM patients with CVD (P = 0,026) and abnormal carotid IMT (P = 0,038) had higher DKK1 concentrations. DKK1 was related to the presence of CVD in T2DM, independently of the presence of risk factors for atherosclerosis. Therefore, for each increase of 28 pg/ml of serum DKK1 there was a 6,2% increase in the risk of CVD in T2DM patients. The ROC curve analysis to evaluate the usefulness of DKK1 as a marker for high risk of CVD showed an area under the curve of 0,667 (95% CI: 0,538–0,795; P = 0,016). In addition, there was a positive correlation between serum DKK1 and spine bone mineral density in the total sample (r = 0,183; P = 0,048).ConclusionIn summary, circulating DKK1 levels are higher in T2DM with CVD and are associated with an abnormal carotid IMT in this cross-sectional study. DKK1 may be involved in vascular disease of T2DM patients.     

Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.     

Dickkopf-1 is involved in invasive growth of esophageal cancer cells

Dickkopf-1 (DKK1) is an inhibitor of Wnt/β-catenin signaling pathway. High levels of DKK1 protein were found in a series of cancers. However, the role of DKK1 in the progression of esophageal carcinoma is not fully understood. In the present study, RT-PCR and Western blot were used to detect the expression of DKK1 in esophageal carcinoma tissues, matched adjacent normal esophageal tissues, and esophageal carcinoma cell lines. Our results showed that the expression of DKK1 was upregulated on both mRNA and protein levels in esophageal carcinoma tissues compared with the adjacent normal esophageal tissues, meanwhile, in four esophageal carcinoma cell lines analyzed, expression of DKK1 was detected with different levels. Immunohistochemistry and immunofluoresence revealed that the distribution of DKK1 was mainly in the cytoplasm in both carcinoma tissues and cell lines. To further explore the biological effects of DKK1 on proliferation, cell cycle and invasion capability, we constructed the eukaryotic expression vector pCMV-Tab-2b-DKK1 which can effectively overexpress DKK1. Subsequently, we observed that exogenous expression of DKK1 in EC9706 cell line resulted in an increased rate of proliferation, and S stage and G2/M stage ratio whereas G0/G1 ratio was decreased. In order to evaluate the invasion capability Boyden chamber was analyzed which implied that overexpression of DKK1 resulted in an increase in the invasion ability in EC9706 cell line. Taken together, the study indicates that DKK1 might be a key regulator in the progression of esophageal carcinoma and a potential therapeutic target in esophageal carcinoma.     

Transforming growth factor-beta1 attenuates expression of both the progesterone receptor and Dickkopf in differentiated human endometrial stromal cells

TGFbeta1 is thought to be intimately involved in cyclic tissue remodeling and inflammatory events associated with menstruation. Menstruation is initiated by progesterone withdrawal; however, the underlying mechanisms are not well understood. In the present study, we have tested the hypothesis that locally produced TGFbeta1 may influence expression of progesterone receptor (PR) or the Wnt antagonist Dickkopf-1 (DKK) with consequential impact on regulation of menstruation. Endometrial stromal cells (ESC) were isolated from endometrial biopsy samples collected from patients undergoing gynecological procedures for benign indications. Treatment of differentiated ESC with TGFbeta1 (10 ng/ml) significantly inhibited the expression of mRNAs encoding PR and DKK. TGFbeta1 also attenuated the protein expression of PR and secretion of DKK proteins in culture supernatants. Neutralization of endogenous TGFbeta1 signaling abolished the TGFbeta1-induced effects, significantly increased expression of PR, and increased DKK protein release levels to that of differentiated ESCs, confirming the specificity of the TGFbeta1 effect. Additionally, in vitro decidualization of ESCs significantly augmented DKK protein release. Moreover, although TGFbeta1 was capable of signaling via the Sma- and mothers against decapentaplegic (MAD)-related protein (SMAD) pathway, the inhibitory effect on DKK was SMAD independent. Conversely, the inhibitory effect of TGFbeta1 on PR was dependent on SMAD signal transduction. In conclusion, these results suggest that local TGFbeta1 signaling can potentiate progesterone withdrawal by suppressing expression of PR and may coordinate tissue remodeling associated with menstruation by inducing Wnt-signaling via inhibition of DKK, which we found to be up-regulated as a consequence of decidualization of ESCs.     

Elevated Levels of Dickkopf-1 Are Associated with β-Catenin Accumulation and Poor Prognosis in Patients with Chondrosarcoma

Background

Dickkopf-1 (DKK1) is an antagonist of Wnt/β-catenin signaling implicated in tumorigenesis. However, the biological role of DKK1 and β-catenin involved in chondrosarcoma has not been sufficiently investigated. This study was designed to investigate the expression profiles of DKK1 and β-catenin, and to clarify their clinical values in chondrosarcoma.

Methods

The mRNA and protein levels of DKK1 and β-catenin in fresh chondrosarcoma and the corresponding non-tumor tissues were analyzed by Real-time PCR and Western blot, respectively. The protein expression patterns of DKK1 and β-catenin were investigated by immunohistochemistry. The associations among DKK1 level, β-catenin accumulation, clinicopathological factors and the overall survival were separately evaluated.

Results

Both DKK1 and β-catenin levels were remarkably elevated in chondrosarcoma compared with the corresponding non-tumor tissues. High DKK1 level and positive β-catenin accumulation in chondrosarcoma specimens were 58.7% and 53.9%, respectively. Elevated DKK1 level significantly correlated with positive β-catenin accumulation, and they were remarkably associated with histological grade and Musculoskeletal Tumor Society stage. Furthermore, DKK1 level and β-catenin accumulation had significant impacts on the prognosis of chondrosarcoma patients. Multivariate analysis revealed that DKK1 level was an independent prognostic factor for overall survival.

Conclusions

Elevated DKK1 levels associated with β-catenin accumulation play a crucial role in chondrosarcoma. DKK1 can serve as a novel predictor of poor prognosis in patients with chondrosarcoma.     

Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway

Objective: CA125/MUC16 is an O-glycosylated protein that is expressed on the surfaces of ovarian epithelial cells. This molecule is a widely used tumor-associated marker for diagnosis of ovarian cancer. Recently, CA125 was shown to be involved in ovarian cancer metastasis. The purpose of this study was to investigate the mechanism of CA125 during ovarian cancer metastasis.Methods: We analyzed the Oncomine and CSIOVDB databases to determine the expression levels of DKK1 in ovarian cancer. DKK1 expression levels were upregulated or downregulated and applied with CA125 to Transwell and Western blot assays to ascertain the underlying mechanism by which CA125 stimulates cell migration via the SGK3/FOXO3 pathway. Anti-mesothelin antibodies (anti-MSLN) were used to block CA125 stimulation. Then the expression levels of DKK1were tested by enzyme-linked immunosorbent assay (ELISA) to eliminate the blocking effect of anti-MSLN to CA125 stimulation. Xenograft mouse models were used to detect the effects of CA125 and anti-MSLN on ovarian cancer metastasis in vivo.Results: DKK1 levels were downregulated in ovarian tumor tissues according to the analyses of two databases and significantly correlated with FIGO stage, grade and disease-free survival in ovarian cancer patients. DKK1 levels were downregulated by CA125 stimulation in vitro. Overexpression of DKK1 reversed the ability of exogenous CA125 to mediate cell migration by activating the SGK3/FOXO3 signaling pathway. Anti-MSLN abrogated the DKK1 reduction and increased the apoptosis of ovarian cancer cells. The use of anti-MSLN in xenograft mouse models significantly reduced tumor growth and metastasis accelerated by CA125.Conclusions: These experiments revealed that the SGK3/FOXO3 pathway was activated, wherein decreased expression of DKK1 was caused by CA125, which fuels ovarian cancer cell migration. Mesothelin is a potential therapeutic target for the treatment of ovarian cancer metastasis.     

R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling

R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.     

Robust circadian rhythms in organoid cultures from PERIOD2::LUCIFERASE mouse small intestine

Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial ‘miniguts’), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role of circadian rhythms in GI stem cell dynamics, epithelial homeostasis and disease.KEY WORDS: Circadian rhythm, Intestinal organoid, PERIOD2, R-spondin, RU486     

Rspo1/Wnt signaling promotes angiogenesis via Vegfc/Vegfr3

Here, we show that a novel Rspo1-Wnt-Vegfc-Vegfr3 signaling pathway plays an essential role in developmental angiogenesis. A mutation in R-spondin1 (rspo1), a Wnt signaling regulator, was uncovered during a forward-genetic screen for angiogenesis-deficient mutants in the zebrafish. Embryos lacking rspo1 or the proposed rspo1 receptor kremen form primary vessels by vasculogenesis, but are defective in subsequent angiogenesis. Endothelial cell-autonomous inhibition of canonical Wnt signaling also blocks angiogenesis in vivo. The pro-angiogenic effects of Rspo1/Wnt signaling are mediated by Vegfc/Vegfr3(Flt4) signaling. Vegfc expression is dependent on Rspo1 and Wnt, and Vegfc and Vegfr3 are necessary to promote angiogenesis downstream from Rspo1-Wnt. As all of these molecules are expressed by the endothelium during sprouting stages, these results suggest that Rspo1-Wnt-VegfC-Vegfr3 signaling plays a crucial role as an endothelial-autonomous permissive cue for developmental angiogenesis.     

Regulation of p53 by Jagged1 Contributes to Angiotensin II-Induced Impairment of Myocardial Angiogenesis

Angiotensin II (AngII) is a major contributor to the development of heart failure, however, the molecular and cellular mechanisms still remain elucidative. Inadequate angiogenesis in myocardium leads to transition from cardiac hypertrophy to dysfunction, this study was therefore conducted to examine the effects of AngII on myocardial angiogenesis and the underlying mechanisms. AngII treatment significantly impaired angiogenetic responses, which were determined by counting the capillaries either in matrigel formed by cultured cardiac microvascular endothelial cells (CMVECs) or in myocardium of mice and by measuring the in vitro and in vivo production of VEGF proteins, and stimulated accumulation and phosphorylation of cytosolic p53 which led to increases in phosphorylated p53 and decreases of hypoxia inducible factor (Hif-1) in nucleus. All of these cellular and molecular events induced by AngII in CEMCs and hearts of mice were largely reduced by a p53 inhibitor, pifithrin-α (PFT-α). Interestingly, AngII stimulated the upregulation of Jagged1, a ligand of Notch, but it didn’t affect the expression of Delta-like 4 (Dll-4), another ligand of Notch. Inhibition of p53 by PFT-α partly abolished this effect of AngII. Further experiments showed that knockdown ofJagged1 by addition of siRNA to cultured CMVECs dramatically declined AngII-stimulated accumulation and phosphorylation of p53 in cytosol, upregulation of phosphorylated p53 and downregulation of Hif-1 expression in nucleus, decrease of VEGF production and impairment of capillary-like tube formation by the cells. Our data collectively suggest that AngII impairs myocardial angiogenetic responses through p53-dependent downregulation of Hif-1 which is regulated by Jagged1/Notch1 signaling.