GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.     

The Wnt antagonist Dickkopf-1 is regulated by Bmp signaling and c-Jun and modulates programmed cell death

Dickkopf-1 (Dkk-1) has been shown to be a potent inhibitor of Wnt/beta-catenin signaling in a variety of assays and organisms. In this study, we show that expression of Dkk-1 overlaps significantly with the sites of programmed cell death in normal as well as mutant vertebrate limb development, and identify several of its upstream regulators, one of which is Bmp-4. Interestingly, Bmp-4 only activates Dkk-1 when it concomitantly induces apoptosis. Moreover, Dkk-1 is heavily up-regulated by UV irradiation and several other genotoxic stimuli. We further show that normal expression of Dkk-1 is dependent on the Ap-1 family member c-Jun and that overexpression of Dkk-1 enhances Bmp-triggered apoptosis in the vertebrate limb. Taken together, our results provide evidence for an important role of Dkk-1-mediated inhibition of Wnt/beta-catenin signaling in response to different stress signals that all converge on the activation of c-Jun in vivo.     

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.     

Secreted Wnt antagonist Dickkopf-1 controls kidney papilla development coordinated by Wnt-7b signalling

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.     

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.     

Dickkopf-1 antagonizes Wnt signaling independent of beta-catenin in human mesothelioma

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/beta-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in beta-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through beta-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway).     

Cyclooxygenase-1 is involved in the inhibition of hippocampal neurogenesis after lipopolysaccharide-induced neuroinflammation

Growing evidence indicates that neuroinflammation can alter adult neurogenesis by mechanisms as yet unclear. We have previously demonstrated that the neuroinflammatory response and neuronal damage after lipopolysaccharide (LPS) injection is reduced in cyclooxygenase-1 deficient (COX-1^-/-) mice. In this study, we investigated the role of CoX-1 on hippocampal neurogenesis during LPS-induced neuroinflammation, using COX-1^-/- and wild-type (WT) mice. We found that LPS-induced neuroinflammation resulted in the decrease of proliferation, survival and differentiation of hippocampal progenitor cells in WT but not in COX-1^-/- mice. Thus, we demonstrate for the first time that COX-1 is involved in the inhibition of BrdU progenitor cells in proliferation and hippocampal neurogenesis after LPS. These results suggest that COX-1 may represent a viable therapeutic target to reduce neuroinflammation and promote neurogenesis in neurodegenerative diseases with a strong inflammatory component.Key words: neurogenesis, cyclooxygenase-1, lipopolysaccharide, inflammation, brain     

Metastasis tumor antigen 1 is involved in the resistance to heat stress-induced testicular apoptosis

Our previous study documented the expression of Mta1 during spermatogenesis. Here, we present evidence for a possible involvement of Mta1 in the regulation of testicular function, possibly by interacting with p53. A notable decrease of Mta1 expression was revealed at postsurgical day 6, consistent with the previously reported upregulation of p53 in mouse cryptorchidism. Furthermore, in vitro over-expression of Mta1 could remarkably elevate the resistance capability of spermatogenic tumor cells against heat-induced apoptosis with a marked impairment of p53 expression. These findings indicate that Mta1 may operate as a negative modifier of apoptosis by interacting with p53 during gametogenesis.     

Cyclooxygenase-1 is involved in the inhibition of hippocampal neurogenesis after lipopolysaccharide-induced neuroinflammation

Growing evidence indicates that neuroinflammation can alter adult neurogenesis by mechanisms as yet unclear. We have previously demonstrated that the neuroinflammatory response and neuronal damage after lipopolysaccharide (LPS) injection is reduced in cyclooxygenase-1 deficient (COX-1-/-) mice. In this study, we investigated the role of COX-1 on hippocampal neurogenesis during LPS-induced neuroinflammation, using COX-1-/- and wild type (WT) mice. We found that LPS-induced neuroinflammation resulted in the decrease of proliferation, survival and differentiation of hippocampal progenitor cells in WT but not in COX-1-/- mice. Thus, we demonstrate for the first time that COX-1 is involved in the inhibition of BrdU progenitor cells in proliferation and hippocampal neurogenesis after LPS. These results suggest that COX-1 may represent a viable therapeutic target to reduce neuroinflammation and promote neurogenesis in neurodegenerative diseases with a strong inflammatory component.     

Wnt inhibitory factor-1, a Wnt antagonist, is silenced by promoter hypermethylation in malignant pleural mesothelioma

Wnt inhibitory factor-1 (WIF-1) is a secreted protein that antagonizes Wnt signaling. We recently demonstrated the importance of aberrant activation of the Wnt signaling pathway in various cancers including malignant pleural mesothelioma. In this study, we revealed downregulated WIF-1 expression in cell lines and primary tissue when compared to normal mesothelial cell lines and adjacent pleura, respectively. We observed hypermethylation in four of four mesothelioma cell lines, but not in two normal mesothelial cell lines. In primary tissue samples, we observed methylation in three paired tumor specimens compared to their adjacent normal pleura and methylation in eight of nine unpaired tumor tissue samples. Taken together, our studies suggest that WIF-1 silencing due to its promoter hypermethylation is an important mechanism underlying the constitutively activated Wnt signaling in mesothelioma. New therapies toward inhibition of the Wnt pathway through WIF-1 might be promising for the future treatment of malignant mesothelioma.     

Brain prolactin is involved in stress-induced REM sleep rebound

REM sleep rebound is a common behavioural response to some stressors and represents an adaptive coping strategy. Animals submitted to multiple, intermittent, footshock stress (FS) sessions during 96 h of REM sleep deprivation (REMSD) display increased REM sleep rebound (when compared to the only REMSD ones, without FS), which is correlated to high plasma prolactin levels. To investigate whether brain prolactin plays a role in stress-induced REM sleep rebound two experiments were carried out. In experiment 1, rats were either not sleep-deprived (NSD) or submitted to 96 h of REMSD associated or not to FS and brains were evaluated for PRL immunoreactivity (PRL-ir) and determination of PRL concentrations in the lateral hypothalamus and dorsal raphe nucleus. In experiment 2, rats were implanted with cannulas in the dorsal raphe nucleus for prolactin infusion and were sleep-recorded. REMSD associated with FS increased PRL-ir and content in the lateral hypothalamus and all manipulations increased prolactin content in the dorsal raphe nucleus compared to the NSD group. Prolactin infusion in the dorsal raphe nucleus increased the time and length of REM sleep episodes 3 h after the infusion until the end of the light phase of the day cycle. Based on these results we concluded that brain prolactin is a major mediator of stress-induced REMS. The effect of PRL infusion in the dorsal raphe nucleus is discussed in light of the existence of a bidirectional relationship between this hormone and serotonin as regulators of stress-induced REM sleep rebound.     

Dickkopf-1 is a master regulator of joint remodeling

Degenerative and inflammatory joint diseases lead to a destruction of the joint architecture. Whereas degenerative osteoarthritis results in the formation of new bone, rheumatoid arthritis leads to bone resorption. The molecular basis of these different patterns of joint disease is unknown. By inhibiting Dickkopf-1 (DKK-1), a regulatory molecule of the Wnt pathway, we were able to reverse the bone-destructive pattern of a mouse model of rheumatoid arthritis to the bone-forming pattern of osteoarthritis. In this way, no overall bone erosion resulted, although bony nodules, so-called osteophytes, did form. We identified tumor necrosis factor-alpha (TNF) as a key inducer of DKK-1 in the mouse inflammatory arthritis model and in human rheumatoid arthritis. These results suggest that the Wnt pathway is a key regulator of joint remodeling.     

The Wnt Antagonist, Dickkopf-1, as a Target for the Treatment of Neurodegenerative Disorders

The canonical Wnt pathway contributes to the regulation of neuronal survival and homeostasis in the CNS. Recent evidence suggests that an increased expression of Dickkopf-1 (Dkk-1), a secreted protein that negatively modulates the canonical Wnt pathway, is causally related to processes of neurodegeneration in a number of CNS disorders, including Alzheimer’s disease (AD), brain ischemia and temporal lobe epilepsy (TLE). Dkk-1 induction precedes neuronal death in cellular and animal models of excitotoxicity, β-amyloid toxicity, transient global ischemia, and kainate-induced epilepsy. In addition, Dkk-1, which is barely visible in the healthy brain, is strongly induced in brain tissue from AD patients or from patients with TLE associated with hippocampal sclerosis. These data raise the attractive possibility that Dkk-1 antagonists or neutralizing antibodies behave as neuroprotective agents by rescuing the activity of the canonical Wnt pathway. Special issue article in honor of Anna Maria Giuffrida-Stella. Agata Copani and Ferdinando Nicoletti—Co-senior authors. Filippo Caraci—PhD Program in Neuropharmacology.     

Targeting the Wnt pathway in cancer: The emerging role of Dickkopf-3

Aberrant activation of the Wnt signaling pathway is a major trait of many human cancers. Due to its vast implications in tumorigenesis and progression, the Wnt pathway has attracted considerable attention at several molecular levels, also with respect to developing novel cancer therapeutics. Indeed, research in Wnt biology has recently provided numerous clues, and evidence is accumulating that the secreted Wnt antagonist Dickkopf-related protein 3 (Dkk-3) and its regulators may constitute interesting therapeutic targets in the most important human cancers. Based on the currently available literature, we here review the knowledge on the biological role of Dkk-3 as an antagonist of the Wnt signaling pathway, the involvement of Dkk-3 in several stages of tumor development, the genetic and epigenetic mechanisms disrupting DKK3 gene function in cancerous cells, and the potential clinical value of Dkk-3 expression/DKK3 promoter methylation as a biomarker and molecular target in cancer diseases.In conclusion, Dkk-3 rapidly emerges as a key player in human cancer with auspicious tumor suppressive capacities, most of all affecting apoptosis and proliferation. Its gene expression is frequently downregulated by promoter methylation in almost any solid and hematological tumor entity. Clinically, evidence is accumulating of Dkk-3 being both a potential tumor biomarker and effective anti-cancer agent. Although further research is needed, re-establishing Dkk-3 expression in cancer cells holds promise as novel targeted molecular tumor therapy.     

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.