Increased Dickkopf-1 expression in breast cancer bone metastases

The aim of this study was to determine whether Dickkopf-1 (Dkk-1) expression in breast cancer was associated with bone metastases. We first analysed Dkk-1 expression by human breast cancer cell lines that induce osteolytic or osteoblastic lesions in animals. Dickkopf-1 levels were then measured in the bone marrow aspirates of hind limbs from eight NMRI mice inoculated with breast cancer cells that induced bone metastases and 11 age-matched non-inoculated control animals. Finally, Dkk-1 was measured in the serum of 17 women with breast cancer in complete remission, 19 women with breast cancer and bone metastases, 16 women with breast cancer and metastases at non-bone sites and 16 healthy women. Only breast cancer cells that induce osteolytic lesions in animals produced Dkk-1. There was a six-fold increase in Dkk-1 levels in the bone marrow from animals inoculated with MDA-B02 cells when compared with that of control non-inoculated animals (P=0.003). Median Dkk-1 levels in the serum of patients with breast cancer and bone metastases were significantly higher than levels of patients in complete remission (P=0.016), patients with breast cancer having metastases at non-bone sites (P<0.0001) and healthy women (P=0.047), although there was a large overlap in individual levels between the different groups. In conclusion, Dkk-1 is secreted by osteolytic human breast cancer cells lines and increased circulating levels are associated with the presence of bone metastases in patients with breast cancer. Measurements of circulating Dkk-1 levels may be useful for the clinical investigation of patients with breast cancer and bone metastases.     

Dickkopf-1与肿瘤

Dickkopf-1(DKK-1)通过Wnt/β-连环蛋白(β-catenin)经典信号传导途径以及Wnt非经典信号传导途径在体内诱导多种肿瘤细胞凋亡,调控多种肿瘤细胞的骨转移以及促进某些肿瘤细胞的浸润和侵袭,从而在肿瘤的发生和发展方面发挥重要作用.     

DKK-1与骨髓瘤骨病发生关系的研究进展

目的:研究DKK-1(Dickkopf-1)在多发性骨髓瘤细胞中的表达水平,以及与骨髓瘤骨病发生的关系。方法:以DKK-1和骨髓瘤为检索词,检索1989-2009年中国知识资源总库CNKI系列数据库和PubMed数据库。纳入标准:1)DKK-1基因结构及生物学特征的研究;2)DKK-1与骨髓瘤发生关系研究。粗选有56篇关于DKK-1结构特点及其与骨髓瘤关系的研究文章,根据纳入标准,精选27篇文献。结果:DKK-1是Wnt/β-cate-nin信号通路的可溶性的抑制剂,目前已被认为在多发性骨髓瘤及骨髓瘤骨病的发生中起到一定作用。结论:中和DKK-1和(或)促进Wnt/β-catenin信号通路能够治疗骨病及抑制骨髓瘤细胞生长,提示DKK-1可能成为骨髓瘤骨病治疗的新靶点。     

Therapeutic approaches to myeloma bone disease: an evolving story

Bone disease is a major morbidity factor in patients with multiple myeloma and significantly affects their overall survival. A complex interplay between malignant plasma cells and other marrow cells results in the generation of a microenvironment capable of enhancing both tumor growth and bone destruction. Bisphosphonates have consistently reduced the incidence of skeletal-related events in patients with multiple myeloma and other osteotropic tumors as well. However, their use is burdened with side-effects, including the risks of osteonecrosis of the jaw and kidney failure, suggesting that they should be discontinued after prolonged administration. New molecular targets of cell cross-talk in myeloma bone marrow are therefore under intensive investigation and new drugs are being explored in preclinical and clinical studies of myeloma bone disease. Compounds targeting osteoclast activation pathways, such as receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin, B-cell activating factor, mitogen-activated protein kinase and macrophage inflammatory protein-1α/chemokine receptor for macrophage inflammatory protein-1α axes, or soluble agents that improve osteoblast differentiation by modulating specific inhibitors such as Dickkopf-1 and transforming growth factor-β, as well as novel approaches of cytotherapy represent a new generation of promising drugs for the treatment of myeloma bone disease.     

Impact of bortezomib on bone health in myeloma: A review of current evidence

Bone disease is a key feature in multiple myeloma (MM) and can have a substantial impact on patient morbidity and quality-of-life. The pathogenesis of lytic bone disease in MM is complex and associated with increased osteoclast activity and impaired osteoblast function. Lytic lesions rarely heal in MM; however, the proteasome inhibitor bortezomib has been linked to increased bone formation and osteoblastic activity. Various clinical studies have reported a positive effect of bortezomib on bone health, including fewer bone disease-related MM progression events, increases in bone volume, and improvements in osteolytic lesions. Alkaline phosphatase (total and bone isoenzyme), a marker of bone formation, is increased during bortezomib treatment; the degree of increase may be associated with treatment response. Bortezomib is associated with a reduction in Dickkopf-1, an inhibitor of osteoblast function. Increases of other bone-formation markers and decreases of bone-resorption markers, have also been observed. These clinical effects are supported by preclinical data suggesting bortezomib is associated with an increase in bone formation and osteoblast numbers/activity, arising from direct effects of bortezomib and proteasome inhibition. As reviewed here, a growing body of evidence indicates that bortezomib exerts a positive effect on bone metabolism in MM and has a bone anabolic effect.     

p21CIP-1/WAF-1 induction is required to inhibit prostate cancer growth elicited by deficient expression of the Wnt inhibitor Dickkopf-1

Osteoblastic bone metastases are the most common metastases produced by human prostate cancers (PCa). Deregulated activity of Wnt growth factors resulting from overexpression of the Wnt inhibitor Dickkopf-1 (DKK-1) is known to contribute to formation of the osteoblastic component of PCa skeletal bone metastases. In this study, we report that DKK-1 knockdown in osteolytic human PCa cells unexpectedly delays the development of both soft tissue and osseous lesions. PCa cells deficient in DKK-1 expression did not increase canonical Wnt signaling in target osteoblast cell lines; however, DKK-1 knockdown PCa cells exhibited increased expression of the CDK inhibitor p21(CIP1/WAF1) and a 32% increase in G(1) arrest compared with control cells. Ablating p21(CIP1/WAF1) in PCa cells deficient in DKK-1 was sufficient to rescue tumor growth. Collectively, our findings demonstrate that DKK-1 overexpression supports tumor growth in part by restricting expression of p21(CIP1/WAF1) through a mechanism independent of canonical Wnt signaling.     

Low-density lipoprotein receptor-related protein 5 (LRP5) mediates the prostate cancer-induced formation of new bone

The tendency of prostate cancer to produce osteoblastic bone metastases suggests that cancer cells and osteoblasts interact in ways that contribute to cancer progression. To identify factors that mediate these interactions, we compared gene expression patterns between two bone-derived prostate cancer cell lines that produce osteoblastic (MDA PCa 2b) or osteolytic lesions (PC-3). Both cell lines expressed Wnt ligands, including WNT7b, a canonical Wnt implicated in osteogenesis. PC-3 cells expressed 50 times more Dickkopf-1 (DKK1), an inhibitor of Wnt pathways, than did MDA PCa 2b cells. Evaluation of the functional role of these factors (in cocultures of prostate cancer cells with primary mouse osteoblasts (PMOs) or in bone organ cultures) showed that MDA PCa 2b cells activated Wnt canonical signaling in PMOs and that DKK1 blocked osteoblast proliferation and new bone formation induced by MDA PCa 2b cells. MDA PCa 2b cells did not induce bone formation in calvaria from mice lacking the Wnt co-receptor Lrp5. In human specimens, WNT7b was not expressed in normal prostate but was expressed in areas of high-grade prostate intraepithelial neoplasia, in three of nine primary prostate tumor specimens and in 16 of 38 samples of bone metastases from prostate cancer. DKK1 was not expressed in normal or cancerous tissue but was expressed in two of three specimens of osteolytic bone metastases (P=0.0119). We conclude that MDA PCa 2b induces new bone formation through Wnt canonical signaling, that LRP5 mediates this effect, and that DKK1 is involved in the balance between bone formation and resorption that determines lesion phenotype.     

Production of Wnt inhibitors by myeloma cells: potential effects on canonical Wnt pathway in the bone microenvironment

Osteoblast impairment occurs within multiple myeloma cell infiltration into the bone marrow. Canonical Wnt signaling activation in osteoprogenitor cells is involved in osteoblast formation through the stabilization of dephosphorylated beta-catenin and its nuclear translocation. The effects of multiple myeloma cells on Wnt signaling in human mesenchymal/osteoprogenitor cells are unclear. In 60 multiple myeloma patients checked, we found that among the Wnt inhibitors, Dickkopf-1 and secreted frizzled-related protein-3 were produced by multiple myeloma cells. However, although multiple myeloma cells or multiple myeloma bone marrow plasma affected expression of genes in the canonical Wnt signaling and inhibited beta-catenin stabilization in murine osteoprogenitor cells, they failed to block the canonical Wnt pathway in human mesenchymal or osteoprogenitor cells. Consistently, Wnt3a stimulation in human osteoprogenitor cells did not blunt the inhibitory effect of multiple myeloma cells on osteoblast formation. Consequently, despite the higher Wnt antagonist bone marrow levels in osteolytic multiple myeloma patients compared with nonosteolytic ones, beta-catenin immunostaining was not significantly different. Our results support the link between the production of Wnt antagonists by multiple myeloma cells and the presence of bone lesions in multiple myeloma patients but show that myeloma cells do not inhibit canonical Wnt signaling in human bone microenvironment.     

Serum concentrations of Dickkopf-1 protein are increased in patients with multiple myeloma and reduced after autologous stem cell transplantation

Dickkopf-1 (DKK-1) protein, a soluble inhibitor of Wnt signalling, has been implicated in the pathogenesis of myeloma bone disease through the suppression of osteoblast differentiation. In this study, serum concentrations of DKK-1 were measured in 50 myeloma patients (32 at diagnosis and 18 before and after autologous stem cell transplantation (ASCT), 18 patients with monoclonal gammopathy of undetermined significance (MGUS), and 22 healthy controls. Serum DKK-1 levels were increased in MM at diagnosis compared with MGUS (mean +/- SD: 67 +/- 54 ng/mL vs. 38 +/- 13 ng/mL; p = 0.006) and controls (31 +/- 11 ng/mL; p = 0.02), while there was no difference between MGUS patients and controls. Although patients with stage 2 and 3 myeloma had higher DKK-1 values than stage 1 patients (79 +/- 63 vs. 40 +/- 13; p = 0.005), no significant correlation between serum DKK-1 and myeloma bone disease was observed. Myeloma patients before ASCT also had increased levels of DKK-1 (63 +/- 77 ng/mL; p = 0.03) compared with controls, supporting the notion that DKK-1 may be responsible for the suppressed osteoblast activity even in patients with low tumor burden. After ASCT, there was a sustained decrease in DKK-1 levels over time, while bone formation markers elevated, suggesting that the reduction of DKK-1 levels after ASCT may correlate with the normalization of osteoblast function. These results could provide the basis for developing agents that block DKK-1, thus restoring osteoblast function and counteracting the increased osteoclastogenesis observed in myeloma.     

A phase II clinical trial does not show that high dose simvastatin has beneficial effect on markers of bone turnover in multiple myeloma

Several studies have evaluated the impact of low dose statin (20-80 mg/day) on bone metabolism with inconclusive results despite promising data of preclinical studies. In this study, we investigated the effect of high dose simvastatin (HD-Sim) on biochemical markers of bone turnover and disease activity in six heavily pretreated patients with multiple myeloma (MM). These patients were treated with simvastatin (15 mg/kg/day) for 7 days followed by a rest period of 21 days in two 4-week cycles. Endpoints were changes in the level of biochemical markers of (i) osteoclast activity (tartrate resistant acid phosphatase, TRACP); (ii) bone resorption (collagen fragments CTX and NTX); (iii) bone formation (osteocalcin and aminoterminal propeptide of type I collagen PINP); (iv) cholesterol; (v) regulators of bone metabolism [osteoprotegerin (OPG) and Dickkopf-1 (DKK-1)] and (vi) disease activity (monoclonal proteins or free light chains in serum). TRACP activity in serum and levels of collagen fragments (NTX) in urine increased for all patients temporarily during the 7 days of treatment with HD-Sim indicating that osteoclasts may have been stimulated rather than inhibited. The other markers of bone metabolism showed no change. None of the patients showed any reduction in free monoclonal light chains or monoclonal proteins in serum during treatment with HD-Sim. In spite of the fact that bone turn over effects of HD-Sim may have been blunted by concomitant treatment of patients with other drugs we observed a transient increase in markers of osteoclast activity. This sign of a transient stimulation of osteoclast activity suggests that HD-Sim may be harmful rather than beneficial for MM patients. For this reason and because of gastro-intestinal side effects the study was stopped prematurely.     

Dickkopf-1相关肿瘤转移研究进展

作为一种分泌型糖蛋白,Dickkopf-1(DKK1)与肿瘤转移关系密切,在肿瘤中高表达或低表达,其异常高表达促进肝癌、胃癌、非小细胞肺癌和乳腺癌等多种恶性肿瘤的转移.最新研究发现,DKK1与肿瘤的潜在转移关系密切.     

Dickkopf-1基因与肿瘤

国内外大量研究证实,Dickkopf-1(Dkk1)在不同肿瘤以及同一肿瘤发生发展的不同阶段表达情况存在很大差异.Dkk1表达水平的高低与某些恶性肿瘤的预后密切相关,可望成为某些肿瘤早期诊断及预测预后、判断治疗敏感性的新的肿瘤标记物.     

人源DKK1真核悬浮分泌表达系统的建立及其特异性抗体的制备和鉴定

目的:利用哺乳动物细胞悬浮培养表达系统分泌表达人源Dickkopf-1(DKK1)蛋白,纯化蛋白并制备特异性抗体。方法:构建DKK1真核表达载体pCMVcStrep-DKK1,并借助脂质体将载体瞬时转染入Free-Style293-F细胞(无血清培养),ELISA和蛋白印迹法检测DKK1蛋白表达量。利用亲和层析法纯化DKK1蛋白,Wnt信号通路荧光素酶报告系统检测其生理活性。以纯化的DKK1蛋白免疫BALB/c小鼠获得相应抗体,并初步鉴定其抗原特异性。结果:DKK1蛋白分泌表达在Free-Style293-F细胞培养液中,蛋白浓度在转染后第5天达最高(20mg/L)。所纯化的DKK1蛋白能够抑制Wnt3a蛋白诱导的报告基因的表达。抗DKK1小鼠抗体能特异性识别细胞内源性DKK1蛋白。结论:建立了在哺乳动物细胞悬浮培养系统中高效分泌表达人源DKK1重组蛋白的表达系统,并获得相应的特异性抗体,为其下一步结构与功能研究及在肿瘤中的应用奠定了实验基础。     

Dickkopf-4 is frequently down-regulated and inhibits growth of colorectal cancer cells

Like Dickkopf-1 (DKK1), DKK4 is a target of β-catenin/Tcf-4 in colorectal cancer. However, as a negative regulator of Wnt signalling its function in colorectal cancer cells is not well understood. We report that DKK4 is frequently down-regulated in colorectal cancer cell lines with deregulated β-catenin/Tcf-4 and in primary colorectal cancers. Exposure of cancer cells to DKK4 strongly inhibits basal β-catenin/Tcf-4 signalling activity, cancer cell growth and cell cycle progression. Therefore, loss of this negative feed-back loop provides Wnt factor expressing cancer cells with a growth advantage. Our data demonstrate that DKK4 is an important negative regulator of colon cancer cell growth.     

Synchronous alterations of Wnt and epidermal growth factor receptor signaling pathways through aberrant methylation and mutation in non small cell lung cancer.

PURPOSE: The Wnt and epidermal growth factor receptor (EGFR) signaling pathways play crucial roles in the pathogenesis of a variety of malignant tumors. Although the details of each cascade are understood, very little is known about their collective effects in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: A total of 238 NSCLC samples were examined for methylation of Wnt antagonists [secreted frizzled-related protein (sFRP)-1, sFRP-2, sFRP-5, Wnt inhibitory factor-1, and Dickkopf-3] and for EGFR and KRAS mutations. Protein expression levels of beta-catenin were assayed in 91 of the 238 NSCLCs. RESULTS: We found that (a) aberrant methylation of Wnt antagonists is common in NSCLCs; (b) methylation of sFRP-2 is more prevalent in females, nonsmokers, and adenocarcinoma cases; (c) Dickkopf-3 methylation is significantly associated with a poor prognosis in adenocarcinomas; (d) there is a positive correlation between activated EGFR mutation and nuclear accumulation of beta-catenin; (e) KRAS mutation and aberrant methylation of Wnt antagonists are positively correlated; and (f) EGFR mutation is significantly associated with a good prognosis in tumors lacking methylated Wnt antagonist genes. CONCLUSIONS: These results contribute to a better understanding of the cross-talk between the Wnt and EGFR signaling pathways and help foster development of chemotherapeutic treatments in NSCLCs.     

Lack of diagnostic potential of Dickkopf-1 in colon and rectum cancers

The Wnt/b-catenin signalling pathway plays crucial roles in development and its aberrant activation is an initial and crucial event in the majority of colon cancers. The Dickkopf-1 (Dkk-1) gene encodes an extracellular Wnt inhibitor that blocks the formation of signalling receptor complexes at the plasma membrane. Here, we report the serum levels of Dkk1 in colorectal cancer patients without any therapy. The levels were determined by enzyme-linked immunosorbent assay (ELISA) in 135 colon and 160 rectum cancer patients, as well as 90 healthy subjects. Data analyses were performed using SPSS software (SPSS 16, Chicago, IL). There were no significant differences among the groups for Dkk-1 (p=0.363). In conclusion, the present study did not confirm that serum Dkk-1 levels could have any diagnostic potential in colon and rectum cancers.     

Dickkopf-1 Levels in Turkish Patients with Bladder Cancer and its Association with Clinicopathological Features

Background: Evidence indicates that Dickkopf-1 (DKK-1) levels may be a biomarker for cancer risk. Theaim of this study was to assess DKK-1 and its correlation with clinic-pathological features in patients withbladder cancer. Materials and Methods: DKK-1 levels were determined in serum samples from 90 patients withbladder cancer before transurethral tumor resection. The concentrations of DKK-1 were determined by usingenzyme linked immune-sorbent assay (ELISA). Results: Elevated preoperative DKK-1 levels were associatedwith tumor stage (p<0.001), grade (p<0.001) and histological grade (p<0.001). Conclusions: The results of ourstudy demonstrated that the level of serum DKK-1 is correlated with both disease progression and increase inthe tumor grade. Preoperative serum DKK-1 elevation may thus represent a novel marker for the determinationof bladder cancer and the detection of patients with a likely poor clinical outcome.