The EF-hand calcium-binding protein tescalcin is a potential oncotarget in colorectal cancer

Tescalcin (TESC) is an EF-hand calcium binding protein that is differentially expressed in several tissues, however it is not reported that the expression and functional roles of TESC in colorectal cancer. Levels of messenger RNA (mRNA) and protein expression of TESC in colorectal cancer tissues were assessed using RT-PCR, real time PCR, immunohistochemistry, and clinicopathologic analyses. Quantitative analysis of TESC levels in serum specimens was performed using sandwich ELISA. Colorectal cancer cells transfected with TESC small interfering RNA and short hairpin RNA were examined in cell proliferation assays, phospho-MAPK array, and mouse xenograft models. Here we demonstrated that TESC is overexpressed in colorectal cancer (CRC), but was not expressed in normal mucosa and premalignant dysplastic lesions. Furthermore, serum TESC levels were elevated in patients with CRC. Knockdown of TESC inhibited the Akt-dependent NF-κB pathway and decreased cell survival in vitro. Depletion of TESC reduced tumor growth in a CRC xenograft model. Thus, TESC is a potential diagnostic marker and oncotarget in colorectal cancer.     

RANKL-induced migration of MDA-MB-231 human breast cancer cells via Src and MAPK activation

Accumulating studies have shown that the receptor activator of nuclear factor-κB ligand (RANKL)/RANK pathway plays an important role in tumor metastasis. However, the involvement of the RANKL/RANK signal transduction pathway in breast cancer metastasis remains unclear. The present study, therefore, investigated the role of downstream molecules of RANKL/RANK signaling in breast cancer cells using Transwell chemotaxis assays. RANKL was shown to direct the migration of MDA-MB-231 breast cancer cells. Osteoprotegerin (OPG; soluble decoy receptor of RANKL) inhibited RANKL-induced migration. RANKL activated Src kinase in MDA-MB-231 cells, as shown by Western blotting, and pretreatment with a Src inhibitor abrogated RANKL-induced cell migration, in a similar manner to OPG. Short-hairpin RNA against RANK, delivered via a lentiviral vector, significantly abolished the expression of phosphorylated Src. Stimulation by RANKL induced the phosphorylation of mitogen-activated protein kinases (MAPKs) (ERK, p38, JNK), and specific inhibitors of MAPKs blocked RANKL-induced cell migration. Furthermore, the expression of phosphorylated MAPKs could be blocked by a Src inhibitor and by small interfering RNA against Src. These findings suggest that Src and MAPK pathways may be involved in RANKL-induced MDA-MB-231 breast cancer cell migration.     

Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer

Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-κB-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared with WT-YBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-κB activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer.     

PI3K/Akt信号通路在RANKL诱导的MCF-7乳腺癌细胞迁移中的作用

目的 核因子-κB受体活化因子配体(RANKL)能促进表达RANK的上皮癌细胞迁移至骨,与乳腺癌骨转移密切相关.本文探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(phosphoinositide3-kinase/serine/threonine protein kinase PI3K/Akt)信号通路在RANKL诱导的乳腺癌MCF-7细胞迁移中的作用.方法 流式细胞仪检测MCF-7细胞表面RANK蛋白的表达;Transwell法测定RANKL刺激后MCF-7细胞迁移能力的改变;Western-blot检测MCF-7细胞RANKL刺激后p-Akt及Akt的表达.SPSS 16.0软件分析实验数据.结果 MCF-7细胞表达RANK 蛋白,RANKL诱导MCF-7细胞迁移能力显著增强,RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移.RANKL刺激后MCF-7细胞p-Akt表达在1、5分钟时一过性升高,PI3K抑制剂LY294002显著抑制RANKL诱导的细胞迁移.结论 PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞系MCF-7迁移.     

The novel IκB kinase β inhibitor IMD-0560 prevents bone invasion by oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) cells display significantly augmented nuclear factor-κB (NF-κB) activity, and inhibiting this activity suppresses malignant tumor characteristics. Thus, we evaluated the effect of IMD-0560, a novel inhibitor of IκB kinase (IKK) β that is under assessment in a clinical trial of rheumatoid arthritis, on bone invasion by the mouse OSCC cell line SCCVII. We examined the inhibitory effects of IMD-0560 on NF-κB activity and tumor invasion using human OSCC cell lines and SCCVII cells in vitro. Using a mouse model of jaw bone invasion by SCCVII cells, we assessed the inhibitory effect of IMD-0560 on jaw bone invasion, tumor growth, and matrix degradation in vivo. IMD-0560 suppressed the nuclear translocation of NF-κB and the degradation of IκBα in OSCC cells. IMD-0560 also inhibited invasion by suppressing matrix metalloproteinase-9 (MMP-9) production in OSCC cells. IMD-0560 protected against zygoma and mandible destruction by SCCVII cells, reduced the number of osteoclasts by inhibiting receptor activator of NF-κB ligand (RANKL) expression in osteoblastic cells and SCCVII cells, increased SCCVII cell death and suppressed cell proliferation and MMP-9 production in SCCVII cells. Based on these results, IMD-0560 may represent a new therapeutic agent for bone invasion by OSCC cells.     

miR-302a对结直肠癌细胞奥沙利铂化疗敏感性的影响及机制

目的:探究miR-302a对结直肠癌细胞奥沙利铂化疗敏感性的影响及机制。方法:在四株结直肠癌细胞系HT29、HCT8、SW480、SW1463中,通过转染miR-302a mimic构建miR-302a过表达模型,RT-PCR检测过表达效果;CCK-8法检测转染48 h后高表达miR-302a细胞的增殖能力及对奥沙利铂的敏感性;蛋白质印迹法检测转染后P-gp蛋白及Wnt/β-catenin通路相关蛋白MMP-7、c-Jun、c-myc、β-catenin、LEF1的变化。结果:相比正常小肠上皮细胞HIEC,miR-302a在结直肠癌细胞系HT29、HCT8、SW480、SW1463中的表达较低。转染mimic后,miR-302a的表达明显上调（P<0.001）。增殖实验发现miR-302a的上调并不影响结直肠癌细胞的增殖,而在转染miR-302a的细胞中加入奥沙利铂,miR-302a组HT29、HCT8、SW480和SW1463细胞存活率相比miR-NC组分别降低2.46、1.89、2.39、2.86倍。进一步探究miR-302a增加奥沙利铂敏感性的机制,发现miR-302a可抑制P-gp蛋白的表达,并且抑制Wnt/β-catenin通路相关蛋白MMP-7、c-Jun、c-myc、β-catenin、LEF1的表达。结论:miR-302a可增加结直肠癌细胞奥沙利铂化疗敏感性,其机制可能是通过抑制P-gp的蛋白表达,并抑制Wnt/β-catenin信号通路相关蛋白表达而实现。     

Simvastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, suppresses osteoclastogenesis induced by receptor activator of nuclear factor-kappaB ligand through modulation of NF-kappaB pathway

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is a cholesterol-lowering drug that may play a role in bone metabolism through a mechanism that is not fully understood. Recently, receptor activator of NF-kappaB ligand (RANKL), a member of the TNF superfamily, has emerged as a major mediator of bone loss via activation of osteoclastogenesis. The latter is also associated with certain cancers such as multiple myeloma and breast cancer. Whether simvastatin can modulate RANKL-induced or cancer induced osteoclastogenesis was investigated. The effect of simvastatin on RANKL signaling and consequent osteoclastogenesis was investigated. RANKL induced NF-kappaB activation, whereas pretreatment with simvastatin completely suppressed such activation and correlated with suppression of RANKL-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation and IkappaBalpha degradation. Similarly, RANKL induced the differentiation of monocytic cells to osteoclasts, whereas simvastatin suppressed it. The inhibition was maximal when cells were exposed to both simvastatin and RANKL simultaneously and minimal when simvastatin was added 1 day after RANKL treatment. Simvastatin also inhibited the osteoclastogenesis induced by human breast cancer and by multiple myeloma cells. Together, our results indicate that simvastatin inhibits the RANKL-induced NF-kappaB activation pathway that leads to suppression of osteoclastogenesis induced by RANKL and by tumor cells, thereby suggesting its therapeutic potential in osteoporosis and in cancer-related bone loss.     

Gene silencing of the BDNF/TrkB axis in multiple myeloma blocks bone destruction and tumor burden in vitro and in vivo

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF‐κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain‐derived neurotrophic factor (BDNF) was identified as a MM‐derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma–osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM‐derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time‐ and dose‐dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID‐RPMI8226 mice model was constructed using lentiviral short hairpin RNA‐transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma–myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.     

PI3K/Akt信号通路在RANKL诱导的MCF-7乳腺癌细胞迁移中的作用

目的：核因子-κB受体活化因子配体（RANKL）能促进表达RANK的上皮癌细胞迁移至骨,与乳腺癌骨转移密切相关。本文探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶（phosphoinositide3-kinase/serine/threonine protein kinase PI3K/Akt）信号通路在RANKL诱导的乳腺癌MCF-7细胞迁移中的作用。方法：流式细胞仪检测MCF-7细胞表面RANK蛋白的表达;Transwell法测定RANKL刺激后MCF-7细胞迁移能力的改变;Western-blot检测MCF-7细胞RANKL刺激后p-Akt及Akt的表达。SPSS 16.0软件分析实验数据。结果：MCF-7细胞表达RANK蛋白,RANKL诱导MCF-7细胞迁移能力显著增强,RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移。RANKL刺激后MCF-7细胞p-Akt表达在1、5分钟时一过性升高,PI3K抑制剂LY294002显著抑制RANKL诱导的细胞迁移。结论：PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞系MCF-7迁移。     

Mechanism of bone metastasis: The role of osteoprotegerin and of the host-tissue microenvironment-related survival factors

Osteoprotegerin (OPG), member of tumor necrosis factor (TNF) receptor superfamily, has various biological functions including bone remodeling. OPG binds to receptor activator of nuclear factor-kB ligand (RANKL) and prevents osteoclastic bone resorption. Recently, OPG has gained more clinical interest as its role in cancer-mediated bone destruction and the potential of RANKL inhibition could act as a novel treatment in tumor-induced bone disease. OPG protects prostate cancer cells from apoptotic effects of TRAIL and therefore provides tumor cells producing OPG with survival advantages. Additionally, the increased RANKL/OPG ratio in metastatic breast cancer results in severe osteolysis. Thus, bone formation and resorption are the crux of cancer metastasis, resulting in bone pain and pathological fractures. This review provides an overview of the role of OPG in cancer-induced bone disease.     

Plasma levels of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in patients with neuroblastoma

Earlier reports showed that the balance between receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy-receptor osteoprotegerin (OPG) plays an important role in the pathogenesis of metastatic osteolysis induced by neuroblastoma cells. In this study, we investigated whether circulating levels of OPG, RANKL and their ratio were associated to the presence of osteolytic lesions in advanced neuroblastoma, as well as whether they provided additional information on the severity and prognosis of the disease. Plasma levels of RANKL and OPG were measured in 54 newly diagnosed neuroblastomas; 27 of them showed metastatic disease (stage IV), including 19 bone dissemination. Thirty-five children who were admitted to the pediatric department for minor surgical problems served as control group. OPG was significantly lower in all patients compared with controls, while RANKL levels were significantly increased in advanced neuroblastoma. OPG-to-RANKL ratio decreased in stage-IV patients, and particularly in those who had bone metastases. The diagnostic accuracy of the OPG-to-RANKL ratio in discriminating the presence of osteolytic lesions was not confirmed statistically. OPG correlated significantly with other prognostic factors, namely, ferritin and neurone-specific enolase. In addition, an inverse relationship was found between OPG and event-free survival, and it was more significant in patients who had bone metastasis. This pilot study confirms that the production of OPG and RANKL is disregulated in neuroblastoma. Although the OPG-to-RANKL ratio does not have a predictive value in detecting bone metastasis, the measurement of the previously mentioned markers could be useful in decisions regarding the use of adjuvant therapies.     

Paclitaxel resistance is mediated by NF-κB on mesenchymal primary breast cancer cells

Paclitaxel has been used widely to treat breast cancer and other types of cancer. However, resistance is a major cause of failure for treatment and results in cancer progression. The present study investigated the association between paclitaxel resistance and the mesenchymal phenotype, using a model of primary breast cancer cells and employing four different cultures, two with an epithelial phenotype (MBCDF and MBCD17) and two with a mesenchymal phenotype (MBCDF-D5 and MBCD3). Epithelial-mesenchymal markers were evaluated by western blotting; MBCDF and MBCD17 cells expressed E-cadherin, SNAIL, Slug, and Twist, low levels of N-cadherin, but not vimentin. MBCDF-D5 and MBCD3 cells expressed N-cadherin, vimentin, and higher levels of SNAIL, and low levels of E-cadherin, Slug, and Twist. Cell viability was evaluated using a crystal violet assay after paclitaxel treatment; primary breast cancer cells with mesenchymal phenotype were resistant to paclitaxel compared with the epithelial primary breast cancer cells. Furthermore, using western blotting, it was revealed that mesenchymal cells had elevated levels of nuclear factor-κΒ (NF-κB) p65 and IκB kinase (IKK). Additionally, it was demonstrated that paclitaxel-induced degradation of the inhibitor of NF-κB, activation of NF-κB in a dose-dependent manner, and Bcl-2 and Bcl-xL upregulation. Finally, employing western blotting and crystal violet assays, the effects of the proteasome inhibitor ALLN were assessed. ALLN inhibited paclitaxel-induced NF-κB activation and restored the sensitivity to paclitaxel. Together, these data suggest that targeting the NF-κB/IKK axis might be a promising strategy to overcome paclitaxel resistance.     

TRAF6 regulates the signaling pathway influencing colorectal cancer function through ubiquitination mechanisms

Tumor necrosis factor receptor‐associated factor‐6 (TRAF6) is a ubiquitin E3 ligase. TRAF6 plays an important role in tumor invasion and metastasis. However, the specific mechanism by which TRAF6 promotes colorectal cancer (CRC) metastasis is incompletely understood. This study aimed to determine whether TRAF6 affects the LPS‐NF‐κB‐VEGF‐C signaling pathway through ubiquitination, which plays a role in colorectal cancer metastasis. Here, our results showed that TRAF6 affected lymphangiogenesis through the LPS‐NF‐κB‐VEGF‐C signaling pathway. Using ubiquitination experiments, we found that TRAF6 was mainly ubiquitinated with the K63‐linked chains, and LPS promoted ubiquitination of TRAF6 and K63‐linked chains. More importantly, TRAF6 124mut is the main ubiquitination site of TRAF6 interacting with K63‐linked chains. TRAF6 affected the migration, invasion, and lymphatic metastasis of colorectal cancer through its ubiquitination. In subcutaneous xenograft models, TRAF6 124mut inhibited tumor growth. In conclusion, our results provide new insight for studying the mechanism of lymphangiogenesis in colorectal cancer to promote cancer metastasis, which may provide new ideas for tumor immunotherapy.     

The role of RANK/RANKL/osteoprotegerin (OPG) triad in cancer-induced bone diseases: physiopathology and clinical implications

Bone homeostasis is maintained by the remodelling of bone which depends on a balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Malignant bone lesions are very common in patients with cancer; whether they result from a tumor in bone (giant cell tumour of bone, osteosarcoma, multiple myeloma...) or they are bony metastases from advanced cancers of which the most osteotropic are breast and prostate cancer. Malignant cells within the bone disrupt the normal bone remodelling process, leading to increased bone destruction and occurence of pathological fractures. Receptor activator of NF-kB (RANK) and its ligand (RANKL) play a pivotal role in the regulation of bone remodelling; by binding to RANK, RANKL stimulates osteoclastogenesis and bone resorption, whereas its cognate decoy receptor osteoprotegerin (OPG) blocks this process by interacting with RANKL. Tumour cells produce different factors that manipulate the RANK/RANKL/OPG pathway in order to stimulate bone destruction. Furthermore, pending on the tumour type, RANKL plays a role in the migration, invasion and proliferation of malignant cells within the bone, while OPG increases survival of tumour cells. Inhibition of RANK/RANKL system may therefore offer new therapeutic perspectives for the treatment of primitive and secondary bone cancers.