Serum osteopontin, an enhancer of tumor metastasis to bone, promotes B16 melanoma cell migration

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Since tumor metastasis shortens patients' lifetime, establishment of therapy for anti-metastasis is very important. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, extracellular matrix (ECM) invasion and cell proliferation via interaction with its receptor, that is, alphavbeta3 integrin. OPN is believed to be a positive regulator of tumor metastasis in vivo. However, how OPN regulates metastasis is largely unknown. Here, we explore the role of OPN in cell migration. Serum from wild-type mice induced cell migration of B16 melanoma cells, while serum from OPN-deficient mouse suppressed this event. The presence of recombinant OPN significantly enhanced cell migration compared to albumin containing medium. OPN-induced cell migration was suppressed by inhibiting the ERK/MAPK pathway indicating that OPN-induced cell migration depends on this pathway. Overexpression of OPN in these cancer cells per se promoted cell proliferation and tended to increase B16 cell migration suggesting that OPN promotes bone metastasis by playing dual roles both in host microenvironment and in tumor cell itself. In conclusion, the elevated OPN expression in host tissue and tumor cell itself promotes tumor cell migration reading to tumor metastasis, suggesting that neutralization of OPN-induced signal might be effective in suppression of tumor metastasis.     

Ecto-5′-Nucleotidase (eN,CD73) is Coexpressed with Metastasis Promoting Antigens in Human Melanoma Cells

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: α₅-, β₁-, β₃-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. α_v-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.     

Angiogenic factor thymidine phosphorylase increases cancer cell invasion activity in patients with gastric adenocarcinoma

We investigated the biological role of thymidine phosphorylase (TP), an angiogenic factor, in gastric cancer cell migration and invasion and explored a therapeutic approach for high TP-expressing tumors using TP enzymatic inhibitor (TPI) and rapamycin. We established TP cDNA overexpressing gastric cancer cell lines (MKN-45/TP and YCC-3/TP) and did invasion and adhesion assays with Matrigel-coated transwell membranes. The related signal pathway using recombinant human TP (rhTP), deoxy-d-ribose (D-dRib), and signal pathway inhibitors (wortmannin, LY294002, and rapamycin) was investigated. First, AGS and MKN-1 gastric cancer cell lines showed dose-dependent up-regulation of invasiveness through Matrigel following treatment with rhTP or D-dRib. TP-overexpressing cancer cell lines displayed increased migration and invasion activity, which doubled with rhTP and D-dRib treatment. This activity depended on the enzymatic activity of TP, and TP stimulated the adhesion of cancer cells onto Matrigel and induced actin filament remodeling. Finally, we showed that this activity is related to increased phosphatidylinositol 3-kinase activity in TP-overexpressing cells and that combination treatment with rapamycin and TP enzymatic inhibitor produces an additive effect to abrogate TP-induced invasion. Taken together, TP increases the migration and invasion of gastric cancer cells, especially in TP-expressing cells. Therapies targeting TP might diminish the propensity for invasion and metastasis in gastric cancer.     

Inhibition of hyaluronan export attenuates cell migration and metastasis of human melanoma

When secreted from malignant cells, hyaluronan facilitates tumor invasion and metastasis, as inhibition of its export by zaprinast inhibited metastasis formation in mice. However, the precise steps of the metastatic cascade, which were influenced by zaprinast, have not been identified as yet. Here we analyzed the cell biological effects of the inhibitor on three human melanoma cell lines that differed in their hyaluronan production and their metastatic capability when xenografted into SCID mice. We measured the influence of zaprinast on cellular hyaluronan export, surface coat formation, proliferation, random migration, colony formation in soft agar, adhesion, and transepithelial resistance. Concentrations of zaprinast not affecting cell proliferation, adhesion and transepithelial resistance, nevertheless reduced hyaluronan export by 50%, surface coat formation, random migration, and colony formation in soft agar. These results indicate that hyaluronan enhances metastasis formation primarily in those steps of the metastatic cascade, which involves tumor cell migration.     

Pyruvate kinase M2 facilitates colon cancer cell migration via the modulation of STAT3 signalling

Understanding the mechanisms of colorectal cancer (CRC) metastatic progression is essential to reducing its morbidity and mortality. Pyruvate kinase (PK) catalyses the final step of glycolysis and has been identified as a critical regulator of glucose consumption. However, the mechanisms and roles of PKM1 and PKM2 in the regulation of CRC cell migration and cell adhesion remain elusive. Here, we report that PKM2 rather than PKM1 drives CRC cell migration and cell adhesion, whereas PKM attenuation reverses these phenomena. Furthermore, the overexpression of PKM2 significantly increases the expression of N-cadherin, MMP-2, MMP-9, STAT3, Snail-2, pFAK and active β1-integrin, while E-cadherin expression is suppressed. More importantly, the results indicated that PKM2 overexpression facilitates STAT3 nuclear translocation, and it is required for PKM2 function in the regulation of migration and adhesion associated signalling. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities but possesses protein kinase activity, is critical for CRC cell migration and cell adhesion. Overall, this study suggests that PKM2 overexpression promotes CRC cell migration and cell adhesion by regulating STAT3-associated signalling and that PKM2 may serve as a therapeutic target for CRC metastasis.     

Loss of maspin expression contributes to a more invasive potential in malignant melanoma

Deregulation of protease expression and activity is known to play an important role in tumour progression of malignant melanoma. The serpin maspin, a tumour suppressor in breast and prostate cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumour metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other cancers. However, little is known about expression, regulation and function of maspin in malignant melanoma. In this study, we found loss of maspin expression in malignant melanoma cells compared with normal human epidermal melanocytes, which was analysed by quantitative real-time PCR, Western blot analysis, immunohistochemistry and microarray. For functional studies, melanoma cell clones stably transfected with a maspin expression vector were tested for changes in proliferation, migration and invasion. Although we could not see differences in proliferation and migration, we detected strongly reduced invasive capacity in the melanoma cell clones in which maspin is re-expressed compared with control. Reduced invasive potential was also detected in three different melanoma cell lines transiently transfected with a maspin expression vector. Furthermore, exogenously added maspin alone was sufficient to reduce invasion in MelIm significantly, indicating that maspin directly inhibits invasion on the cell surface. In summary, we believe that maspin is a tumour suppressor in malignant melanoma.     

CD10-Equipped Melanoma Cells Acquire Highly Potent Tumorigenic Activity: A Plausible Explanation of Their Significance for a Poor Prognosis

CD10 has been widely used in cancer diagnosis. We previously demonstrated that its expression in melanoma increased with tumor progression and predicted poor patient survival. However, the mechanism by which CD10 promotes melanoma progression remains unclear. In order to elucidate the role of CD10 in melanoma, we established CD10-overexpressing A375 melanoma cells and performed DNA microarray and qRT–PCR analyses to identify changes in the gene expression profile. The microarray analysis revealed that up-regulated genes in CD10-A375 were mostly involved in cell proliferation, angiogenesis, and resistance to apoptosis; down-regulated genes mostly belonged to the categories associated with cell adhesion and migration. Accordingly, in functional experiments, CD10-A375 showed significantly greater cell proliferation in vitro and higher tumorigenicity in vivo; CD10 enzymatic inhibitors, thiorphan and phosphoramidon, significantly blocked the tumor growth of CD10-A375 in mice. In migration and invasion assays, CD10-A375 displayed lower migratory and invasive capacity than mock-A375. CD10 augmented melanoma cell resistance to apoptosis mediated by etoposide and gemcitabine. These findings indicate that CD10 may promote tumor progression by regulating the expression profiles of genes related to cell proliferation, angiogenesis, and resistance to apoptosis.     

信号转导和转录活化因子3通过趋化因子CX3C配体1促进血管内皮细胞增殖迁移

信号转导和转录活化因子3 (STAT3)与趋化因子CX3C配体1 (Fractalkine/CX3CL1)在血管炎症和损伤中起重要作用,为了探讨STAT3是否通过CX3CL1促进血管内皮细胞增殖和迁移,在血管内皮细胞(HUVEC)中过表达或敲降STAT3,通过quantitative real-time PCR、Western blotting实验确定STAT3对CX3CL1表达的影响。构建含有STAT3结合位点及突变STAT3结合位点的CX3CL1启动子荧光素酶报告基因质粒,利用荧光素酶活性分析实验研究STAT3对CX3CL1启动子转录活性的作用。利用MTT实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞增殖率的影响。利用划痕实验检测过表达或敲降STAT3或CX3CL1对血管内皮细胞迁移率的影响。结果显示,过表达STAT3可以促进CX3CL1表达,敲降STAT3可以使CX3CL1表达下调。STAT3可以直接结合到CX3CL1的启动子促进其转录激活,其促进作用依赖于CX3CL1启动子上的GAS位点。敲降STAT3可以抑制血管内皮细胞的迁移,过表达CX3CL1拮抗该抑制作用。总结得出,STAT3通过结合到CXCL1启动子促进CX3CL1转录与表达进而促进血管内皮的增殖与迁移。     

转染人核糖核酸酶抑制因子基因对B16黑色瘤细胞及肿瘤转移的影响(英)

人核糖核酸酶抑制因子(human ribonuclease inhibitor, RI)是一种细胞质中分子质量为50 ku的酸性糖蛋白.RI能抑制核糖核酸酶A（RNase A）的活性, RNase A与血管生成因子（angiogenin,Ang）的氨基酸有着高度保守的同源序列.Ang是RNase A超家族的一员,RI通过与RNase A和Ang的紧密结合而抑制其活性.血管生成及新血管的形成, 是肿瘤发生和转移的必要条件.所以抗血管生成将是一种很有希望的对抑制肿瘤生长和转移的有效方法.实验显示RI能有效地抑制肿瘤诱导血管的生成.RI由含有许多亮氨酸重复序列的多肽组成.含有这样重复序列的100多种蛋白质显示了广泛的功能,包括细胞周期调节,DNA修复,对细胞外基质相互作用以及抑制酶活性等.RI被认为是胚胎发育,创伤愈合及肿瘤发生中新血管形成的一种调节因子.RI定位于染色体的11p15.5,与ras基因邻近,在肿瘤病人中经常存在染色体11p15.5部位的变异和异常.RI可能与细胞的生长和分化有关, 因此,RI 可能还具有尚未知的生物学作用.为了进一步了解RI的潜在功能以及探讨RI与肿瘤浸润、转移的关系, 将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317,并转染到B16小鼠黑色瘤细胞中, 用转染空载体和未转染的B16细胞作为对照.通过PCR, RT-PCR, 蛋白质免疫印迹, 免疫荧光分析鉴定,获得稳定表达人核糖核酸酶抑制因子的细胞株.结果显示, 转染的RI基因在体外能显著地抑制细胞增殖和细胞迁移,增加了细胞的粘附以及改善细胞的恶性形态,B16,B16 pLNCX,B16 pLNCX-RI 3种细胞的倍增时间分别为(24.98±0.16) h, (25.62±0.28) h, (32.64±1.11) h.与对照组相比,转RI的细胞粘附率增加17.8%和19.5%而迁移降低了61.4%和60%.转RI的细胞比对照组细胞较平展,核仁和分裂相较少,胞质嗜碱性减弱,提示细胞增殖活性降低和恶性表型的改善. 将3种B16细胞静脉注射到C57BL/6小鼠中, 结果表明, 转染RI基因的实验组显著地抑制了肿瘤的转移, 与两个对照组相比,荷瘤小鼠有更长的存活时间, 少得多的转移节结, 更低的肿瘤血管密度和肺重量.结果显示,RI的表达可能与黑色瘤的转移有关, 提示RI能显著地抑制肿瘤的转移,可能由于其与抑制血管作用,增加细胞粘附,降低细胞迁移及增殖有关.     

Ecto-5'-nucleotidase (eN, CD73) is coexpressed with metastasis promoting antigens in human melanoma cells

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: alpha5-, beta1-, beta3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. alphav-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.     

Conditioned media from adipocytes promote proliferation,migration, and invasion in melanoma and colorectal cancer cells

Epidemiological evidence suggests that obesity can significantly increase the risk of various cancers, although the mechanisms underlying this link are completely unknown. Here, we analyzed the effect of adipocytes on melanoma and colon cancer cells proliferation, migration, and invasion. The potential effects of conditioned media (CM) obtained from differentiated mouse 3T3-L1 cells and human adipose tissue-derived mesenchymal stem cells (hAMSC) on the proliferation, migration, and invasion of B16BL6 melanoma and colon 26-L5 cancer cells were investigated. The 3T3-L1 and hAMSC CM increased cell proliferation, migration, and invasion in both the cell lines. In addition, adipocytes CM increased matrix metalloproteinase 9 (MMP-9) and MMP-2 activity in both B16BL6 and colon 26-L5 cells. These effects were found to be associated with an increased expression of various oncogenic proteins in B16BL6 and colon 26-L5 cells. Also, adipocyte CM induced Akt and mTOR activation in both tumor cell lines, and the pharmacological inhibition of Akt and mTOR blocked the CM induced Akt as well as mTOR activation and CM-stimulated melanoma and colon cancer cell proliferation, migration, and invasion. These data suggest that adipocyte promotes melanoma and colon cancer progression through modulating the expression of diverse proteins associated with cancer growth and metastasis as well as modulation of the Akt/mTOR signaling.     

A novel sialyltransferase inhibitor AL10 suppresses invasion and metastasis of lung cancer cells by inhibiting integrin‐mediated signaling

Aberrant sialylation catalyzed by sialyltransferases (STs) is frequently found in cancer cells and is associated with increased cancer metastasis. However, ST inhibitors developed till now are not applicable for clinical use because of their poor cell permeability. In this study, a novel ST inhibitor AL10 derived from the lead compound lithocholic acid identified in our previous study is synthesized and the anti‐cancer effect of this compound is studied. AL10 is cell‐permeable and effectively attenuates total sialylation on cell surface. This inhibitor shows no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of α‐2,3‐ST‐overexpressing A549 and CL1.5 human lung cells. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of various integrin molecules and attenuated activation of the integrin downstream signaling mediator focal adhesion kinase. More importantly, AL10 significantly suppresses experimental lung metastasis in vivo without affecting liver and kidney function of experimental animals as determined by serum biochemical assays. Taken together, AL10 is the first ST inhibitor, which exhibits potent anti‐metastatic activity in vivo and may be useful for clinical cancer treatment. J. Cell. Physiol. 223: 492–499, 2010. © 2010 Wiley‐Liss, Inc.     

Rhodanine-based PRL-3 inhibitors blocked the migration and invasion of metastatic cancer cells

PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-1, which inhibited PRL-3 enzymatic activity with IC₅₀ values of 0.8 μM and 1.1 μM, respectively. CG-707 and BR-1 strongly inhibited the migration and invasion of PRL-3 overexpressing colon cancer cells without exhibiting cytotoxicity. The specificity of the inhibitors on PRL-3 phosphatase activity was confirmed by the phosphorylation recovery of known PRL-3 substrates such as ezrin and cytokeratin 8. The compounds selectively inhibited PRL-3 in comparison with other phosphatases, and CG-707 regulated epithelial-to-mesenchymal transition (EMT) marker proteins. The results of the present study reveal that rhodanine is a specific PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.     

Expression of kindlin-3 in melanoma cells impedes cell migration and metastasis

Kindlins are a small family of 4.1-ezrin-radixin-moesin (FERM)-containing cytoplasmic proteins. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Kindlin-3 promotes integrin activation, clustering and outside-in signaling. Aberrant expression of kindlin-3 was reported in melanoma and breast cancer. Intriguingly, kindlin-3 has been reported to either positively or negatively regulate cancer cell metastasis. In this study, we sought to clarify the expression of kindlin-3 in melanoma cells and its role in melanoma metastasis. Two widely used metastatic mouse and human melanoma cell lines B16-F10 and M10, respectively, were examined and found to lack kindlin-3 mRNA and protein expression. When kindlin-3 was ectopically expressed in these cells, cell migration was markedly reduced. These are attributed to aberrant Rac1 and RhoA activation and overt membrane ruffling. Our data demonstrate for the first time that despite its well established role as a positive regulator of integrin-mediated cell adhesion, aberrant expression of kindlin-3 could lead to imbalanced RhoGTPases signaling that impedes rather than promotes cell migration.     

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.     

In vitro effects of SIKVAV retro and retro-enantio analogues on tumor metastatic events

The SIKVAV peptide, located on the long arm of the laminin alpha1 chain, promotes cell adhesion, invasion and migration of tumor and endothelial cells, resulting in tumor growth, angiogenesis and metastasis. In this paper, we report the synthesis of the SIKVAV peptide and its retro (reverse l-amino acid order) and retro-enantio (reverse d-amino acid order) analogues and their effect on three critical steps in the metastatic process: cell-extracellular matrix protein (ECM) adhesion, cell migration and homotypic cell adhesion, using B16F10 melanoma cells. Results show that all peptides compete with laminin-1 cell attachment, but only SIKVAV induces peptide-cell adhesion. Retro analogue, but not retro-enantio, inhibits cell adhesion to SIKVAV, indicating that retro peptide recognizes the SIKVAV receptors while retro-enantio does not. Retro-enantio peptide is able to inhibit cell migration, by contrast of the SIKVAV chemoattractant activity. All three peptides reduce the homotypic cell adhesion in a dose-dependent manner, but retro-enantio sequence is the most effective reaching a 35% inhibition of controls at the higher concentration. These findings suggest that that both analogues of SIKVAV peptide, especially retro-enantio, may be considered as potential antimetastatic agents.