Inhibition of HGF/SF-induced breast cancer cell motility and invasion by the HGF/SF variant, NK4

NK4 is a variant form of HGF/SF, comprising the N-terminal and subsequent four kringle domains of mature HGF/SF. HGF/SF is a multifunctional cytokine that enhances the metastatic behaviour of tumour cells in vitro by stimulation of the c-met receptor tyrosine kinase and has been implicated in the development of tumour metastasis in vivo. The aims of this study were to further investigate the potential antagonistic effects of the recently described variant form of HGF/SF, NK4, on HGF/SF activity in breast cancer cells. All cell lines used expressed both the HGF/SF receptor gene and protein as shown by RT-PCR and Western blotting. NK4 inhibited HGF/SF-induced tumour cell invasion through an artificial basement membrane. Tumour cell motility and scattering induced by HGF/SF were also dramatically reduced by the inclusion of NK4. Immunoprecipitation studies revealed that NK4 inhibited the phosphorylation of the c-met receptor in response to HGF/SF. Treatment of these cells with NK4 alone did not have any significant effects on their metastatic behaviour. From this data we conclude that NK4 demonstrates significant antagonistic properties towards HGF/SF, inhibiting HGF/SF-stimulated breast tumour cell invasion, motility and migration. NK4 may therefore be of potential benefit in the development of anti-metastasis therapies.     

NK4, a four-kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells

Because of the highly aggressive behaviour, i.e. invasive, disseminative and metastatic properties, the outcome for patients with pancreatic cancer is morbid. A better understanding and interference with the malignant behaviour of pancreatic cancer may provide new directions for treatment. We report here the induction of highly motile and invasive properties in human pancreatic cancer cells by hepatocyte growth factor (HGF) and blockage of these properties by NK4, a newly identified antagonist for HGF. In all of eight human pancreatic cancer cell lines we used (AsPC-1, BxPC-3, H-48N, KP-1N, KP-2, KP-3, MIA PaCa-2 and SUIT-2 cells), the c-Met/HGF receptor was expressed at varying levels. Although weak mitogenic activity of HGF was seen only in SUIT-2 and KP-3 cells, HGF strongly stimulated migration and invasion of these pancreatic cancer cells, except for BxPC-3 and MIA PaCa-2 cells. In contrast, migration and invasion potently induced by HGF in KP-1N, KP-3 and SUIT-2 cells were inhibited by NK4. The invasion of SUIT-2 cells was also potently stimulated with the influence of cocultured pancreatic fibroblasts and by ascitic fluid obtained after pancreatic cancer resection, however, invasiveness of the cancer cells in such conditions was practically abolished by NK4. Consistently, the ascitic fluid in patients who had undergone pancreatic cancer surgery contained high levels of HGF. These findings mean that HGF is probably involved in invasion, dissemination, and metastasis of pancreatic cancer, particularly through tumour-stromal interaction and after resection of the pancreatic cancer. NK4, an effective antagonist of HGF, may prove to have the potential for anti-invasion/metastasis.     

Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist

Hepatocyte growth factor/scatter factor (HGF/SF) is a cytokine primarily produced by stromal fibroblasts and is a known angiogenic and invasion-inducing factor. It is increased in patients with breast cancer. This study examined the effect of NK4, a newly described HGF/SF antagonist, on HGF/SF-promoted growth of a human breast cancer. Both in vitro (invasion and migration assays) and in vivo (murine tumour model) methods were used to ascertain the effect of NK4 on HGF/SF from two sources: human fibroblast-derived HGF/SF and recombinant HGF/SF. In the in vitro invasion assay and migration assay, both HGF/SF and human fibroblasts, which secrete bioactive HGF/SF, increased the invasiveness and migration of the breast cancer cells (MDA MB 231). NK4 significantly reduced this invasiveness and motility. In the animal model, tumour volume and weight was significantly reduced with addition of NK4. It also suppressed HGF/SF-induced growth and markedly retarded tumour growth induced by fibroblasts (MRC5), secreting bioactive HGF/SF. Tumour angiogenesis was assessed by immunohistochemical staining of primary tissue sections using VE-cadherin (an endothelial cell specific cell-cell adhesion molecule). Again, NK4 reduced the effects of both HGF/SF and fibroblasts. We conclude that NK4 has a significant effect on the growth of human breast tumours in nude mice, particularly when stimulated by HGF/SF or fibroblasts. This may occur by decreasing angiogenesis. This gives a clear indication of the therapeutic worth of NK4.     

SH2-B与结肠癌细胞运动和迁移相关性的研究

目的:分析SH2-B对结肠癌细胞侵袭迁移的影响,探讨结肠癌转移的分子机制。方法:免疫荧光筛选SH2-B低表达结肠癌细胞,用Lipofectamine 2000TM将pcDNA3.1-SH2-B质粒转染至HT-29细胞,细胞划痕实验分析SH2-B对结肠癌细胞HT-29爬行迁移运动的影响,用Boden Chamber分析SH2-B对结肠癌细胞HT-29侵袭运动的影响。结果:HT-29为SH2-B低表达结肠癌系,基因转染后HT-29细胞表达SH2-B显著增高;细胞划痕研究结果表明,SH2-B转染组、空载体转染组和未转染母细胞组迁移细胞数倒置显微镜10个视野分别为867±187、349±121和279±158,SH2-B转染组爬行细胞数显著高于空载体组和未转染细胞组,P均<0.05;Trans-well研究结果显示,转染SH2-B组、空载体转染组和未转染细胞组穿过滤膜细胞数分别为278±107、129±88和112±81,转染SH2-B组透过细胞数显著高于未转染组和空载体转染组,P<0.05。结论:SH2-B可能通过增强结肠癌细胞运动和迁移能力参与结肠癌细胞的侵袭和转移。     

The HGF/SF antagonist NK4 reverses fibroblast- and HGF-induced prostate tumor growth and angiogenesis in vivo

Our study examined the in vitro and in vivo responses of a newly discovered HGF/SF antagonist, NK4, on HGF/SF-promoted growth of human prostate cancer cells (PC-3). Nude mice were s.c. injected with either PC-3- and/or HGF/SF-producing fibroblasts (MRC5), and tumor size was measured over a 4-week period. rh-HGF/SF and/or NK4 were introduced by osmotic minipumps. An in vitro study found that NK4 significantly suppressed HGF/SF-induced invasion (HGF/SF; p < 0.01 vs. HGF/SF+NK4) and migration (HGF/SF; p < 0.05 vs. HGF/SF+NK4). Similarly, NK4 also suppressed the invasion (MRC5; p < 0.01 vs. MRC5+NK4) and migration (MRC5; p < 0.05 vs. MRC5+NK4) induced by MRC5 cells. NK4 also suppressed HGF/SF- and MRC5-induced tyrosine phosphorylation of the HGF/SF receptor Met as assessed by immunoprecipitation. Using a nude mouse model, prostate tumor volume (mm(3)) was significantly increased in both HGF/SF- (HGF/SF; p < 0.05 vs. control) and MRC5- (MRC5; p < 0.01 vs. control) treated groups compared to the control. In contrast, NK4 alone significantly reduced the growth of prostate tumors (NK4; p < 0.01 vs. control). In addition, NK4 also suppressed both HGF/SF- (HGF/SF; p < 0.01 vs. HGF/SF+NK4) and MRC5- (MRC5; p < 0.05 vs. MRC5+NK4) induced tumor growth in vivo by significantly reducing (p < 0.05) the degree of tumor angiogenesis using a recently discovered family of tumor endothelial markers (TEMs) by Q-RT-PCR analysis. In conclusion, NK4 suppresses both HGF/SF- and MRC5-induced invasion/migration of PC-3 cells in vitro. Furthermore, the HGF/SF antagonist NK4 significantly reduces prostate tumor growth in vivo by inhibiting the degree of tumor angiogenesis as determined by TEM-1 and TEM-8. Finally, our study provides evidence of the therapeutic potential of NK4 in prostate cancer development by antagonising HGF/SF-mediated events.     

HGF/SF activates glycolysis and oxidative phosphorylation in DA3 murine mammary cancer cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine growth factor which increases cellular motility and has also been implicated in tumor development and progression and in angiogenesis. Little is known about the metabolic alteration induced in cells following Met-HGF/SF signal transduction. The hypothesis that HGF/SF alters the energy metabolism of cancer cells was investigated in perfused DA3 murine mammary cancer cells by nuclear magnetic resonance (NMR) spectroscopy, oxygen and glucose consumption assays and confocal laser scanning microscopy (CLSM). 31P NMR demonstrated that HGF/SF induced remarkable alterations in phospholipid metabolites, and enhanced the rate of glucose phosphorylation (P < .05). 13C NMR measurements, using [13C1]-glucose-enriched medium, showed that HGS/SF reduced the steady state levels of glucose and elevated those of lactate (P < .05). In addition, HGF/SF treatment increased oxygen consumption from 0.58+/-0.02 to 0.71+/-0.03 micromol/hour per milligram protein (P < .05). However, it decreased CO2 levels, and attenuated pH decrease. The mechanisms of these unexpected effects were delineated by CLSM, using NAD(P)H fluorescence measurements, which showed that HGF/SF increased the oxidation of the mitochondrial NAD system. We propose that concomitant with induction of ruffling, HGF/SF enhances both the glycolytic and oxidative phosphorylation pathways of energy production.     

Expression of HGF/SF in mesothelioma cell lines and its effects on cell motility, proliferation and morphology.

The expression of hepatocyte growth factor/scatter factor (HGF/SF) was studied in 12 mesothelioma cell lines characterized by either an epithelioid or a fibroblast-like phenotype. Conditioned media from these lines were analysed by bioassay and ELISA, and HGF/SF was detected in three cell lines, all with a fibroblast-like or mixed morphology. None of eight epithelioid cell lines expressed the factor. Thus, for these cell lines, the ability to secrete HGF/SF correlated with the cell phenotype. Following on from these observations, two cell lines, BR and BT, with a fibroblast-like and an epithelioid phenotype, respectively, were further investigated. Both cell lines expressed the Met receptor but only BR secreted HGF/SF. Both cell lines responded to exogenous HGF/SF treatment by a change of morphology but in different ways: BR became more elongated and bipolar, while BT formed more spread-out cell colonies. HGF/SF acted as a paracrine effector on the epithelioid BT cells and stimulated both cell-spreading and proliferation. Interestingly, BT cells spread but did not scatter in response to exogenous HGF/SF. In contrast BR cells showed only some stimulation of cell motility with HGF/SF and no increase in cell proliferation was observed. Because HGF/SF was previously found in the pleural effusion fluids of patients with malignant mesothelioma and in paraffin-embedded tumour tissues, it is concluded that HGF/SF may well stimulate the growth and spread of malignant mesothelioma in vivo by paracrine and/or autocrine mechanisms.     

HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice

We reported that NK4, composed of the N-terminal hairpin and subsequent four kringle domains of hepatocyte growth factor (HGF), acts as the competitive antagonist for HGF. We now provide the first evidence that NK4 inhibits tumor growth and metastasis as an angiogenesis inhibitor as well as an HGF antagonist. Administration of NK4 suppressed primary tumor growth and lung metastasis of Lewis lung carcinoma and Jyg-MC(A) mammary carcinoma s.c. implanted into mice, although neither HGF nor NK4 affected proliferation and survival of these tumor cells in vitro. NK4 treatment resulted in a remarkable decrease in microvessel density and an increase of apoptotic tumor cells in primary tumors, which suggests that the inhibition of primary tumor growth by NK4 may be achieved by suppression of tumor angiogenesis. In vivo, NK4 inhibited angiogenesis in chick chorioallantoic membranes and in rabbit corneal neovascularization induced by basic fibroblast growth factor (bFGF). In vitro, NK4 inhibited growth and migration of human microvascular endothelial cells induced by bFGF and vascular endothelial growth factor (VEGF) as well as by HGF. HGF and VEGF activated the Met/HGF receptor and the KDR/VEGF receptor, respectively, whereas NK4 inhibited HGF-induced Met tyrosine phosphorylation but not VEGF-induced KDR phosphorylation. NK4 inhibited HGF-induced ERK1/2 (p44/42 mitogen-activated protein kinase) activation, but allowed for bFGF- and VEGF-induced ERK1/2 activation. These results indicate that NK4 is an angiogenesis inhibitor as well as an HGF antagonist, and that the antiangiogenic action of NK4 is independent of its activity as HGF antagonist. The bifunctional properties of NK4 to act as an angiogenesis inhibitor and as an HGF antagonist raises the possibility that NK4 may prove therapeutic for cancer patients.     

Oncogenic K-RAS is required to maintain changes in cytoskeletal organization, adhesion, and motility in colon cancer cells

RAS oncogenes are thought to play a role at multiple stages of tumorigenesis. The role and mechanisms by which RAS oncogenes maintain the transformed state of human cancer cells are poorly understood. Here, we have studied the role of oncogenic K-RAS in maintaining cytoskeletal disruption, cell adhesion and motility in metastatic colon carcinoma cells. Targeted deletion of K-RAS(G13D) from HCT116 colon carcinoma cells restored their ability to assemble stress fibers and focal adhesions/complexes, accompanied by increased cell-matrix adhesion and reduced motility. We further show that oncogenic K-Ras induces high Rho activity, but uncouples Rho from stress fiber formation. This uncoupling required the maintenance of high levels of the activator protein-1 family member, Fra-1, via a mitogen-activated protein/extracellular signal-regulated kinase-dependent pathway. We also show that PI3-kinase signaling is required for the motility of HCT116 cells downstream of oncogenic K-Ras. Our findings suggest that mutated K-RAS oncogenes are essential for maintenance of the transformed and invasive phenotype of human colon cancer cells.     

NDRG2调控HGF/c-MET通路抑制结肠癌细胞的增殖

目的:探讨抑癌基因NDRG2通过调节HGF/c-MET信号通路,抑制结肠癌HT29细胞增殖的机制。方法:建立NDRG2过表达细胞株HT29-NDRG2及对照组HT29-Cherry,并于第1、2、3、4、5天以MTT法检测细胞增殖;以不同浓度的HGF（0ng/ml、1ng/ml、2ng/ml、4ng/ml、8ng/ml、10ng/ml）刺激HT29亲本细胞,并于第1、2、3天以MTT法检测HT29细胞的增殖;采用qPCR法检测NDRG2过表达细胞株HT29-NDRG2与对照组HT29-Cherry两组细胞中HGF的表达水平;Western blot检测HT29-Cherry和HT29-NDRG2两组细胞以及添加HGF（10ng/ml）刺激HT29亲本细胞后各组细胞中HGF/c-MET通路中关键分子的表达。结果:过表达NDRG2以及HGF刺激均可以显著抑制HT29细胞的增殖能力;NDRG2的高表达可以上调HGF的转录水平;HGF可以刺激c-MET和p-ERK1/2,并上调p 21和p 27,抑制HT29细胞的增殖;过表达NDRG2明显上调c-MET、p-ERK1/2,并诱导p 21和p 27的高表达,从而抑制HT29细胞的增殖。结论:NDRG2可以通过调节HGF/c-MET信号通路从而抑制结肠癌细胞HT29细胞的增殖能力。     

Survivin对子宫内膜癌细胞运动和侵袭能力影响的研究

目的:探讨Survivin对子宫内膜癌细胞运动、侵袭能力的调节作用及其可能机制。方法:将含有Survivin特异性siRNA的重组质粒pRNAT-suv和阴性对照质粒pR-NAT-neg分别转染子宫内膜癌细胞株Ish-ikawa,G418筛选稳定并有效表达pRNAT-suv和pRNAT-neg的克隆。用蛋白质印迹法检测稳定表达克隆中Survivin的干扰效果,并用划痕实验、Matrigel体外侵袭实验和蛋白质印迹法评价稳定并有效表达pR-NAT-suv的细胞与阴性对照组细胞运动、侵袭能力以及E-钙黏蛋白表达的差异。结果:稳定表达pRNAT-suv的细胞其Survivin的表达水平低于未转染的细胞和稳定表达pR-NAT-neg的细胞。且稳定表达pRNAT-suv的细胞其运动和侵袭能力均低于未转染的细胞和稳定表达pRNAT-neg的细胞,3组细胞穿透Matrigel基质的细胞数分别为18.60±1.08、50.97±4.87和52.67±4.32,差异有统计学意义,F=63.83,P<0.01。RNA干扰抑制Survivin亦上调了E-钙黏蛋白的表达,差异有统计学意义,F=201.40,P<0.01。结论:RNA...     

Up-regulation of METCAM/MUC18 promotes motility,invasion, and tumorigenesis of human breast cancer cells

Background  

Conflicting research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as both a tumor promoter and a tumor suppressor in the development of breast cancer. To resolve this, we have re-investigated the role of this CAM in the progression of human breast cancer cells.     

Tumor suppression effect using NK4, a molecule acting as an antagonist of HGF,on human gastric carcinomas

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator of tumor-stromal interactions, facilitating tumor invasion and metastasis. We have investigated whether a blockade of HGF using recombinant NK4, an HGF antagonist, would lead to growth inhibition of the human gastric carcinoma cell line, TMK1. To evaluate the function of endogenous NK4 and investigate its potential inhibitory effect, TMK1 cells were transfected with NK4 plasmid. After selection, NK4-expressing cells (T11) were obtained, and cell growth was evaluated. Significant growth inhibition was observed in the T11-group compared to the control both in vitro and in vivo. Moreover, we investigated the effect of exogenous NK4 transferred by an adenovirus vector (AdCMV.NK4). Cell proliferation of AdCMV.NK4 infected TMK1 cells was significantly inhibited compared with the control group. We also assessed the in vivo tumor suppression effect of AdCMV.NK4. The tumor volume following treatment with AdCMV.NK4 was significantly inhibited compared to that of the control group. These findings indicate that NK4 gene expression has a potential role in controlling proliferation of cancer cells. In conclusion, NK4 is a promising therapeutic agent and its gene delivery may be a new approach to treating patients with advanced gastric cancer.     

Inhibition of hepatocyte growth factor-induced motility and in vitro invasion of human colon cancer cells by gamma-linolenic acid.

In this study we have determined the effects of the n-6 essential fatty acid gamma-linolenic acid (GLA) on the motility and invasive/metastatic nature of the human colon cancer cell lines HT115, HT29 and HRT18. Cell motility was induced by hepatocyte growth factor/scatter factor (HGF/SF) and measured by both colony scattering and dissociation from carrier beads. Invasiveness was measured in vitro by cellular invasion into extracellular matrix. At concentrations up to 100 microM (which had no effect on cell growth over the duration of the experiments) both cell motility and invasion induced by HGF/SF were markedly reduced by GLA and its lithium salt. The attachment of these cells to the extracellular matrix components (Matrigel and fibronectin) was also inhibited. There were also changes in the cell-surface E-cadherin, but not fibronectin receptor at similar concentrations. It is concluded that n-6 essential fatty acids have the ability to inhibit both motility and invasiveness of human colon cancer cells, perhaps by modifying cell-surface adhesion molecules.     

TMIGD1在结肠癌中的表达及对结肠癌细胞增殖和侵袭的影响

目的 检测TMIGD1在结肠癌及其相应的癌旁正常结肠上皮组织中的表达情况及过表达TMIGD1对结肠癌细胞生物学行为的影响,并探讨潜在分子机制.方法 通过生物信息学数据库和免疫组织化学染色检测TMIGD1在结肠癌与癌旁正常组织中的表达情况,并分析其与不同临床病理因素之间的关系.对结肠癌细胞系SW480通过慢病毒包装系统使...     

COX-2 overexpression increases motility and invasion of breast cancer cells

Cyclooxygenase-2 (COX-2), an inducible enzyme involved in prostaglandin (including PGE(2)) biosynthesis, is overexpressed in several epithelial malignancies including breast cancer. We tested the hypothesis that COX-2 overexpression in breast cancer cells results in increased cell motility and invasion. COX-2 overproducing cells were generated by stable transfection of several human breast cancer cells with pSG5-COX2 vector. We confirmed the overexpression of COX-2 protein by western blotting, and by measuring PGE(2) in the medium with an immunoassay. We measured cell motility by counting the number of cells crossing an 8-micron pore size PET membrane, and cell invasion by counting the number of cells invading through a Matrigel-coated membrane that simulates basement membrane. COX-2 transfected MDA-231 cells produced 30-43-fold more PGE2 as compared to parental cells. COX-2 overexpression increased cell migration approximately 2.2-fold and cell invasion through Matrigel approximately 5.1-fold. Addition of 50 microM NS-398, a COX-2 inhibitor, inhibited Matrigel invasion of MDA-231 cells by 54% as compared to solvent confirming the role of COX-2 in cell invasion. It is known that an increase in cell migration and invasion can be brought about by cytoskeletal alterations and basement membrane degradation due to increased expression of pro-urokinase plasminogen activator (pro-uPA). To investigate the mechanism of our observed increase in cell invasion by COX-2, we found by western blotting that the level of pro-uPA was significantly higher (approximately 5-fold) in COX-2 transfected MDA-231 cells than untransfected MDA-231 cells. Similar to our observations in cell culture, we found evidence that increased COX-2 activity correlates with uPA in a mouse model of breast cancer metastasis to bone. In this study, we conclude that COX-2 overexpression in human breast cancer cells enhances cell motility and invasiveness thus suggesting a mechanism of COX-2 mediated metastasis.     

TRIM21 is a novel regulator of Par-4 in colon and pancreatic cancer cells

The prostate apoptosis response protein 4 (Par-4) is a tumor-suppressor that has been shown to induce cancer-cell selective apoptosis in a variety of cancers. The regulation of Par-4 expression and activity is a relatively understudied area, and identifying novel regulators of Par-4 may serve as novel therapeutic targets. To identify novel regulators of Par-4, a co-immunoprecipitation was performed in colon cancer cells, and co-precipitated proteins were identified by mass-spectometry. TRIM21 was identified as a novel interacting partner of Par-4, and further shown to interact with Par-4 endogenously and through its PRY-SPRY domain. Additional studies show that TRIM21 downregulates Par-4 levels in response to cisplatin, and that TRIM21 can increase the resistance of colon cancer cells to cisplatin. Furthermore, forced Par-4 expression can sensitize pancreatic cancer cells to cisplatin. Finally, we demonstrate that TRIM21 expression predicts survival in pancreatic cancer patients. Our work highlights a novel mechanism of Par-4 regulation, and identifies a novel prognostic marker and potential therapeutic target for pancreatic cancer.