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.     

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.     

Suppression of metastasis of human pancreatic cancer to the liver by transportal injection of recombinant adenoviral NK4 in nude mice

NK4, a 4-kringle fragment of hepatocyte growth factor (HGF), is an HGF antagonist that also acts as an angiogenesis inhibitor. NK4 strongly inhibits the infiltration, metastasis, and tumor growth of pancreatic cancer. The aim of our study was to evaluate the antitumor effect of adenovirus-mediated NK4 gene transfer to the liver on hepatic metastasis of pancreatic cancer in vivo. We constructed recombinant adenoviral NK4 (Ad-NK4), which encodes a secreted form of human NK4. Intrasplenic injection of Ad-NK4 induced high and relatively maintained expression of NK4 protein in the liver and suppressed the number and growth of metastatic foci in the liver in a nude mouse model. Microscopically, central necrosis was found even in small metastatic foci in Ad-NK4 treated mice. Immunohistochemical analysis of metastatic tumors showed a remarkable decrease in microvessel density and an increase in the number of apoptotic tumor cells after treatment with Ad-NK4. These results indicate that intraportal injection of Ad-NK4 may be a useful therapeutic modality for the clinical control of hepatic metastasis in pancreatic cancer.     

HGF、c-Met及p-Erk蛋白在胃癌组织中的表达及意义

目的:本研究主要探讨肝细胞生长因子(hepatocyte growth factor,HGF)和肝细胞生长因子受体(mes-enchymal epithelial transition factor,c-Met)及下游信号通路分子磷酸化的Erk(p-Erk)表达与临床病理参数间的关系及其意义.研究HGF、c-Met、p...     

SMAD4-dependent polysome RNA recruitment in human pancreatic cancer cells

Pancreatic cancer is the fourth leading cause of cancer death in the United States because most patients are diagnosed too late in the course of the disease to be treated effectively. Thus, there is a pressing need to more clearly understand how gene expression is regulated in cancer cells and to identify new biomarkers and therapeutic targets. Translational regulation is thought to occur primarily through non‐SMAD directed signaling pathways. We tested the hypothesis that SMAD4‐dependent signaling does play a role in the regulation of mRNA entry into polysomes and that novel candidate genes in pancreatic cancer could be identified using polysome RNA from the human pancreatic cancer cell line BxPC3 with or without a functional SMAD4 gene. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for most of the remaining polysome RNA, levels are regulated via a “tagging” of the RNAs in the nucleus for rapid entry into the polysomes; (iv) a SMAD4‐dependent pathway appears to indeed play a role in regulating mRNA entry into polysomes; and (v) a gene list derived from differentially expressed polysome RNA in BxPC3 cells generated new candidate genes and cell pathways potentially related to pancreatic cancer. © 2011 Wiley Periodicals, Inc.     

shRNA-mediated Slc38a1 silencing inhibits migration, but not invasiveness of human pancreatic cancer cells

Objective：Early metastasis is a major biological feature of pancreatic cancer.The current study examined whether silencing Slc38a1,a gene involved in energy metabolism,using short hairpin RNA （shRNA） could inhibit the growth,migration,and invasiveness of pancreatic cancer cells.Methods：A series of Slc38a1 shRNAs were designed and cloned into the pGPU6/GFP/Neo vectors.An shRNA with the most efficacious inhibitory action on SCL38A1 expression （65％ inhibition） upon screening in DH5α bacteria was used to transfect SW1990 human pancreatic cancer cells.Cell growth,migration,and invasiveness were examined using cell counting kit-8,Boyden chamber without and with Matrigel,respectively.Results：Transfection of SW1990 cells with the SLCs38A1 shRNA significantly decreased the proliferation （P＜0.0001） and migratory potential （by 46.7％,P=0.0399） of the cancer cells.Invasiveness,however,was not affected.Conclusions：Inhibiting Slc38a1 using shRNA technology could decrease the growth and migration of representative pancreatic cancer cells.However,the fact that invasiveness was not affected suggested that SLC38A1 is unlikely to be responsible for early metastasis.     

白桦脂酸对人NK细胞杀伤SW1990胰腺癌细胞影响及机制探讨

目的观察中药白桦脂酸对人NK细胞杀伤SW1990胰腺癌细胞的影响,并初步探讨可能机制。方法分离健康人外周血单个核细胞,细胞因子诱导培养NK细胞达实验要求;不同浓度(0.025、0.05、0.1、0.2、0.4、0.8、1.6、3.2、6.25、12.5、25、50和100μg/mL)的白桦脂酸分别诱导NK细胞、SW1990胰腺癌细胞48h后,CCK8法检测白桦脂酸对NK细胞增殖的影响;四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)比色法检测白桦脂酸对SW1990胰腺癌细胞株增殖的影响;流式细胞术(flow cymetory,FCM)检测白桦脂酸作用48h后NK细胞颗粒酶B、穿孔素、IFN-γ和CD107a的表达;乳酸脱氢酶(lactate dehydrogenase,LDH)释放法检测白桦脂酸对NK细胞杀伤SW1990胰腺癌细胞的影响;蛋白质印迹法检测白桦脂酸对NK细胞表达磷酸化的ERK1/2(phosphorylation-ERK1/2,p-ERK1/2)的影响。结果培养10d的NK细胞比例达74.43%。与对照组比较,白桦脂酸在其浓度为0.4、0.8、1.6、3.2、6.25、12.5和25μg/mL时显著促进NK细胞增殖,F=129.476,P<0.01;并抑制胰腺癌细胞增殖,F=278.222,P<0.01。浓度为0.05、0.2、0.8和3.2μg/mL的白桦脂酸作用NK细胞48h后,与对照组比较均能促进NK细胞对SW1990胰腺癌细胞的杀伤,F=36.451,P<0.05。与对照组比较,白桦脂酸在浓度为0.2和0.8μg/mL时不同程度促进NK细胞颗粒酶B、穿孔素、IFN-γ和CD107a的表达,P<0.05。与对照组比较,浓度为0.8、3.2和12.5μg/mL的白桦脂酸可促进NK细胞表达p-ERK1/2,F=83.708,P<0.05。结论白桦脂酸在一定的浓度范围内能够增强NK细胞对SW1990胰腺癌细胞的杀伤作用,其作用机制可能通过促进NK细胞增殖,抑制胰腺癌细胞增殖,激活NK细胞内ERK信号通路,提高NK细胞颗粒酶B、穿孔素、IFN-γ和CD107a的表达。     

miR-130a-3p通过HGF/MET信号通路抑制乳腺癌细胞MCF-7 侵袭

目的：探究miR-130a-3p 通过HGF/MET信号通路调控上皮间质转化（epithelial-mesenchymal transition,EMT）影响乳腺癌细胞侵袭转移的分子机制。方法：收集承德医学院附属医院2018 年1 月至10 月收治的22 例乳腺癌患者癌组织和配对癌旁组织标本,乳腺癌细胞系（MCF-7、MDA-MB-231 和MDA-MB-453）和正常乳腺上皮细胞MCF10A来自承德医学院基础研究所,然后采用qPCR检测组织和细胞系中miR-130a-3p 的表达情况;将实验分为对照组、miR-130a-3p mimics 组、miR-130a-3p inhibitor组、PHA665752（MET小分子抑制剂）转染组及共转PHA665752+miR-130a-3p inhibitor 组,然后采用CCK-8 法和Transwell 实验分别检测MCF-7 细胞增殖活力、侵袭和迁移能力;WB实验检测MCF-7 细胞EMT和HGF/MET信号通路相关蛋白的表达情况;此外,采用双荧光素酶报告基因检测miR-130a-3p 与MET之间的靶向关系。结果：miR-130a-3p 在乳腺癌组织和细胞系中呈低表达;过表达miR-130a-3p 可抑制MCF-7 细胞增殖、侵袭、迁移和EMT;而抑制miR-130a-3p 出现相反的结果。双荧光素酶报告基因结果证实miR-130a-3p 靶向下调MET的表达水平,且miR-130a-3p 负调控HGF/MET信号通路的表达;进一步实验证明,miR-130a-3p 通过阻断HGF/MET信号通路抑制MCF-7 细胞增殖、侵袭、迁移和EMT。结论：miR-130a-3p 通过阻断HGF/MET信号通路抑制MCF-7 细胞EMT过程,进而抑制MCF-7 细胞侵袭转移。     

Human MUC4 mucin induces ultra-structural changes and tumorigenicity in pancreatic cancer cells

MUC4 is a type-1 transmembrane glycoprotein and is overexpressed in many carcinomas. It is a heterodimeric protein of 930 kDa, composed of a mucin-type subunit, MUC4alpha, and a membrane-bound growth factor-like subunit, MUC4beta. MUC4 mRNA contains unique 5' and 3' coding sequences along with a large variable number of tandem repeat (VNTR) domain of 7-19 kb. A direct association of MUC4 overexpression has been established with the degree of invasiveness and poor prognosis of pancreatic cancer. To understand the precise role of MUC4 in pancreatic cancer, we engineered a MUC4 complementary DNA construct, mini-MUC4, whose deduced protein (320 kDa) is comparable with that of wild-type MUC4 (930 kDa) but represents only 10% of VNTR. Stable ectopic expression of mini-MUC4 in two human pancreatic cancer cell lines, Panc1 and MiaPaCa, showed that MUC4 minigene expression follows a biosynthesis and localisation pattern similar to the wild-type MUC4. Expression of MUC4 resulted in increased growth, motility, and invasiveness of the pancreatic cancer cells in vitro. Ultra-structural examination of MUC4-transfected cells showed the presence of increased number and size of mitochondria. The MUC4-expressing cells also demonstrated an enhanced tumorigenicity in an orthotopic xenograft nude mice model, further supporting a direct role of MUC4 in inducing the cancer properties. In conclusion, our results suggest that MUC4 promotes tumorigenicity and is directly involved in growth and survival of the cancer cells.     

NK4基因转染对裸鼠胰腺癌移植瘤生长的影响

背景与目的:NK4不仅是肝细胞生长因子的拮抗剂,而且是血管形成的抑制剂。研究已经证实NK4可以抑制肿瘤的生长和转移,但有关其在胰腺癌中的作用,目前少见文献报道。为此,本研究旨在探讨NK4基因在裸鼠体内的抗胰腺癌作用及其可能的机制。方法:建立裸鼠胰腺癌皮下移植瘤模型,构建NK4基因真核细胞表达载体并转染入瘤体内,转染前后称其体重、瘤重和测肿瘤体积,采用免疫组织化学和脱氧核糖核酸末端转移酶介导的缺口末端标记技术对裸鼠胰腺癌组织中的凋亡细胞、增殖抗原和微血管密度进行观察和比较。结果:4周后,NK4转染组裸鼠移植瘤体积为(1.39±0.33)cm3,明显小于对照组和空载体组[(2.06±0.55)cm3和(1.90±0.36)cm3,P<0.01];其瘤重为(1.30±0.81)g,也显著低于对照组和空载体组[(3.45±1.88)g和(3.14±1.51)g,P<0.01],抑瘤率为62.29%。NK4转染组肿瘤细胞的凋亡指数为9.34±0.91,显著高于对照组和空载体组(4.13±0.79和3.94±1.03,P<0.001);而NK4转染组肿瘤细胞的增殖指数为53.88±4.30,与对照组间和空载体组比较无显著性差异(56.24±4.03和54.33±5.41,P>0.05);NK4转染组肿瘤组织的微血管密度为(12.24±4.63),明显低于对照组和空载体组(20.13±7.00和19.70±6.15,P<0.05)。结论:转染NK4基因可显著抑制裸鼠胰腺癌移植瘤的生长,其作用机制可能是通过抑制肿瘤新生血管的形成,促进肿瘤细胞凋亡。     

转染NK4基因对人胰腺癌SW1990细胞生物学特性的影响

背景与目的:肝细胞生长因子(hepatocytegrowthfactor,HGF)通过其受体c-Met的激活,在调节肿瘤侵袭和转移和血管形成中具有重要作用。NK4不仅是HGF的拮抗剂,也是血管形成的抑制剂,阻断HGF/c-Met途径和肿瘤血管的形成可成为抗肿瘤治疗的策略之一。为此,我们构建NK4基因真核细胞表达载体并进行转染研究,探讨NK4基因在胰腺癌细胞中的表达及其对胰腺癌细胞生物学特性的影响。方法:对重组pcDNA3/hNK4质粒进行酶切,将NK4基因克隆到真核表达载体pRC/CMV2,应用脂质体将重组pRC/CMV2-hNK4质粒转入胰腺癌SW1990细胞中,采用逆转录聚合酶链反应(RT-PCR)及免疫印迹(Westernblot)分别检测转染的肿瘤细胞中NK4mRNA和蛋白的表达并筛选出高表达的细胞克隆。采用Transwall小室和Matrigel侵袭小室测定转染NK4基因对胰腺癌细胞运动和侵袭的影响。结果:转导NK4基因的SW1990细胞可表达并分泌NK4,RT-PCR扩增出预期的453bp片段,Westernblot显示有50000的NK4蛋白,转染NK4基因对胰腺癌细胞SW1990的生长无抑制作用(P>0.05),但可显著抑制HGF或成纤维细胞所诱导胰腺癌细胞的运动和侵袭(P<0.01),而转染空载体则无此作用(P>0.05)。结论:转染NK4基因可抑制胰腺癌细胞的运动和侵袭,在胰腺癌抗转移治疗中可能具有重要作用。     

microRNA-122抑制胰腺癌细胞增殖、迁移和侵袭

目的 明确microRNA-122(miR-122)在胰腺癌中的表达及miR-122对胰腺癌细胞增殖、迁移和侵袭的作用.方法 采用RT-qPCR方法检测20例胰腺癌组织、胰腺癌细胞株(ASPC1、PANC1、MP和SW1990)和人源正常胰腺导管上皮细胞株(HPDE)中miR-122的表达量.将miR-122模拟类似物...     

肝细胞生长因子在肿瘤侵袭转移中的作用

肝细胞生长因子(HGF)是一种多功能的细胞因子,通过与其特异性受体c-Met蛋白结合引发细胞内的信号传导途径,参与肿瘤的侵袭转移过程.对HGF及其受体在肿瘤侵袭转移过程中作用机制的研究为抗肿瘤转移治疗提供了一个极有希望的新靶点.     

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.     

Hepatocyte growth factor inhibition: a novel therapeutic approach in pancreatic cancer

Background:

Pancreatic stellate cells (PSCs, which produce the stroma of pancreatic cancer (PC)) interact with cancer cells to facilitate PC growth. A candidate growth factor pathway that may mediate this interaction is the HGF–c-MET pathway.

Methods:

Effects of HGF inhibition (using a neutralising antibody AMG102) alone or in combination with gemcitabine were assessed (i) in vivo using an orthotopic model of PC, and (ii) in vitro using cultured PC cells (AsPC-1) and human PSCs.

Results:

We have shown that human PSCs (hPSCs) secrete HGF but do not express the receptor c-MET, which is present predominantly on cancer cells. HGF inhibition was as effective as standard chemotherapy in inhibiting local tumour growth but was significantly more effective than gemcitabine in reducing tumour angiogenesis and metastasis. HGF inhibition has resulted in reduced metastasis; however, interestingly this antimetastatic effect was lost when combined with gemcitabine. This suggests that gemcitabine treatment selects out a subpopulation of cancer cells with increased epithelial–mesenchymal transition (EMT) and stem-cell characteristics, as supported by our findings of increased expression of EMT and stem-cell markers in tumour sections from our animal model. In vitro studies showed that hPSC secretions induced proliferation and migration, but inhibited apoptosis, of cancer cells. These effects were countered by pretreatment of hPSC secretions with a HGF-neutralising antibody but not by gemcitabine, indicating a key role for HGF in PSC–PC interactions.

Conclusions:

Our studies suggest that targeted therapy to inhibit stromal–tumour interactions mediated by the HGF–c-MET pathway may represent a novel therapeutic approach in PC that will require careful modelling for optimal integration with existing treatment modalities.     

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.     

Nerve growth factor and enhancement of proliferation,invasion, and tumorigenicity of pancreatic cancer cells

Nerve growth factor (NGF) exerts both stimulatory and inhibitory effects on neuronal and certain non-neuronal tumors. In pancreatic cancer NGF is overexpressed, and this overexpression is associated with increased perineural invasion. NGF has the potential to stimulate the growth of some pancreatic cancer cell lines, and this effect is mediated by the phosphorylation of tyrosine kinase receptor A and mitogen-activated protein kinase activation; it is dependent on the expression levels of tyrosine kinase receptor A and p75 receptors. To determine whether cancer cell-derived NGF can participate in the regulation of pancreatic cancer cell proliferation, PANC-1 human pancreatic cancer cells were stably transfected with a full-length human beta-NGF expression vector. In vitro and in vivo growth characteristics were analyzed by proliferation assays and invasion assays and in a nude mouse tumor model. Stable transfection of NGF in PANC-1 cells resulted in enhanced anchorage-dependent growth, with a decrease in doubling times of up to 50%, and in an approximately twofold increase in anchorage-independent cell growth and cell invasion. Furthermore, stably transfected PANC-1 cells showed enhanced tumorigenicity in nude mice. These results suggest that NGF has the capacity to act in a paracrine and/or an autocrine manner in pancreatic cancer and that it enhances cancer cell growth and invasion in vivo, thereby contributing to the aggressiveness and poor prognosis of this disease.