肝细胞生长因子拮抗剂NK4抑制肿瘤的研究进展

肝细胞生长因子(HGF)是一种多功能细胞因子,其生物学活性由c-Met蛋白所介导,通过多种途径调节细胞的生长,发挥重要的生理效应.Met活化失调在许多肿瘤的发生及侵袭中都有重要的作用.目前,对HGF/c-Met系统的研究已取得了很大进展.c-Met抑制剂成为抗肿瘤药物的研究热点,其中NK4能显著抑制肿瘤的生长、增殖和转移,本文对NK4在抗肿瘤方面的作用做一综述.     

基于分子对接的小分子 c-Met 抑制剂 结构特征及其研究进展

受体酪氨酸激酶 c-Met 是抗肿瘤治疗的一个重要靶点,c-Met/HGF 通路在肿瘤的发生、发展、转移及血管再生中发挥重要作用。 综述 c-Met 及与配体 HGF 的复合物结构特征、c-Met/HGF 通路的生物学作用以及靶向 c-Met 抗肿瘤小分子抑制剂的研究进展。     

VEGF和Notch通路在肝癌血管形成中的作用

血管内皮生长因子(vascular endothelial growth factor,VEGF)和Notch信号通路在血管新生及肿瘤血管生成过程发挥极重要的作用。VEGF被认为是血管生成的刺激因子,可启动血管生成,而Notch信号通路则在肿瘤血管生成过程发挥负反馈作用,防止血管过度生成,两者的协调作用保证形成的血管具有一定功能,确保肿瘤生长的氧供。同时阻断DLL4/Notch和VEGF具有协同作用,既能降低肿瘤血管的密度及功能,又抑制肿瘤生长。如果能证实VEGF/Notch信号网络在肝癌新生血管中的作用,可以为肝癌的治疗提供新的思路。     

抗VEGF/VEGFR靶向肿瘤血管药物的研究进展

研究表明,肿瘤的生长转移和新血管的生成有密切关系,其中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)及其信号途径在肿瘤血管生成中起关键作用。阻断该途径的任何环节均可有效抑制肿瘤血管的生成,进而抑制肿瘤的生长和转移。近年来,已有多种以VEGF/VEGFR为靶点的抗肿瘤血管生成药物投入临床应用,其中bevacizumab为第一个获批上市的抗肿瘤血管生成药物。继bevacizumab后,一种以基因工程手段获得的人Fc融合蛋白Zaltrap也成功在美国上市,这种杂交分子的药代动力学明显优于单克隆抗体,能更好的遏制肿瘤血管的发生并消退已形成的肿瘤血管。在肿瘤的临床治疗中,Zaltrap比bevacizumab显示出更大的优势。此外,VEGFC/D Trap及小分子酪氨酸激酶抑制剂也能有效抑制肿瘤血管的生成。在此对以VEGF/VEGFR为靶点的抗肿瘤血管生成药物进行综述。     

基于血管生成素的药物研发

血管生成素（angiogenin,ANG）在伤口愈合、月经周期、妊娠、胚胎发育、先天性免疫、细胞应激保护和维持机体稳态等生理病理过程,特别是肿瘤的生存与进展、神经细胞的存活和生长中扮演着重要角色,是药物研发的重要靶点.本文综述了ANG在功能上的特殊性及其药物研发潜力.在肿瘤中,ANG扮演促进肿瘤细胞增生和促进血管生成的双重角色,且是其它血管生成因子如血管内皮生长因子（vascular endothelial growth factor, VEGF）、酸性成纤维生长因子（acidic fibroblast growth factor, aFGF）、碱性成纤维生长因子（basic fibroblast growth factor, bFGF）和表皮生长因子（epidermal growth factor, EGF）发挥作用的必需准许因子. ANG的抗肿瘤治疗较之目前常用的针对单一血管生成因子的抑制剂更有效,具有良好的药物研发和临床运用前景.由于ANG通过核转位促进rRNA转录是发挥促进肿瘤细胞增生和血管生成活性所必须的,因此,它的核转位抑制剂如新霉胺,将有望首先获得抗肿瘤临床应用.另外,业已证明重组ANG能促进体内外运动神经元的存活,且可明显改善肌萎缩侧索硬化症（amyotrophic lateral sclerosi, ALS）模型小鼠的行为,其在神经退行性疾病治疗方面也将有良好的研发前景.     

小分子c-Met激酶抑制剂的研究进展

受体酪氨酸激酶c-Met即肝细胞生长因子HGF受体。HGF/c-Met信号通路在肿瘤形成、生长和转移过程中被频繁激活,因此, c-Met 已成为抗癌药物研究中一个重要靶标。重点介绍近年来基于c-Met通路的抗癌药物研究进展。     

以VEGF及VEGFR2为靶位的抗肿瘤血管生成主动免疫治疗的研究进展

肿瘤细胞通过刺激新生血管生成来满足对营养及供氧的不断增长的需求,因此,肿瘤组织生长对于新生血管形成的依赖性使得抗血肿瘤管生成已经成为肿瘤学基础研究与临床治疗领域中最吸引人的策略之一.在众多的促血管生成因子中,血管内皮生长因子(VEGF)及其受体VEGFR2(鼠和人中也分别称为Flk-1和KDR)对于与肿瘤生长、转移及复发相关的血管生成是至关重要的.此外,通过打破肿瘤组织自身介导的免疫耐受与逃避,主动免疫治疗已成为一种崭新的抗肿瘤治疗方法.通过将这两种策略联合应用,抗血管生成主动免疫治疗使得更加有效地抑制肿瘤血管生成成为可能.这种免疫治疗与抗血管生成的联合应用有望成为一种有良好前景的研究方案.本文总结了通过打破VEGF/VEGFR2信号通路实现的抗肿瘤血管生成主动免疫治疗方面最新研究进展.本文讨论了旨在抑制血管生成的三种不同形式的抗肿瘤疫苗-细胞疫苗、蛋白质/多肽疫苗及基因/DNA疫苗,以及这一领域未来的研究方向.     

血管内皮生长因子受体信号转导通路与肿瘤血管生成

血管内皮生长因子是促进血管生成的重要调节因子.它能促进内皮细胞增殖、迁移,阻止内皮细胞凋亡、管腔网状结构退化,增加血管渗透性.所有这些作用都是通过血管内皮生长因子受体信号转导通路实现的.它们在肿瘤血管生成、肿瘤生长中起着重要的作用.以血管内皮生长因子受体信号转导通路为靶点是开发肿瘤血管生成抑制剂的理想策略.     

血管内皮生长因子受体的信号转导通路

血管内皮生长因子受体(VEGFR)是VEGF的特异性受体,由于在刺激血管内皮细胞增殖、迁移、管腔形成,促进肿瘤生长和转移过程中起着重要的作用,而成为抗肿瘤新生血管生成的热点。该主要围绕VEGF及其不同受体的信号转导通路作一综述。     

肿瘤血管生成抑制剂的作用机制研究进展

肿瘤血管生成抑制剂批能破坏或抑制血管生成,有效地阻止肿瘤生长和转移的药物,可分为特异性和非特异性两大类。其作用机制主要有：（１）调控血管形成生长因子;（２）抑制基底膜降解;（３）影响信号转导通路;（４）调控细胞生长周期;（５）调控肿瘤机关基因。本文对其作用机制的进展作一综述。     

Mechanisms and significance of bifunctional NK4 in cancer treatment

Based on the background that hepatocyte growth factor (HGF) and c-Met/HGF receptor tyrosine kinase play a definite role in tumor invasion and metastasis, NK4, four-kringles containing intramolecular fragment of HGF, was isolated as a competitive antagonist for the HGF-c-Met system. Independent of its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF, indicating that NK4 is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. Interestingly, kringle domains in distinct types of proteins, e.g., plasminogen, prothrombin, plasminogen activators, apolipoprotein(a), and HGF, share angioinhibitory actions. In experimental models of distinct types of cancers, NK4 protein administration or NK4 gene therapy inhibited tumor invasion, metastasis, and angiogenesis-dependent tumor growth. Cancer treatment with NK4 may prove to suppress malignant tumors to be 'static' in both tumor growth and spreading, as based on biological characteristics of malignant tumors.     

NK4 therapy: a new approach to target angiogenesis and inflammation in rheumatoid arthritis

Rheumatoid arthritis (RA) is a progressive autoimmune disease characterized by synovial membrane hyperplasia, inflammation, and angiogenesis. Hepatocyte growth factor (HGF) and its receptor, c-Met, are both overexpressed in the RA synovium. NK4 is an antagonist of HGF which has been shown to inhibit tumor growth, metastasis, and angiogenesis. In an experimental model of RA, NK4 gene therapy inhibited joint damage and inflammation in both preventative and therapeutic models. NK4 treatment therefore represents a possible therapeutic option in combating RA.Rheumatoid arthritis (RA) is a progressive, inflammatory autoimmune disease characterized by an erosive synovitis. In addition to being an inflammatory condition, RA is also considered to be a member of the angiogenic family of diseases. Angiogenesis is growth of new blood vessels from pre-existing blood vessels. As the disease progresses, the hyperplastic synovial pannus creates a hypoxic, inflammatory environment that induces angiogenesis. Further vascularization of the synovial tissue promotes pannus growth and continued infiltration of inflammatory leukocytes, thus perpetuating the disease.In the previous issue of Arthritis Research & Therapy, Tsunemi and colleagues [1] reported on the targeting of hepatocyte growth factor (HGF) by NK4 in the treatment of RA. HGF is a pleiotropic growth factor that is expressed by mesenchymal cells and promotes processes such as mitogenesis, differentiation, and angiogenesis [2]. It mediates these functions via binding to its unique receptor c-Met, a receptor tyrosine kinase. c-Met is expressed by a variety of cell types, including endothelial cells (ECs) [3].We have previously shown that HGF is elevated in the synovial fluid of patients with RA [4]. More recently, Grandaunet and colleagues [5] found that plasma levels of HGF predict the severity of joint damage in patients with RA. In the joint, we found that HGF and c-Met are elevated in the RA synovial lining compared with normal controls [4]. The report by Tsunemi and colleagues [1] supports these findings and further shows that c-Met is expressed on fibroblasts, mononuclear cells, and ECs in the RA synovium.HGF is a heterodimeric protein composed of an ?-chain, which contains four kringle domains, and a ?-chain [6]. The ?-chain binds c-Met with high affinity, whereas the ?-chain is responsible for activation of c-Met. In an attempt to inhibit HGF, Date and colleagues [7] generated a cleavage product of HGF termed NK4, which contains the four kringle domains of the HGF ?-chain. Therefore, NK4 serves as an antagonist of HGF and can bind c-Met with high affinity without activating it.As described above, one of the primary functions of HGF is to induce angiogenesis by binding to c-Met on the surface of ECs. Therefore, it was postulated that NK4 would act as a competitive inhibitor of HGF, thus inhibiting angiogenesis. Indeed, NK4 has been shown to inhibit angiogenesis in vitro and in various in vivo cancer models [6,8,9]. However, in addition to having antagonistic action against HGF, NK4 inhibits angiogenesis induced by vascular endothelial growth factor and basic fibroblast growth factor in a c-Met-independent fashion [9]. In addition to c-Met, NK4 binds to perlecan, a sulfate proteoglycan that interacts with the vascular endothelial basement membrane. Sakai and colleagues [9] found, specifically, that NK4 binds perlecan and prevents proper fibronectin assembly in the basement membrane, which inhibits several facets of angiogenesis.These features of NK4 make it an attractive potential adjunctive therapy in angiogenic diseases. Over the past decade, numerous studies have been performed to assess the efficacy of either a recombinant NK4 protein or NK4 gene expression vector in many experimental cancer models [3,6]. Collectively, these studies have indicated that NK4 treatment has the potential to inhibit tumor growth, angiogenesis, and metastasis [3,6]. Much of the preclinical success of NK4 can be attributed to its ability to inhibit multiple pathways involved in growth and angiogenesis.RA is driven by inflammation and angiogenesis, and thus much work has been aimed at identifying and testing potential angiogenesis inhibitors in models of experimental arthritis [10]. Tsunemi and colleagues [1] have now adopted their approach of studying the antiangiogenic properties of NK4 in cancer to experimental arthritis. Using an adenovirus vector containing the NK4 gene, they found that NK4 inhibited the development of ?-glucan-induced arthritis [1]. NK4 was able to inhibit inflammation, joint swelling, and bone erosion. However, the authors did not show direct evidence of NK4 inhibiting synovial blood vessel density. Importantly, they also showed that NK4 gene therapy was effective when given therapeutically, after the onset of the experimental arthritis [1].These results are highly encouraging in the application of NK4 as a potential adjunctive RA therapy. This report, coupled with the high expression levels of HGF and c-Met in the RA synovium, makes NK4 treatment an intriguing possibility. In the future, it will be of great interest to determine whether these effects of NK4 are observable in other animal models of RA, as not all facets of RA are represented in a singular model of the disease. Moreover, many of the effects of NK4 observed by Tsunemi and colleagues [1] are attributed to a reduction in inflammation and inflammatory cytokines. Therefore, elucidating the anti-inflammatory and antiangiogenic mechanisms of NK4 will be paramount to transitioning from an interesting candidate to a possible RA therapy.     

Molecular imaging of c-Met tyrosine kinase activity

The receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), modulate signaling cascades implicated in cellular proliferation, survival, migration, invasion, and angiogenesis. Therefore, dysregulation of HGF/c-Met signaling can compromise the cellular capacity to moderate these activities and can lead to tumorigenesis, metastasis, and therapeutic resistance in various human malignancies. To facilitate studies investigating HGF/c-Met receptor coupling or c-Met signaling events in real time and in living cells and animals, here we describe a genetically engineered reporter where bioluminescence can be used as a surrogate for c-Met tyrosine kinase activity. c-Met kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to the activation or inhibition of the HGF/c-Met signaling pathway. Treatment of tumor-bearing animals with a c-Met inhibitor and the HGF neutralizing antibody stimulated the reporter’s bioluminescence activity in a dose-dependent manner and led to a regression of U-87 MG tumor xenografts. Results obtained from these studies provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate c-Met activity and validate c-Met as a target for human glioblastoma therapy.     

Hepatocyte growth factor and Met in tumor biology and therapeutic approach with NK4

Hepatocyte growth factor (HGF) and Met/HGF receptor tyrosine kinase play a role in the progression to invasive and metastatic cancers. A variety of cancer cells secrete molecules that enhance HGF expression in stromal fibroblasts, while fibroblast-derived HGF, in turn, is a potent stimulator of the invasion of cancer cells. In addition to the ligand-dependent activation, Met receptor activation is negatively regulated by cell-cell contact and Ser985 phosphorylation in the juxtamembrane of Met. The loss of intercellular junctions may facilitate an escape from the cell-cell contact-dependent suppression of Met-signaling. Significance of juxtamembrane mutations found in human cancers is assumed to be a loss-of-function in the negative regulation of Met. In attempts to block the malignant behavior of cancers, NK4 was isolated as a competitive antagonist against HGF-Met signaling. Independently on its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF. In experimental models of distinct types of cancers, NK4 inhibited Met activation and this was associated with inhibition of tumor invasion and metastasis. NK4 inhibited tumor angiogenesis, thereby suppressing angiogenesis-dependent tumor growth. Cancer treatment with NK4 suppresses malignant tumors to be "static" in both tumor growth and spreading.     

Angioinhibitory Action of NK4 Involves Impaired Extracellular Assembly of Fibronectin Mediated by Perlecan-NK4 Association

NK4, a fragment of hepatocyte growth factor (HGF), exerts bifunctional action as a competitive antagonist against HGF and its receptor c-Met and an angiogenesis inhibitor. Here we studied the anti-angiogenic mechanism of NK4. In cultured human endothelial cells, NK4 inhibited DNA synthesis induced not only by HGF but also by either basic fibroblast growth factor or vascular endothelial growth factor. Even if c-Met expression was diminished by small interference RNA, NK4 inhibited basic fibroblast growth factor-induced DNA synthesis, indicating that anti-angiogenic action of NK4 is c-Met-independent. Affinity purification with NK4-immobilized beads revealed that NK4 binds to perlecan. Consistent with this, NK4 colocalized with perlecan in endothelial cells. Perlecan is a multidomain heparan sulfate proteoglycan that interacts with basement membrane components such as fibronectin. NK4 inhibited extracellular assembly of fibronectin, by which fibronectin-dependent endothelial cell spreading was inhibited by NK4. Knockdown of perlecan expression by small interference RNA significantly abrogated the inhibitory effect of NK4 on fibronectin assembly and cell spreading. In NK4-treated endothelial cells, tyrosine phosphorylation of focal adhesion kinase and Rac activation were reduced, whereas overexpression of activated Rac recovered the DNA synthesis in NK4-treated endothelial cells. These results indicate that the association between NK4 and perlecan impairs fibronectin assembly, thereby inhibiting anchorage-dependent signaling. The identified mechanism for angiostatic action provides further proof of significance for NK4 in the treatment of cancer and potentially for vascular regulation as well.The manipulation of angiogenesis has potential therapeutic value for the treatment of a variety of diseases including cancer, arthritis, and cardiovascular disease (1, 2). In addition to endothelial cell migration and proliferation, angiogenesis is a process involving dynamic matrix transition (3). During angiogenesis, the vascular basement membrane undergoes proteolytic degradation and transit to the provisional matrix consisting of fibronectin, etc., followed by an intermediate and mature new vascular basement membrane. Growing evidence has shown that such an extracellular matrix (ECM)² not only provides mechanical support to the cells but also essentially regulates cell growth, migration, and survival. The fact that a number of endogenous inhibitors of angiogenesis have been identified from proteolytic fragments of ECM molecules also highlights the important regulatory roles of ECM in angiogenesis (3).NK4 is a proteolytic fragment of hepatocyte growth factor, HGF (4), consisting of an N-terminal hairpin domain and four kringle domains of the α-chain of HGF (5). By competitively binding to HGF receptor c-Met, NK4 acts as an HGF antagonist (5, 6). The NK4 fragment seems to be physiologically generated by mast cells and neutrophils peptidases during inflammation (7). Because HGF regulates malignant behavior in a variety of tumors by inducing invasive, angiogenic, and metastatic responses (8, 9), the blockade of HGF-c-Met signaling by NK4 is a strategy to inhibit tumor invasion and metastasis (6, 9–11). During investigation of a therapeutic approach with NK4 in experimental cancer models, we unexpectedly found that NK4 functions as an angiogenesis inhibitor (12). Based on the bifunctional characteristic as HGF antagonist and angiogenesis inhibitor, NK4 suppressed malignant behavior of cancers, including invasion, metastasis, and angiogenesis-dependent tumor growth (9–12).The angiostatic activity of NK4 is probably independent of its original activity as an HGF antagonist because an anti-HGF antibody capable of preventing HGF-c-Met association did not inhibit human endothelial cell growth stimulated by either bFGF or VEGF (12). However, the mechanism by which NK4 inhibits angiogenic responses in endothelial cells remains to be addressed. In the present study we newly identified perlecan to be an NK4 binding molecule and found that in vascular endothelial cells the association of NK4 with perlecan inhibited extracellular fibronectin assembly, fibronectin-dependent cell spreading, and the subsequent anchorage-dependent signals. Together with our finding that c-Met/HGF receptor is not required for the inhibition of DNA synthesis by NK4, we propose that the association of NK4 with perlecan plays a key role in angiogenesis inhibition by NK4.     

Improvement in oral bioavailability of 2,4-diaminopyrimidine c-Met inhibitors by incorporation of a 3-amidobenzazepin-2-one group

The hepatocyte growth factor (HGF)-c-Met signaling axis is involved in the mediation of many biological activities, including angiogenesis, proliferation, cell survival, cell motility, and morphogenesis. Dysregulation of c-Met signaling (e.g., overexpression or increased activation) is associated with the proliferation and metastasis of a wide range of tumor types, including breast, liver, lung, colorectal, gastric, bladder, and prostate, among others. Inhibiting the HGF-c-Met pathway is predicted to lead to anti-tumor effects in many cancers. Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which pharmaceutical properties were modulated by substituents appended on the C2-benzazepinone ring. In particular, certain-3-amidobenzazepin-2-one analogs had improved oral bioavailability and were evaluated in PK/PD and efficacy models. Lead compounds demonstrated tumor stasis with partial regressions when evaluated in a GTL-16 tumor xenograft mouse model.     

Differential mitogenic effects of single chain hepatocyte growth factor (HGF)/scatter factor and HGF/NK1 following cleavage by factor Xa

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF.     

Inhibition of colon cancer growth and metastasis by NK4 gene repetitive delivery in mice

NK4, originally prepared as a competitive antagonist for hepatocyte growth factor (HGF), is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. When the expression plasmid for NK4 gene was administered into mice by hydrodynamics-based delivery, the repetitive increase in the plasma NK4 protein level was achieved by repetitive administration of NK4 gene. Mice were subcutaneously implanted with colon cancer cells and weekly given with the NK4 plasmid. The repetitive delivery and expression of NK4 gene inhibited angiogenesis and invasiveness of colon cancer cells in subcutaneous tumor tissue and this was associated with suppression of primary tumor growth. By fifty days after tumor implantation, cancer cells naturally metastasized to the liver, whereas NK4 gene expression potently inhibited liver metastasis. Inhibition of the HGF-Met receptor pathway and tumor angiogenesis by NK4 gene expression has potential therapeutic value toward inhibition of invasion, growth, and metastasis of colon cancer.