Inhibition of invasion and metastasis in oral cancer by targeting urokinase-type plasminogen activator receptor

There have been reports of strong correlations between poor prognosis in various cancers and concomitant expression of urokinase-type plasminogen activator (uPA) and its surface receptor (uPAR). We and others have previously shown that the uPA system plays a significant role in a subset of head and neck squamous cell carcinoma. In the present study, we found that uPAR is required for invasion and metastasis of highly malignant oral cancer cells (OSC-19). Treating OSC-19 cells with antisense oligonucleotides (AS) targeting uPAR resulted in a dramatic decrease of uPAR mRNA expression. Furthermore, pretreatment with AS or siRNA targeting uPAR inhibited progression of OSC-19 cells in experimental models. These results suggest that overexpression of uPAR increases the invasiveness and metastasis of OSC-19 cells, and that uPAR is a promising therapeutic target for regulation of progression of oral cancer.     

Adenovirus-mediated delivery of antisense gene to urokinase-type plasminogen activator receptor suppresses glioma invasion and tumor growth.

The urokinase-type plasminogen activator (uPA) and uPA receptor (UPAR) play important roles in the proteolytic cascade involved in the invasiveness of gliomas and other invasive tumors. High-level expression of uPAR has been correlated with high-grade glioma cell lines and tumors We report here that down-regulating uPAR levels by antisense strategy using an adenovirus construct (Ad-uPAR) inhibited glioma invasion in Matrigel and spheroid in vitro models. sc. (U87-MG) and intracranial (SNB19) injections of Ad-uPAR-infected glioma cells did not produce tumors in nude mice. However, injection of the Ad-uPAR construct into previously established so U87-MG tumors in nude mice caused regression of those tumors. Our results support the therapeutic potential of targeting the uPA-uPAR system for the treatment of gliomas and other cancers.     

Monoclonal antibodies against vascular endothelial growth factor and epidermal growth factor receptor in advanced colorectal cancers: present and future directions

PURPOSE OF REVIEW: Targeted therapies are emerging as potentially important therapeutic interventions in the treatment of advanced colorectal cancer. There have been considerable developments in this field within the past few years, with some agents entering the clinic and others soon to follow. Monoclonal antibodies against important tumorigenic targets have been in development for over a decade. Recently, cetuximab and bevacizumab have generated significant interest and are the focus of this review. RECENT FINDINGS: Cetuximab, a monoclonal antibody against the epidermal growth factor receptor, and bevacizumab, a monoclonal antibody against vascular endothelial growth factor, have now been subject to rigorous assessment within several clinical studies, including a large randomized phase II study of cetuximab and a randomized phase III study of bevacizumab. The demonstration of efficacy and safety with these agents has led to the issue of licenses for cetuximab in Switzerland and bevacizumab in the United States. SUMMARY: These results have led to a rapid expansion of further studies to define the role of these antibodies and to aid their future integration with conventional approaches for the management of advanced colorectal cancer.     

Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer

Background  

Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (uPA) are involved in colorectal cancer invasion and metastasis. There is still debate whether the activity of MMP-2 and MMP-9 differs between tumors located in the colon and rectum. We designed this study to determine any differences in the expression of MMP-2, MMP-9 and uPA system between colon and rectal cancer tissues.     

Significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) expression in human colorectal carcinomas.

Despite the advances in the medical care of colorectal carcinoma patients, the prognosis has improved only marginally over recent decades. Thus, additional prognostic indicators would be of great clinical value to select patients for adjuvant therapy. In the present study, the antigen levels of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1, and their immunohistochemical staining were compared in paired colorectal tumor (n = 64) and background colon tissue of the same patients with clinical and pathological staging. The antigen levels, measured with an ELISA method, were found to be significantly higher in cancer tissue (mean 1.92 ng/mg protein for uPA and 7.08 for PAI-1) than in corresponding normal mucosa (0.29 ng/mg protein for uPA and 1.11 ng/mg protein for PAI-1). There was a positive correlation between uPA and PAI-1 antigen levels and clinicopathological parameters such as grade (p < 0.001 and p = 0.01, respectively), while for Dukes' stage, only PAI-1 correlated positively (p = 0.018). Nodal status correlated positively with uPA but not with PAI-1 antigen levels. Immunohistochemical localization of both antigens was observed mainly in cancer cells and much less in stromal cells. Staining intensity increased from adenoma to adenocarcinoma. The degree of staining was associated with grade, Dukes' stage and nodal status for uPA (p < 0.001, p = 0.002, p < 0.001, respectively) and only with grade for PAI-1 (p = 0.007).     

尿纤溶酶原激活物及血管内皮生长因子在食管癌中的表达及对肿瘤血管形成的影响

目的探讨尿纤溶酶原激活物（uPA）、血管内皮生长因子（VEGF）在食管癌中的表达及对肿瘤血管生成的影响。方法采用免疫组织化学sP法检测正常食管黏膜上皮组织（18例）及食管癌组织（68例）中uPA、VEGF的表达,检测CD。用以标记肿瘤微血管密度（MVD）,根据MVD均值分为高、低MVD组,分析食管癌uPA、VEGF的表达和临床病理特征的关系及对肿瘤血管形成的影响。结果uPA蛋白在正常食管黏膜上皮组织、食管癌组织中的阳性率分别为27．8％（5／18）和70．6％（48／68）,差异有统计学意义（X^2=11．63,P〈0．05）;VEGF蛋白在正常食管黏膜上皮组织、食管癌组织中的阳性率分别为22．2％（4／18）和63．2％（43／68）,差异有统计学意义（X^2=9．78,P〈0．05）。食管癌组织中uPA与VEGF表达有一致性（X^2=9．72,P〈0．05）。MVD平均为42．38±11．62,高MVD组uPA、VEGF蛋白表达显著高于低MVD组（X^2值分别为6．13和10．12,均P〈0．05）。uPA、VEGF蛋白表达与年龄、性别、病理类型无关（均P〉0．05）,均与临床病理分期、分化程度和淋巴结转移相关（P〈0．05）。结论食管癌组织中uPA、VEGF蛋白高表达,可能促进肿瘤血管形成,提示预后不良。     

微血管密度和血管内皮生长因子与大肠癌肝转移的关系研究

目的研究微血管密度(MVD)和血管内皮生长因子(VEGF)与大肠癌术后发生肝转移的关系。方法检测160例大肠癌标本中VEGF、和MVD的表达并结合随访结果。结果 VEGF、和MVD与大肠癌的临床分期密切相关:Duke C期的vEGF和MVD表达高于B期,差异有显著性(p<0.01);术后发生肝转移组VEGF和MVD表达高于无转移组,差异有显著性(P<0.01)。结论检测大肠癌病灶中MVD和VEGF的表达能预测患者的临床分期,MVD和VEGF过度表达的患者,术后发生肝转移的危险性大。     

血管内皮生长因子及其受体与骨肉瘤关系的研究进展

血管内皮生长因子（ＶＥＧＦ）是一种序列高度保守、高度特异性的促血管内皮细胞生长因子,广泛分布于人和动物体内的大脑、肾脏、肝脏、脾脏、胰腺和骨骼等组织中,对内皮细胞具有强烈的促有丝分裂作用,刺激血管内皮细胞增殖和血管通透性增加,促进新生血管形成。ＶＥＧＦ通过与血管内皮细胞表面受体（ＶＥＧＦＲ）特异性结合发挥生物学效应。抑制ＶＥＧＦ及ＶＥＧＦＲ的活性可以减缓或阻滞骨肉瘤侵袭和转移。研究表明,ＶＥＧＦ及ＶＥＧＦＲ对肿瘤血管及淋巴管的生成及肿瘤侵袭和转移起重要作用。本文对ＶＥＧＦ及ＶＥＧＦＲ与骨肉瘤血管与淋巴管生成及其侵袭与转移的关系作一综述。     

Physiological and pathological changes of plasma urokinase-type plasminogen activator, plasminogen activator inhibitor-1, and urokinase-type plasminogen activator receptor levels in healthy females and breast cancer patients

The plasma urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and urokinase-type plasminogen activator receptor (uPAR) levels were measured in healthy volunteers and breast cancer patients. In pre-menopause healthy females, blood was sampled weekly during one menstruation cycle and menstruation phases (follicular, ovulatory, luteal) were determined by FSH/LH levels. uPA, PAI-1, and uPAR levels were at the nadir during ovulatory phase. uPA level was highest at follicular phase while PAI-1 level was highest at luteal phase. In comparison between pre- and post-menopause states, uPA and uPAR levels were higher in post-menopause state while PAI-1 level was higher in pre-menopause state. In breast cancer patients, uPA, PAI-1, and uPAR positive rates were low when we use the menopause-state-unmatched cut-off points. As we adjusted the cut-off points by menopause states, the PAI-1 positivity increased mainly in post-menopause cancer patients. These findings suggest that there is a minor but possible sequential change of these molecules during menstruation cycle which might blur the pathological positivity in pre-menopause cancer patients. The pathological elevation of PAI-1 was well detected in post-menopause cancer patients, but this elevation did not correlate with tumor burden such as number of metastatic sites or metastatic location. In conclusion, adjustment of physiological changes of uPA, PAI-1, and uPAR is required in determining pathological elevation of the plasma levels in cancer patients, especially in females.This revised version was published online in October 2005 with corrections to the Cover Date.     

大肠癌血管内皮生长因子及其受体的表达和临床意义

目的　探讨血管内皮生长因子 (VEGF)及其受体 (KDR)在大肠癌组织中的表达及其与临床特征之间的关系 ,为通过阻断VEGF及其受体KDR的作用治疗大肠癌提供依据。方法　应用免疫组织化学SABC法检测 6 8例大肠癌组织和癌旁组织VEGF及KDR蛋白的表达 ,2 0例正常组织作对照。结果　 2 0例正常组织中未发现VEGF及KDR的表达 ,6 8例大肠癌组织中VEGF表达阳性率为 5 5 .9%( 38/6 8) ,KDR表达阳性率为 45 .6 %( 31/6 8) ,均明显高于癌旁组织 ( 11.8%,8/6 8;8.8%,6 /6 8) (P <0 .0 1)。大肠癌组织VEGF及KDR的表达与肿瘤浸润深度、淋巴结转移、远处转移、血管侵犯、Dukes′分期密切相关 ,而与组织学分型无关。VEGF和KDR的表达密切相关。结论　VEGF及其受体KDR在大肠癌的生长、进展和转移中起了重要作用。     

Identification and localization of urokinase-type plasminogen activator in human NK-cells

We have studied the presence of plasminogen activators in large granular lymphocytes and other peripheral blood cells. After immunofluorescence staining with polyclonal antiserum against urokinase-type plasminogen activator, the majority of LGLs showed granular staining which was located in Golgiderived vesicles. LGLs were negative for tissue-type activator. The presence of only urokinase-type PA in LGLs was also confirmed by determining the molecular weight of the intracellular activator and by immunoblotting the antigen from solubilized cell preparations. Monocytes and granulocytes were more intensely stained with anti-u-PA than LGLs. In LGL/K562 cell conjugates the fluorescence was often located close to the contact area and the vesicular fluorescence polarized during conjugate formation. Inhibitors of PA and other serine proteinases are known to abolish NK-cell activity. We now show that they affect a later stage than programming for lysis in the cytotoxic action, suggesting a role for u-PA or other serine proteases in the lethal-hit stage of NK activity.     

Differential gene expression of urokinase-type plasminogen activator and its receptor in human renal cell carcinoma

The urokinase-type plasminogen activator (uPA) system plays a central role in extracellular matrix degradation, cell migration, and invasion. uPA belongs to the family of serine proteases. It has been shown that its proteolytic activity is involved in the metastatic process by activation and binding to its receptor (uPAR). Previous studies in several organ systems have elucidated a higher uPA expression in malignant tissue in comparison to normal tissue. In this study uPA and uPAR gene expression were investigated in 18 human renal cell carcinoma (RCC) specimens in comparison with adjacent non-malignant renal tissues. mRNA in situ hybridisation and immunohistochemical staining were performed. mRNA of uPA and uPAR was significantly higher expressed in 56% (10/18) and 72% (13/18) of the RCC specimens in comparison to the adjacent non-malignant renal tissue (p<0.0001), respectively. uPA-mRNA and uPAR-mRNA were expressed predominantly in malignant renal cells and in very few surrounding stromal cells. The elevated expression of uPAR-protein in RCC reached statistical significance compared to adjacent normal tissue (p=0.007). uPAR genes were higher expressed in comparison to uPA alone. There was a statistical trend that higher expression of uPA and uPAR corresponded with TNM tumour stage and grade in RCC. Further investigations need to be done with larger sample sizes to prove a correlation of expression between uPA and uPAR to a more aggressive phenotype. We conclude that uPA- and uPAR are overexpressed in RCC and could function as tumour markers.     

卵巢交界性肿瘤血管内皮生长因子表达和微血管密度的研究

背景与目的:血管生成是实体肿瘤生长、侵袭、扩散转移的关键,微血管密度(microvesselofdensity,MVD)可反映肿瘤的血管形成情况。血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)是具有重要意义的促血管生长因子,它与肿瘤的生长密切相关。本研究检测卵巢交界性肿瘤组织中VEGF的表达和MVD,探讨它们与临床病理特征及预后的关系。方法:采用免疫组化SP法和原位杂交技术检测69例卵巢交界性肿瘤、18例卵巢良性肿瘤和27例卵巢恶性肿瘤组织中VEGF蛋白及VEGFmRNA的表达,用FⅧ因子单克隆抗体标记新生血管内皮,计数并计算MVD。结果:VEGF蛋白、VEGFmRNA在卵巢交界性肿瘤组织中的表达均介于卵巢良性肿瘤与卵巢恶性肿瘤之间,其结果差异均有显著性(P<0.05);也与卵巢交界性肿瘤的临床分期、MVD值密切相关(P<0.05);但VEGF与卵巢交界性肿瘤的组织分型、有无腹腔种植无关(P>0.05)。结论:VEGF与卵巢肿瘤的生长、侵袭、转移密切相关;检测VEGF有助于判断卵巢肿瘤的恶性程度,为判断卵巢交界性肿瘤患者的预后提供依据。     

Frequent expression of the vascular endothelial growth factor in human non-small-cell lung cancers

BACKGROUND: Angiogenesis is an essential factor for progression and metastases in solid tumors. It has been reported that several angiogenic factors play a role in the regulation of angiogenesis. Vascular endothelial growth factor (VEGF) is one of the most important molecules in angiogenesis. We investigated expressions of VEGF in a series of lung carcinomas with regard to clinicopathological factors. METHOD: VEGF expression was investigated by use of immunohistochemical studies and Northern blot analysis, using 155 primary and 26 metastatic lung carcinomas for the immunohistochemical studies and 10 primary and two metastatic lung carcinomas for the Northern blot analysis. All lesions were resected at surgery. RESULTS: The frequencies for positive VEGF expression were 64 of 74 (86.5%) adenocarcinomas, 38 of 67 (56.7%) squamous cell carcinomas, four of four (100%) large cell carcinomas, two of three (66.7%) adenosquamous carcinomas and one of five (20%) small-cell carcinomas, the degree of positivity generally being greater in well differentiated tumors. The majority of metastatic foci from adenocarcinomas and squamous cell carcinomas at other sites were also positive (76.5 and 66.7%, respectively). VEGF expression did not correlate with clinicopathological factors such as tumor size or pathological stage, but pathological stage I adenocarcinoma cases positive for VEGF demonstrated a shorter disease-free period when followed up for 48 months than those cases expressing VEGF negatively. CONCLUSIONS: The results indicated that VEGF expression was frequently detected in non-small-cell lung cancers and suggested that VEGF might relate to the disease-free period of the patients with early adenocarcinomas.      

Tobacco smoke induces urokinase-type plasminogen activator and cell invasiveness: evidence for an epidermal growth factor receptor dependent mechanism

Multiple tobacco smoke-related premalignant and malignant lesions develop synchronously or metachronously in various organ sites, including the oral cavity. Both field cancerization and clonal migration seem to contribute to the occurrence of multiple tumors. Although the importance of endogenous factors (e.g., oncogenes) in regulating clonal migration is well established, little is known about the role of exogenous factors. Hence, the main objective of this study was to elucidate the mechanism by which tobacco smoke stimulated the migration of cells through extracellular matrix (ECM). Treatment of MSK-Leuk1 cells with a saline extract of tobacco smoke induced the migration of cells through ECM. Tobacco smoke induced the expression of urokinase-type plasminogen activator (uPA), resulting in plasmin-dependent degradation of ECM and increased cell migration. AG1478, a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, a neutralizing antibody to EGFR, or an antibody to amphiregulin, an EGFR ligand, also blocked tobacco smoke-mediated induction of uPA and cell migration through ECM. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase activity, caused similar inhibitory effects. Taken together, these results suggest that tobacco smoke activated the EGFR-->extracellular signal-regulated kinase 1/2 MAPK pathway, causing induction of uPA. This led, in turn, to increased plasmin-dependent degradation of matrix proteins and enhanced cell migration through ECM. These data strongly suggest that chemicals in tobacco smoke can mimic the effects of oncogenes in regulating uPA-dependent cell invasion through ECM. These findings also strengthen the rationale for determining whether inhibitors of EGFR tyrosine kinase reduce the risk of tobacco smoke-related second primary tumors.     

Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

MicroRNAs (miRNAs) are involved in a number of biological processes, including tumour biology. Pre-clinical studies have shown that miRNA-126 regulates signalling downstream of vascular endothelial growth factor receptor 2 (VEGFR-2) and, consequently, angiogenesis. The aim of this study was to analyse the possible relationship between miRNA-126, VEGFR-2 and angiogenesis in tumour tissue from patients with colorectal cancer (CRC). Tumour tissue was obtained from 81 patients. The miRNA-126 and VEGFR-2 gene expression levels were analysed by PCR and the protein concentrations of VEGFR-2 were analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA-126-expressing using the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24-0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28-0.60), p=0.02. A positive association was observed with VEGFR-2 protein concentrations, p=0.06. The median MVD was significantly lower in the tumours expressing low levels of miRNA-126, 5.8 (95% CI, 5.33-6.67), compared to those expressing high levels, 8.0 (95% CI, 6.33-9.00), p<0.01. miRNA-126 was detected in endothelial cells by in situ hybridisation analysis. These results suggest that high levels of miRNA-126 in CRC are associated with high VEGFR-2 mRNA and protein levels and a higher density of newly formed microvessels. However, further studies should be conducted to analyse the clinical value of miRNA-126 in CRC.     

Effects of hypoxia and reoxygenation on the expression levels of the urokinase-type plasminogen activator, its inhibitor plasminogen activator inhibitor type-1 and the urokinase-type plasminogen activator receptor in human head and neck tumour cells

One aim during oncological radiation therapy is to induce reoxygenation in hypoxic tumours in order to enhance radiosensitivity and ultimately increase cell death. In squamous cell carcinomas of the head and neck (SCCHN), hypoxia is considered a pivotal physiological modulator for malignant progression, whereby the plasminogen activation system is involved in overlapping functions such as the shaping of the extracellular matrix, cell proliferation and signal transduction. Since little is known about reoxygenation and the plasminogen activation system in SCCHN, three human SCCHN cell lines (BHY, FaDu, and CAL27) and a non-transformed control cell line (VH7) were exposed to hypoxic (<0.5% O2) conditions for up to 72 h and subsequently reoxygenated for 24 h at normoxic conditions. The mRNA expression of the urokinase-type plasminogen activator (uPA), the plasminogen activator inhibitor type-1 (PAI-1) and the urokinase-type plasminogen activator receptor (uPAR) was assessed by means of real-time semi-quantitative RT-PCR, and the protein expression was determined by immunoenzymometric quantification (ELISA). Both hypoxia and reoxygenation induced statistically significant changes in uPA, PAI-1 and uPAR mRNA and protein levels in the various cell lines investigated, showing that oxygen tension is a strong modulator of the plasminogen activation system in vitro. However, no uniform correlation pattern was found between the mRNA and protein levels analysed over all three time-points (24, 48, and 72 h) and oxygen treatment variants (N, H, R) nor according to oxygen treatment conditions over all three time-points. Changes in oxygen tension could therefore be modulating the fragile balance between the various components of the plasminogen activation system in SSCHN ultimately leading to an increased tumour matrix disruption, alterations in cell invasiveness, and the dissemination of tumour cells to distant organs.