A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa serine protease inhibitor found at high levels in extracellular matrix. Recombinant human TFPI-2 has recently been shown to be a strong inhibitor of trypsin, plasmin, plasma kallikrein, and factor XIa amidolytic activity. Earlier studies in our laboratory showed that the expression of TFPI-2 is lost during tumor progression in human gliomas. We stably transfected this protease inhibitor in multiform glioblastoma cell line (SNB-19) and in low-grade glioma cell line (Hs683) in sense and antisense orientation respectively. This confirmed that the upregulation/down-regulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas both in vitro and in vivo models. Collectively, these results suggested an idea to determine whether TFPI-2 is necessary for cell survival and inhibition of tumor formation in nude mice, due to apoptosis of intracerebrally injected SNB-19 cells. In the present study we determined p-ERK levels and found that they are decreased in TFPI-2 over-expressed clones (SNB-19) and increased in TFPI-2 down-regulated clones (Hs683). We also checked the levels of BAX/BCl-2, caspases (for e.g., 9, 7, 3, 8), PARP, cytochrome-c and Apaf-1. Moreover, the increase of apoptosis in vitro is associated with increased and decreased expression of apoptotic protein BAX in sense clones (SNB-19) and antisense clones (Hs683) respectively, when compared to controls and vice versa with Bcl-2 the anti-apoptotic protein. Caspases (9, 7 and 3), cytochrome-c, Apaf-1 and PARP levels are increased in SNB-19 and decreased in Hs683. Caspase 8 was not expressed in either cell line. Caspases 9 and 3 activity assay revealed higher activity in sense clones (SNB-19) but lesser in antisense clones (Hs683) compared to controls. This is the first report of TFPI-2 playing a novel role in cell survival in human gliomas.     

Overexpression of tissue factor pathway inhibitor-2 (TFPI-2), decreases the invasiveness of prostate cancer cells in vitro

Human tissue factor pathway inhibitor-2 (TFPI-2) is a 32-kDa serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase and tissue-type plasminogen activators or thrombin. After discovering that TFPI-2 expression is down-regulated or lost during tumor progression, we investigated the role of TFPI-2 in the invasiveness of the prostate cancer cell line (LNCaP). We stably transfected LNCaP cells with a 0.7-kb vector expressing TFPI-2 in the sense orientation and measured the expression of TFPI-2 protein and mRNA by these cells by western and northern blotting. Neither TFPI-2 protein nor mRNA was expressed by parental LNCaP cells or vector-transfected controls, but levels of both protein and mRNA were significantly increased in the sense-TFPI-2 clones. The sense clones were less invasive than the control cells in Matrigel invasion and spheroid migration assays. This is the first demonstration that upregulation of TFPI-2 plays a significant role in the invasive behavior of human prostate cancer cells.     

组织因子途径抑制物-2表达与肝癌细胞体外侵袭能力的实验研究

目的:研究肝癌组织中组织因子途径抑制物-2(tissue factor pathway inhibitor-2 ,TFPI-2)基因的表达,同时探讨TFPI-2对肝癌细胞侵袭能力的影响.方法:应用原位杂交法和免疫组织化学法检测30例肝癌患者的癌组织和癌旁正常肝组织中TFPI-2基因和蛋白的表达;通过脂质体法将TFPI-2基因转染人HepG2细胞,RT-PCR检测转染前后TFPI-2 mRNA的表达;Transwell小室法测定TFPI-2基因转染前后肝癌细胞体外侵袭迁移能力的变化.结果: 肝癌组织中TFPI-2 mRNA与蛋白的表达水平明显低于癌旁正常肝组织(P < 0.05);成功转染TFPI-2基因的肝癌细胞HepG2中证实有TFPI-2 mRNA的表达;与未转染的细胞相比,转染TFPI-2的细胞体外侵袭能力明显下降,穿膜细胞数分别为45.2±5.6和 87.8±4.5 ,差异有统计学意义(P < 0.05);而迁移能力无明显变化,转染前后HepG2细胞的穿膜细胞数分别为140.4±7.4和152.8±9.6,差异无统计学意义( P > 0.05).结论:肝癌组织中TFPI-2表达明显降低,TFPI-2基因表达可明显抑制肝癌细胞的体外侵袭能力.     

Silencing of tissue factor pathway inhibitor-2 gene in malignant melanomas

To identify tumor-suppressor genes inactivated by aberrant methylation of promoter CpG islands (CGIs) in human malignant melanomas, genes upregulated by treatment of cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), were searched for using oligonucleotide microarrays in melanoma cell lines, HMV-I, MeWo and WM-115. Seventy-nine known genes with CGIs were identified as being upregulated (>or=16-fold), and 18 of them had methylation of their putative promoter CGIs in 1 or more of 8 melanoma cell lines. Among the 18 genes, TFPI-2, which is involved in repression of the invasive potential of malignant melanomas, was further analyzed. Its expression was repressed in a melanoma cell line with its complete methylation, and was restored by 5-aza-dC treatment. It was unmethylated in cultured neonatal normal epidermal melanocyte, and was induced by ultraviolet B. In surgical melanoma specimens, TFPI-2 methylation was detected in 5 of 17 metastatic site specimens (29%), while it was not detected in 20 primary site specimens (0%) (p=0.009). By immunohistochemistry, the 5 specimens with promoter methylation lacked immunoreactivity for TFPI-2. The results showed that TFPI-2 is silenced in human malignant melanomas by methylation of its promoter CGI and suggested that its silencing is involved in melanoma metastasis.     

Prognostic significance of tissue factor pathway inhibitor-2 in pancreatic carcinoma and its effect on tumor invasion and metastasis

Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated kunitz-type serine proteinase inhibitor that plays an important role in plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor angiogenesis, invasion and metastasis. Earlier studies have shown that the production of TFPI-2 is downregulated during the progression of various tumors. To detect whether TFPI-2 can be expressed in human pancreatic carcinoma samples, to evaluate its prognostic significance on pancreatic carcinoma and to investigate its effect on tumor invasion and metastasis, we collected 9 normal pancreatic tissue samples and 41 pancreatic carcinoma samples and stably transfected the human pancreatic carcinoma cell line Panc-1 with a vector capable of expressing TFPI-2 gene. RT-PCR and Western blot analysis revealed that the expression of TFPI-2 in pancreatic carcinoma samples was markedly lower than that in normal pancreas samples, and there was no TFPI-2 expression in Panc-1 cell. Its expression was related with biological characters of pancreatic carcinoma. The results of Boyden chamber assay and orthotopic pancreatic carcinoma model showed that TFPI-2 could inhibit invasion and metastasis ability of pancreatic carcinoma in vitro and in vivo. Kaplan–Meier survival curve and Cox proportional hazards model assay identified TFPI-2 as an independent prognostic factor for pancreatic carcinoma. Our data suggest that TFPI-2 plays a significant role in the invasion and metastasis of pancreatic carcinoma cell in vitro and in vivo and is determined to be an important prognostic factor for pancreatic carcinoma patients.     

组织因子途径抑制物-2基因与非小细胞肺癌关系的研究进展

组织因子途径抑制物-2（TFPI-2）是一种广谱的丝氨酸蛋白酶抑制物,可抑制包括纤溶酶、胰蛋白酶、基质金属蛋白酶在内的多种蛋白酶.TFPI-2可通过维持细胞外基质的完整、调节肿瘤血管生成、调控肿瘤细胞凋亡等机制调节非小细胞肺癌的侵袭转移,并成为肿瘤基因治疗的新靶点.     

Sequential 5-Aza 2'-deoxycytidine/depsipeptide FK228 treatment induces tissue factor pathway inhibitor 2 (TFPI-2) expression in cancer cells

cDNA arrays were used to examine gene induction in CALU-6 and H460 lung cancer cells mediated by sequential 5-aza 2'-deoxycytidine (DAC)/depsipeptide FK228 (DP) exposure in order to identify translational end points for clinical trials evaluating these agents. In both cell lines, sequential DAC/DP treatment induced expression of tissue factor pathway inhibitor-2 (TFPI-2), an inhibitor of Factor VII: tissue factor signal transduction known to diminish the malignant phenotype of cancer cells. TFPI-2 expression was diminished or absent in 16 of 32 cell lines established from thoracic malignancies. Sequential DAC/DP treatment induced TFPI-2 in cancer cells deficient for TFPI-2 expression in the basal state. Promoter methylation coincided with loss of TFPI-2 expression in a number of cancer lines. TFPI-2 promoter methylation was observed in one of five pulmonary adenocarcinomas, and seven of seven esophageal adenocarcinomas, but not corresponding normal tissues. DP enhanced acetylation of TFPI-2-associated histones in CALU-6 cells. DP or PDBU, alone, induced TFPI-2 expression in cancer cells deficient for TFPI-2 expression in the absence of promoter methylation. In these cells, DP-mediated TFPI-2 induction was abrogated by calphostin. Induction of TFPI-2 by distinct, yet cooperative mechanisms involving chromatin remodeling and PKC signaling strengthens the preclinical rationale for sequential administration of DNA demethylating agents and HDAC inhibitors in cancer patients. Furthermore, induction of TFPI-2 may be a useful surrogate marker of treatment response in individuals receiving sequential DAC/DP infusions.     

Tissue factor pathway inhibitor-2 inhibits the growth and invasion of hepatocellular carcinoma cells and is inactivated in human hepatocellular carcinoma

Human tissue factor pathway inhibitor-2 (TFPI-2) is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor that inhibits the plasmin- and trypsin-mediated activation of matrix metalloproteinases and inhibits tumor progression, invasion and metastasis. Previous studies have shown that TFPI-2 is downregulated in the progression of various tumors. The purpose of this study was to investigate the expression and function of TFPI-2 in hepatocellular carcinoma (HCC). In situ hybridization was used to detect human TFPI-2 mRNA and immunohistochemistry was performed to examine the role of TFPI-2 expression in hepatocarcinoma tissues. Cell proliferation was assessed using MTT assay. In?situ hybridization and immunohistochemical analyses revealed that the expression of TFPI-2 in hepatocarcinoma tissues was markedly lower than that in tumor-adjacent normal hepatic tissues. Restored expression of TFPI-2 in HepG(2) cells inhibits cell proliferation and invasion. Taken together, the results suggest that TFPI-2 has a tumor-suppression action and its inactivation may contribute to HCC.     

Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with non-small-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4-120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P=0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P=0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P=0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC.     

组织因子及其抑制剂-1、2与肿瘤转移及血栓形成的相关性研究

 目的　探讨组织因子（TF）、组织因子抑制剂（TFPI）-1、2在肿瘤转移和血栓形成中的作用。方法　收集292例恶性肿瘤患者血液和组织标本,以130位门诊体检人员作对照组。ELISA法检测血浆TF、TFPI-1和TFPI-2浓度;免疫组织化学染色观察TF、TFPI-1和TFPI-2的表达。结果　各肿瘤组TF以及TFPI-1血浆浓度均高于对照组（P＜0.05）,肺癌组TF和TFPI-1血浆浓度分别为（3.34±1.12）U／L、（156.13±57.12）μg／L,高于其他类型肿瘤组;各组TFPI-2差异无统计学意义（P＞0.05）。各肿瘤组TF组织表达均较瘤旁组织增高（P < 0.05）;TF在肺癌组织表达显著高于其他肿瘤组织（P < 0.05）。肿瘤合并血栓形成组与无血栓形成组TF、TFPI-1血浆浓度均较对照组增高（P < 0.05）,TF在血栓形成组较无血栓形成组明显增高（P < 0.05）。结论　多种肿瘤高表达TF,不同肿瘤类型表达部位不同,类型不同的肿瘤表达强度不尽相同。血循环中TFPI-1和TF的失衡状态可能与肿瘤的高凝状态有关,促进了肿瘤血栓形成。     

Trypsinogen 4 boosts tumor endothelial cells migration through proteolysis of tissue factor pathway inhibitor-2

Proteasescontribute to cancer in many ways, including tumor vascularization and metastasis, and their pharmacological inhibition is a potential anticancer strategy.We report that human endothelial cells (EC) express the trypsinogen 4 isoform of the serine protease 3 (PRSS3), and lack both PRSS2 and PRSS1. Trypsinogen 4 expression was upregulated by the combined action of VEGF-A, FGF-2 and EGF, angiogenic factors representative of the tumor microenvironment. Suppression of trypsinogen 4 expression by siRNA inhibited the angiogenic milieu-induced migration of EC from cancer specimens (tumor-EC), but did not affect EC from normal tissues. We identified tissue factor pathway inhibitor-2 (TFPI-2), a matrix associated inhibitor of cell motility, as the functional target of trypsinogen 4, which cleaved TFPI-2 and removed it from the matrix put down by tumor-EC. Silencing tumor-EC for trypsinogen 4 accumulated TFPI2 in the matrix.Showing that angiogenic factors stimulate trypsinogen 4 expression, which hydrolyses TFPI-2 favoring a pro-migratory situation, our study suggests a new pathway linking tumor microenvironment signals to endothelial cell migration, which is essential for angiogenesis and blood vessel remodeling. Abolishing trypsinogen 4 functions might be an exploitable strategy as anticancer, particularly anti-vascular, therapy.     

Tyrosine phosphorylation of PYK2 mediates heregulin-induced glioma invasion: novel heregulin/HER3-stimulated signaling pathway in glioma

Receptor tyrosine kinases of the EGFR family transmit extracellular signals that control diverse cellular functions such as proliferation, differentiation and survival. Signaling function of a member of this family, HER3, is believed to be impaired due to deviations in its kinase consensus motifs. Here we address the functional role and signaling mechanisms of HER3. HER3 preferentially forms heterodimers with HER2 inducing the most potent mitogenic signal among EGFR family members. Our data show that in a glioma-derived cell line the cytoplasmic tyrosine kinase PYK2 is constitutively associated with HER3 and that stimulation with Heregulin results in PYK2 tyrosine phosphorylation. HER3, but not HER2, mediates the phosphorylation of the C-terminal region of PYK2 to promote a mitogenic response through activation of the MAPK pathway. A central role of PYK2 in signaling downstream of HER3 is substantiated by the demonstration that expression of a dominant-negative PYK2-KM construct abrogates the Heregulin-induced MAPK activity and inhibits the invasive potential of glioma cells. These results suggest a novel Heregulin/HER3-stimulated signaling pathway in glioblastoma-derived cell lines that involves phosphorylation of PYK2 and mediates invasiveness of glioma cells.     

MicroRNA-616 induces androgen-independent growth of prostate cancer cells by suppressing expression of tissue factor pathway inhibitor TFPI-2

Expression of microRNA genes is profoundly altered in cancer but their role in the development of androgen-independent prostate cancer has received limited attention as yet. In this study, we report a functional impact in prostate cancer cells for overexpression of the microRNA miR-616, which occurred consistently in cells that were androgen-independent (AI) versus androgen-dependent (AD). miR-616 overexpression was confirmed in malignant prostate tissues as opposed to benign prostate specimens. Stable miR-616 overexpression in LNCaP cells by a lentiviral-based approach stimulated AI prostate cancer cell proliferation in vitro whereas concomitantly reducing androgen-induced cell growth. More importantly, miR-616 overexpressing LNCaP cells overcame castration resistance as shown by an enhanced ability to proliferate in vivo after bilateral orchiectomy. Conversely, antagonizing miR-616 in AI prostate cancer cells yielded opposite effects. Microarray profiling and bioinformatics analysis identified the tissue factor pathway inhibitor TFPI-2 mRNA as a candidate downstream target of miR-616. In support of this candidacy, we documented interactions between miR-616 and the 3'UTR of TFPI-2 and determined TFPI-2 expression to be inversely correlated to miR-616 in a series of prostate cell lines and clinical specimens. Notably, reexpression of TFPI-2 in LNCaP cells with stable miR-616 overexpression rescued the AD phenotype, as shown by a restoration of androgen dependence and cell growth inhibition. Taken together, our findings define a functional involvement for miR-616 and TFPI-2 in the development and maintenance of androgen-independent prostate cancer.     

组织因子途径抑制物-2和γ-神经突触核蛋白在食管癌中的表达及其与肿瘤细胞浸润、转移和凋亡之间的关系

目的:探讨组织因子途径抑制物-2(tissue factor pathway inhibitor-2 ,TFPI-2)和γ-神经突触核蛋白(synuclein gamma,SNCG)在食管癌组织中的表达及其与肿瘤浸润、转移和细胞凋亡之间的关系.方法:应用免疫组织化学法检测82例食管癌组织及20例相应癌旁不典型增生组织和54例相应癌旁正常食管组织中TFPI-2、SNCG和基质金属蛋白酶-9(matrix metalloproteinase-9 ,MMP-9)的表达,应用TUNEL法检测肿瘤细胞的凋亡并计算细胞凋亡指数(apoptosis index,AI).结果: TFPI-2和SNCG在食管癌组织、癌旁不典型增生组织及正常食管组织中的阳性表达率分别为30.4%、60.0%、87.0%和63.4%、30.0%、3.7%,3组间两两比较差异均有统计学意义(P<0.01).TFPI-2和SNCG的表达与肿瘤淋巴结转移、浸润深度、TNM分期和分化程度密切相关(P<0.01),但与患者年龄、性别、肿瘤大小、肿瘤部位和肿瘤大体分型无关(P>0.05).食管癌组织中TFPI-2与MMP-9的表达呈负相关(r=-0.636,P=0.000),而SNCG与MMP-9的表达呈正相关(r=0.393,P=0.000).TFPI-2和SNCG的表达与AI有关 (P<0.05).结论:TFPI-2可能通过抑制MMP-9的表达诱导细胞凋亡,抑制食管癌细胞的生长和转移,而SNCG可能在此过程中起着相反的作用, TFPI-2和SNCG有望成为有效的肿瘤标志物和肿瘤基因治疗的新靶点.