Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation

VEGFR-1 is devoid of ligand-dependent tyrosine autophosphorylation and its activation is not associated with proliferation of endothelial cells. The molecular mechanism responsible for this characteristic of VEGFR-1 is not known. In this study, we show that VEGFR-1 is devoid of ligand-dependent downregulation and failed to stimulate intracellular calcium release, cell migration and angiogenesis in vitro. To understand the molecular mechanisms responsible for the poor tyrosine autophosphorylation of VEGFR-1, we have either deleted the carboxyl terminus of VEGFR-1 or exchanged it with the carboxyl terminus of VEGFR-2. The deletion of carboxyl terminus of VEGFR-1 did not reverse its defective ligand-dependent autophosphorylation. The carboxyl terminus-swapped VEGFR-1, however, displayed ligand-dependent autophosphorylation, downregulation and also conveyed strong mitogenic responses. Thus, the carboxyl tail of VEGFR-1 restrains the ligand-dependent kinase activation and downregulation of VEGFR-1 and its ability to convey the angiogenic responses in endothelial cells.     

Inhibition of multiple vascular endothelial growth factor receptors (VEGFR) blocks lymph node metastases but inhibition of VEGFR-2 is sufficient to sensitize tumor cells to platinum-based chemotherapeutics

Vascular endothelial growth factor receptors (VEGFR) have important roles in cancer, affecting blood and lymphatic vessel functionality as well as tumor cells themselves. We compared the efficacy of a VEGFR tyrosine kinase inhibitor, PTK787/ZK222584 (PTK/ZK), which targets the three VEGFRs, with blocking antibodies directed against VEGFR-2 (DC101) or VEGF-A (Pab85618) in a metastatic melanoma model. Although all inhibitors exerted comparable effects on primary tumor growth, only PTK/ZK significantly reduced lymph node metastasis formation. A comparable decrease in lymphatic vessel density following blockade of VEGFR-2 (DC101) or the three VEGFRs (PTK/ZK) was observed in the metastases. However, the functionality of lymphatics surrounding the primary tumor was more significantly disrupted by PTK/ZK, indicating the importance of multiple VEGFRs in the metastatic process. The antimetastatic properties of PTK/ZK were confirmed in a breast carcinoma model. B16/BL6 tumor cells express VEGF ligands and their receptors. Blockade of a VEGFR-1 autocrine loop with PTK/ZK inhibited tumor cell migration. Furthermore, the tumor cells also showed enhanced sensitivity to platinum-based chemotherapy in combination with PTK/ZK, indicating that autocrine VEGFRs are promoting tumor cell migration and survival. In summary, our results suggest that, in addition to blocking angiogenesis, combined inhibition of the three VEGFRs may more efficiently target other aspects of tumor pathophysiology, including lymphatic vessel functionality, tumor cell dissemination, survival pathways, and response to chemotherapeutic compounds.     

Cyclooxygenase-2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs.     

口服型VEGFR-2 DNA疫苗的研制及其对小鼠的免疫效应

目的:血管内皮生长因子受体-2(vascular endothelial factor recepto-2,VEGFR-2)在肿瘤生长和转移过程中起着重要作用,制备口服型VEGFR-2 DNA疫苗并研究其生物免疫效应.方法:采用基因重组技术构建VEGFR-2胞外区基因表达载体pcDNA3.1-VR-2, DNA序列分析证实后,脂质体法转染COS-7细胞, Western blotting检测其VEGFR蛋白表达;以氯化钙法将pcDNA3.1-VR-2转化减毒沙门菌SL3261,制备口服型VEGFR-2 DNA疫苗.口服型疫苗免疫C57BL/6小鼠,ELISA法检测免疫后小鼠外周血VEGFR-2抗体水平,MTT法检测小鼠脾淋巴细胞对小鼠血管内皮细胞的体外杀伤活性.结果:成功制备口服型VEGFR-2 DNA疫苗.ELISA法检测显示,疫苗组小鼠抗体滴度随着免疫时间和免疫次数的增加而增高,在免疫6周后产生了高水平抗VEGFR-2 IgG类抗体(1.07±0.018),明显高于生理盐水组(0.14±0.033)和质粒对照组(0.14±0.038),差异均有统计学意义(F=853.17,P=0.000).MTT法检测显示,疫苗组小鼠脾淋巴细胞对靶细胞MS1的杀伤率随着效靶比的增加而增加,在效靶比8∶1时CTL活性(89.38±1.51)明显高于生理盐水组(15.17±2.54)和空质粒对照组(12.99±4.31),差异有统计学意义(F=315.42,P=0.000).结论:成功制备了口服型VEGFR-2 DNA疫苗,该疫苗可激活小鼠特异性的细胞免疫和体液免疫,为进一步抗肿瘤研究奠定了基础.     

Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2

Lymph nodes are the first site of metastases for most types of cancer, and lymph node status is a key indicator of patient prognosis. Induction of tumor lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) has been shown to play an important role in promoting tumor metastases to lymph nodes. Here, we employed receptor-specific antagonist antibodies in an orthotopic spontaneous breast cancer metastasis model to provide direct evidence for the key role of VEGFR-3 activation in metastasis. Inhibition of VEGFR-3 activation more potently suppressed regional and distant metastases than inactivation of VEGFR-2, although VEGFR-2 blockade was more effective in inhibiting angiogenesis and tumor growth. Despite prominent proliferation, metastases were not vascularized in any of the control and treatment groups, indicating that the growth of metastases was not dependent on angiogenesis at the secondary site for the duration of the experiment. Systemic treatment with either VEGFR-2 or VEGFR-3 antagonistic antibodies suppressed tumor lymphangiogenesis, indicating that VEGFR-3 signaling affects the rate of tumor cell entry into lymphatic vessels through both lymphangiogenesis-dependent and independent mechanisms. Combination treatment with the anti-VEGFR-2 and anti-VEGFR-3 antibodies more potently decreased lymph node and lung metastases than each antibody alone. These results validate the concept of targeting the lymphatic dissemination and thereby very early steps of the metastatic process for metastasis control and suggest that a combination therapy with antiangiogenic agents may be a particularly promising approach for controlling metastases.     

Comparison of the prognosis indication of VEGFR-1 and VEGFR-2 and Tie2 receptor expression in breast carcinoma

The degree of angiogenesis in breast cancer has previously been shown to be an indicator of prognosis, and tumor microvasculature is a candidate target for new antiangiogenic therapies. The aim of this study was to investigate the prognostic value of vascular endothelial growth factor (VEGF) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and Tie2/tek receptor tyrosine kinase in breast carcinoma. VEGF receptors and Tie2 expression was investigated using immunohistochemical assays with monoclonal antibodies on frozen sections in a series of 918 and 909 patients respectively. VEGFR-1 and VEGFR-2 and Tie2 were correlated with long-term (median, 11.3 years) patients' outcome. Univariate (Kaplan-Meier) analysis showed that VEGFR-1 positive tumor surface (cutoff = 5%) was significantly correlated with high metastasis risk (p=0.03) and relapse (p<0.01) in all patients, and in those with node negative tumors (p<0.001 and p<0.01 respectively), but not with overall survival. In contrast Tie2 positive tumor surface (cutoff = 7%) was significantly correlated with poor overall survival (p=0.025) and also with high metastasis risk particularly among node negative patients (p<0.01). Moreover, Tie2 immunoexpression was significantly predictive of relapse (p=0.003) in the node negative subgroup (p=0.02). In multivariate analysis (Cox model), VEGFR-1 and Tie2 immunoexpressions were identified as independent prognostic indicators. In contrast, univariate analysis showed that VEGFR-2 positive tumor surface (cutoff = 10%) was not correlated with survival or with metastasis and relapse risk. Our results suggest that VEGFR-1 and Tie2 immunohistochemical expression permits the identification of patients with poor outcome, and particularly node negative ones with a high risk for metastasis and relapse. VEGFR-1 and Tie2 immunodetection may also be considered as potential tools for selecting patients who could benefit in the future from specific antiangiogenic therapy interfering with VEGFR-1 and Tie2 activation pathways.     

Potentiation and inhibition of tumor cell invasion by host cells and mediators

A culture model for invasion of rat mesothelial cell layer by rat ascites hepatoma cells has been developed. By using this quantitative model, the preculture with macrophages (0.1 less than macrophage/tumor cell less than 1.0) was found to enhance both the in vitro and in vivo invasive potentials of the tumor cells. This potentiation appears to be mediated partly by oxygen radicals generated by the cocultured macrophages. The in vitro invasive capacity was also augmented by pretreating the tumor cells with TGF-beta or with activated platelets. A factor with anti-invasive potential (IIF) was extracted from rat liver. It inhibited the directed migration but not the growth of the tumor cells and was effective on their in vivo invasion and metastasis, as well.     

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression ofmdr1 mRNA. However, the organ-specific mechanism for upregulatingmdr1 and P-glycoprotein has yet to be elucidated.     

Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells

Angiogenesis is regulated by the balance between pro- and antiangiogenic factors. Vascular endothelial growth factor (VEGF), acting via the receptors VEGFR-1 and VEGFR-2, is a key mediator of tumour angiogenesis. The soluble form of the VEGF receptor-1 (sVEGFR-1) is an important negative regulator of VEGF-mediated angiogenesis. The majority of breast cancers are oestrogen dependent, but it is not fully understood how oestrogen and the antioestrogen, tamoxifen, affect the balance of angiogenic factors. Angiogenesis is a result of the interplay between cancer and endothelial cells, and sex steroids may exert effects on both cell types. In this study we show that oestradiol decreased secreted sVEGFR-1, increased secreted VEGF, and decreased the ratio of sVEGFR-1/VEGF in MCF-7 human breast cancer cells. The addition of tamoxifen opposed these effects. Moreover, human umbilical vein endothelial cells (HUVEC) incubated with supernatants from oestradiol-treated MCF-7 cells exhibited higher VEGFR-2 levels than controls. In vivo, MCF-7 tumours from oestradiol+tamoxifen-treated nude mice exhibited decreased tumour vasculature. Our results suggest that tamoxifen and oestradiol exert dual effects on the angiogenic environment in breast cancer by regulating cancer cell-secreted angiogenic ligands such as VEGF and sVEGFR-1 and by affecting VEGFR-2 expression of endothelial cells.     

Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants

Tumor growth is angiogenesis-dependent. Current evidence suggests that vascular endothelial growth factor (VEGF), a major regulator of embryonic and hypoxia-mediated angiogenesis, is necessary for tumor angiogenesis. VEGF is expressed in tumor cells in vivo, and its tyrosine kinase receptors VEGFR-1 and VEGFR-2 are up-regulated in the tumor endothelium. A second endothelial cell-specific ligand/receptor tyrosine kinase system, consisting of the tie2 receptor, its activating ligand angiopoietin-1 and the inhibitory ligand angiopoietin-2, has been characterized. We have examined 6 human primary breast-cancer samples and 4 murine breast-cancer cell lines (M6363, M6378, M6444, M6468), transplanted into nude mice, by in situ hybridization and/or Northern analysis. Expression of angiopoietin-1, angiopoietin-2 and tie2 was compared to VEGF and VEGFR-2 expression. Human tumors expressed VEGFR-2 and tie2 but varied considerably in VEGF and angiopoietin-1/-2 expression. In the murine tumor models, we observed high heterogeneity of receptor and ligand expression. M6363 and M6378 tumors were analyzed in detail because they showed different expression of components of the tie2/angiopoietin signaling system. M6363 tumors expressed VEGF, VEGFR-2 and angiopoietin-2 but not tie2 or angiopoietin-1, suggesting activation of VEGFR-2 and inhibition of tie2 signaling pathways, whereas M6378 tumors expressed VEGF, VEGFR-2, tie2 and angiopoietin-1 but little angiopoietin-2, suggesting activation of both VEGFR-2 and tie2 signaling pathways. In vivo studies using truncated dominant-negative tie2 and VEGFR-2 mutants revealed inhibition of M6363 tumor growth by 15% (truncated tie2) and 36% (truncated VEGFR-2), respectively. In contrast, M6378 tumor growth was inhibited by 57% (truncated tie2) and 47% (truncated VEGFR-2), respectively. These findings support the hypothesis that tumor angiogenesis is dependent on VEGFR-2 but suggest that, in addition, tie2-dependent pathways of tumor angiogenesis may exist. For adequate application of angiogenesis inhibitors in tumor patients, analysis of prevailing angiogenesis pathways may be a prerequisite.     

The effects of VEGF and VEGFR-2 on survival in patients with gastric cancer

Prognostic criteria of the patients with gastric cancer are of critical importance in their management and follow-up. Angiogenesis is essential for the growth and metastasis of solid tumors. Tumor angiogenesis is a multi-step interactive process, and vascular endothelial growth factor (VEGF) and its receptors have a major role in tumor angiogenesis. Thus, we investigated the effects of VEGF and VEGF receptor-2 (VEGFR-2, KDR) on survival in patients with gastric cancer. We analyzed 51 patients who had undergone total or subtotal gastric resection. The patients were divided into two subgroups according to their VEGF and VEGFR-2 (KDR) expression in resected specimens. There was no significant difference between sex, surgical method, lymph node metastasis, serosal invasion, hematogenous metastasis, chemotherapy status of the two subgroups. Mean follow-up time was 24.22 +/- 15.38 months. We found the survival rates of the patients with VEGF positive tumors to be significantly shorter than those of the patients with VEGF negative tumors. There was no significant difference between the survival rates of VEGFR-2 (KDR) positive and negative patients. It was established that the presence of VEGF expression was significantly associated with the short survival rates in patients with gastric cancer. Analysis of VEGF expression in resected specimens may provide additional guidance in determining the prognosis of such patients. If more extensive studies confirm the significance of VEGF and its receptors in gastric cancer, new therapeutic approaches targeting VEGF and its receptors may be considered in gastric cancer management.     

Differential effects of p21(WAF1/CIP1) deficiency on MMTV-ras and MMTV-myc mammary tumor properties

p21(WAF1/CIP1) (p21) functions as a cyclin-dependent kinase (CDK) inhibitor and is a key mediator of p53-dependent growth arrest. However, its role in cell cycle regulation is complex, because it also appears to promote CDK activity in certain experimental contexts. Its potential role in tumor suppression was evaluated in MMTV-ras and MMTV-myc transgenic mice that were interbred to p21(WAF1/CIP1) knockout mice (p21-/-). p21 deficiency had differential effects on tumor incidence and age of onset, proliferation, and apoptosis in the presence of these two oncogenes. Tumors arising in MMTV-ras/p21-/- mice displayed higher S-phase fractions and correspondingly increased cyclin D1 and E/CDK activity than MMTV-ras tumors. In contrast, MMTV-myc/p21-/- tumors had lower S-phase fractions and levels of cyclin D1 and E/CDK activity than MMTV-myc tumors. In both tumor types, changes in cyclin D1 and E/CDK activity were paralleled by changes in the corresponding cyclin protein levels. Tumor cell apoptosis was also differentially influenced by p21 deficiency in the two models. MMTV-ras/p21-/- tumors exhibited a significant increase in spontaneous apoptosis as compared with MMTV-ras tumors, whereas p21 deficiency had minimal effect on apoptosis in MMTV-myc tumors. These results indicate that the effects of p21 expression on cellular proliferation are differentially affected by the expression of different oncogenes, and that p21 may play a role in promoting either growth arrest or proliferation, depending on the specific cellular context.     

Differential effects of JNK1 and JNK2 on signal specific induction of apoptosis

The c-Jun N-terminal kinases (JNKs) are activated by a variety of stress inducing agents and are thought to regulate apoptosis in a cell type and signal-specific manner. We have used fibroblasts lacking JNK1 or JNK2 to define their roles in response to different stress signals. Lack of JNK1 results in reduced c-Jun phosphorylation and resistance to UV-induced cell death. JNK2 deficient cells show increased sensitivity to UV irradiation which correlates with elevated and sustained phosphorylation of JNK1 and c-Jun. On the contrary, both Jnk1-/- and Jnk2-/- cells were more sensitive to tumor necrosis factor - alpha (TNF-alpha) and sorbitol-induced cell death. Treatment of Jnk1-/- cells with these reagents resulted in reduced JNK activity and a concomitant reduction of c-Jun phosphorylation, suggesting that phosphorylation of c-Jun does not influence TNF-alpha and sorbitol-induced apoptosis in fibroblasts. Moreover, both JNK1 and JNK2 appear to negatively regulate apoptosis independent of c-Jun phosphorylation. These data provide genetic evidence that although the JNK pathway is activated by a plethora of signals, it is required only for the induction of UV-induced cell death in a c-Jun phosphorylation-dependent manner, but not for TNF-alpha and sorbitol-induced apoptosis.     

Influence of neoadjuvant therapy on VEGFR-2 levels in tumor and blood serum from breast cancer patients

Degree and trends of changes taking place in the angiogenic factors VEGF and VEGFR-2 were studied in cytosols and blood serum sampled from breast cancer patients in the course of neoadjuvant therapy. Such evidence was evaluated vis-alpha-vis basic indices for clinic-morphological features of the disease. The study included 30 cases of locally-advanced tumors. Our results pointed to an influence produced by preoperative therapy on the levels of angiogenic factors both in tumor and serum. However, trends of development of such parameters are different and independent of therapeutic modality. Correlation was established between VEGF and VEGFR-2 levels in residual tumor, on the one hand, and degree of therapeutic pathomorphosis, on the other.     

Additive effects of Cox-1 and Cox-2 inhibition on breast cancer in vitro

We hypothesized that combined treatment with Cox-1 and Cox-2 specific inhibitiors would exhibit synergistic effects against breast cancer in vitro. Two human breast cancer cell lines (HTB26, MCF-7) were treated with catechin (Cox-1 inhibitor) or NS398 (Cox-2 inhibitor) at 100 microM as both single and combined treatments. Reductions in cell growth were observed in both cell lines at 24 and 72 h in both single and combined treatments (p<0.001). Combined treatment produced a significantly greater inhibition as compared to single agents alone. Upon cell cycle evaluation, Cox-1 and -2 antagonism increased G1 and G2 phase fractions in MCF-7 cells (p<0.001 and p<0.05 respectively). No additive changes were observed when the two agents were combined. An increase in the G2 phase was observed in the HTB26 cells when treated with NS398 alone (p<0.001). However, a decrease in the S-phase was observed when these cells were treated with NS398, as a single agent (p<0.01) or when the two agents were combined (p<0.01). The significant and additive effects exhibited by the combination of Cox-1 and -2 inhibitors and their effects on cell cycle suggest that these agents could become an effective treatment modality for carcinoma of the breast.