Relationship between Expression of beta-catenin and VEGFs （VEGFA, VEGF-C）, VEGF Receptors-2 （VEGFR-2） in Medulloblastoma

Objective： To investigate the expression of beta-catenin and VEGFs （VEGF-A, VEGF-C） and VEGF receptor-2 （VEGFR-2） protein in medulloblastoma. Methods： Immunohistochemical staining with SP method was conducted to determine the expression of beta-catenin and VEGFs （VEGF-A, VEGF-C） and VEGFR-2 in 33 cases of medulloblastoma and 10 normal cerebellar tissues. Results： The expression rate of beta-catenin, and VEGFs （VEGF-A, VEGF-C） and VEGFR-2 in medulloblastoma were significantly higher than that in normal tissue. A significant positive correlation was found between beta-catenin and VEGFs （VEGF-A, VEGF-C） and VEGFR-2 protein in medulloblastoma. Conclusion： There was a correlation between beta-catenin and VEGFs （VEGF-A, VEGF-C） and VEGFR-2 in medulloblastoma, which may play a role in the pathogenesis and development of medulloblastoma.     

Vascular endothelial growth factor-D is an independent prognostic factor in epithelial ovarian carcinoma

We assessed the presence of vascular endothelial growth factor (VEGF)-C, VEGF-D and their receptor VEGFR-3 by immunohistochemistry in 59 epithelial ovarian carcinomas, 11 borderline tumours and 20 benign cystadenomas. VEGF-C and VEGF-D were generally expressed in tumour cells and also in endothelia adjacent to tumour nests which showed a strong staining for them. VEGFR-3 was expressed in lymphatic and vascular endothelial cells adjacent to tumour nests. Immunoreactivity was significantly more frequent as lesions progressed from a benign tumour to advanced carcinoma. A strong correlation was found between VEGF-C and VEGF-D detected in carcinoma and VEGFR-3 detected in neighbouring endothelial cells. Increased expression of VEGF-C, VEGF-D and VEGFR-3 was significantly associated with lymph node metastasis and peritoneal metastasis outside the pelvis. There was a significant correlation between the high levels of VEGF-C and VEGF-D proteins, and poor survival. The presence of VEGF-D was an independent prognostic indicator by multivariate analysis. We conclude that VEGF-C, VEGF-D and VEGFR-3 play an important role in lymphatic spread and intraperitoneal tumour development in ovarian carcinoma. Since VEGF-D was found to be an independent predictor of poor outcome, its measurement, together with other prognostic markers may improve prospective identification of patients with a poor prognosis.     

Crosstalk between angiogenesis and lymphangiogenesis in tumor progression.

Extensive studies have identified reliable markers of lymphatic endothelial cells, and mechanisms and molecules that regulate development and growth of the lymphatic vessels. Vascular endothelial growth factors (VEGF)-C and VEGF-D, and their cognate receptor tyrosine kinase, VEGF receptor-3 (VEGFR-3), are critical regulators of lymphangiogenesis. By binding to its endothelial cell surface receptors VEGFR-1 and VEGFR-2, VEGF-A mediates vascular leakage, endothelial proliferation and migration. Angiopoietin-2 (Ang-2) is expressed at sites of blood vessel remodeling and invasion, and factors that induce angiogenesis in vivo, such as VEGF-A, upregulate Ang-2 in endothelial cells. In this review, we summarize the literature concerning the crosstalk between angiogenesis and lymphangiogenesis in tumor progression, that is, involvement of VEGF-C, VEGF-D and VEGFR-3 in angiogenesis, and the role played by VEGF-A and Ang-2 in lymphangiogenesis, respectively.     

Expression of VEGF-C, VEGF-D and their receptor VEGFR-3 in diffuse large B-cell lymphomas

Lymphangiogenesis-the new growth of lymphatic vessels is an important route for the metastatic spread of human cancer. The receptor tyrosine kinase VEGFR-3 is expressed predominantly on lymphatic endothelium, and activation by its ligands VEGF-C and VEGF-D induces lymhpangiogenesis. VEGF-C, VEGF-D and VEGFR-3 have been found to play an important role in the lymphangiogenesis of several cancers. The present study investigated the expression of these factors by immunohistochemical staining of diagnosis specimens from 38 patients with diffuse large B-cell lymphoma (DLBCL). VEGF-C, VEGF-D and VEGFR-3 were expressed in both lymphoma cells and endothelial cells of blood and lymphatic tissue in all but one patient (who was negative for VEGF-D in lymphoma). There was a significant correlation in the intensity of staining between VEGF-C and -D in lymphoma and blood vessels (P < 0.001), and between the intensity of staining of VEGF-D and the patient International Prognostic Index score (P = 0.049) and borderline significance with overall survival (P = 0.051). Mean microvessel count was 58 (range 23-120), and it increased in association with high-intensity VEGF-C staining in lymphoma cells. Our findings indicate the importance of lymphangiogenic factors in the pathogenesis of DLBCL and suggest a potential therapeutic role for antilymphangiogenesis agents.     

Expression of the vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligand VEGF-C in human colorectal adenocarcinoma

Vascular endothelial growth factors (VEGF) are secreted by many tumor types, and are believed to affect tumor growth by promoting angiogenesis through binding to their receptors present on vascular endothelium. Recently, mRNA for VEGF-C the ligand for VEGFR-3, was found to be up-regulated in colorectal adenocarcinoma (CRC). The aim of this work was to determine: 1) the distribution of VEGF-C and VEGFR-3 in CRC, and 2) the biological significance of such expression. Sections of formalin-fixed and paraffin-embedded tissues from 56 CRC were immunohistochemically stained for VEGF-C and VEGFR-3. The type and percent of positive cells was recorded. Survival analysis was performed using the Kaplan-Meier method. All CRC were positive for VEGF-C which was present in the cancer cells themselves, as well as in stromal cells. Normal colon epithelium was usually negative. Only ten (17%) of the 56 CRC completely lacked VEGFR-3 expression. VEGFR-3 immunoreactivity was detected in <25% of the cancer cells in 22 cases and in >25% of the cells in 34 cases. Expression of VEGFR-3 in >25% of the cancer cells was associated with significantly poorer overall survival (p<0.05), but not with lymph node metastasis or depth of tumor invasion. Our results suggest that VEGFs promote cancer growth not only by stimulating angiogenesis, but also by acting on receptors present on the cancer cells themselves.     

VEGF-A and VEGFR-3 correlate with nodal status in operable non-small cell lung cancer: inverse correlation between expression in tumor and stromal cells

BACKGROUND: Lymph node metastasis is an essential determinant for stage and clinical management of non-small cell lung cancer (NSCLC). The vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are fundamental molecules in angiogenesis and lymphangiogenesis. We aimed to explore the correlations between nodal metastasis and the expression of VEGFs and VEGFRs in tumor cells and in tumor-related stroma. PATIENTS AND METHODS: Tumor tissue samples from 335 resected patients with stage I-IIIA NSCLC were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and surrounding stromal tissue from each resected specimen. Immunohistochemistry was used to evaluate the expression of VEGF-A, VEGF-C, and VEGF-D and VEGFR-1, VEGFR-2 and VEGFR-3. RESULTS: There were 232 N0 and 103 N+ patients (76 N1, 27 N2). In multivariate analyses, low stromal VEGF-A expression (P=0.018) is associated with N+ status. In tumor cells, strong correlations exist between high VEGF-A expression (P=0.032) and N+ status, and high VEGFR-3 expression (P<0.001) and N2-status. CONCLUSION: The converse impact by stromal VEGF-A versus tumor cell VEGF-A expression on nodal metastasis may allude the importance of the tumor-stroma interaction when trying to understand lymphatic metastasis in NSCLC.     

Lymphangiogenesis in kidney cancer: expression of VEGF-C, VEGF-D and VEGFR-3 in clear cell and papillary renal cell carcinoma

The vascular endothelial growth factors VEGF-C, VEGF-D and its receptor, VEGFR-3, are overexpressed in different malignancies and associated with lymph node metastasis and poor prognosis. We analysed these factors in clear cell (ccRCC) and papillary (pRCC) renal cell carcinoma (RCC). The results were correlated with various clinicopathological parameters (CPP). We constructed a tissue microarray with tumor samples of 135 (81%) ccRCC and 31 (19%) pRCC. After immunohistochemical staining using polyclonal antibodies for VEGF-C, VEGF-D and VEGFR-3, a semiquantitative analysis was performed to determine the levels of expression. The results were compared between the two subgroups and were correlated with CPP. In the two subgroups the expression of VEGF-C was significantly correlated with that of VEGF-D (p<0.001). There was an increased expression of VEGF-C in 11% of ccRCC and 36% of pRCC (p=0.002). VEGF-D expression was positive by means of analysis in 22% of ccRCC and 42% of pRCC (p=0.039). There was no significant difference regarding the expression of VEGFR-3 between the subgroups (44% ccRCC and 61% pRCC, p=0.11). No correlation was found between the expression of the analysed parameters and CPP (TNM, grading, progression-free survival and overall survival) in either the entire group or in the two subgroups. In summary, ccRCC and pRCC show a different expression pattern of the analysed lymphangiogenic factors. Further studies are necessary to confirm these results and to determine whether the VEGF-C/VEGF-D/VEGFR-3-axis can play a role as a prognostic tool or a target for therapeutic intervention in renal cell carcinoma.     

Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival. A number of clinical and experimental studies suggest that tumor-induced lymphangiogenesis driven by vascular endothelial growth factor (VEGF)-C- and/or VEGF-D-induced activation of VEGF receptor (VEGFR)-3 may promote metastasis to regional lymph nodes. Here we show that constitutive VEGF-C and VEGF-D expression by tumor cells of diverse origin grown in tissue culture does not correlate with metastatic potential in vivo. However, tumors derived from cell lines that do not constitutively express VEGF-C or VEGF-D in tissue culture can nevertheless express one or both of these factors. We demonstrate that both tumor and stromal cells can contribute to this expression, suggesting that tumor cell-host interactions determine tumor expression of VEGF-C and VEGF-D. Using immunocompetent rat mammary tumor models, we show in two ways that this expression can promote metastasis via the lymphatics. Firstly, ectopic expression of a soluble VEGFR-3 receptor globulin protein in MT-450 tumor cells that are highly metastatic via the lymphatics blocked VEGF-C and VEGF-D activity and suppressed metastasis formation in both the regional lymph nodes and the lungs. Secondly, ectopic expression in the weakly metastatic NM-081 cell line of a mutant form of VEGF-C that is only able to activate VEGFR-3 strongly promoted metastasis of these cells to the regional lymph nodes and lung. These data show that expression of VEGF-C and VEGF-D in tissue culture does not reflect expression in vivo and that activation of VEGFR-3 in the absence of VEGFR-2 activation is sufficient to promote tumor-induced lymphangiogenesis and metastasis, and they support the notion that blockade of VEGFR-3 activation will be useful as a novel form of cancer therapy.     

The relevance of cell type- and tumor zone-specific VEGFR-2 activation in locally advanced colon cancer

Background

For the successful therapeutic use of inhibitors of the vascular endothelial growth factor receptor (VEGFR) pathway detailed knowledge of the mechanisms leading to tumor progression is indispensable. The main goal of this study was to determine the relevance of the VEGFR-2 activating pathway for colon carcinoma (CC) metastasis. The initial event is ligand-induced receptor activation through tyrosine autophosphorylation.

Methods

VEGFR-2, its ligands VEGF-C and VEGF-D and the phosphorylated (activated) receptor forms pVEGFR-2^Tyr1175 and pVEGFR-2^Tyr1214 were investigated immunohistochemically in different tumor compartments (intratumoral (zone 1) - invasive front (zone 2) – extratumoral soft tissue (zone 3)) and various cell types (tumor cells, inflammatory cells, macro- and microvasculature) in 84 non-metastatic, lymphogenous-metastatic and haematogenous-metastatic CC.

Results

VEGF-D produced by tumor cells has an autocrine affinity for its receptor VEGFR-2. In tumor budding regions VEGF-D-induced receptor activation by autophosphorylation at Tyr1214 seems to be a possible initial event of the VEGFR-2-mediated signaling pathway, but without effect on metastatic behaviour. In inflammatory cells of almost all CC VEGFR-2 phosphorylation at Tyr 1175 and Tyr 1214 was detectable without accompanying receptor expression, suggesting receptor activation without cell surface expression. Peritumoral inflammatory cells also expressed paracrine acting VEGF-C. The autocrine VEGF-D/VEGFR-2 signaling axis and receptor autophosphorylation at Tyr1214 appear to be main events for capillaries in all three tumor zones and for small vessels in zone 1 and 2. Independent of the metastatic status a large number of cases with capillary immunopositivity in the angiogenically active invasive front was documented, especially for VEGF-D, VEGFR-2 and pVEGFR-2^Tyr1214. VEGFR-2 positive extratumoral capillaries were significantly more common in distant metastatic CC. In all tumor compartments the investigated biomolecules were also detected in different frequencies in the macrovasculature, which is responsible for sufficient tumor vascularization. In addition, vascular paracrine-acting VEGF-C production was widely detected, but without zone and vessel-type dependence.

Conclusions

The VEGFR-2 activating pathway is closely involved in tumor cell-associated, vessel-mediated and immuno-inflammatory processes in colon carcinoma and appears to contribute to tumor survival and growth as well as maintenance of the infiltrative phenotype rather than to promote metastasis.     

Clinicopathological and prognostic significance of vascular endothelial growth factors (VEGF)-C and -D and VEGF receptor 3 in invasive breast carcinoma.

AIMS: Vascular endothelial growth factors C and D (VEGF-C and VEGF-D) play a major role in lymphangiogenesis and activate VEGF receptor 3 (VEGFR-3). Our purpose was to study the clinicopathologic and clinical value of VEGF-C, VEGF-D and VEGFR-3 in invasive breast carcinoma. MATERIAL AND METHODS: Immunohistochemistry was performed in paraffin-embedded tissue specimens from 177 invasive breast carcinomas to detect the proteins VEGF-C, VEGF-D, VEGFR-3, p53, Ki67, c-erbB-2, topoII alpha and ER/PR. The results were statistically processed. RESULTS: VEGF-C, VEGF-D and VEGFR-3 were found to be predominantly expressed in the cytoplasm of the malignant cells. VEGF-C occasionally showed a submembranous intensification. VEGF-D and VEGFR-3 were also immunodetected in the nuclei of the malignant cells. Nuclear VEGF-D was positively correlated to p53, Ki67 and topoII alpha proteins' expression (p=0.003, p=0.009 and p=0.017 respectively) and nuclear VEGFR-3 to topoII alpha (p=0.034). Cytoplasmic expression of VEGF-C and its submembranous intensification were found to be independent indicators of patients' overall and disease-free survival, respectively (p=0.003 and p=0.044 respectively). The group with high expression of both cytoplasmic VEGF-C and stromal VEGFR-3 showed poor overall survival (p=0.024) and the group with both submembranous VEGF-C and stromal VEGFR-3 immunostaining showed poor both disease-free and overall survival (p=0.012 and p=0.038 respectively). CONCLUSION: VEGF-D and VEGFR-3 seem to exert proliferative activity in invasive breast carcinomas. VEGF-C was found to be an independent indicator of patient's poor prognosis and the simultaneous expression of tumor VEGF-C and stromal VEGFR-3 yielded additional prognostic information.     

The association between vascular endothelial growth factor-C, its corresponding receptor, VEGFR-3, and prognosis in primary breast cancer: a study with 193 cases

Lymphangiogenesis plays an important role in several normal and pathological conditions, such as wound healing, pathogen infection, inflammation or the metastasis formation of endothelial malignancies. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. Both growth factors mediate their biological activity mainly by VEGF receptor-3 (VEGFR-3, Flt-4). In this study, we measured intratumoral levels of VEGF-C and VEGFR-3 through enzyme-linked immunosorbent assay (ELISA) in 193 primary breast cancer tissues and examined their prognostic values. A significant correlation was found between the VEGF-C and VEGFR-3 protein levels. High VEGF-C levels were associated with low-grade tumors and a smaller size. Univariate analysis showed that high VEGF-C was significantly associated with a favourable prognosis for disease-free survival (DFS) and overall survival (OS). No significant prognostic value of VEGFR-3 was detected. Multivariate analysis confirmed the independent prognostic value of VEGF-C. The intratumoral VEGF-C level is a significant prognostic indicator of primary breast cancer. An investigation of the mechanisms of VEGF-C protein processing in human cancer tissue should be carried out in the future.     

ErbB2 overexpression correlates with increased expression of vascular endothelial growth factors A,C, and D in human breast carcinoma

BACKGROUND: The angiogenic factor vascular endothelial growth factor (VEGF)-A plays an important role in breast cancer progression. However, the involvement of VEGF-C and VEGF-D, two newer members of the VEGF family, in breast carcinoma and their relationship with clinicopathologic parameters have not been clearly demonstrated. METHODS: In this study, the expression levels of VEGF-A, VEGF-C, and VEGF-D protein in 107 breast carcinoma cases and 22 nonmalignant breast tissue samples were examined by immunohistochemistry and quantitated by image analysis. RESULTS: Higher expression of VEGF-C and VEGF-D was found in breast carcinomas than in nonmalignant breast tissue samples. Moreover, expression of VEGF-A, VEGF-C, and VEGF-D was significantly and positively correlated with ErbB2 expression. High levels of VEGF-A expression were associated with shorter disease-free survival (DFS). Patients with tumors expressing high levels of VEGF-C or VEGF-D showed a notable trend for worse DFS, however, it was not statistically significant. The combination of VEGF-A and VEGF-C status predicted survival better than either marker alone. CONCLUSIONS: Our study suggests that expression of the angiogenic and lymphangiogenic factors (i.e., VEGFs) might be regulated at least in part by ErbB2. In addition, the combination of VEGF-A and VEGF-C status may better predict prognosis of patients with breast carcinoma than VEGF-A alone.     

The role of the VEGF-C/VEGFR-3 axis in cancer progression

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) (also called VEGFR-3) is activated by its specific ligand, VEGF-C, which promotes cancer progression. The VEGF-C/VEGFR-3 axis is expressed not only by lymphatic endothelial cells but also by a variety of human tumour cells. Activation of the VEGF-C/VEGFR-3 axis in lymphatic endothelial cells can facilitate metastasis by increasing the formation of lymphatic vessels (lymphangiogenesis) within and around tumours. The VEGF-C/VEGFR-3 axis plays a critical role in leukaemic cell proliferation, survival, and resistance to chemotherapy. Moreover, activation of the VEGF-C/VEGFR-3 axis in several types of solid tumours enhances cancer cell mobility and invasion capabilities, promoting cancer cell metastasis. In this review, we discuss the novel function and molecular mechanism of the VEGF-C/VEGFR-3 axis in cancer progression.