2324

FLT4

fms-related tyrosine kinase 4;FLT4;fms-related tyrosine kinase 4

FLT-4|VEGFR-3|fms-like tyrosine kinase 4|soluble VEGFR3 variant 1|soluble VEGFR3 variant 2|soluble VEGFR3 variant 3|tyrosine-protein kinase receptor FLT4|vascular endothelial growth factor receptor 3

5q35.3

protein-coding

BACKGROUND & OBJECTIVE: Vascular endothelial growth factor-C(VEGF-C) is a member of VEGF family and the only factor that can combine receptor VEGFR-3 (fms-like tyrosine kinase, Flt-4) located at endothelium of lymphatic vessel and modulates the physiological function of lymphatic vessel. Previous study showed that VEGF-C/Flt-4 system play a modulating role in metastasis of many kinds of tumors, but there were few reports about its function for primary breast cancer at home and abroad. The objective of this study was to identify the function of VEGF-C/Flt-4 system in proliferation and metastasis of primary breast cancer and its significance. METHODS: A series of 101 primary breast cancer specimens were detected for the expression of VEGF-C, Flt-4,and PCNA by Immunohistochemical methods. RESULTS: Among 101 cases of breast cancer, the positive rate of VEGF-C was 93.1% (94/101), the positive rate of flt-4 was 86.1% (87/101). With the increase of the expression of VEGF-C, the positive index of flt-4 increased (r=0.816,P< 0.001). The positive rate of PCNA was 88.8% (89/101). With the increase of the expression of VEGF-C, the proliferation activity of PCNA was stronger (r=0.673,P< 0.001). The positive index of VEGF-C in lymph node metastases group (61.89+/-17.79) was significantly higher than that of no lymph node metastasis group (44.28+/-17.87)(P< 0.05). With the increase of VEGF-C protein level, the number of flt-4 positive vessels increased and significant differences among these groups were observed (P< 0.001). The number of flt-4 positive vessels in lymph node metastases group (15.55+/-3.63)was significantly higher than that of no lymph node metastasis group (10.71+/-2.90 ) (P< 0.05). CONCLUSION: VEGF-C and Flt-4 are overexpressed in primary breast cancer and related to lymph node metastasis. VEGF-C can promote proliferation of breast cancer cell. VEGF-C/flt-4 system can promote vasculogenesis in stroma of breast cancer. The number of Flt-4 positive vessels is closely related to lymph node metastasis.

metastasis to regional lymph nodes is an important and early event in many tumors. Vascular endothelial growth factor-C (VEGF-C), VEGF-D and their receptor VEGFR-3, play a role in tumor spread via the lymphatics, although the timing of their involvement is not understood. In contrast, VEGFR-2, activated by VEGF-A, VEGF-C and VEGF-D, is a mediator of angiogenesis and drives primary tumor growth. We demonstrate the critical role for VEGFR-3, but not VEGFR-2, in the early events of metastasis. In a tumor model exhibiting both VEGF-D-dependent angiogenesis and lymphangiogenesis, an antibody to VEGFR-2 (DC101) was capable of inhibiting angiogenesis (79 % reduction in PECAM + blood vessels) and growth (93 % reduction in tumor volume). However, unlike an anti-VEGFR-3 Mab (mF4-31C1), DC101 was not capable of eliminating either tumor lymphangiogenesis or lymphogenous metastasis (60 % reduction of lymph node metastasis by DC101 vs 95 % by mF4-31C1). Early excision of the primary tumors demonstrated that VEGF-D-mediated tumor spread precedes angiogenesis-induced growth. Small but highly metastatic primary human breast cancers had significantly higher lymphatic vessel density (23.1 vessels/mm(2)) than size-matched (11.7) or larger non-metastatic tumors (12.4) thus supporting the importance of lymphatic vessels, as opposed to angiogenesis-mediated primary tumor growth, for nodal metastasis. These results suggest that lymphangiogenesis via VEGF-D is more critical than angiogenesis for nodal metastasis.

Vascular endothelial growth factor receptor 3 (VEGFR-3) is a major inducer of lymphangiogenic signalling and seems to be involved also in angiogenesis. Since both processes are closely linked with tumor metastasis this study investigated the expression of VEGFR-3 in tumor-associated vessels in colorectal carcinomas and evaluated its relevance for lymphogenous and hematogenous metastasis. In a comparative study between microvascular endothelial cells isolated from the tumor (HCTEC) and the corresponding non-neoplastic tissue (HCMEC) from five patients with colorectal cancer VEGFR-3 expression was measured using a specific ELISA. The expression pattern was individually different, with cases showing reduced, elevated and unchanged protein levels. Under hypoxic culture conditions (3% O2 for 24 h), which are more realistic for the tumor situation, the levels remained unchanged. In contrast, hypoxia exposure of macrovascular human umbilical vein endothelial cells (HUVEC) led to a consistent downregulation of VEGFR-3 protein. These data indicate a 'hypoxia-resistant' behaviour of VEGFR-3 in colonic microvasculature. Using immunohistochemistry the endothelial expression pattern of VEGFR-3 in 74 non-metastatic, lymphogenously-metastatic and hematogenously-metastatic colorectal carcinoma specimens was assessed. Positive VEGFR-3 expression was highly significantly associated with those cases showing distant metastasis (p=0.0003). In contrast, significant differences in the expression of VEGFR-3 between non-metastatic tumors and carcinomas with lymph node metastasis were not found. The majority of the detectable intratumoral VEGFR-3-positive vessels were of blood vascular origin (CD31 positive, D2-40 negative). Whereas intratumoral lymphatic vessels were collapsed, VEGFR-3 positive peritumoral lymphatic vessels had mostly open lumina. These morphological observations provide evidence for a predominant significance of VEGFR-3-positive, possibly angiogenesis-mediated, tumor-associated blood vessels in hematogenous metastasis of colorectal cancer. In addition, due to their patency VEGFR-3-positive peritumoral, but not intratumoral lymphatics could be the vascular substrate functionally mediating lymphogenous metastasis.

Aberrant activation of the hedgehog (Hh) signaling pathway is associated with tumorigenesis in various tissues. In colorectal cancer (CRC), evidence for Hh activation is inconsistent, and the relationship between the Hh signaling pathway and lymphangiogenesis has not been studied. The aim of this study was to determine the relationship between Hh signaling and lymphangiogenesis and the association of this relationship with lymph node metastasis in CRC. We investigated 189 patients who underwent curative surgical resection for CRC between 2002 and 2004 at Korea University Guro Hospital. Paraffin-embedded specimens of colorectal adenocarcinoma and adjacent normal mucosa were evaluated. Immunohistochemical staining for Sonic hedgehog (Shh), Gli1, vascular endothelial growth factor C (VEGFC), and VEGF receptor 3 (VEGFR3) was performed for each specimen. tumor specimen showed significantly strong staining of Shh, Gli1, VEGFC, and VEGFR3 compared with a normal specimen. Shh expression was not associated with Gli1 expression. Gli1 expression correlated positively with VEGFC and VEGFR3 expression (P < 0.05 in both) but not with lymph node metastasis. Activation of the Hh signaling pathway associated with Gli1 promotes expression of lymphangiogenesis proteins, VEGFC and VEGFR3, in CRC. Further studies are necessary to determine the association of this relationship with lymph node metastasis in CRC.

AIM: To investigate the expression of pathological factors of VEGF-C and its receptor FLT-4 in primary gastric cancer and adjacent normal tissues. METHODS: The expression of VEGF-C and FLT-4 was studied in 80 primary gastric cancers and adjacent normal tissues from the same patients by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immumohistochemistry. RESULTS: Both primary gastric cancer and adjacent normal tissue could express VEGF-C and FLT-4, and FLT-4 expression was also detected in endothelial cells of stromal blood vessels and lymphatic vessels. There was a significant difference in expression of VEGF-C between primary tumor and adjacent normal tissue samples (P=0.01), and a statistical correlation between VEGF-C and FLT-4 expression in tumors (P=0.00886). With regard to VEGF-C expression, there was a significant difference between moderate-poor differential type and high differential type (P=0.032), and a significant difference between positive and negative lymph node metastases (P=0.024). However, there was no significant difference between positive and negative serosal invasions (P=0.219). CONCLUSION: VEGF-C and its receptor FLT-4 play a role in the development of gastric cancer, and the tumors with expression of VEGF-C and FLT-4 are more likely to have lymph node metastasis.

AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their levels and clinicopathological parameters in gastric cancer. METHODS: Tissue samples were obtained from 33 patients (8 females) with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS: LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic features such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the distribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION: LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.

OBJECTIVE: To investigate the expression of VEGF-C and its receptor FLT-4 in laryngeal and hypopharyngeal squamous cell carcinoma, and to analyse its role in lymphatic metastasis and micrometastasis. METHOD: Forty nine cases postoperative specimens verified by histopathology were studied. The expression of VEGF-C, FLT-4 were detected with immunohistochemical SP method, image analysis method. The clinicopathological parameters of tumor were compared with the immunohistochemical results of VEGF-C and FLT-4, followed by the further analysis on the relationship between VEGF-C/FLT-4 expression and lymph nodal metastasis or micrometastasis. RESULT: There was a statistical correlation between the expression of VEGF-C and grades of pathological differentiation in laryngeal and hypopharyngeal squamous cell carcinoma. Moreover there was also a statistical correlation between the expression of VEGF-C and lymph node metastases. But no correlation was observed between the expression of VEGF-C and clinical T grades. The statistical correlation also existed between VEGF-C and FLT-4 expression in laryngeal or hypopharyngeal tumors. There was a significant difference of expression of FLT-4 in laryngeal and hypopharyngeal tumours with positive and negative lymph node metastases. No correlation was observed between FLT-4 and grades of pathological differentiation or clinical T grades. There was no statistical correlation between the expression of VEGF-C/FLT-4 and nodal micrometastasis (P > 0.05). CONCLUSION: VEGF-C and its receptor FLT-4 play an important role in the lymphatic metastasis of laryngeal and hypopharyngeal squamous cell carcinoma. VEGF-C/FLT-4 may be a useful biological marker to foresee the cervical lymph node metastasis and prognosis.

OBJECTIVES: expression of vascular endothelial growth factor C (VEGF-C)and vascular endothelial growth factor receptor-3 (VEGFR-3) in laryngeal squamous carcinoma and its relationship to lymph node metastasis were investigated. METHODS: VEGF-C and VEGFR-3 gene expression in 30 cases of normal laryngeal mucosa tissue (NLM), primary laryngeal carcinoma cell carcinomas (PLC) and cervical lymph nodes (CLN) was examined by reverse transcription polymerase chain reaction (RT-PCR). Protein levels of VEGF-C expression were determined by immunohistochemical staining in 60 cases of PLC. RESULTS: expression of VEGF-C and VEGFR-3 different among NLM, PLC and CLN in the same patient. In PLC, expression was significantly higher in lymph node positive group than in the lymph node negative group and associated with histological grade of differentiation; expression of VEGF-C and VEGFR-3 was not linked with age, sex, site or T stage. CONCLUSIONS: A close correlation was found between VEGF-C/VEGFR-3 expression and lymph node metastasis in PLC, suggesting a role in metastasis of laryngeal carcinomas.

Regional lymph node metastasis is one of the important indicators of cutaneous malignant melanoma. Newly formed lymphatic vessels are considered to provide a route whereby tumor cells can migrate to the lymph nodes. Both vascular endothelial growth factors (VEGF) -C and -D have been confirmed to participate in tumor lymphangiogenesis, but the prognostic significance of VEGF-C, VEGF-D, and lymphangiogenesis in cutaneous malignant melanoma remains controversial. To clarify the effects of these factors and to evaluate the relationships between lymphangiogenesis, lymph node metastasis, and prognosis in patients with malignant melanoma, the expressions of VEGF-C, VEGF-D, and their receptor (VEGFR) -3 were detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction. The expressions of both VEGF-C and VEGF-D proteins were concomitantly detected in the cytoplasm of the malignant cells. VEGF-C and VEGF-D expressions were associated with VEGFR-3 expression and were significantly correlated with both peritumoral lymphangiogenesis and lymph node metastasis. The incidence of peritumoral lymphatic vessels was significantly higher in lymph node metastatic melanomas than that in nonmetastatic melanomas. Univariate and multivariate analyses indicated that VEGF-C and VEGF-D were independent prognostic factors for overall survival and disease-free survival in patients with malignant melanoma. This study suggests that both VEGF-C and VEGF-D are involved in peritumoral lymphangiogenesis and lymphatic metastasis. VEGF-C and VEGF-D expression may be clinically useful indicators for prognostic evaluation in patients with cutaneous malignant melanoma.

Endothelin receptor B (ET(B)R) is a G-protein-coupled receptor overexpressed in melanoma, blood, and lymphatic endothelial cells. Given that aberrant signal transduction can be mediated through cross talk between receptors, here, we explore the functional relationship between ET(B)R and the vascular endothelial growth factor receptor (VEGFR)-3 system and how this cross talk might influence the aggressive behavior of melanoma cells. The expression of VEGFR-3 and its ligands, VEGF-C and VEGF-D, significantly increased after activating ET(B)R by ET-1 in primary and metastatic melanoma cell lines. These effects, similarly to those induced by hypoxia, were mediated by hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha. ET-1 caused the phosphorylation of VEGFR-3, which was accompanied by the activation of the downstream signaling molecules, such as MAPK and AKT. Inhibition of c-Src activity or silencing of the scaffold protein beta-arrestin-1 reduced ET-1-induced VEGFR-3 phosphorylation, demonstrating that, upon ET-1 stimulus, beta-arrestin-1 is involved with c-Src in the ET(B)R-mediated VEGFR-3 transactivation. Moreover, ET-1 in combination with VEGF-C further increased VEGFR-3, MAPK, and AKT phosphorylation and markedly promoted cell migration and vasculogenic mimicry. Dual inhibition of ET(B)R and VEGFR-3 was required for the effective inhibition of these effects, as well as for VEGFR-3 phosphorylation, demonstrating that ET(B)R cross talk with VEGFR-3 enhances cell plasticity and motility. Finally, in melanoma xenografts, ET(B)R antagonist inhibited tumor growth and the activation of the VEGF-C/VEGFR-3 axis, indicating that targeting ET(B)R may improve melanoma treatment acting directly or indirectly by impairing ET(B)R cross talk with VEGFR-3.

A positive association between vascular endothelial growth factor-C (VEGF-C) expression and lymph node metastasis has been reported in several cancers. However, the relationship of VEGF-C and lymph node metastasis in some cancers, including non-small cell lung cancer (NSCLC), is controversial. We evaluated the VEGF-C and vascular endothelial growth factor receptor-3 (VEGFR-3) expression in NSCLC samples from patients who had undergone surgery between 1998 and 2002 using real-time quantitative RT-PCR and immunohistochemical staining. We failed to find a positive association between VEGF-C and VEGFR-3 mRNA expression and lymph node metastasis in NSCLC. An immunohistological study demonstrated that VEGF-C was expressed not only in cancer cells, but also in macrophages in NSCLC, and that VEGFR-3 was expressed in cancer cells, macrophages, type II pneumocytes and lymph vessels. The VEGF-C/VEGFR-3 ratio of the node-positive group was significantly higher than that of the node-negative group. Immunohistochemical staining showed that VEGFR-3 was mainly expressed in cancer cells. The immunoreactivity of VEGF-C and VEGFR-3 was roughly correlated to the mRNA levels of VEGF-C and VEGFR-3 in real-time PCR. VEGF-C mRNA alone has no positive association with lymph node metastasis in NSCLC. The VEGF-C/VEGFR-3 ratio was positively associated with lymph node metastasis in NSCLC. This suggests that VEGF-C promotes lymph node metastasis while being influenced by the strength of the VEGF-C autocrine loop, and the VEGF-C/VEGFR-3 ratio can be a useful predictor of lymph node metastasis in NSCLC.

OBJECTIVE: To explore the role of vascular endothelial growth factor C (VEGF-C), VEGFR-3 and nm23-H(1) in lymphatic metastasis of oral squamous cell carcinoma (OSCC). METHODS: The expression of VEGF-C, VEGFR-3 and nm23-H(1) were examined by immunohistochemical SP method. The lymphatic vessels density (LVD) in tumors was counted and analyzed with clinicopathologic parameters. RESULTS: VEGF-C, LVD and nm23-H(1) expressions were significantly different between OSCC and benign and precancerous lesions (P < 0.05). VEGF-C was correlated with lymph node metastasis and LVD (P < 0.01). nm23-H(1) was related to clinical stage, histological grade and lymphatic metastasis (P < 0.05). CONCLUSIONS: The high expressions of VEGF-C/VEGFR-3 and inactivation of nm23-H(1) may play an important role in lymphatic metastasis in OSCC.

AIM: Lymph node metastasis is one of the predictive factors associated with poor prognosis of epithelial ovarian cancer. To clarify the role of CD34 and vascular endothelial growth factor receptor-3-positive (CD34+/VEGFR3+) lymphatic/vascular endothelial progenitor cells (LVEPC) in patients with lymph node metastasis and epithelial ovarian cancer progression, the levels of circulating CD34+/VEGFR3+ LVEPC in epithelial ovarian cancer patients were detected. We also tested the plasma protein levels of VEGF and stromal cell-derived factor to find out their possible relationships with lymph node metastasis in our epithelial ovarian cancer cohort. MATERIAL AND METHODS: Peripheral blood samples were collected from 54 patients diagnosed as epithelial ovarian cancer, and 31 normal samples as control. The circulating levels of LVEPC were carried out by flow cytometry, and blood protein levels of biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of circulating LVEPC was significantly higher in patients with ovarian cancer compared with that of healthy controls. There was also a statistically significant correlation between LVEPC levels and surgical staging of epithelial ovarian cancer (P < 0.01). CONCLUSION: The circulating levels of bone marrow-derived LVEPC are significantly increased in epithelial ovarian cancer patients and these levels correlate with lymph node metastasis too.

PURPOSE: The molecular mechanisms underlying lymph node metastasis are poorly understood, despite the well-established clinical importance of lymph node status in many human cancers. Recently, vascular endothelial growth factor (VEGF)-C and VEGF-D have been implicated in the regulation of tumor lymphangiogenesis and enhancement of lymphatic invasion via activation of VEGF receptor-3. The purpose of this study was to determine the expression pattern of the VEGF-C/VEGF-D/VEGF receptor-3 axis in prostate cancer and its relationship with lymph node metastasis. EXPERIMENTAL DESIGN: The expression pattern of VEGF-C, VEGF-D, and VEGF receptor-3 in localized prostate cancer specimens (n = 37) was determined using immunohistochemistry. RESULTS: Widespread, heterogeneous staining for VEGF-C and VEGF-D was observed in all cancer specimens. Intensity of VEGF-C staining was lower in benign prostate epithelium than in adjacent carcinoma, whereas no difference between benign epithelium and carcinoma was observed for VEGF-D staining. VEGF receptor-3 immunostaining was detected in endothelial cells of lymphatic vessels in 18 of 37 tissue samples. The presence of VEGF receptor-3-positive vessels was associated with lymph node metastasis (P = 0.0002), Gleason grade (P < 0.0001), extracapsular extension (P = 0.0382), and surgical margin status (P = 0.0069). In addition, VEGF receptor-3 staining highlighted lymphatic invasion by VEGF-C-positive/VEGF-D-positive carcinoma cells. CONCLUSIONS: Together, these results suggest that paracrine activation of lymphatic endothelial cell VEGF receptor-3 by VEGF-C and/or VEGF-D may be involved in lymphatic metastasis. Thus the VEGF-C/VEGF-D/VEGF receptor-3 signaling pathway may provide a target for antilymphangiogenic therapy in prostate cancer.

BACKGROUND: Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGFR-3, have been implicated as important factors in the formation of lymphatic vessels and recent evidence suggests that tumor lymphangiogenesis promotes lymphatic metastasis. METHODS: The expression of VEGF-C and VEGFR-3 was examined in 22 human prostate cancer specimens with immunohistochemistry. A semi-quantitative scoring system was used for evaluation of staining. RESULTS: expression of VEGF-C was stronger in prostate cancer areas in comparison to adjacent benign glands. In addition, patients with lymph node metastases had a significantly higher expression of VEGF-C than patients without lymph node metastases. Interestingly, VEGFR-3 was expressed in malignant prostate epithelial cells and its expression was significantly higher in the lymph node positive group compared to the lymph node negative group. CONCLUSIONS: The results of the present study indicate that increased expression of VEGF-C and VEGFR-3 play a role in prostate cancer progression and in metastasis to regional lymph nodes.

OBJECTIVE: To detect the expression of VEGF receptors in papillary renal cell carcinoma and to explore the correlation between their expression and clinical prognosis. METHODS: expression of VEGF receptors and PCNA (proliferating cell nuclear antigen) were evaluated in 82 patients with papillary renal cell carcinoma using tissue microarray and SP immunohistochemical staining. RESULTS: The expression of VEGFR-1 in papillary renal cell carcinoma was 82.93%, VEGFR-2 63.41%, VEGFR-3 34.15% and PCNA 67.07%, respectively. Increased VEGFR-2 expression was significantly correlated with tumor size (P = 0.016), histological grade (P = 0.034) and distant metastasis (P = 0.002). VEGFR-3 expression was correlated with histological grade (P = 0.028), lymph node status (P = 0.010) and distant metastasis (P = 0.018), but not correlated with gender, age, location, tumor size and TNM staging. VEGFR-1 expression had no correlation with any clinic and pathologic factors. PCNA expression was correlated with histological grade (P = 0.011), but not correlated with other factors. The expression of VEGFR-2 and VEGFR-3 in death cases were higher than that in surviving patients. CONCLUSION: Both VEGFR-2 and VEGFR-3 can serve as markers for prognosis of papillary renal cell carcinoma. Differently, VEGFR-3 is a predictor of lymph node metastasis, increased VEGFR-2 expression could be used to predict a potential blood dissemination.