RESUMO
Lymphangiogenesis critically contributes to the lymphatic metastasis of colorectal carcinomas (CRCs), but the underlying mechanism of CRC lymphangiogenesis remains largely elusive. We have previously demonstrated that Semaphorin-3F (SEMA3F) is critically involved in CRC metastasis, and the receptor of SEMA3F, neuropilin-2 (NRP2), originally described as an axon guiding chemorepulsant implicated in nerve development, has been suggested in promoting lymphangiogenesis via acting as an obligate co-receptor of VEGFR3 cooperatively enhancing the activity of VEGF-C. Our present study revealed that in colorectal carcinomas, NRP2 expression levels of tumor-associated lymphatic endothelial cells (LECs) are significantly correlated with the density of tumor lymphatic vessels. In vitro, activation of NRP2 in LECs substantially facilitates their migration, sprouting, and tubulogenesis capacity via regulating the rearrangement of cytoskeleton polarity. In vivo model further showed that in the xenografts generated from SEMA3F knockdown CRC cells, NRP2 is substantially activated in tumor-associated LECs, resulting in a significantly increased tumor lymphangiogenesis. Further evidence demonstrated that CRC cell induces the activation of NRP2 in LECs to promote tumor lymphangiogenesis via integrinα9ß1/FAK/Erk pathway independent VEGF-C/VEGFR3 signaling. Our study for the first time revealed the novel molecular mechanism of NRP2-mediated-lymphangiogenesis in CRCs, suggesting NRP2 as a potential therapeutic target in preventing lymphatic metastasis of CRCs.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Linfangiogênese , Neuropilina-2/metabolismo , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Integrinas/metabolismo , Linfangiogênese/genética , Camundongos , Neuropilina-2/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
PURPOSE: To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma. EXPERIMENTAL DESIGN: SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F. RESULTS: SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvß3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo. CONCLUSIONS: Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells.
Assuntos
Carcinoma/patologia , Proliferação de Células , Neoplasias Colorretais/patologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Axônios/metabolismo , Axônios/fisiologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/prevenção & controle , Linhagem Celular Tumoral , Inibição de Migração Celular/genética , Proliferação de Células/efeitos dos fármacos , Fatores Quimiotáticos/antagonistas & inibidores , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/prevenção & controle , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The objective of this study was to investigate whether tumor derived fibronectin alternatively spliced EDA domain has a lymphangiogenic potency on human lymphatic endothelial cells (LECs) in tumor generation to facilitate tumor lymphatic metastasis. LECs were cultured in three-dimentional culture system and treated with SW480 supernant which was highly rich in EDA, the result demonstrated that SW480 supernant could facilitate tube-like formations of LECs evidently when compared with controls. Integrinalpha9 was identified by immunofluorescence to be a specific receptor for EDA because we found co-locozation of EDA and integrinalpha9 on LECs as well as significant upregulation of integrinalpha9 in SW480 supernant treated group. Western blot and immunofluorescence revealed that EDA also had important roles accommodating the expressions of some key regulators of lymphangiogenesis such as Prox1 and F-actin so as to facilitate motility and sprouting of LECs. In addition, it had been confirmed that all of these effects could be inhibited markedly by EDA antibody (IST-9). Based on these findings, we assert that EDA derived from tumor cells has an important role in facilitating lymphangiogenesis of malignant tumor. Furthermore, EDA pathway may provide a potent target for tumor lymphatic metastasis therapy.