| Background & Objective:Slit, a secreted protein, functions through the Roundabout (Robo) receptor as a chemorepellent in axon guidance and neuronal migration, and as an inhibitor in leukocyte chemotaxis. Recent studies have shown that Slit-Robo signaling can mediate the crosstalk between tumor cells and endothelial cells, thus promotes tumor-induced angiogenesis. Therefore, targeting Slit-Robo signaling may offer a novel molecular target for inhibiting tumor growrh. However, it has not been reported whether Slit-Robo signaling mediates the crosstalk between tumor cells and tumor cells and inhinites tumor growth or not. Furthermore, it is still indefinite of mechanism of Slit-Robo signaling in angiogenesis, proliferation, apoptosis and metastasis at the process of carcinogenesis. The current study aim to reveal the mechanism of Slit-Robo signaling in oral squamous cell carcinogenesis and growth, and provide a novel molecular target for oral squamous cell carcinoma treatment.Methods:We select a multistage model of 7, 12-dimethyl-1,2-benzanthracene (DMBA) -induced squamous cell carcinoma in the hamster buccal pouch, 117 samples of human cheek mucosal squamous cell carcinoma at varying stages of disease progression, chick embryo and a metastatic cell line from brain metastasis of human tongue cancer Tca8113 cells as media. In addition, we make use of spontaneous carcinogenesis animal medel, the methods of molecular pathology, cytology, molecular biology and so on. In addition, the expression of Slit2, von Willbrand factor (vWF) and vascular endothelial growth factor (VEGF) were examined by using human tissue of oral cheek mucosa with oral squamous cell carcinoma (OSCC). In the current study, we took emphasis on approaching the effect of Slit-Robo signaling on oral squamous cell carcinoma and hamster cheek mucosa, and the relation of Slit-Robo signaling to neovascularization. Meanwhile, interference of the Slit-Robo interaction using R5, the function-blocking monoclonal antibodies against the immunoglobulin motif of Robo1, inhibited the growth of hamster cheek mucosa. Furthermore, we investigated the effect of R5 on the proliferation, apoptosis, invasion and migration.Results1. Slit2 was minimally expressed in normal and hyperplastic mucosa, moderately expressed in dysplastic mucosa, and highly expressed in neoplastic mucosa obtained from hamster buccal pouch. Robo1 was expressed on vascular endothelial cells of the varying stage of disease progression, and was relative to expression tendency of Slit2.The enhancement of Slit2 expression in the stage of carcinoma progression positively correlated with the degree of vWF and VEGF levels.2. Slit2 was rarely expressed in the normal and simple hyperplastic mucosa of human oral squamous cell carcinoma tissue. It was with increasing frequency in samples of hyperplastic tissue, dysplastic tissue, carcinoma in situ and carcinoma. The strong positive malignant cells infiltrated the tissue in the way of nest. And it has been found that Slit2 protein was expressed in a periphery -to- center gradient in squamous cell carcinoma tissue. The expression of Slit2 positively enhanced in the boundary of the normal mucosa and the canceration anterior border in the carcinoma in situ. The enhancement of Slit2 expression in the process of oral squamous cell carcinoma progression was siginificantly in relation to the the degree of vWF and VEGF levels.3. Compared with the blank and control IgG group, intraperitoneal administration of R5 for hamster clearly decreased tumor volume. Furthermore, HE staining revealed new vessel numbers of tumor tissue significantly reduced. MVD and BrdU positive cells significantly reduced in the R5 group compared to the blank and control IgG groups. There was no significant difference of leukocytes and VEGF levels among R5, control and blank groups. The topical application of R5 also suppressed the formation of neovasculature chick embryo chorioallantoic membrane (CAM).4. The results showed that TB cell expressed both Slit2 and Robo1 by immunocytochemistry. Along this line investigation, we also detected the expression of Slit2 and Robo1 in TB cell by Western-blot. Protein expression of Slit2 was detected in the supernatants of TB cells culture, showing Slit2 was a kind of secreting protein and localized in cell surface. However protein of Robo1 was expressed in TB cells. After the treatment of TB cells with R5 mAb, the results showed that R5 mAb significantly suppressed the growth of TB cell line, cell survival rate and the ability of TB cells to form colonies , and made PCNA expression decrease as compared with the Blank and control IgG group. Agarose gel electrophoresis of DNA extracted from cells treated with R5 revealed ladders of DNA fragmentation. The cell cycle distribution changed treated with R5 antibody, the proportion of G2/M phase increased significantly. Expression of fas and fasL in TB cells treated with R5 was enhanced. The above results showed that R5 significantly induced apoptosis of TB cells.5. Protein expression of Slit2 and Robo1 in TB cells was more intense than that in Tca8113 by FCM intra-cellular factor detection method. After the treatment of TB and Tca8113 cells with R5 , the results showed that R5 inhibited the adhesion and migration rate of TB and Tca8113 cells and the activity of MMP-2 and MMP-9, and the expression level of E-cadherin protein was increased by Western-blot. And the effect of R5 on adhesion rate of TB cells was more significant than that of Tca8113. However, there was no obvious difference in migration rate between TB and Tca8113 cells. After the treatment of TB and Tca8113 cells with R5, the invasion abilities of TB cells were positively decreased with chicken embryon invasion experiments.Conclusions1. Slit-Robo signaling can be significantly activitated in the stage of hamster cheek pouch and oral carcinoma in situ, plays an important role in neovascularization in the process of carcinoma progression, and has any synergistic effects on the angiogenic activities of VEGF.2. Tumor angiogenesis and growth can be inhibited in chemical-induced hamster squamous carcinogenesis by specific neutralization of the Slit2-Robol interaction with R5, the function-blocking monoclonal antibody for Robo1. Furthermore, the effectiveness of Robo blockade is independent of VEGF signaling. Teatment with R5 has no significant effect on leukocytes levels in vivo model.3. TB and Tca8113 cells express both Slit2 and Robo1 indicating that Slit-Robo signaling mediates the crosstalk between tumor cells and tumor cells and has the autocrine function. Furthermore, block of the expression of Slit2 and Robo1 on TB and Tca8113 cells by R5 mAb inhibites TB cell proliferation and induces apoptosis through down-regulating PCNA expression level of TB cell and up-regulating Fas/FasL signal pathway. These results have indicated that Slit-Robo signaling promotes tumor growth.4. Activation of Slit-Robo signaling is in relation to tumor metastasis and can promote adhesion, migration, chemotaxis, invasive ability and angiogenesis through increasing the activity of MMP-2 and MMP-9 and decreasing E-cadherin signal pathway.Results from our current studies have firstly demonstrated that Slit-Robo signaling playes a critical role in the angiogenic switch in the stages of oral carcinogenesis, especially when pre-carcinoma translates to carcinoma. Furthermore, Slit-Robo signaling can be used as a reliable marker for the diagnosis of early carcinoma. R5 mAb can inhibit neovascularization of hamster cheek pouch in vivo, promotes cell proliferation and migratory ability of tongue cancer in vitro, and induces cell apoptosis of tongue cancer in vitro. These results have indicated that Slit-Robo signaling has an effect on tumor growth and metastasis and R5 can be expected to be an effective drug for patients with oral carcinoma through preventing Slit-Robo signaling.
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