The Study Of CX3CL1and Its Receptor CX3CR1in Migration, Metastasis Of Renal Cell Carcinomas And Its Clinical Significance

Objectives:Renal cell carcinoma (RCC) is the most common cancer of kidney with the majority of cases classified as clear cell renal cell carcinoma (CCRCC). Identification of critical proteins regulating CCRCC metastasis is important for prognostic prediction, prevention of metastasis, and the treatment of this lethal malignancy. In the present study, we evaluated the expression of CX3CL1/CX3CR1in4RCC cell lines and TMAs of primary and metastatic CCRCC, and analysed the roles of CX3CL1/CX3CR1in cellular adhesion, migration and metastasis. We further investigated the relationship between the expression of CX3CR1and the prognosis of CCRCC patients, and the downstream molecular signaling mechanism of CX3CL1-CX3CR1-induced migration and metastasis.Methods:1. The expression and localization of CX3CL1/CX3CR1in RCC cell lines and human nonmalignant kidney epithelial cells293T were assessed by western-blotting and immunofluorescence analysis.2. The impact of CX3CL1on the migration of cancer cells were examined by wound healing assay, and transwell migration assay.3. MTT proliferation assay was used to test the effect of CX3CL1on the proliferation of RCC cells.4. The expression level of CX3CL1/CX3CR1in78CCRCC samples,16normal kidney cortex tissue samples and16cases of metastatic lesions of CCRCC were evaluated using immuno-histochemical analysis on tissue microarray.5. The signal pathways of CX3CL1-CX3CR1induced migration of cancer cells were analyzed by the western-blotting method and inhibitory migration assay.Results:1. The expression level of CX3CR1was evidently higher in the4kidney cancer cells compared to that of293T, but the expression level of fractalkine was negligible in the cancer cells. CX3CR1was localized both on the cell membrane and in the cytoplasm of A-498and786-0cells, but only in the cytoplasm of Caki-1and SN12C cells.2. CX3CL1could induce chemotaxis and migration of human RCC cell lines, in which membrane CX3CR1were strongly expressed.3. In786-O, PI3K/Akt and MEK/ERK1/2were each activated upon the soluble CX3CL1stimulation in a time-dependent manner.4. CX3CL1induced-chemotaxis and migration of human RCC cell lines was by the Go/Gi-CX3CR1activated PI3K/Akt-andMEK/ERK1/2-dependent signal pathways.5. Immuno-histochemical analysis of these CCRCC TMAs revealed a significant correlation of high level of CX3CR1membranous expression with metastasis status (P<0.005), and there was a striking positive correlation between strong CX3CR1expression and poor tumor prognosis for the patients (P<0.01).6. Immuno-histochemical analysis of these metastatic lesions of CCRCC TMAs revealed that the expression levels of CX3CR1in the metastatic lesions was obviously higher than that in the primary lesions.7. The positive rate of CX3CL1expression in the CCRCC TMAs was much lower than that in the normal kidney cortex TMAs, but the endothelial cells of the intatumor blood vessels could express CX3CL1.Conclusions:1. The expression level of CX3CR1was evidently higher in RCC cells lines compared to293T, but the expression level of fractalkine was negligible. And the expression levels of CX3CR1in the metastatic lesions was obviously higher than that in the primary lesions.2. CX3CL1-CX3CR1is associated with the process of cellular migration of CCRCC in vitro.3. CX3CR1expression is associated with the process of tumor metastasis of CCRCC in vivo.4. Both clinical and molecular cellular evidence suggest that CX3CR1is a potential marker and therapeutic target for CCRCC prognostic prediction and treatment.