Purpose: To explore the mechanisms underlying clear-cell renal cell carcinoma (ccRCC) metastasis using transcriptional profiling and bioinformatics analysis of ccRCC samples, and to elucidate the role of FOXO3a in ccRCC metastasis.Experimental Design: Gene expression profiling was performed using four primary metastatic and five primary nonmetastatic ccRCC samples. The mRNA and protein levels of FOXO3a in ccRCC samples were investigated by real-time reverse transcription PCR and immunohistochemistry, respectively. The association between metastasis-free survival of patients with ccRCC and FOXO3a mRNA levels was analyzed. Biologic functions of FOXO3a in renal cancer cell lines were investigated. The influence of FOXO3a on tumor metastasis was also studied in vivo orthotopic xenograft tumor model. Finally, the mechanism by which FOXO3a attenuation could increase invasion and migration of tumor cells was explored.Results: Bioinformatics analysis of the profiling data identified FOXO3a as a key factor in ccRCC metastasis. FOXO3a expression was decreased in primary metastatic ccRCC samples. Patients with low FOXO3a mRNA levels had poor metastasis-free survival (P ¼ 0.003). Knocking down FOXO3a induced tumor cell invasion and migration in the nonmetastatic ccRCC cells. Induced FOXO3a overexpression in SN12-PM6 cells could inhibit tumor metastasis in vivo. Downregulation of FOXO3a increased SNAIL1 expression, thereby activating the epithelial-mesenchymal transition (EMT) of RCC cell lines.Conclusions: The loss of FOXO3a induced EMT of tumor cells by upregulating SNAIL1, which promoted tumor cells metastasis in vitro and in vivo. Thus, FOXO3a could be considered as an independent prognostic factor in ccRCC metastasis and could be a marker of occult metastases. Clin Cancer Res; 20(7); 1779-90. Ó2014 AACR.

BackgroundEndothelial DLL4 plays an important role in controlling of tumor angiogenesis, which is required for tumor invasive growth and metastasis. However, the regulation of DLL4 in clear cell renal cell carcinoma (ccRCC) has not yet been systematically elucidated.MethodologyWe performed bioinformatical analysis to explore miRNAs targeting DLL4. miR-30a was selected as a representative to validate its functional association in endothelial cell. Then, the expressions of DLL4 and mature miR-30a from 90 cases of ccRCC and 28 cases of nonmatched adjacent non-tumor tissues were measured by quantitative real-time PCR. Finally, the expression of miR-30a was correlated with DLL4 expression, tumor features (metastatic condition and microvessel density), and patient metastasis-free survival. The univariate and multivariate analyses were performed to select the risk factors associated with hematogenous metastasis, respectively.Principal FindingsmiR-30a negatively regulated DLL4 and inhibited the proliferation and migration of endothelial cells. DLL4 was up-regulated in ccRCC and further increased in hematogenous metastatic cases, while miR-30a was down-regulated in tumor tissues and further decreased in hematogenous metastatic ccRCC (student t test, all p<0.05). Additionally, expression of miR-30a was inversely correlated with expression of DLL4 and microvessel density (linear correlation analysis, both p<0.05). Low-level miR-30a also indicated a higher probability of developing metastasis (log-rank test, p = 0.010). Most importantly, miR-30a expression was an independent predictor of ccRCC hematogenous metastasis by the univariate analysis and binary logistic regression model (both p<0.05).ConclusionsDown-regulated miR-30a in ccRCC was associated with tumor hematogenous metastasis through increasing microvessel density by targeting angiogenesis-specific DLL4.

The Notch ligand Delta-like 4 (DLL4) plays an important role in tumor angiogenesis, which is required for tumor invasion and metastasis. Here we showed that DLL4 was elevated in endothelium and Notch signaling was activated in renal cell carcinoma (RCC). Exogenous DLL4 induced RCC cell migration and invasion by activating intercellular Notch signaling. Importantly, the DLL4/Notch/Hey1/MMP9 cascades connecting the endothelium to the cancer cells in metastasis were identified. Knockdown of Hey1 decreased expression of MMP9 and attenuated tumor invasion. The clinical investigation on 120 cases of RCC specimens indicated that expressions of Hey1 and MMP9 correlated with DLL4 density. Moreover, univariate and multivariate analyses showed that tumor hematogenous metastasis not only was depended on microvessel density but was also associated with tumor size and DLL4 density. During 4-year surveillance, high-level of DLL4 density was associated with a higher probability of developing metastasis and being sensitive to target therapies. Our data suggest that RCC progression is caused in part by activated DLL4/Notch signaling, interaction of endothelium and cells, which can be therapeutically targeted.

MAL can serve as an accurate and reproducible anatomic landmark for the identification of the renal artery during retroperitoneal laparoscopic renal surgery.