Imaging , Diagnosis , Prognosis Expression of Snail in Upper Urinary Tract Urothelial Carcinoma : Prognostic Significance and Implications for Tumor Invasion

Purpose: There are few molecular markers known to predict upper urinary tract urothelial carcinomas (UTUC) prognosis. Snail, which contributes to epithelial–mesenchymal transition (EMT), has been documented in cancer progression, but not clear yet in UTUC. We therefore addressed the expression and biological significance of Snail in UTUC. Experimental Design: To elucidate the biological significance of Snail in UTUC, we examined the immunohistochemical expression of snail in UTUC and analyzed its clinical significance in 150 patients with UTUC. Biological effects of Snail in EMT and invasion were evaluated by using small interfering RNA (siRNA) specific for Snail in urothelial carcinoma cell lines and the Matrigel invasion assay. Results:Nuclear Snail staining was very weak in superficial UTUC. In contrast, strong Snail staining was observed in many of the nucleus of invasive UTUC. Snail expression was significantly higher in the high tumor stage, high grade, and in tumors showing lymphovascular invasion (LVI). Multivariate Cox regression analysis revealed that elevated Snail expression was a significant and an independent prognostic predictor of recurrence-free survival and cancer-specific survival. Patients with positive LVI and high Snail expression showed the worse outcome. Targeting of Snail mRNA expression in UMUC-3 cells with Snailspecific siRNA downregulated the mRNA expression of Snail, Vimentin, MMP2, and MMP9. Furthermore, the cells with siRNA for Snail showed decreased invasion activity in comparison with the cells transfected with a nontargeting siRNA. Conclusion: Snail-induced EMT represents a clinically relevantmechanism of UTUCprogression and an attractive target for the treatment of patients with UTUC. Clin Cancer Res; 16(23); 5814–23. 2010 AACR. Upper urinary tract urothelial carcinoma (UTUC) accounts for approximately 10% of all renal tumors and for approximately 5% of all urothelial carcinomas (UC; ref. 1). UTUC is a relatively rare tumor and there are few molecular markers known to predict the disease prognosis (2, 3). Radical nephroureterectomy (RNU) with excision of the bladder cuff is the standard of procedure for UTUC. Lymphovascular invasion (LVI) has been recently reported to be an independent predictor in a large, multicenter, international, retrospective dataset of patients treated with RNU (4). Despite the clinical importance, little is known about the molecular mechanism of LVI and the subsequent establishment of markers for predicted LVI that may contribute to improvement of patients with UC. Therefore, it is important to examine the factors playing a significant role in LVI with patient with UTUC. Epithelial to mesenchymal transition (EMT) is a process that was initially observed during embryonic development in which cells lose epithelial characteristics and gain migratory and invasive properties of mesenchymal cells. According to several reports, EMT is a key process in cancer development and progression (5, 6). Recent study has indicated that EMT may contribute to chemoresistance (7, 8). Tumor cells transform from a noninvasive to an invasive phenotype through a series of metastatic steps, whereby epithelial cells lose polarity and invade the lymphovasculature. Recent research has documented the role of Snail in EMT (5). Snail is a member of a super family of zinc-finger transcription factors, which was first identified in Drosophila melanogaster (9). Snail mediates events in the mesoderm, neuroectoderm, and development of other organs in the embryo (10). Snail is considered a key regulator of EMT and thereby of tumor progression and metastasis (11). Snail represses the transcription of E-cadherin (12–14) by binding to E-box elements found in the proximal Ecadherin promoter (5), thereby triggering a complete EMT with acquisition of invasive properties. In squamous cell carcinoma and hepatocarcinoma, Snail expression has Authors' Affiliations: Department of Urology, Division of Diagnostic Pathology, and Department of Pathology, Keio University School of Medicine, Tokyo, Japan Corresponding Author: Eiji Kikuchi, Department of Urology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160–8582, Japan. Phone: 81-3-5363-3825; Fax: 81-3-3225-1985. E-mail: eiji-k@kb3.so-net.ne. jp. doi: 10.1158/1078-0432.CCR-10-023