Protein expression of ZEB2 in renal cell carcinoma and its prognostic significance in patient survival

Abstract

Background: ZEB2 has been reportedly shown to mediate the epithelial-to-mesenchymal transition (EMT) and disease aggressiveness in human tumors. However, the expression status of ZEB2 in renal cell carcinoma (RCC) and ZEB2's clinicopathologic/prognostic significance are poorly understood.

Methodology/principal findings: In this study, tissue microarray, immunohistochemistry (IHC) and western blot analyses were utilized to investigate the ZEB2 expression status in RCC and adjacent renal tissue samples. In our study, samples from 116 RCC patients treated with radical nephrectomy were used as a training set to generate a ZEB2 optimal cut-point for patient outcome by receiver operating characteristic (ROC) analysis. For validation, the correlation of ZEB2 expression with the clinical characteristics and patient outcomes in another set (including 113 patients) was analyzed to validate the obtained cut-point. In the training and validation sets, high expression of ZEB2, defined by ROC analysis, predicted a poorer overall survival and progression-free survival, as evidenced by the univariate and multivariate analyses. In different subsets of overall patients, ZEB2 expression was also a prognostic indicator in patients with stage I/II, stage III/IV, grade 1/2 and grade 3/4 disease (P<0.05). Downregulation of ZEB2 by shRNA decreased the migration and invasion ability of 769-P cells in vitro. Furthermore, high ZEB2 expression was positively correlated with vimentin expression and inversely linked to E-cadherin expression in RCC.

Conclusions/significance: Our findings provide a basis for the concept that high ZEB2 expression in RCC may be important in the acquisition of an aggressive phenotype. This evidence suggests that ZEB2 overexpression (examined by IHC) is an independent biomarker for the poor prognosis of patients with RCC.

Figure 1. Expression of the ZEB2 protein in RCC and adjacent non-malignant renal tissues.

(A) Up-regulated expression of the ZEB2 protein was observed in 8/10 RCC cases using western blot when compared to the adjacent non-malignant renal tissues. T, RCC tissue; N, non-neoplastic renal tissue. (B) High ZEB2 expression was observed in an RCC sample (case 63), in which more than 90% of the tumor cells revealed positive immunostaining of ZEB2 in the cytoplasm and nuclei (upper panel, ×100). (C) A RCC case (case 56) demonstrated low ZEB2 expression, in which fewer than 50% of the tumor cells showed immunoreactivity of the ZEB2 protein (upper panel, ×100). (D) Nearly negative expression of the ZEB2 protein was demonstrated in an RCC case (case 72, upper panel, ×100). The lower panels indicate the higher magnification (×400) from the area of the boxes in (B), (C) and (D), respectively.

Figure 2. Receiver operating characteristic curves were used to determine the cutoff score for high ZEB2 expression in RCC.

The sensitivity and specificity for each outcome were plotted, and the areas under curve (AUCs) were indicated: age (P = 0.683), tumor size (P = 0.561), TNM stage (P = 0.227), Fuhrman grade (P = 0.869), tumor subtype (P = 0.019) and survival status (P = 0.001).

Figure 3. Kaplan-Meier survival analysis of ZEB2 expression in the training and validation cohort of patients with RCC (log-rank test).

Kaplan-Meier survival analysis of ZEB2 expression for overall survival (A) and progression-free survival (B) in the training cohort (log-rank test). Kaplan-Meier survival analysis of ZEB2 expression for overall survival (C) and progression-free survival (D) in the validation cohort (log-rank test).

Figure 4. Kaplan-Meier survival analysis of ZEB2 expression in different subsets of overall patients with RCC (log-rank test).

(A) Stage I/II, the probability of survival of stage I/II patients with RCC: low expression, n = 109; high expression, n = 42. (B) Stage III/IV, probability of survival of stage III/IV patients with RCC: low expression, n = 46; high expression, n = 32. (C) Grade 1/2, the probability of survival of grade 1/2 patients with RCC: low expression, n = 118; high expression, n = 51. (D) Grade 3/4, probability of survival of grade 3/4 patients with RCC: low expression, n = 37; high expression, n = 23.

Figure 5. Silencing of ZEB2 by RNA interference inhibits RCC cell migration and invasion.

(A) Western blotting reveals that ZEB2 was efficiently knocked down by the treatment of ZEB2-shRNA. (B) Cell invasion was evaluated using a matrigel invasion chamber. Silencing of ZEB2 decreased 769-P cell invasive capacity. The numbers of invaded cells in siZEB2 and control siSCR groups are shown in the right panel. Error bars indicate ±SE. (C) Wound-healing assays show that ZEB2-silenced 769-P cells had lower motility compared with that in control cells. Data are the means±SE of three independent experiments. ^* P<0.05 by unpaired two-sided T-test.

Figure 6. Correlations between ZEB2 expression and expression of E-cadherin or vimentin in RCC tissues.

(A) High ZEB2 expression was observed in an RCC (case 87), in which more than 90% of the tumor cells showed positive staining of the ZEB2 protein (upper panel, ×100). High vimentin expression was examined in the same RCC case 87 (lower panel, ×100). (B) Low ZEB2 expression was shown in an RCC (case 102, upper panel, ×100). High E-cadherin expression was observed in the same RCC case 102 (lower panel, ×100).