Expression of Spred2 in the urothelial tumorigenesis of the urinary bladder

Abstract

Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK (mitogen-activated protein kinase) pathway is involved in the progression of cancer, including urothelial carcinoma; but the negative regulation remains unclear. In the present study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA was highly expressed in high-grade non-invasive papillary urothelial carcinoma (HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression, important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly, membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while ERK activation and the expression of the cell proliferation marker Ki67 index were increased. HGPUC with membranous Spred2 expression correlated significantly with lower levels of ERK activation and Ki67 index as compared to those with negative Spred2 expression. Thus, our pathological findings suggest that Spred2 counters cancer progression in non-invasive papillary carcinoma possibly through inhibiting the Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high malignancy. Spred2 appears to be a key regulator in the progression of non-invasive bladder carcinoma.

Fig 1. Spred2 mRNA expression in urothelial tumors.

(A) Representative photographs of HE- (original magnification 400×) and in situ hybridization-sections from each category are shown. Spred2 mRNA expression was presented by red dots. (B) The number of the red-dots per cell was counted under microscope and Spred2 mRNA expression level was shown per one cell from each category (N: non-tumor; n = 10, P: PUNLMP; n = 10, L: LGPUC; n = 15, H: HGPUC; n = 18, C: CIS; n = 18, and I: IUC; n = 14). Data were mean ± SEM. ^#p<0.05, ^§p<0.01, ^¶p<0.001, *p<0.0001 (Dunn’s multiple comparison test).

Fig 2. Immunohistochemical analyses of Spred2 protein expression in urothelial tumors.

(A) Representative photographs of Spred2 immunohistochemistry (original magnification 400×) from each category are shown. (B) Expression levels of Spred2 mRNA in each Spred2 staining pattern were shown. C; cytoplasm, M; membrane. (C-M-; n = 81, C+M-; n = 122, C-M+; n = 54, C+M+; n = 11). Data were mean ± SEM. ^#p<0.05, ^§p<0.01 (Dunn’s multiple comparison test). (C) The positive rate of membranous Spred2 expression in each category was shown (N: non-tumor; n = 101, P: PUNLMP; n = 19, L: LGPUC; n = 41, H: HGPUC; n = 43, C: CIS; n = 39, and I: IUC; n = 32). Data were mean ± SEM. ^#p<0.05, ^§p<0.01 (Multiple Fisher’s exact test).

Fig 3. pERK score in urothelial tumors.

(A) Representative photographs of pERK immunohistochemistry (original magnification 400×) from each category are shown. (B) pERK staining intensity was evaluated and scored (0–5), and pERK score in each category was shown (N: non-tumor; n = 101, P: PUNLMP; n = 19, L: LGPUC; n = 41, H: HGPUC; n = 43, C: CIS; n = 39, and I: IUC; n = 32). Data were mean ± SEM. ^§p<0.01, ^¶p<0.001, *p<0.0001 (Dunn’s multiple comparison test).

Fig 4. Ki67 index in urothelial tumors.

(A) Representative photographs of Ki67 immunohistochemistry (original magnification 400×) from each category are shown. (B) Ki67 index in each category was shown (N: non-tumor; n = 101, P: PUNLMP; n = 19, L: LGPUC; n = 41, H: HGPUC; n = 43, C: CIS; n = 39, and I: IUC; n = 32). Data were mean ± SEM. ^#p<0.05, ^§p<0.01, ^¶p<0.001, *p<0.0001 (Dunn’s multiple comparison test).

Fig 5. Comparison between pERK score/Ki67 index and membranous Spred2 expression.

pERK score (A) and Ki67 index (B) were compared between membranous Spred2 negative (M-) and positive (M+) in cancer categories (LGPUC; n = 41 (M-:21, M+:20), HGPUC; n = 43 (M-:15, M+:28), CIS; n = 39 (N-:33, M+:6), and IUC; n = 32 (M-:22, M+:10)). Bar in each graph represents median. ^#p<0.05, ^§p<0.01 (Mann-Whitney test).

Fig 6. Spred2 expression in overall survival of patients with bladder cancer.

(A) Statistical analyses of Spred2 expression in normal, superficial bladder cancer (superficial) and infiltrating bladder urothelial carcinoma (infiltrating) from 3 different datasets (Sanchez-Carbayo bladder 2, Blaveri bladder 2, and Stransky bladder) were shown. The numbers in parentheses indicates the number of samples. ^§p<0.01, *p<0.0001 (unpaired two-tailed t test). (B, C) Kaplan-Meier analysis of the data in www.kmplot.com was used to determine the survival probability for 30 months (B) and 150 months (C) of patients with high or low Spred2 expression, followed by the log-rank test.