Utility of SAM68 in the progression and prognosis for bladder cancer

Abstract

Background: Muscle invasive bladder cancer (MIBC) is often lethal and non-MIBC (NMIBC) can recur and progress, yet prognostic markers are currently inadequate. SAM68, a member of RNA-binding proteins, has been reported to contribute to progression of other cancers. The aim of this study is to investigate the potential utility of SAM68 in the progression and prognosis of bladder cancer.

Methods: Quantitative PCR and immunohistochemistry were utilized to examine the expression of SAM68 in ten pairs of MIBC and adjacent normal bladder urothelium, and eight pairs of MIBC and non-MIBC (NMIBC) tissues from the same patient. Moreover, SAM68 protein expression level and localization were examined by immunohistochemistry in 129 clinicopathologically characterized MIBC samples. Prognostic associations were determined by multivariable analysis incorporating standard prognostic factors.

Results: SAM68 expression was elevated in MIBC tissues compared with adjacent normal bladder urothelium, and was increased at both transcriptional and translational levels in MIBC tissues compared with NMIBC tissues of the same patient. For MIBC, high expression and nucleus-cytoplasm co-expression of SAM68 were associated with higher T-stage, higher N-stage and worse recurrence-free survival. Five-year recurrence-free survival was 80% and 52.9% for MIBC patients with low and high SAM68 expression, respectively (p = 0.001). SAM68 nucleus-cytoplasm co-expression associated with worse 5-year recurrence-free survival rate (49.2%) than SAM68 expression confined to the nucleus (82.5%) or cytoplasm (75.5%) alone. On multivariable analysis SAM68 expression level, SAM68 nucleus-cytoplasm co-expression, T-stage, and N-stage were all independent prognostic factors for recurrence-free survival of MIBC patients.

Conclusions: SAM68 expression is increased in MIBC when compared to normal urothelium and NMIBC, and appears to be a potentially useful prognostic marker for MIBC.

Figure 1

Expression of SAM68 mRNA and protein in paired muscle invasive bladder cancer tissues and adjacent non-carcinoma tissues. (A) Quantitative PCR showed the mRNA level of SAM68 was much higher in bladder cancer tissues compared with adjacent non-carcinoma tissues. Expression levels were normalized for GAPDH. Error bars represent the standard deviation (SD) calculated from three parallel experiments. (B) Immunohistochemistry analysis was performed to detect the SAM68 protein level in tumor (T) and non-carcinoma tissues (N) from the same patient.

Figure 2

Expression of SAM68 protein was increased when bladder tumor developed from non-muscle invasive to muscle invasive. (A) Increased fold of SAM68 mRNA for muscle invasive tumor comparing to non-muscle invasive tumor from the same patient. (B) Immunohistochemistry study confirmed the expression of SAM68, in the protein level, was much higher in muscle invasive tumor samples than non-muscle invasive tumor from the same patient. For each pair of sample, the left panel represents non-muscle invasive bladder cancer (NMIBC) sample obtained from transurethral resection of bladder tumor and the right panel represents muscle invasive bladder cancer (MIBC) sample gained from radical cystectomy.

Figure 3

Typical images showed the expression level and localization of SAM68 protein as examined by immunohistochemistry (IHC). (A) Series showed the negative, low level and high level expression of SAM68 protein. Left panel: 200×; right panel: 400×. (B) Three types of SAM68 expression according to the intracellular localization: nucleus expression, cytoplasm expression and nucleus-cytoplasm co-expression. Left panel: 400×; right panel: 800 × .

Figure 4

Kaplan-Meier curves with log-rank test comparing recurrent free survival (RFS) in different subgroups (A) Bladder cancer patients with low SAM68 expression (bold line) had a cumulative 5-year RFS rate of 80.0%, compared to 52.9% for patients with high SAM68 expression (dotted line; p = 0.001). (B) Comparison of RFS curves between groups with different SAM68 intracellular localization. The RFS curves were similar between SAM68 nucleus expression and cytoplasm expression groups (p = 0.539). However, the RFS was much worse in the nucleus-cytoplasm co-expression group, no matter compared with nucleus expression group (p = 0.002) or cytoplasm expression group (p = 0.037).