Overexpression of RBBP6, alone or combined with mutant TP53, is predictive of poor prognosis in colon cancer

Abstract

Retinoblastoma binding protein 6 (RBBP6) plays an important role in chaperone-mediated ubiquitination and interacts with TP53 in carcinogenesis. However, the clinicopathologic significance of RBBP6 expression in colon cancer is unknown; in particular, the prognostic value of RBBP6 combined with TP53 expression has not been explored. Therefore, quantitative real-time PCR and western blot analyses were performed to detect RBBP6 expression in colon cancer tissues. RBBP6 and TP53 expression were assessed by immunohistochemistry in a tissue microarray format, in which the primary colon cancer tissue was paired with noncancerous tissue. Tissue specimens were obtained from 203 patients. We found that RBBP6 was overexpressed in colon tumorous tissues and was significantly associated with clinical stage, depth of tumor invasion, lymph node metastasis (LNM), distant metastasis, and histologic grade. Further studies revealed that a corresponding correlation between RBBP6 overexpression and mutant TP53 was evident in colon cancer (r = 0.450; P<0.001). RBBP6 expression was an independent prognostic factor for overall survival (OS) and disease free survival (DFS). Interestingly, patients with tumors that had both RBBP6 overexpression and mutant TP53 protein accumulation relapsed and died within a significantly short period after surgery (P<0.001). Multivariate analysis showed that patients with LNM and patients with both RBBP6- and TP53-positive tumors had extremely poor OS (HR 6.75; 95% CI 2.63-17.35; P<0.001) and DFS (HR 8.08; 95% CI 2.80-23.30; P<0.001). These clinical findings indicate that the assessment of both RBBP6 and mutant TP53 expression will be helpful in predicting colon cancer prognosis.

Figure 1. Expression of RBBP6 in colon tumorous tissues and adjacent normal mucosa.

A. Relative expression of RBBP6 gene in a series of 40 matched colon cancerous tissue specimens compared with that in normal mucosa specimens. A logarithmic scale of 2− ^ΔΔCT was used to represent the fold change in quantitative real-time PCR detection. B. Western blotting analysis of RBBP6 protein expression in representative 4 paired colon tumor tissues. GAPDH was used as the loading control.

Figure 2. Immunohistochemical staining for RBBP6 expression in normal and colon cancer tissues.

(A) Negative RBBP6 expression in normal colonic epithelium and (B) well-differentiated tumor. (C) Weak RBBP6 staining in a well-differentiated colon tumor. (D) Diffused, intense RBBP6 staining in moderately- and (E) poorly differentiated colon tumors. (F) Strong RBBP6 staining in a colon cancer lymph node metastasis sample. Original magnification ×200 (×400 for inset images).

Figure 3. Expression of RBBP6 and

TP53. Representative photographs of RBBP6 and TP53 expression in normal colon, colon tumor, and nodal metastasis specimens. RBBP6 expression was more frequently detected in specimens that stained positively for mutant TP53. Original magnification ×400 (×50 for inset images).

Figure 4. Kaplan-Meier analyses with a log rank test of survival.

A. Disease-free survival (DFS) and overall survival (OS) of patients in relation with RBBP6 expression levels that were determined by immunohistochemical staining. B. DFS was significantly better in patients with TP53-negative tumors than in those with TP53-positive tumors (P = 0.036). C. DFS and OS were significantly lower in patients with RBBP6− and TP53-positive tumors than in those with RBBP6− and TP53-negative tumors (P<0.001 for both).