Regulation of Skp2-p27 axis by the Cdh1/anaphase-promoting complex pathway in colorectal tumorigenesis

Abstract

Abrogated entry into S phase is a common hallmark of cancer cells. Skp2, a subunit of ubiquitin ligase, is critical for regulating the G(1)/S transition. Uncontrolled Skp2 activity is detected frequently in human tumors, often correlated with poor prognosis. Current studies have suggested that the regulation of Skp2 turnover is mediated by another critical ubiquitin ligase, the anaphase-promoting complex (APC), in association with its substrate-specific factor Cdh1. To dissect the potential role of Cdh1/APC in tumorigenesis through the degradation of Skp2, we analyzed the Cdh1/APC-Skp2-p27 axis in colorectal tumorigenesis using a human tumor array and biochemical analyses. Our results show that the percentage of Cdh1- and p27-positive samples in colon cancer tissues was significantly lower than that in adjacent nonmalignant tissue. Conversely, the percentage of Skp2-positive colon cancer samples was significantly higher than that in normal tissue. Furthermore, results from clinicopathological analysis revealed that elevated Cdh1 expression was associated with lower histological grade tumors. In addition, depletion of Cdh1 by RNA interference in nonmalignant colon cells resulted in increased cellular proliferation, whereas knockdown of Skp2 significantly suppressed cancer cell growth. Our result suggests a pathological correlation between Skp2 and Cdh1/APC in colorectal cancer. Thus, Cdh1 may function as a component in tumor suppression via proteolysis of Skp2 in colorectal tumorigenesis and may serve as a prognostic marker in colon cancer patients.

Figure 1

Abrogation of Cdh1, APC, Skp2, and p27 in cancer. A: Expression profile of Cdh1 in 15 different types of malignant tumors with the adjacent nonmalignant tissues as measured by immunohistological analysis (a). As indicated, Cdh1 is expressed relatively higher in brain, breast, colon, esophagus, liver, ovary, rectum, skin, and stomach cancer compared with adjacent normal tissues. Moreover, significant difference of Cdh1 expression is detected between cancer and normal tissue of colon and breast, where expression of Cdh1 is significantly lower in cancer tissues than normal tissues. Each value (b) shows mean score of the positive Cdh1 staining from at least six different samples. B: Cdh1 and p27 are down-regulated, whereas Skp2 is up-regulated in both breast and colon cancer cells (a). Quantification of protein expression in normal and cancer cells (b). C: Immunocytochemical analysis of Cdh1 and Skp2 in breast (a) and colon cancer cells (b). Both Cdh1 (red) and Skp2 (green) are expressed and co-localized in the nucleus. Estimation of Cdh1 and Skp2 expression is reflected by fluorescent signal in nonmalignant and cancer cells using surface plotting. Each value (c) shows relative intensity/pixel from at least five independent views.

Figure 2

Effect of overexpression of Skp2 or depletion of Cdh1 in nonmalignant colon cells. A: Overexpression of Skp2 results in down-regulation of p27 in nonmalignant colon cells (a). Quantification of expression of Skp2 and p27 from three replicate experiments is shown (b). B: Depletion of Cdh1 by shRNA (a). Knockdown of Cdh1 leads to up-regulation of Skp2 and down-regulation of p27 (b). Quantification of Cdh1 knockdown experiment from three replicate experiments (c). C: Depletion of Cdh1 or overexpression of Skp2 promotes anchorage-independent growth in nonmalignant colon cells (a). Quantification of anchorage-independent growth analysis from three replicate experiments (b). D: Effect of Cdh1 knockdown and overexpression of Skp2 on the cell cycle profile of nonmalignant colon cells (a). Effect of Cdh1 knockdown or overexpression of Skp2 on cellular proliferation of nonmalignant cells measured by BrdU analysis (b). Quantification of BrdU analysis from three replicate experiments (c).

Figure 3

Effect of overexpression of Cdh1 or depletion of Skp2 in colon cancer cells. A: Overexpression of Cdh1 results in down-regulation of Skp2 in colon cancer cells (a). Quantification of expression of Cdh1, Skp2, and p27 from three replicate experiments (b). B: Engineer of Skp2 shRNA clones (a). Knockdown of Skp2 results in elevation of p27 (b). Quantification of Skp2 knockdown experiment (c). C: Depletion of Skp2 or overexpression of Cdh1 suppresses anchorage-independent growth in breast cancer cells (a). Quantification of anchorage-independent growth analysis from three replicate experiments (b). D: Effect of Skp2 knockdown or overexpression of Cdh1 on the cell cycle profile of colon cells (a). Effect of Skp2 knockdown or overexpression of Cdh1 on cellular proliferation of colon cancer cells measured by BrdU analysis (b). Quantification of BrdU analysis from three replicate experiments (c).

Figure 4

Human tissue array analysis of proteins in the Cdh1/APC-Skp2-p27 cascade. A: Representative picture of negative or positive staining of array sample. B: Significant reduction of Cdh1 protein levels was measured in colon cancer tissue, while abundant Cdh1 protein was detected in adjacent nonmalignant tissue. Accumulation of Skp2 was observed in colon cancer tissue, whereas moderate Skp2 protein levels were shown in adjacent tissue. Attenuation of p27 protein levels was examined in cancer tissue, whereas abundant p27 protein was tested in normal tissue. C: Significant inverse correlation (r = −0.297, P = 0.0255) is observed between the level of Cdh1 and Skp2 (a). Levels of Cdh1 and Skp2 are plotted according to its IHC score using 80 different tissues. Statistically significant correlation (r = 0.4584, P = 0.00026) is found between the levels of Cdh1 and p27 (b). r, Correlation coefficient; P < 0.05 correlations are statistically significant.

Figure 5

Pathological relevance of Cdh1 in colon cancer. Correlation between percentage of Cdh1-positive colon cancer and histological grade. Analytical result indicates that higher numbers of Cdh1-positive cells correlates with lower tumor histological grade in colon cancer.

Figure 6

Regulation of G₁/S transition by the Cdh1/APC-Skp2-p27 cascade in colon cancer Cdh1/APC is critical in regulating Skp2-p27-cyclin E/CDK2 axis, resulting in averting abnormal entry of S phase.