Targeting the Ajuba/Notch axis increases the sensitivity of colon cancer cells to 5-fluorouracil

Abstract

Objective: Colorectal cancer is severely challenging because of the insufficient understanding of the mechanism underlying its resistance to clinical chemotherapy. The purpose of our study is to investigate the role of the LIM protein Ajuba (JUB) in the chemoresistance of colon cancer and its potential effect on clinical treatment. Material and Methods: The protein levels of JUB in colon cancer tissues were evaluated using Western blot analysis and immunohistochemistry assays. The correlation between JUB and the prognosis of patients with colorectal cancer was determined using Kaplan–Meier plot analysis. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were employed to determine the 50% inhibitory concentration of 5-fluorouracil (5-FU) and thus assess the effect of JUB on the effectiveness of 5-FU. In addition, the rate of cellular apoptosis was measured using fluorescence-activated cell sorting assays. Side population and sphere formation analyses were conducted to determine the role of JUB in promoting the stem cell-like traits of colon cancer cells. In vivo assays were performed and detect whether the downregulation of JUB induces 5-FU sensitivity. Moreover, luciferase and Western blot assays were employed to uncover the mechanism through which JUB promotes chemoresistance in colon cancer. Results: JUB expression was upregulated in chemoresistant colon cancer (P < 0.001) and correlated with relapse-free survival (P = 0.000002). Functionally, the upregulation of JUB conferred 5-FU resistance to colon cancer cells in vitro, whereas the downregulation of JUB induced 5-FU sensitivity in colon cancer cells in vivo. The high expression of JUB promoted the tumorigenic capability of colon cancer cells. Furthermore, the increased expression of JUB activated multiple downstream genes of the Notch signaling pathway with increased expression in JUB-overexpressing cells but reduced expression in JUB-silenced cells. Importantly, the inhibition of Notch signaling using a small-molecule inhibitor significantly suppressed JUB-induced chemoresistance. Conclusion: Results suggest that JUB plays an important role and may serve as a biomarker for the clinical treatment of patients with 5-FU-resistant colon cancer.