The oncogenic cytoplasmic p21 contributes to cancer aggressiveness and chemotherapeutic failure. However, the molecular mechanisms remain obscure. Here, we show for the first time that cytoplasmic p21 mediates 5-Fluorouracil (5FU) resistance by shuttling p-Chk2 out of the nucleus to protect the tumor cells from its pro-apoptotic functions. We observed that cytoplasmic p21 levels were up-regulated in 5FU-resistant colorectal cancer cells in vitro and the in vivo Chorioallantoic membrane (CAM) model. Kinase array analysis revealed that p-Chk2 is a key target of cytoplasmic p21. Importantly, cytoplasmic form of p21 mediated by p21^T145D transfection diminished p-Chk2-mediated activation of E2F1 and apoptosis induction. Co-immunoprecipitation, immunofluorescence, and proximity ligation assay showed that p21 forms a complex with p-Chk2 under 5FU exposure. Using in silico computer modeling, we suggest that the p21/p-Chk2 interaction hindered the nuclear localization signal of p-Chk2, and therefore, the complex is exported out of the nucleus. These findings unravel a novel mechanism regarding an oncogenic role of p21 in regulation of resistance to 5FU-based chemotherapy. We suggest a possible value of cytoplasmic p21 as a prognosis marker and a therapeutic target in colorectal cancer patients.

Purpose Enhancer of zeste homolog 2 (EZH2) is associated with epigenetic gene silencing and aggressiveness in many tumor types. However, the prognostic impact of high EZH2 expression is controversially discussed for colorectal cancer. For this reason, we immunohistochemically analyzed EZH2 expression in 105 specimens from colon cancer patients separately for tumor center and invasion front. Methods All sections from tissue microarrays were evaluated manually and digitally using Definiens Tissue Studio software (TSS). To mirror-image the EZH2 status at the tumor invasion front, we treated HCT116 colon cancer cells with the EZH2 inhibitor 3-Deazaneplanocin A (DZNep) and studied the growth of in ovo xenografts in the chorioallantoic membrane (CAM) assay. Results We showed a significant decrease in EZH2 expression and the repressive H3K27me3 code at the tumor invasion front as supported by the TSS-constructed heatmaps. Loss of EZH2 at tumor invasion front, but not in tumor center was correlated with unfavorable prognosis and more advanced tumor stages. The observed cell cycle arrest in vitro and in vivo was associated with higher tumor aggressiveness. Xenografts formed by DZNep-treated HCT116 cells showed loosely packed tumor masses, infiltrative growth into the CAM, and high vessel density. Conclusion The differences in EZH2 expression between tumor center and invasion front as well as different scoring and cutoff values can most likely explain controversial literature data concerning the prognostic value of EZH2. Epigenetic therapies using EZH2 inhibitors have to be carefully evaluated for each specific tumor type, since alterations in cell differentiation might lead to unfavorable results.