Abstract 2569: Disruption of ceramide glycosylation restores p53-dependent apoptosis in p53 mutant drug-resistant cancer cells: Alternative splicing determines the feature of p53 activity

Abstract

Abstract Mutation of tumor suppressor p53 is frequently detected in many different types of cancers. p53 mutants acts as oncogenic proteins preventing programmed cell death and promoting cancer progression. Restoration of p53 that is to increase the expression of wild-type p53 and to eliminate mutant expression can substantially increase the efficacy of chemotherapy and stop malignant progression. However, incomplete understanding the molecular mechanism underlying p53 gene regulation limits the success in p53 restoration. We, for the first time, report that disruption of ceramide glycosylation restores p53 dependent-apoptosis in p53 mutant drug-resistant cancer cells. A lose of 21 base-pair in the mRNA of exon-5 can result in the accumulation of mutant p53 and cells insensitiveness to induced-apoptosis in human NCI/ADR-RE ovarian cancer cells. We found that treatment of mixed-backbone oligonucleotide against human glucosylceramide synthase (MBO-asGCS) increased the levels of wild-type p53 mRNA (containing the 21 bp in exon-5) and protein of wild-type p53 in dose-dependent manner (50-200 nM). In response to doxorubicin-induced DNA damage, phosphorylated p53 (Ser15 in the exon-5) was significantly increased, that in turn increased the protein levels of p21 and Bax, and apoptosis (52% vs. 3%) after MBO-asGCS treatment compared to control in NCI/ADR-RE cells. Silencing of GCS using MBO-asGCS (1 mg/kg) restored wild-type p53 expression and substantially sensitizes NCI/ADR-RE tumors to doxorubicin-induced apoptosis in vivo (10 mice per group), as compared to MBO-scramble control or other treatments. In contrast, introduction of GCS gene in NCI-ADR-RE cells reduces wild-type p53 expression and p53-depedent apoptosis. Further mechanic studies indicated that increased ceramide after GCS silencing modulates the function of SRp30a (ASF/SF2) regulating alternative pre-mRNA splicing. Increasing endogenous ceramide by inhibition of its glycosylation using PDMP or enhanced its production using sphingomylinase promoted wild-type p53 expression at the levels of mRNA, protein and phosphorylation; decreasing ceramide by inhibition of ceramide synthesis using fumonisin (FB1) repressed wild-type p53 expression. Present study indicates that cancer cells marked with mutant p53 phenotype are capable of expressing functional p53; ceramide glycosylation is one molecular mechanism that shifts expression to wild-type form of p53 at alternative splicing of post-transcription processing. MBO-asGCS, a specific agent silencing GCS restores p53-dependent apoptosis in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2569.