Abstract 4839: Double knockdown of MDM4 and MDM2 modulates the antitumor effects of cytotoxic drugs in colon and gastric cancer cells expressing high levels of MDM4

Abstract

Introduction and Aim: Inactivation of the TP53 tumor suppressor gene is important for cancer development and progression. Wild-type (wt) TP53 is inactivated in various types of cancers including colon and gastric cancers by upregulated murine double minute homolog 2 (MDM2) and MDM4. We previously reported that simultaneous knockdown of MDM4 and MDM2 using synthetic DNA-modified siRNAs revived p53 activity and synergistically inhibited in vitro cell growth in cancer cells with wild-type TP53 and high MDM4 expression (wtTP53/highMDM4), whereas only MDM2 knockdown but not MDM4 knockdown suppressed that of high MDM2 expression cancer cells. In the present study, we investigated the combined activity of MDM4/MDM2 double knockdown and cytotoxic drugs (5-fluorouracil (5-FU), cisplatin, oxaliplatin) to develop a new therapy for gastric and colon cancers with wtTP53/highMDM4. Experimental procedure: Two colon (HCT116, LoVo) and two gastric cancer cell lines (NUGC-4, SNU-1) were used. Respective synthetic DNA-modified siRNAs against MDM4 (chiMDM4) and MDM2 (chiMDM2) were transfected using Lipofectamine RNAiMAX. Cell viability was determined by WST-8 assays. Expression of p53, p21, MDM2, MDM4, and β-actin were analyzed by immunoblotting. Cell cycle distribution was analyzed by flow cytometry. In vivo antitumor effects were examined using nude mice carrying HCT116 xenograft tumors. siRNAs with atelocollagen were intratumorally injected weekly and 5-FU was intraperitoneally administered three times per week. Results:MDM4/MDM2 double knockdown with the siRNAs enhanced in vitro antitumor activity of 5-FU in all four wtTP53/highMDM4 cell lines (HCT116, LoVo, SNU-1, and NUGC-4) and cisplatin and oxaliplatin in gastric (NUGC-4) and colon cancer cell lines (HCT116, LoVo), respectively. Exposure to 5-FU increased p53 and its downstream gene products (p21, PUMA, MDM2), and induced cell cycle arrest in the G1 phase. MDM4/MDM2 double knockdown suppressed MDM2, and enhanced expression of p53 and p21 and arrested G1. p53-MDM2 forms a negative feedback loop. Enhanced antitumor effects of 5-FU by MDM4/MDM2 double knockdown may be attributed to blocking this feedback loop, in addition to direct suppression of MDM4/MDM2. Since MDM2 interacts directly with molecules like RB, p21, and E2F1, other than p53, MDM2 knockdown may have some advantages over peptides or small molecular inhibitors of p53-MDM2 interaction in such combination therapies. Intratumor injection of chiMDM4/chiMDM2 suppressed in vivo tumor growth and boosted the antitumor effects of 5-FU in a xenograft model using HCT116 cells. Conclusion: Combining MDM4/MDM2 knockdown and conventional cytotoxic drugs may be a promising treatment strategy for wtTP53/highMDM4 gastrointestinal cancers. Citation Format: Mamiko Imanishi, Yoshiyuki Yamamoto, Shinji Endo, Kenji Yamato, Ichinosuke Hyodo. Double knockdown of MDM4 and MDM2 modulates the antitumor effects of cytotoxic drugs in colon and gastric cancer cells expressing high levels of MDM4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4839.