RUNX3 Modulates DNA Damage-mediated Phosphorylation of Tumor Suppressor p53 at Ser-15 and Acts as a Co-activator for p53

Chizu Yamada,Toshinori Ozaki,Kiyohiro Ando,Yusuke Suenaga,Ken-Ichi Inoue,Yoshiaki Ito,Rintaro Okoshi,Hajime Kageyama,Hideki Kimura,Masaru Miyazaki,Akira Nakagawara

doi:10.1074/jbc.m109.055525

Abstract

Although it has been shown that the gastric tumor suppressor RUNX3 has a growth inhibitory activity, the precise molecular mechanisms behind RUNX3-mediated tumor suppression remained unclear. In this study, we found that RUNX3 is closely involved in DNA damage-dependent phosphorylation of tumor suppressor p53 at Ser-15 and acts as a co-activator for p53. The small interference RNA-mediated knockdown of RUNX3 inhibited adriamycin (ADR)-dependent apoptosis in p53-proficient cells but not in p53-deficient cells in association with a significant reduction of p53-target gene expression as well as phosphorylation of p53 at Ser-15. In response to ADR, RUNX3 was induced to accumulate in the cell nucleus and co-localized with p53. Immunoprecipitation experiments demonstrated that RUNX3 forms a complex with p53 in cells. In vitro pulldown assays revealed that the COOH-terminal portion of p53 is required for the interaction with RUNX3. Forced expression of RUNX3 enhanced p53-mediated transcriptional activation. Additionally, RUNX3 had an ability to induce the phosphorylation of p53 at Ser-15, thereby promoting p53-dependent apoptosis. Intriguingly, RUNX3 interacted with phosphorylated forms of ataxia telangiectasia-mutated in response to ADR; however, it did not affect the extent of DNA damage. From the clinical point of view, coordinated p53 mutation and decreased expression of RUNX3 in 105 human lung adenocarcinomas were significantly associated with the poor outcome of patients (p = 0.0203). Thus, our present results strongly suggest that RUNX3 acts as a novel co-activator for p53 through regulating its DNA damage-induced phosphorylation at Ser-15 and also provide a clue to understanding the molecular mechanisms underlying RUNX3-mediated tumor suppression.

Highlights

RUNX3, which is mapped to human chromosome 1p36, is one of the RUNX family of transcription factors, including RUNX1–3 [1]
The RUNX3 gene was rarely mutated in primary gastric cancers; its expression levels were significantly down-regulated in primary gastric cancers and gastric cancer-derived cell lines, which might be due to the combination of its hemizygous deletion and the hypermethylation of its promoter region
Subsequent studies revealed that the frequent reduction of RUNX3 expression levels is observed in several human cancers such as lung cancer, breast cancer, colon cancer, pancreatic cancer, and prostate cancer, which might be attributed to promoter hypermethylation [7,8,9,10,11,12,13], indicating that the down-regulation of RUNX3 is not restricted to gastric cancer

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—African green monkey embryonic kidney COS7, human cervical carcinoma HeLa, human osteosarcoma U2OS, and SAOS-2 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% of heat-inactivated fetal bovine serum (Invitrogen), penicillin (100 IU/ml), and streptomycin (100 ␮g/ml). Cell lysates prepared from COS7 cells transfected with the expression plasmid encoding RUNX3 were mixed and incubated overnight at 4 °C. Forty eight hours after ADR treatment, cells were washed in ice-cold PBS and lysed in lysis buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% Nonidet P-40, and protease inhibitor mixture for 30 min at 4 °C. Forty eight hours after transfection, cells were lysed, and both firefly and Renilla luciferase activities were measured with Dual-Luciferase reporter assay system (Promega), according to the manufacturer’s instructions. For knocking down of the endogenous p53, U2OS cells were transiently transfected with the empty plasmid (pSUPER, OligoEngine, Seattle, WA) or with pSUPER expression plasmid encoding siRNA against p53 (pSUPER-siRNA-p53) by using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instruction. After 14 days, viable colonies were washed in PBS and stained with Giemsa solution

RESULTS

DISCUSSION

Akira Nakagawara