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

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.RUNX3, which is mapped to human chromosome 1p36, is one of the RUNX family of transcription factors, including RUNX1-3 (1). The RUNX family contains the well conserved 128-amino acid region (Runt domain) and forms a stable complex with PEBP2␤/CBF␤ to exert its transactivation ability. Extensive studies demonstrated that RUNX1 plays an important role in the regulation of hematopoiesis (2, 3), whereas RUNX2 contributes to the generation and maturation of osteoblasts (4, 5). In contrast to RUNX1 and RUNX2, it has been shown that RUNX3 acts as a candidate tumor suppressor for human gastric cancers (6). According to their results, the RUNX3 gene was rarely mutated in primary gastric cancers; however, 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. Additionally, a mutation (R122C) found within the Runt domain of RUNX3 resulted in a complete lack of its tumor suppressive activity.Subsequent studies revealed that the frequent reduction of RUNX3 expression levels is also observed in several human cancers such as lung cancer, breast cancer, colon cancer, pancreatic cancer, and prostate cancer...

Runt-related Transcription Factor 1 (RUNX1) Stimulates Tumor Suppressor p53 Protein in Response to DNA Damage through Complex Formation and Acetylation

“…Although the molecular mechanisms underlying the pro-apoptotic effects of TGF␤ appear to be cell type-dependent, it is likely that RUNX family members such as RUNX1 and RUNX3 are the key components during this cellular process. Recently, we have found that RUNX3 participates in p53-dependent DNA damage response (56). Based on our results, RUNX3 was induced to access the cell nucleus following DNA damage and formed a complex with p53 to enhance its transcriptional as well as pro-apoptotic activity.…”

Genome profiling of pancreatic adenocarcinoma

“…Hypermethylation and loss are common mechanisms of RUNX3 inactivation in pancreatic cancer (Wada et al, 2004;Nomoto et al, 2008). RUNX3 could act as a coactivator for TP53 by regulating its DNA damage-induced phosphorylation (Yamada et al, 2010). Rearrangements of chromosome arm 8p are one of the most common genetic events in breast, pancreatic and many other epithelial carcinomas (Emi et al, 1992;Tirkkonen et al, 1998;Davidson et al, 2000;Loo et al, 2004;Bashyam et al, 2005), suggesting the presence of candidate tumor suppressor genes; ARHGEF10 (8p23) and TUSC3 (8p22) have been proposed as potential 8p tumor suppressor genes (Cooke et al, 2008).…”

Heterogeneity of liver cancer and personalized therapy

“…By checking different time-points of DEN induced generation of liver cancer, Michael R. Speicher et al found that chromosomal gains and losses were early observed in week 32 and increased significantly in week 56. Moreover, the loss of distal chromosome 4q, which contained several HCC suppressor genes such as Runx3 [31] and Nr0b2/Shp [32], occurred early and persisted during all tumor stages. Intriguingly, the mutations and activation of Wnt/β-catenin occurred at late stages, but were not involved in tumor initiation in this model.…”