MDM4 as a prognostic factor for patients with gastric cancer with low expression of p53:immunohistochemical study of p53, MDM2, and MDM4

概要

Purpose: The oncoproteins murine double minute (MDM) 2 and MDM4 inactivate the tumor suppressor protein p53 and play essential roles in tumorigenesis of TP53-wild type tumors. The mutual relationship of p53, MDM2, and MDM4 with the prognosis of gastric cancer (GC) remains unknown. The purpose of this study is to investigate the relationship among p53, MDM2 and MDM4 and evaluate the prognostic significance in GC.

Material and method: We retrospectively collected the clinical data and tumor tissue samples of patients with stage I–IV GC who underwent surgery or received chemotherapy at the University of Tsukuba Hospital from January 2006 to December 2018. The following clinical data were extracted from the medical records: age, sex, Eastern Cooperative Oncology Group (ECOG) PS, TNM stage , adjuvant chemotherapy, histologic type, serum carcinoembryonic antigen (CEA) level, and overall survival (OS). The primary tumor size and disease-free survival (DFS) in stage I–III patients and the number of metastatic organs and length of progression-free survival (PFS) in stage IV patients were also investigated. p53, MDM2, and MDM4 were immunohistochemically stained. High expression of p53 was defined as more than 25% of tumor nuclei stained. High expression of MDM2 and MDM4 were defined as more than 50% of tumor nuclei stained. Statistical analyses were performed using IBM SPSS software, version 25 (SPSS, Chicago, IL, USA). In an in vitro study, we transduced MDM4 into a gastric cancer cell line NUGC4 harboring wild type TP53 and evaluated the effect of MDM4 overexpression on tumor growth properties and sensitivity to cytotoxic drugs by the soft-agar colony formation assay and drug sensitivity analysis, respectively.

Result: The number of patients were 47, 20, 79, and 95 in stage I, II, III, and IV, respectively. The multivariable analysis for overall survival in all patients showed p53 was an independent prognostic factor, but not MDM2 and MDM4. Focusing on 173 patients with low expression of p53 (implying non-mutated and intact p53), the proportions of MDM2- and MDM4-high expression patients were 48% and 40% in stage I-III, and 61% and 26% in stage IV. In these patients with low expression of p53, MDM4 overexpression was an independent prognostic factor in stage I-III (hazard ration [HR] 2.7, 95% confidence interval [CI] 1.1-6.4; p = 0.027) and stage IV (HR 6.2, 95% CI 2.2-17.6; p = 0.001). In in vitro studies, MDM4-transduced NUGC4 cells expressed a nearly 24-fold higher level of MDM4 than that of EGFP-NUGC4 cells (control cells), formed two-fold more colonies, and showed higher IC50 for 5- fluorouracil and oxaliplatin than control, but not for cisplatin.

Discussion: In patients with stage I–IV GC, the multivariable analysis showed that the patients with high expression of p53 had significantly shorter OS, DFS, and PFS than those of patients with low expression of p53, suggesting that p53-positive staining indicates poor prognosis. This was consistent with the findings of other reports. In tumors with mutant TP53, MDM2 and MDM4 cannot exert their main role of p53 degradation and their physiological relationship was disrupted. Therefore, we focused on low p53 expression (suggestion intact p53) tumors. We showed that MDM4 was a poor prognostic marker in GC patients with low expression of p53. To our knowledge, this is the first report to show that MDM4 is a poor prognostic marker in GC patients with low expression of p53.
To strengthen the clinical impact of MDM4 as a prognostic factor, we evaluated MDM4 in in vitro studies, MDM4 transduction enhanced the anchorage-independent cell growth, showing that MDM4 overexpression induced a potent tumor-forming activity. Furthermore, MDM4 overexpression provided NUGC4 cells with resistance to cytotoxic drugs including 5-FU and oxaliplatin, but not cisplatin. Oxaliplatin contains the 1,2-diaminocyclohexane (DACH) carrier ligand, whereas cisplatin does not. This might suggest that chemotherapy including cisplatin benefits p53-low and MDM4-high expression GC patients. This is an important clinical question to be solved.

Conclusion: We demonstrated the role of MDM4 as a poor prognostic factor for GC patients with low expression of p53. MDM4 may be a promising target in GC patients with p53 low and MDM4 high expression.