Purpose: Unresectable or metastatic hepatoblastoma generally confers a poor survival. MDM4 and MDM2 are the major negative regulators of p53, which has a predominantly wild type status in hepatoblastoma. Having previously shown that MDM4 has copy gain/amplification associated with the 1q32.1 amplicon in hepatoblastoma, we hypothesize that MDM4 is the predominant negative regulator of p53 function in hepatoblastoma and that blocking MDM4 will cause tumor cell death due to uninhibited p53 tumor suppressor activity.

Methods: An MTT assay was used to measure NSC207895 (MDM4 inhibitor) and Nutlin-3a (MDM2 inhibitor) cytotoxicity on hepatoblastoma cell lines (HepG2, HepT1, and Huh-6) as well as a patient-derived hepatoblastoma cell line (PDCL-1). CCK-8 was used to assess effects of low dose NSC207895 on HB cell line proliferation. Soft agar was used to assess the effect of NSC207895 on hepatoblastoma colony formation. Immunoblotting and quantitative RT-PCR was used to measure expression of p53 downstream targets in hepatoblastoma after treatment with NSC207895. P53 knockdowns were created to assess the effect of NSC207895 in hepatoblastoma in the absence of p53. Cell cycle analysis was performed using BRDU.

Results: Huh-6, HepG2, and HepT1 were all tested with Nutlin-3a and did not show significant cell death (IC50 >10μΜ) inhibition with NSC207895 caused significant cell death in Huh-6 (IC50=1.27μΜ), HepG2 (1.62μΜ), and HepT1 (2.05μΜ). A decrease in cell proliferation was noted with concentrations of NSC207895 as low as 0.05μΜ, 0.1μΜ and 0.3μΜ in HepG2, HepT1, and Huh-6 respectively. An increase in IC50 was seen in the p53 knockdowns compared to each respective control (HepG2: 12.6μΜ vs 3.38μΜ; HepT1: >50μΜ vs 1.45μΜ; Huh-6: 20.2μΜ vs 7.52μΜ). Increased PARP cleavage products and decreased MDM4 expression were seen on immunoblotting after treatment with NSC207895. By both immunoblotting and qPCR, the p53 downstream transcriptional targets, p21, BAX and PUMA, were increased after exposure to NSC207895 in all three cell lines. Cell cycle analysis showed an increased percentage of cells in G1/G0-phase and decreased percentage of actively replicating S-phase cells in HepG2 with NSC207895 treatment.

Conclusion: Our data supports the hypothesis that in hepatoblastoma, MDM4 may be a dominant inhibitor of p53 and inhibition of MDM4 causes p53-mediated cell death and growth inhibition. Therefore, inhibition of MDM4 may be a viable drug target for treating hepatoblastoma.

Citation Format: Yan Shi, Roma Patel, Emporia Hollingsworth, Stephen Sarabia, Jingling Jin, Dolores Lopez-Terrada, Sanjeev Vasudevan. MDM4 is a major p53 regulator in hepatoblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B29.