Abstract 2842: Dual-specificity phosphatase 12 as a key modulator of DNA damage response and nucleolar stress in hepatocellular carcinoma

Abstract The dual-specificity phosphatase 12 (DUSP12) has been implicated in liver pathologies, such as hepatocellular carcinoma (HCC), through molecular mechanisms poorly understood. Our group identified protein partners of DUSP12 in tumor cells under genotoxic stress, including the nucleolar proteins NAT10 and TCOF1, well-recognized for their roles in ribosome biogenesis, but which involvement in DNA damage response (DDR) mechanisms has gained attention. Given these proteins are associated with HCC progression and poor prognosis, this study aimed to explore the role of DUSP12 in the hepatic cell lines HepaRG and HuH-7 under treatments with cisplatin (CP) or doxorubicin (DX). Datamining the TCGA database revealed frequent amplification of DUSP12 in HCC patients, resulting in elevated mRNA and protein expression levels, which were strongly correlated with NAT10 and TCOF1 expression. Gene ontology analyses of DUSP12-correlated genes in HCC patients highlighted novel functional relationships between DUSP12 and processes related to DNA repair and DDR. In vitro, DX treatment produced marked cytotoxic and antiproliferative effects in both monolayer cultures of HepaRG and HuH-7 cells. However, noteworthy resistance was observed in 3D spheroid cultures of these cell lines. At the molecular level, DX treatment elicited an early and robust activation of DDR pathways compared to CP treatment, as evidenced by increased levels of p-Chk2 (Thr68), p-p53 (Ser15), and γH2AX (Ser139) in both cell lines. Intriguingly, DX treatment also induced distinct nucleolar stress features, nuclear accumulation of DUSP12, and the redistribution of NAT10 and TCOF1 to the nucleoplasm, although their expression levels remained unchanged. CRISPR-Cas9-mediated knockdown (KD) of DUSP12 in HuH-7 cells sensitized these cells to DX treatment in both monolayer and spheroid cultures in a manner proportional to DUSP12 expression levels. This sensitization to DX occurs independently of reactive oxygen species (ROS) modulation and was accompanied by increased levels of p-p53 (Ser15) and γH2AX, as well as the modulation of double-strand break levels as assessed by comet assay, suggesting that DUSP12 KD affects DDR signaling and DNA repair in these cells. Similarly, transfection with DUSP12 siRNA enhanced γH2AX focus formation in HepaRG cells treated with DX. Notably, DUSP12 KD in HuH-7 cells induced nucleolar stress characteristics, as evidenced by the mislocalization of NAT10 and TCOF1, as well as hyperploidy, increased micronuclei frequency, and a higher mitotic cell population, indicated by elevated p-H3 (Ser10) levels. Altogether, our findings reveal that DUSP12 plays a pivotal role in hepatocellular carcinoma by affecting genomic stability and modulating DNA damage response and nucleolar stress pathways under genotoxic conditions, underscoring its potential as a therapeutic target in HCC. Citation Format: Viktor Kalbermatter Boell, Fabio Luis Forti. Dual-specificity phosphatase 12 as a key modulator of DNA damage response and nucleolar stress in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2842.