Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, has an overall 5-year survival rate of around 20%, making it the third leading cause of cancer-related death worldwide. Upregulated nucleotide metabolism is frequently identified in HCC and represents a metabolic weakness of tumor cells. The enzyme dUTPase (DUT) catalyzes the synthesis of nucleotide precursor and prevents undesired uracil misincorporation into DNA, and thus plays an important role in the maintenance of DNA integrity and cell viability. Although common upregulation of DUT has been reported in cancers, its role in HCC remains largely unknown. In this study, we investigated the mechanism underlying DUT biology in HCC and tumor susceptibility to drug targeting dUTPase. Overexpression of DUT was found in 42% of HCC cases and significantly correlated with advanced stage tumors. CRISPR-Cas9 mediated knockout of DUT resulted in growth retardation, cell cycle arrest and a spontaneous induction of DNA damage in multiple HCC cell lines. A protective effect from oxidative stress was also demonstrated in both knockout and overexpression DUT assays. Metabolomics analysis showed altered DUT expression in HCC cells resulted in profound impact on pyrimidine and purine metabolism. In addition, levels of DUT protein strongly correlate with cellular level of dUTP and dTDP, which suggested its critical role in dNTP homeostasis. Interestingly, hepatic organoids overexpressing DUT showed drug resistance to tyrosine kinase inhibitor (TKI) Sorafenib. Both in vitro and in vivo assays confirmed that targeting dUTPase activity with TAS-114 synergized the effect of Sorafenib in suppressing HCC growth. In conclusion, our study showed that upregulated DUT conferred growth advantage to HCC cells by reducing uracil misincorporation and favoring nucleotide synthesis. Targeting DUT with its first-in-class inhibitor TAS-114 in combination with Sorafenib represents an effective treatment regime for HCC. Citation Format: Yue Liu, Mingjing Xu, Ho Lee Wan, Alissa M. Wong, Xiaofan Ding, Kelvin K. Ng, Nathalie Wong. Targeting nucleotide synthesizing enzyme dUTPase (DUT) represents a metabolic weakness and therapeutic opportunity in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3987.
The Ubiquitin-Proteasome System (UPS) is the major pathway in eukaryotic cells that regulates intracellular protein degradation and turnover. Cumulative evidence has suggested that defects in the UPS machinery are involved in the pathogenesis of human diseases, including cancer. The proteasome-associated polyubiquitin receptor ADRM1 (also known as Rpn13) orchestrates the process of substrate recruitment with deubiquitination in the proteasome through its N-terminal ubiquitin-binding domain and C-terminal activation of deubiquitinating enzyme. Using long-read SMRT-seq to profile the RNA splicing landscape in hepatocellular carcinoma (HCC), we previously reported on an unannotated alternative spliced variant of ADRM1 in human hepatocellular carcinoma (HCC) (Hepatology 2019). This novel isoform exhibits an exon 9 skipping (ADRM1-ΔEx9) that resulted in an altered C-terminus of the protein. In this study, we undertook further investigations to characterize the detailed functional effects of ADRM1-ΔEx9 in HCC biology and the underlying mechanism. Initial study by junction-specific Taqman PCR assay showed that ADRM1-ΔEx9 is more frequently upregulated in HCC tumors than the canonical full-length counterpart (ADRM1-FL). There is an interchange between the two isoforms as tumor develops which underscores an isoform-switch in favor of HCC development. Indeed, our result showed that ADRM1-ΔEx9, but not ADRM1-FL, correlated significantly with inferior patient survival. Functionally, ADRM1-ΔEx9 knockdown profoundly suppressed proliferation by inducing spontaneous apoptosis in HCC cell lines and patient-derived HCC organoids. The corresponding effects were less apparent from ADRM1-FL knockdown. Consistently, overexpression of ADRM1-ΔEx9 in normal human liver organoids promoted pre-malignant features alongside increased in-vitro propagation time. Ubiquitin Proteome profiling revealed that ADRM1-ΔEx9 led to a reduced expression of the pivotal tumor suppressor p53. A parallel transcriptome also substantiated that ADRM1-ΔEx9 modulated genes involved in the p53 and apoptosis pathways. Collectively, our study demonstrated that the non-canonical spliced isoform ADRM1-ΔEx9 confers growth and pro-survival advantages in HCC through mediating selective degradation of tumor suppressor p53 protein. Citation Format: Yanmei Sun, Mingjing Xu, Ho Lee Wan, Alissa M. Wong, Kelvin K. Ng, Nathalie Wong. Novel spliced isoform of the proteasome subunit ADRM1/Rpn13 promotes hepatocellular carcinoma (HCC) development through selective degradation of tumor suppressor p53. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3959.
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