ASS1 inhibits liver cancer by promoting CAD ubiquitination and reversing the urea cycle and pyrimidine synthesis imbalance

Sep 23, 2026

Uploaded by Dipper

Background: The urea cycle and pyrimidine synthesis in the liver undergo opposite shifts during hepatocarcinogenesis. Argininosuccinate synthase 1 (ASS1) and carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) are key enzymes in these pathways, sharing aspartate. ASS1 is low in some cancers, whereas CAD is high, but their roles in liver cancer are unclear. Methods: Expression was assessed in tissue microarrays; ASS1/CAD were modulated by lentivirus in cell models, validated by Co-IP/GST pull-down and BALB/c nude mice, with metabolites measured by LC–MS/MS. Results: ASS1 was low and CAD high; recurrent cases showed negative correlation, and low ASS1 with high CAD predicted poor prognosis. ASS1 directly promoted CAD ubiquitination via STUB1 and CAD overexpression reduced the tumor-suppressive effects of ASS1. Pyrimidine supplementation restored growth. Conclusions: ASS1 deficiency disrupts urea cycle and pyrimidine synthesis balance, suppressing liver cancer through direct CAD ubiquitination control; ASS1 is a druggable target.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages16

Reading Time40 min

AccessFree to download

Abstract

Background

Methods

Results

Conclusions

Introduction

DOI: 10.1097/HC9.0000000000000769 O R IG INAL ART ICL E ASS1 inhibits liver cancer by promoting CAD ubiquitination and reversing the urea cycle and pyrimidine synthesis imbalance Zhengnan Ming 1 | Tiao luo2 | Zizheng Zou 1 | Wensong Luo1 | Xiyuan Hu1 | Ling Chen 1 | Jiang Zhou1 | Xiaohe Liu1 | Mingquan Liu1 | Jijia Li3 | Junli Luo4 | Dayou Ma5 | Suyou Liu5 | Zhiyong Luo1 1Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University, Changsha, China 2Xiangya Stomatological Hospital, Central South University, Changsha, China 3Center of Stomatology, Xiangya Hospital, Central South University, Changsha, China 4Hengyang Medical School, University of South China, Hengyang, China 5Xiangya School of Pharmaceutical Science, Central South University, Changsha, China Correspondence Zhiyong Luo, Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University, Changsha 410000, China. Email: [luozhiyong@csu.edu.cn](luozhiyong@csu.edu.cn) | | Abstract | | | --- | --- | --- | | | Background: The urea cycle and pyrimidine synthesis occur mainly in the liver and undergo opposite changes during hepatocarcinogenesis. Argininosuccinate synthase 1 (ASS1) and carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) are key enzymes in the urea cycle and pyrimidine synthesis, respectively, and compete for the common substrate, aspartate. Moreover, ASS1 is lowly expressed in certain cancers, while CAD is highly expressed. However, the role of ASS1 and CAD in liver cancer still remains unclear.
Methods: ASS1 and CAD expression in liver cancer were detected by tissue microarrays. Overexpression of ASS1 and CAD was achieved via lentivirus methods. All in vitro experiments were conducted in cells. The interactions of ASS1 and CAD were detected by co-immunoprecipitation (Co-IP) and GSTpull down. The in vivo study was conducted in a BALB/c nude mouse model. Intracellular metabolites were detected by LC–MS/MS.
Results: ASS1 was lowly expressed in liver cancer, while CAD was highly expressed. In patients with recurrent liver cancer, ASS1 and CAD were significantly negatively correlated. Moreover, liver cancer patients with low ASS1 expression and high CAD expression had a poor prognosis. ASS1 and CAD interacted directly and promoted CAD ubiquitination through STUB1 . In addition, Overexpression of CAD attenuated the tumor-suppressive effect of ASS1 in liver cancer cells. Pyrimidine supplementation enhanced the growth | | | | | | Abbreviations: ARG1, arginase 1; ASP, aspartate; ASS1, argininosuccinate synthase 1; BSA, bovine serum albumin; CAD, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; CHX, cycloheximide; CMP, cytidine 5′-monophosphate; Co-IP, co-immunoprecipitation; CPS1, carbamoyl phosphate synthetase 1; FBS, fetal bovine serum; HE, hematoxylin–eosin; IARC, International Agency for Research on Cancer; IHC, immunohistochemistry; LC–MS/ MS, liquid chromatography–mass spectrometry; NC, negative control; oe, overexpression; OS, overall survival; OTC, ornithine transcarbamylase; PALA, N-(phosphonacetyl)-L-aspartate; PBST, PBS containing 0 . 1% Tween-20; TMP, thymidine 5′-monophosphate; UMP, uridine 5 ′-monophosphate; UMPS, uridine monophosphate synthase; WCL, whole cell lysis. Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal's website, www.hepcommjournal.com. -- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- - This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially witho

FAQs

What is the study’s central focus regarding ASS1 and CAD in liver cancer?

The study examines how ASS1 deficiency disrupts the balance between the urea cycle and pyrimidine synthesis, and how ASS1 and CAD relate during liver cancer development and progression.

How were ASS1 and CAD expressions and interactions investigated?

ASS1 and CAD expression were detected in liver cancer tissue microarrays. ASS1/CAD interactions were examined using co-immunoprecipitation (Co-IP) and GST pull-down, with intracellular metabolites measured by LC–MS/MS.

What mechanistic pathway did the study identify as linking ASS1 to CAD?

ASS1 directly promoted CAD ubiquitination through STUB1. Overexpressing CAD attenuated the tumor-suppressive effect of ASS1 in liver cancer cells.

Sep 23, 2026

Uploaded by Dipper