Zhiyong Luo

Metabolic reprogramming, particularly enhanced lipid anabolism, is a hallmark of oral squamous cell carcinoma (OSCC), yet effective therapies targeting this process remain lacking. Here, we identify concurrent activation of acetyl-CoA synthetase 2 (ACSS2) and ATP-citrate lyase (ACLY) as a cooperative metabolic driver of OSCC progression. Whereas previous efforts have focused on single-enzyme inhibition, we show that ACSS2 and ACLY form a compensatory network that sustains lipid anabolism and drives OSCC progression, providing a rationale for their dual inhibition. Integrative transcriptomic and lipidomic analyses reveal a metabolically aggressive OSCC subset defined by concurrent ACSS2 and ACLY upregulation, which was associated with poor clinical outcomes. Dual inhibition of ACSS2 and ACLY depleted acetyl-CoA, suppressed lipogenesis, and impaired tumor growth in vitro and in vivo. We further identified S8588, a quinoxaline-based small molecule, as a dual-target degrader that induces ubiquitin–proteasome-dependent degradation of both ACSS2 and ACLY, thereby blocking OSCC proliferation. Mechanistically, S8588 directly binds key residues of both enzymes, promoting their proteasomal turnover. Supplementation with acetate and citrate reverses its anti-tumor effect, confirming acetyl-CoA depletion as the primary mechanism. Collectively, our findings establish ACSS2/ACLY co-activation as a therapeutic vulnerability in OSCC and nominate S8588 as a promising dual-target therapeutic to dismantle metabolic adaptability for improved treatment.

Argininosuccinate synthase 1 (ASS1) is a key rate-limiting enzyme in the urea cycle and is highly conserved at the protein level. Its catalytic activity directly determines the efficiency of ammonia clearance. Hyperammonemia is a clinical syndrome caused by abnormally elevated blood ammonia levels and is characterized mainly by central nervous system dysfunction, with neurotoxicity and a high mortality rate. When liver function is impaired or key enzymes of the urea cycle are deficient, ammonia clearance is reduced, leading to ammonia accumulation and passage across the blood-brain barrier, resulting in disturbances of consciousness, coma, or even death. Among currently used ammonia-lowering agents, L-ornithine-L-aspartate (LOLA) can promote the urea cycle to reduce blood ammonia levels and protect the liver; however, high doses are prone to gastrointestinal adverse effects, limiting its clinical application. This study obtained biologically active human ASS1 protein through prokaryotic expression and analyzed its enzymatic kinetic properties, aiming to explore the ammonia-lowering effect of ASS1 combined with LOLA and to provide new ideas and potential enzymatic intervention strategies for the clinical treatment of hyperammonemia. The full-length open reading frame of human ASS1 was amplified by reverse transcription polymerase chain reaction (RT-PCR), and both the PCR product and the pET-28a vector were digested with restriction enzymes. After purification by agarose gel electrophoresis, the pET-28a-ASS1 prokaryotic expression vector was successfully constructed using homologous recombination technology. The recombinant plasmid was transformed into Escherichia coli, and expression of the 6×His-ASS1 recombinant protein was induced by isopropyl β-D-thiogalactopyranoside (IPTG). The protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. In vitro enzyme activity assays were performed to verify the biological activity of the recombinant protein. An enzymatic reaction system using citrulline (Cit) and aspartate (Asp) as substrates was established to determine the optimal temperature and pH conditions for ASS1 activity and to further analyze its enzymatic kinetic parameters. A hyperammonemia mouse model was established in C57BL/6 mice by intraperitoneal injection of NH₄Cl (400 mg/kg). The ammonia-lowering effects of ASS1 combined with LOLA were evaluated. Mice were randomly grouped, and 4 hours after modeling, ASS1, LOLA, or combined treatment was administered via the tail vein for 4 hours, followed by measurement of blood ammonia and urea levels. In addition, normal C57BL/6 mice were treated with combined ASS1 and LOLA for 24 hours, after which liver and renal function indices were assessed. The pET28a-ASS1 vector was successfully constructed using homologous recombination, and soluble recombinant ASS1 protein was obtained in a prokaryotic expression system. After purification and concentration by Ni-NTA affinity chromatography, the protein purity reached 95.4%±1.2%, with a concentration of (10.5± 0.8) mg/mL. In vitro enzyme activity assays systematically characterized the enzymatic properties of ASS1, showing an optimal reaction temperature of 37 ℃ and an optimal pH of 8.0. Using Asp and Cit as substrates, the enzymatic kinetic parameters were determined. The K_m and k_cat/K_m values of ASS1 for Asp and Cit were (31.00±1.24) μmol/L and 2.07 L/(μmol·s), and (46.53±2.75) μmol/L and 1.35 L/(μmol·s), respectively. In vivo experiments showed that in an acute hyperammonemia mouse model induced by intraperitoneal injection of NH₄Cl (400 mg/kg), ASS1 (50 mg/kg) reduced blood ammonia levels by 48.86% by accelerating the urea cycle. When ASS1 (50 mg/kg) was combined with low-dose LOLA (50 mg/kg), the ammonia-lowering efficiency was further enhanced, with a reduction of 76.31%, restoring blood ammonia levels to the normal range in hyperammonemic mice. Recombinant ASS1 protein obtained by prokaryotic expression exhibits stable and well-defined biological activity, with catalytic function significantly influenced by temperature and pH and displaying clear enzymatic kinetic characteristics. In an NH4Cl-induced mouse model of hyperammonemia, combined application of ASS1 and LOLA showed a more stable and effective ammonia-lowering effect than monotherapy, demonstrating good synergistic effects. These findings provide a new enzymatic intervention strategy for the treatment of hyperammonemia, lay a theoretical foundation for the development of combination ammonia-lowering therapies, and indicate potential application prospects.

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 GST-pull 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 of liver cancer cells with ASS1 overexpression. Conclusions: ASS1 deficiency causes an imbalance in the urea cycle and pyrimidine synthesis in liver cancer. ASS1 directly controls the ubiquitination of CAD via STUB1, rather than just competing with aspartate, thereby suppressing liver cancer. Thus, ASS1 has potential as a druggable target in liver cancer.

Background: Skin cutaneous melanoma (SKCM) is a malignant tumor characterized by aggressive invasion and a high tendency for metastasis. This study explores the potential of MCM4 as a biomarker for SKCM and its impact on the tumor microenvironment (TME).

Triple-negative breast cancer (TNBC) is the most malignant molecular subtype of breast cancer and is characterized by aggressiveness, high mortality, significant heterogeneity, and poor prognosis. AMPK plays a critical role in maintaining the cellular energy balance, and its inactivation is associated with malignant breast cancer. Here, we identified the pharmacological mechanism of the 1,4-naphthoquinone derivative ZSW-4B. MTT, colony formation, and nude mouse xenograft tumour models demonstrated that ZSW-4B selectively inhibits the proliferation of TNBC cells both in vitro and in vivo. Flow cytometry and Western blot analysis revealed that ZSW-4B induces apoptosis in TNBC cells. Phosphoproteomic analysis revealed activation of the AMPK signalling pathway by ZSW-4B. Additionally, the application of the CRISPR-Cas9 system to genetically knockout AMPK in TNBC cell lines was demonstrated to reverse the antitumour effects elicited by ZSW-4B both in vitro and in vivo. In summary, ZSW-4B inhibits TNBC by inducing cellular apoptosis through the activation of AMPK.

Argininosuccinate synthase (ASS1), a critical enzyme in the urea cycle, acts as a tumor suppressor in many cancers. To date, the anticancer mechanism of ASS1 has not been fully elucidated. Here, we found that phosphoglycerate dehydrogenase (PHGDH), a key rate-limiting enzyme in serine synthesis, is a pivotal protein that interacts with ASS1. Our results showed that ASS1 directly binds to PHGDH and promotes its ubiquitination-mediated degradation to inhibit serine synthesis, consequently suppressing tumorigenesis. Importantly, the tumor suppressive effects of ASS1 were strongly abrogated by PHGDH knockout. In addition, ASS1 knockout and knockdown partially rescued cell proliferation when serine and glycine were depleted, while the inhibitory effect of ASS1 overexpression on cell proliferation was restored by the addition of serine and glycine. These findings unveil a novel role of ASS1 and suggest that the ASS1/PHGDH serine synthesis pathway is a promising target for cancer therapy.

Ginsenosides, the primary bioactive constituents in ginseng (Panax ginseng), possess substantial pharmacological potential and are in high demand in the market. The plant hormone methyl jasmonate (MeJA) effectively elicits ginsenoside biosynthesis in P. ginseng, though the regulatory mechanism remains largely unexplored. NAC transcription factors are critical in intricate plant regulatory networks and participate in numerous plant physiological activities. In this study, we identified a MeJA-responsive NAC transcription factor gene, PgNAC72, from a transcriptome library produced from MeJA-treated P. ginseng callus. Predominantly expressed in P. ginseng flowers, PgNAC72 localizes to the nucleus. Overexpressing PgNAC72 (OE-PgNAC72) in P. ginseng callus notably elevated total saponin levels, particularly dammarane-type ginsenosides, by upregulating dammarenediol synthase (PgDDS), encoding a key enzyme in the ginsenoside biosynthesis pathway. Electrophoretic mobility shift assays and dual-luciferase assays confirmed that PgNAC72 binds to the NAC-binding elements in the PgDDS promoter, thereby activating its transcription. Further RNA-seq and terpenoid metabolomic data in the OE-PgNAC72 line confirmed that PgNAC72 enhances ginsenoside biosynthesis. These findings uncover a regulatory role of PgNAC72 in MeJA-mediated ginsenoside biosynthesis, providing insights into the ginsenoside regulatory network and presenting a valuable target gene for metabolic engineering.