A phase II clinical study on the efficacy and predictive biomarker of pegylated recombinant arginase on hepatocellular carcinoma.

BACKGROUND: Pegylated recombinant human arginase (PEG-BCT-100) is an arginine depleting drug. Preclinical studies showed that HCC is reliant on exogenous arginine for growth due to the under-expression of the arginine regenerating enzymes argininosuccinate synthetase (ASS) and ornithine transcarbamylase (OTC). METHODS: This is a single arm open-label Phase II trial to assess the potential clinical efficacy of PEG-BCT-100 in chemo naïve sorafenib-failure HCC patients. Pre-treatment tumour biopsy was mandated for ASS and OTC expression by immunohistochemistry (IHC). Weekly intravenous PEG-BCT-100 at 2.7 mg/kg was given. Primary endpoint was time to progression (TTP); secondary endpoints included radiological response as per RECIST1.1, progression free survival (PFS) and overall survival (OS). Treatment outcomes were correlated with tumour immunohistochemical expressions of ASS and OTC. RESULTS: In total 27 patients were recruited. The median TTP and PFS were both 6 weeks (95% CI, 5.9-6.0 weeks). The disease control rate (DCR) was 21.7% (5 stable disease). The drug was well tolerated. Post hoc analysis showed that duration of arginine depletion correlated with OS. For patients with available IHC results, 10 patients with ASS-negative tumour had OS of 35 weeks (95% CI: 8.3-78.0 weeks) vs. 15.14 weeks (95% CI: 13.4-15.1 weeks) in 3 with ASS-positive tumour; expression of OTC did not correlate with treatment outcomes. CONCLUSIONS: PEG-BCT-100 in chemo naïve post-sorafenib HCC is well tolerated with moderate DCR. ASS-negative confers OS advantage over ASS-positive HCC. ASS-negativity is a potential biomarker for OS in HCC and possibly for other ASS-negative arginine auxotrophic cancers. TRIAL REGISTRATION NUMBER: NCT01092091. Date of registration: March 23, 2010.

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

Argininosuccinate synthetase 1 (ASS1) is a rate-limited enzyme in arginine biosynthesis. The oncogenic potential of ASS1 in terms of prognosis and cancer metastasis in arginine prototrophic gastric cancer (GC) remains unclear at present. We identify differentially expressed proteins in microdissected GC tumor cells relative to adjacent nontumor epithelia by isobaric mass tag for relative and absolute quantitation proteomics analysis. GC cells with stable expression or depletion of ASS1 were further analyzed to identify downstream molecules. We investigated their effects on chemoresistance and cell invasion in the presence or absence of arginine. ASS1 was highly expressed in GC and positively correlated with GC aggressiveness and poor outcome. Depletion of ASS1 led to inhibition of tumor growth and decreased cell invasion via induction of autophagy-lysosome machinery, resulting in degradation of active ß-catenin, Snail, and Twist. Ectopic expression of ASS1 in GC cells reversed these effects and protected cancer cells from chemotherapy drug-induced apoptosis via activation of the AKT-mammalian target of rapamycin signaling pathway. ASS1 contributes to GC progression by enhancing aggressive potential resulting from active ß-catenin, Snail, and Twist accumulation. Our results propose that ASS1 might contribute to GC metastasis and support its utility as a prognostic predictor of GC.-Tsai, C.-Y., Chi, H.-C., Chi, L.-M., Yang, H.-Y., Tsai, M.-M., Lee, K.-F., Huang, H.-W., Chou, L.-F., Cheng, A.-J., Yang, C.-W., Wang, C.-S., Lin, K.-H. Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism.

A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma ß-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism.

Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo.

The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

A transgenic approach to study argininosuccinate synthetase gene expression.

BACKGROUND: Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. RESULTS: Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene. CONCLUSIONS: We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene.

Proteomic analysis of oropharyngeal carcinomas reveals novel HPV-associated biological pathways.

Oropharyngeal carcinoma (OPC) can be classified into two equally prevalent subtypes depending on the presence of human papillomavirus (HPV). Patients with HPV-positive (HPV+) OPC represent a unique cohort with a distinct tumor biology and clinical behavior compared to HPV-negative (HPV-) OPC. Genetic studies have demonstrated chromosomal and gene expression changes associated with distinct subclasses of OPC; however, the proteomic consequences of HPV infection are not known. We analyzed sets of ten HPV+ and ten HPV- OPCs and ten normal adult oral epithelia using a standardized global proteomic analysis platform. This analysis yielded a total of 2,653 confidently identified proteins from which we chose 31 proteins on the basis of expression differences between HPV+, HPV- and normal epithelium for targeted protein quantitation. Analysis of differentially expressed proteins by HPV status revealed enrichment of proteins involved in epithelial cell development, keratinization and extracellular matrix organization in HPV- OPC, whereas enrichment of proteins in DNA initiation and replication and cell cycle control was found for HPV+ OPC. Enrichment analysis for transcription factor targets identified transcription factors E2F1 and E2F4 to be highly expressed in HPV+ OPC. We also found high expression of argininosuccinate synthase 1 in HPV+ OPC, suggesting that HPV+ OPC is more dependent on conditionally essential amino acid, arginine, and this was confirmed on a OPC-specific tissue microarray. These identified proteomic changes reveal novel driving molecular pathways for HPV+ and HPV- OPCs that may be pertinent in therapeutic strategies and outcomes of OPC.

Endothelial argininosuccinate synthetase 1 regulates nitric oxide production and monocyte adhesion under static and laminar shear stress conditions.

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.

A regulatory role of Kruppel-like factor 4 in endothelial argininosuccinate synthetase 1 expression in response to laminar shear stress.

Endothelial argininosuccinate synthetase 1 (ASS1) regulates the provision of l-arginine to nitric oxide synthase 3 (NOS3). Previous studies demonstrated that endothelial ASS1 expression was induced by laminar shear stress (LSS) and that this enzyme plays a role in maintaining anti-inflammatory microenvironments through enhancing NO production. However, differently from the case of NOS3, the regulatory mechanism for the endothelial ASS1 expression in response to LSS is not well understood. This study addressed a specific issue whether endothelial ASS1 expression is regulated by Kruppel-like factors (KLFs) that are presumed to coordinate endothelial gene expressions in response to LSS. The cDNA microarray data indicated that LSS stimulated the expression of numerous KLFs in human umbilical vein endothelial cells. KLF4 showed the highest fold increase and LSS-dependent increases of KLF4 and most other KLFs were similar in young versus senescent endothelial cells. LSS-induced KLF4 expression was verified by RT-PCR and Western blotting. LSS-induced ASS1 expression and NO production were suppressed by a small interfering RNA for KLF4. The ectopic expression of KLF4 led to the increase of ASS1 expression and NO production. The present study demonstrated a key regulatory role of KLF4 in the endothelial ASS1 expression and NO production in response to LSS.

Combined lysosomal protein transmembrane 4 beta-35 and argininosuccinate synthetase expression predicts clinical outcome in hepatocellular carcinoma patients.

PURPOSE: The newly-identified lysosomal protein transmembrane 4 beta-35 (LAPTM4B-35) plays important roles in tumor progression and metastasis, while argininosuccinate synthetase (ASS) provides arginine as an indispensable nutrient for hepatocellular carcinoma (HCC). The present study investigated the clinical significance of the coexpression of LAPTM4B-35 and ASS in HCC patients on determining the prognosis. METHODS: Immunohistochemistry was used to evaluate the expression of LAPTM4B-35 and ASS in HCC tissues and paired noncancerous liver samples from 71 patients. The correlation of combined LAPTM4B-35 and ASS expression with selected clinicopathologic parameters was assessed with the chi-squared test. Patient survival and differences in survival were determined by the Kaplan-Meier method and the log-rank test. A Cox regression analysis was adopted for a multivariate analysis of the prognostic factors. RESULTS: Combined LAPTM4B-35 and ASS expression was significantly associated with TNM stage and portal vein invasion. In addition, patients with HCCs expressing both LAPTM4B-35 and ASS exhibited both markedly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001). According to the multivariate analyses, combined LAPTM4B-35 and ASS expression was found to be an independent prognostic factor for OS and DFS (P = 0.039 and P = 0.035, respectively). CONCLUSION: The overexpression of LAPTM4B-35 in combination with positive ASS expression is a negative prognostic marker for HCC.