Despite progress in the treatment of non-visceral malignancies, the prognosis remains poor for malignancies of visceral organs and novel therapeutic approaches are urgently required. We evaluated a novel therapeutic regimen based on treatment with Se-methylselenocysteine (MSC) and concomitant tumor-specific induction of Kynurenine aminotransferase 1 (KYAT1) in hepatocellular carcinoma (HCC) cell lines, using either vector-based and/or lipid nanoparticle-mediated delivery of mRNA. Supplementation of MSC in KYAT1 overexpressed cells resulted in significantly increased cytotoxicity, due to ROS formation, as compared to MSC alone. Furthermore, microRNA antisense-targeted sites for miR122, known to be widely expressed in normal hepatocytes while downregulated in hepatocellular carcinoma, were added to specifically limit cytotoxicity in HCC cells, thereby limiting the off-target effects. KYAT1 expression was significantly reduced in cells with high levels of miR122 supporting the concept of miR-guided induction of tumor-specific cytotoxicity. The addition of alpha-ketoacid favored the production of methylselenol, enhancing the cytotoxic efficacy of MSC in HCC cells, with no effects on primary human hepatocytes. Altogether, the proposed regimen offers great potential to safely and specifically target hepatic tumors that are currently untreatable.

Ultrasound screening for hepatocellular carcinoma (HCC) in patients with liver cirrhosis has a poor sensitivity for small tumors. Circulating microRNAs (miRNAs) have been explored as HCC biomarkers, but results are diverging. Here, we evaluate if miRNAs up-regulated in HCC tissue can be detected in plasma and used as screening biomarkers for HCC. In this cross-sectional study, plasma, HCC tissue and surrounding non-tumorous liver tissue were collected from liver resections. Tissue miRNAs were identified and quantitated by RNA-sequencing analysis, and the fold-changes between HCC and surrounding liver tissue were calculated. The miRNAs up-regulated in HCCs were then re-analyzed in plasma from the same patients, and the miRNAs with the highest plasma levels were subsequently measured in plasma from an independent cohort of patients with cirrhosis or HCC. In tissues from 84 resected patients, RNA-sequencing detected 197 differentially expressed miRNAs, 40 of which had a raw count above 200 and were analyzed in plasma from the same cohort. Thirty-one miRNAs were selected for further analysis in 200 patients with HCC or cirrhosis. Of these, eleven miRNAs were significantly increased in HCC as compared to cirrhosis patients. Only miR-93-5p and miR-151a-3p were significantly associated with HCC, with an AUC of 0.662. In comparison, alpha-fetoprotein and des-gamma-carboxy prothrombin yielded an AUC of 0.816, which increased to 0.832 if miR-93-5p and miR-151a-3p were added. When including sex and age, the addition of miR-93-5p and miR-151a-3p did not further improve the AUC (from 0.910 to 0.911). In conclusion, micro-RNAs up-regulated in HCCs are detectable in plasma but have a poor performance as screening biomarkers of HCC.

Human kynurenine aminotransferase 1 (hKYAT1) plays a crucial role in the transamination of aromatic amino acids and kynurenine. This promiscuous homodimeric enzyme transaminates various amino acids into their corresponding alpha-keto acids. Additionally, hKYAT1 is known to catalyze the beta-elimination of cysteine-conjugates and cysteine-Se-conjugates. In this study, we performed mutational analyses of hKYAT1, targeting its catalytic, ligand-binding, and substrate-binding sites. The transamination activity of thirteen mutant variants was systematically evaluated against sixteen different amino acid substrates, including kynurenine, selenomethionine (SeMet), and Se-methylselenocysteine (MSC), as well as for the -elimination of SeMet and MSC. Our results demonstrate that mutations of residues E27 in the catalytic site and H279 in the substrate-stabilizing site significantly enhanced the transamination of several amino acids, including phenylalanine, tryptophan, histidine, and MSC. The H279F mutation increased transamination and -elimination of MSC by two- and 1.5-fold, respectively. Furthermore, mutation at the ligand-binding residues R398, F125 and N185 substantially reduced MSC transamination activity of hKYAT1. Interestingly, none of the tested mutations affected the transamination of L-kynurenine, a natural substrate of hKYAT1. Altogether, these findings support future investigation into hKYAT1 as a modifiable target in selenium-mediated anticancer approaches.