LANCL1-FAM49B AXIS PROMOTES CANCER STEMNESS OF HEPATOCELLULAR CARCINOMA BY REDUCING NADPH OXIDASE-MEDIATED OXIDATIVE STRESS

Abstract

<p>Background:</p><p>Hepatocellular carcinoma (HCC) is a common cancer worldwide. Tumor-initiating cells<br>(TICs) belong to a small subset of cancer cells with the capacity for self-renewal,<br>chemoresistance, and tumorigenicity. Notably, TICs in some human tumors contain<br>lower reactive oxygen species (ROS) levels than corresponding non-tumorigenic cells to<br>maintain the self-renewal potential and stemness. In this study, we identi ed a novel<br>cell membrane protein that could promote TIC properties by ne-tuning the balance<br>between ROS-generation and antioxidant defense systems. This may help understand<br>HCC development and progression.</p><p>Methods: Combining RNA sequencing data from the HKU-QMH cohort and TCGA<br>database, we selected all membrane protein-encoding genes that were upregulated in<br>human HCCs. Tailor-made siRNA library, shRNA, and literature review screening were<br>used to narrow down the candidates, based on consistent suppression of self-renewal<br>ability in sphere formation assays upon the gene knockdown, and novelty with no<br>publications for liver cancer. Selected targets have membranous localization and tumor<br>initiation ability con rmed by Immuno uorescence and limiting dilution assay<br>respectively. Knockdown and overexpression approaches were used for functional<br>characterization. Mass spectrometry was conducted to look for potential binding<br>partners of the selected target in HCC cells.</p><p>Results:</p><p>Lanthionine synthase C-like protein 1 (LANCL1) was selected as a potential liver TICs<br>surface marker for further functional characterization. Stable LANCL1 knockdown<br>inhibited stemness phenotypes including sphere formation, chemoresistance, and in<br>vivo tumorigenicity of HCC cells, while overexpressing LANCL1 reversed the inhibitory<br>e ect of LANCL1 knockdown on these phenotypes. Furthermore, LANCL1 knockdown<br>signi cantly increased the intracellular ROS levels of HCC cells, and this was reversed by<br>supplementation of antioxidants as well as LANCL1 rescue, which reversed the<br>functional suppression in sphere formation and tumor initiation by LANCL1 knockdown.<br>Mass spectrometry analysis together with functional assay screening revealed that<br>FAM49B and TRIM21 were the functional potential binding partners of LANCL1 in<br>regulating HCC cell stemness through regulating intracellular ROS levels. LANCL1 blocks<br>the interaction of FAM49B with an E3 ligase, TRIM21, thus protecting FAM49B from<br>ubiquitination and degradation. Enrichment of FAM49B inhibits RAC1 activation, which<br>in turn reduces NADPH oxidase (NOX)-mediated ROS production. Targeting the LANCL1-<br>FAM49B axis may be a potential strategy for HCC treatment.</p><p>Conclusion:</p><p>LANCL1 is a potential novel TIC surface marker for HCC and it is a functional protein by<br>binding to and protecting FAM49B from TRIM21-mediated ubiquitination degradation,<br>thus reducing ROS levels in HCC cells to promote HCC initiation.</p>