LANCL1-FAM49B axis promotes cancer stemness of hepatocellular carcinoma by reducing NADPH oxidase-mediated oxidative stress

Abstract

Hepatocellular carcinoma (HCC) is typical cancer worldwide. Tumor-initiating cells (TICs) belong to a small batch of tumor cells with the features of self-renewal, chemoresistance, and tumorigenicity. Notably, TICs in some human tumors harbor lower levels of reactive oxygen species (ROS) levels than those of corresponding non-tumorigenic cells to maintain self-renewal potential and stemness. This study identified a novel cell membrane protein that could promote TIC properties by fine-tuning the balance between antioxidant defense and ROS-production systems. This may help understand HCC development and progression.

Combining RNA sequencing data from the HKU-QMH cohort and TCGA database, we selected all membrane protein-encoding genes that were upregulated in human HCCs. Tailor-made siRNA library, shRNA, and literature review screening were used to narrow down the candidates, based on consistent suppression of self-renewal ability in sphere formation assays upon the gene knockdown, and novelty with no publications for liver cancer. Selected targets have membranous localization and tumor initiation ability confirmed by immunofluorescence and limiting dilution assay respectively. Knockdown and overexpression approaches were used for functional characterization. Mass spectrometry was conducted to look for potential binding partners of the selected target in HCC cells. Rescued function assays were used to validate downstream and upstream proteins of LANCL1 in vitro and in vivo.

Lanthionine synthase C-like protein 1 (LANCL1) was selected to be a potential liver TICs surface marker for further functional characterization. Stable LANCL1 knockdown inhibited stemness features like sphere formation, chemoresistance, as well as in vivo tumorigenicity of HCC cells while overexpressing LANCL1 reversed the inhibitory effect of LANCL1 knockdown on these phenotypes. Furthermore, LANCL1 knockdown significantly promoted the ROS levels in HCC cells, and this was reversed by supplementation of antioxidants as well as LANCL1 rescue, which reversed the functional suppression in sphere formation and tumor initiation by LANCL1 knockdown. Mass spectrometry analysis together with functional assay screening revealed that FAM49B and TRIM21 were the functional potential binding partners of LANCL1 in regulating HCC cell stemness. LANCL1 blocks the interaction of FAM49B with an E3 ligase, TRIM21, thus protecting FAM49B from ubiquitination and degradation. Enrichment of FAM49B inhibits Rac1 activation, which in turn reduces NADPH oxidase (NOX)-mediated ROS production. Targeting the LANCL1-FAM49B axis could be a potential and promising strategy for HCC therapeutics.