VIRULENCE-PROTEIN-MEDIATED CROSS-TALK OF ENTEROCLOSTER BOLTEAE AND CANCER CELLS PROMOTES HEPATOCELLULAR CARCINOMA PROGRESSION

Background & Aims: Gut microbiota plays an important regulatory role in tumor progression. Previous studies have shown that there are bacteria in hepatocellular carcinoma (HCC). However, the role of these bacteria in the development and progression of HCC remains unclear. In this study, we aimed to explore the biological role of gut microbiota in HCC.
Methods: Feces and matched tumor tissues from 132 HCC patients and feces from 50 healthy individuals were collected and subjected to metagenomic, bulk RNA and 16S r-RNA sequencing. The pro-tumorigenic effects of Enterocloster bolteae were assessed in vitro and in vivo, including orthotopic HCC model, SPFdiethylnitrosamine (DEN)-induced HCC model and germ-free DEN-induced HCC model. Fluorescent tracer technology and transmission electron microscopy (TEM) were used to evaluate the translocation of E.bolteae to liver tumor and the bacterium-cell adhesion, respectively. Protein profiling, liquid chromatography, Far-western and metagenomic sequencing data were harnessed to identify tumor-promoting proteins on the surface of E.bolteae. Survival analysis of HCC patients based on the E.bolteae and its virulence protein were performed.
RESULTS: Our metagenomic data revealed that E.bolteae was significantly enriched in the feces and tumors of HCC patients. E.bolteae significantly promoted liver tumor load and disrupted the intestinal barrier in our different kinds of mouse models. In addition，E.bolteae could adhere to the surface of tumor cells and promote cell viability, while its conditional medium had no pro-cancer effects in vitro. Fluorescent tracer assay confirmed that E.bolteae could colonize the mouse intestine and translocate to the liver tumors. Mechanistically, E.bolteae promoted tumor cell growth via its membrane proteins, among which three membrane proteins (SecA, HMA domain-containing protein and Penicillin-binding transpeptidase domain-containing protein) were significantly enriched in the feces of HCC patients. Clinically, survival analysis showed that not only high abundance of E.bolteae but also high expression of these three membrane proteins indicate poor prognosis of HCC patients.
Conclusion: Our research identified a novel bacterium E.bolteae, which was enriched in HCC patients’ stool and significantly affects patients’ prognosis. E.bolteae could translocate to liver and interact with tumor cells through its virulence proteins, promoting HCC progression. Eliminating E.bolteae or its virulence protein may be a novel treatment strategy for HCC patients.