<i>LACTOBACILLUS ACIDOPHILUS </i>SUPPRESSES NON-ALCOHOLIC FATTY LIVER DISEASE-ASSOCIATED HEPATOCELLULAR CARCINOMA THROUGH PRODUCING METABOLITE VALERIC ACID

Background: Through shotgun metagenomic sequencing, we identified the depletion of probiotic species Lactobacillus acidophilus in stools of mice with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we aimed to investigate the prophylactic potential and protective mechanism of L. acidophilus against NAFLD-HCC.

Methods: NAFLD-HCC tumorigenesis models were established in mice with a single injection of diethylnitrosamine (DEN) at day 14 and feeding of high-fat high-cholesterol (HFHC) diet for 8 months, or choline-deficient high-fat diet (CD-HFD) for 6 months. Orthotopic NAFLD-HCC xenografts were established in mice with intrahepatic injection of mouse HCC cell line Hepa 1-6 and feeding of HFHC diet for 4 months. Metabolomic profiling of stool, portal vein serum and liver tissue of mice, and culture medium of L. acidophilus (LA-CM) were evaluated by non-targeted and targeted liquid chromatography-mass spectrometry. Human NAFLD-HCC cell lines (HKCI-2, HKCI-10) and mouse NAFLD-HCC organoids were used for bio-functional assays of L. acidophilus and its metabolite. RNA-sequencing was performed on NAFLD-HCC cells treated with candidate metabolite.

Results: Oral gavage of L. acidophilus suppressed NAFLD-HCC tumorigenesis in mice induced by DEN and HFHC diet, and also in orthotopic mouse xenografts. L. acidophilus was undetectable in liver tissues but was detected in stools of L. acidophilus-treated mice, implicating that the antitumor function of L. acidophilus is attributed to its products. Co-culture with LA-CM significantly reduced viability (P<0.0001) and proliferation (P<0.01) of human NAFLD-HCC cells, and suppressed mouse NAFLD-HCC organoid growth (P<0.0001). The tumor-suppressing effect of L. acidophilus was attributed to its non-protein small molecules with a molecular weight of <3kDa. By metabolomic profiling, valeric acid which is a short-chain fatty acid, was identified as the top differential metabolite in LA-CM, and its upregulation was confirmed in stool and portal vein serum of L. acidophilus-treated mice. Oral gavage of valeric acid significantly suppressed tumor number (P<0.01) and tumor load (P<0.05) in DEN-treated mice fed with HFHC diet, accompanied with improved gut barrier integrity. This was confirmed in DEN-treated mice fed with CD-HFD diet. Valeric acid also reduced viability (P<0.01) and proliferation (P<0.001) of human NAFLD-HCC cells, and inhibited mouse NAFLD-HCC organoid growth (P<0.0001). Mechanistically, valeric acid bound to G protein-coupled receptor (GPR)41 and GPR43 of hepatocytes to induce their expression, which in turn inhibiting the oncogenic Rho-GTPase pathway to ablate NAFLD-HCC development.

Conclusion: L. acidophilus protects against hepatocarcinogenesis by secreting antitumor metabolite valeric acid. Probiotic supplementation is therefore a potential prophylactic of NAFLD-HCC.