Hepatocellular carcinoma (HCC), the third leading cause of cancer-related deaths worldwide, remains a major focus and challenge in medical research. Recent advances in understanding the interplay between bile acid metabolism, gut microbiota, and immune cell function have revealed their critical roles in HCC progression. A recent study published in Cell Metabolism titled "AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity" has unveiled the mechanism by which AKR1D1 influences HCC progression through the "bile acid-gut microbiota-NK cell" axis, providing novel insights and therapeutic targets for HCC treatment.

 

The liver, as the primary site of bile acid synthesis, plays a crucial role in maintaining metabolic homeostasis of lipids, cholesterol, and carbohydrates. Bile acids synthesized in the liver are metabolized by gut microbiota into secondary bile acids, forming the "gut-liver axis" that regulates metabolic and immune status in both organs.

 

Increasing evidence suggests that dysregulated bile acid metabolism and gut microbiota imbalance are closely associated with HCC development. AKR1D1, a key bile acid synthesis enzyme, has been implicated in non-alcoholic fatty liver disease (NAFLD) and neonatal cholestasis, but its role in HCC and whether it affects tumor progression through bile acid metabolism, gut microbiota, and NK cell function remained unclear. Concurrently, NK cells, as essential components of the innate immune system, have gained increasing attention for their anti-tumor role in HCC. HCC tissues often exhibit reduced NK cell numbers and impaired function, but the specific metabolic mechanisms underlying this immunosuppressive state were unknown. This study aimed to investigate the role of AKR1D1 in HCC development, focusing on whether it regulates HCC progression through bile acid metabolism, gut microbiota composition, and NK cell function.

(Image: AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity)

 

Materials and Methods
 

Researchers first examined the expression of primary bile acid metabolism-related genes in HCC patient tumor tissues and adjacent non-tumor tissues, finding that AKR1D1 was significantly downregulated. To further investigate AKR1D1 function, they constructed AKR1D1 knockout mouse models (Akr1d1-KO) and utilized multiple HCC mouse models, including DEN-induced HCC, Hepa1-6-induced HCC, and RIL-175-induced HCC models, to observe the effects of AKR1D1 deletion on tumor incidence and tumor burden. Additionally, the researchers employed various techniques such as RNA-seq analysis, flow cytometry, LC-MS targeted metabolomics, and metagenomic sequencing to comprehensively investigate the impact of AKR1D1 deficiency on NK cell function, bile acid metabolism, and gut microbiota composition, and to elucidate the underlying molecular mechanisms.

 

Research Results
 

1) AKR1D1 Deficiency Promotes HCC Progression: Analysis of HCC patient tissues revealed significantly reduced AKR1D1 expression in tumor tissues compared to adjacent non-tumor tissues. In AKR1D1 knockout mouse models, AKR1D1 deletion significantly increased tumor incidence and tumor burden. Approximately 60% of naturally housed AKR1D1 knockout mice spontaneously developed HCC at 18-24 months, while wild-type mice showed no tumor formation. Furthermore, analysis of the TCGA database demonstrated that higher AKR1D1 expression levels correlated with longer overall survival in HCC patients, further confirming the tumor-suppressive role of AKR1D1 in HCC.
 

(Image: AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity)

 

2) AKR1D1 Deficiency Impairs NK Cell Anti-Tumor Effects: In Hepa1-6-induced HCC mouse models, RNA-seq analysis suggested that AKR1D1 exerts anti-tumor effects by influencing immune cell function. Flow cytometry assessment revealed significantly reduced NK cell numbers in the livers of AKR1D1 knockout mice, with impaired cytotoxic function manifested by decreased levels of IFN-γ, TNF-α, and granzyme B. Further experiments showed that NK cell depletion using anti-NK1.1 antibodies significantly increased tumor burden in wild-type mice, eliminating the difference with AKR1D1 knockout groups, confirming that NK cells are key mediators of AKR1D1's anti-tumor effects.
 

(Image: AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity)

 

3) AKR1D1 Regulates iso-LCA via CREB1 Pathway to Suppress NK Cell Function: LC-MS targeted metabolomics revealed that AKR1D1 deficiency significantly altered the hepatic bile acid profile, particularly increasing levels of six secondary bile acids including iso-LCA. Among these, iso-LCA exhibited the strongest inhibitory effect on NK cells, significantly reducing IFN-γ and TNF-α secretion and inducing NK cell apoptosis. Mechanistic studies showed that iso-LCA specifically inhibited CREB1 phosphorylation in NK cells without affecting total protein expression. In AKR1D1 knockout mice, tumor-infiltrating NK cells showed significantly reduced p-CREB1 levels, further confirming that iso-LCA impairs NK cell function by inhibiting p-CREB1, thereby promoting tumor immune escape.

(Image: AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity)

 

4)  B. ovatus Promotes iso-LCA Generation and Impairs NK Cell Anti-Tumor Capacity: Metagenomic sequencing analysis revealed significant changes in gut microbiota structure in AKR1D1 knockout mice, with increased abundance of Bacteroidetes phylum, particularly Bacteroides genus and B. ovatus species. Antibiotic cocktail treatment to clear gut microbiota attenuated the pro-tumor effects of AKR1D1 deficiency and significantly enhanced NK cell IFN-γ secretion. Correlation analysis showed a positive correlation between B. ovatus abundance and iso-LCA levels. Further experiments demonstrated that B. ovatus can convert primary bile acid CDCA into iso-LCA, and culture supernatant containing iso-LCA significantly inhibited NK cell function. This indicates that B. ovatus promotes tumor progression by producing iso-LCA and impairs NK cell anti-tumor capacity.

(Image: AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity)

 

5) iso-LCA Antagonist SPI Enhances Anti-Tumor Efficacy of PD-1 Antibody: Researchers screened compounds structurally similar to iso-LCA and found that spironolactone (SPI) most effectively restored NK cell IFN-γ and TNF-α secretion. In RIL-175 and Hepa1-6 HCC models, SPI significantly enhanced the anti-tumor effect of PD-1 antibody, with clear synergistic tumor suppression and no abnormal blood cell ratios or renal toxicity, demonstrating good safety.

(Image: AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity)

Research Conclusion
 

This study reveals the critical role of AKR1D1 in regulating HCC progression through the "bile acid-gut microbiota-NK cell" axis. AKR1D1 is significantly downregulated in HCC tissues, and its deficiency disrupts the gut-liver bile acid cycle, significantly increasing hepatic levels of secondary bile acid iso-LCA. Bacteroides ovatus converts primary bile acid CDCA into immunosuppressive iso-LCA, which inhibits NK cell cytotoxicity by activating the p-CREB1 pathway. Spironolactone (SPI) can partially reverse iso-LCA-mediated NK cell suppression and synergize with immune checkpoint inhibitors to enhance anti-HCC immune responses, providing a potential new strategy for HCC treatment. Furthermore, the significant correlation between decreased AKR1D1 expression, elevated iso-LCA levels, and increased B. ovatus abundance in HCC patients validates the clinical translational significance of these findings.

 

MingCeler Biotech Facilitates Mechanism Research
 

Leveraging its self-developed TurboMice™ technology, MingCeler Biotech has established multiple disease mouse models. The TurboMice™ technology overcomes the challenges of long modeling cycles and low success rates for complex models, enabling editing at virtually any target genomic locus and generating complete homozygous gene-edited mouse models directly from embryonic stem cells in as little as two months.

MingCeler Biotech can customize various HCC research-related mouse models according to client needs, such as Akr1d1 KO mice and NK cell-specific knockout mice (Eomes flox/flox NKp46-Cre+). We welcome inquiries!

 

References:

[1] AKR1D1 suppresses liver cancer progression by promoting bile acid metabolism-mediated NK cell cytotoxicity

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