Simultaneously targeting SOAT1 and CPT1A ameliorates hepatocellular carcinoma by disrupting lipid homeostasis

Hepatocellular carcinoma (HCC) exhibits metabolic reprogramming, including altered fatty acid metabolism. While cancer cells utilize fatty acids for energy and growth, excessive free fatty acids (FFAs) can cause lipotoxicity. Maintaining FFA homeostasis is crucial, especially in steatohepatitis HCC (SH-HCC), which is often associated with obesity and high FFA intake. This study focuses on the role of sterol O-acyltransferase 1 (SOAT1) and carnitine palmitoyltransferase 1A (CPT1A) in regulating lipid homeostasis in HCC. SOAT1 facilitates fatty acid storage as cholesterol esters (CEs) in lipid droplets (LDs), while CPT1A is a rate-limiting enzyme in fatty acid β-oxidation (FAO), which generates energy through the TCA cycle and ketone bodies. Previous studies have shown SOAT1 or CPT1A overexpression in various cancers, suggesting their potential as therapeutic targets. However, the interplay between these two pathways in HCC and their role in preventing lipotoxicity remain largely unexplored. This research investigates the interaction between SOAT1-mediated fatty acid storage and CPT1A-mediated FAO, aiming to identify a novel therapeutic strategy for HCC treatment by targeting lipid metabolism.

The literature review section extensively cites previous research on metabolic reprogramming in cancer, highlighting the Warburg effect and altered fatty acid metabolism in tumor cells. It discusses the role of SOAT1 and SOAT2 in cholesterol esterification and lipid droplet formation, as well as the function of CPT1A, CPT1B, and CPT1C in fatty acid oxidation and energy production. Existing studies demonstrate that SOAT1 or CPT1A overexpression is observed in various cancers, including HCC, breast cancer, and glioblastoma. The review also addresses the impact of fatty acid esterification and oxidation on tumor growth, showing that blocking either pathway can suppress tumor growth, although research on their combined effects is limited. This sets the stage for the current study's investigation into the interplay between SOAT1 and CPT1A in regulating lipid homeostasis and their potential as combined therapeutic targets in HCC.

The study employed both in vitro and in vivo models. In vivo, diethylnitrosamine (DEN)-induced HCC in mice was used, with half the mice fed a high-fat diet (HFD). The impact of HFD on SOAT1 and CPT1A expression was assessed using Western blot and immunohistochemistry (IHC). In vitro studies utilized HepG2, HUH7, and PLC/PRF/5 HCC cell lines. The effects of SOAT1 and CPT1A inhibition (through siRNA knockdown or small molecule inhibitors avasimibe and etomoxir, respectively) were evaluated using CCK8 assays, colony formation assays, EdU assays, Western blot analysis to assess cell viability, proliferation, and protein expression levels. Lipid droplet (LD) formation was analyzed using BODIPY staining and quantified using ImageJ software. Levels of free fatty acids (FFAs), cholesterol, acetyl-CoA, ATP, and ketone bodies were measured using commercially available kits. Bioinformatic analysis, including cBioPortal, UALCAN, LinkedOmics, and GSEA, was used to analyze gene expression data from the TCGA database to determine the relationship between SOAT1, CPT1A, and other genes involved in lipid metabolism. In vivo efficacy of avasimibe and the combination of avasimibe and etomoxir was evaluated in DEN-induced HCC mice using tumor volume and weight measurements and H&E staining. Synergistic effects of the drug combination were analyzed using Calcusyn software.

High-fat diet (HFD) increased SOAT1 and CPT1A expression in DEN-induced HCC in mice. Bioinformatics analysis revealed that SOAT1 and CPT1A form a double-negative feedback loop regulating lipid homeostasis. SOAT1 inhibition upregulated CPT1A protein, leading to increased fatty acid oxidation and ketone body production. Conversely, CPT1A inhibition upregulated SOAT1, leading to increased lipid droplet formation. Avasimibe (SOAT1 inhibitor) significantly suppressed HCC growth in HFD-fed mice in vivo. The combination of avasimibe and etomoxir showed synergistic anti-cancer efficacy in vitro and in vivo, reducing cell proliferation more effectively than either drug alone. This synergistic effect was attributed to the disruption of the SOAT1-CPT1A feedback loop, leading to an overload of FFAs and subsequent lipotoxicity. The combination therapy was able to partially counteract the increase in acetyl-CoA, ATP, and β-hydroxybutyrate caused by avasimibe alone, highlighting the interaction between the two pathways.

The study's findings demonstrate that SOAT1 and CPT1A are crucial regulators of lipid homeostasis in HCC, preventing lipotoxicity in the lipid-rich environment often associated with the disease. The double-negative feedback loop between these two pathways allows HCC cells to adapt to varying lipid levels. Targeting SOAT1 alone may upregulate CPT1A, mitigating the therapeutic effect, suggesting that a combination therapy is necessary. The synergistic effect of avasimibe and etomoxir provides a promising novel strategy for HCC treatment. The combination disrupts the delicate balance of lipid homeostasis, leading to an overload of FFAs and causing lipotoxicity, ultimately leading to enhanced cytotoxic effects. The good safety profile of avasimibe and etomoxir in previous clinical trials makes this combination a promising candidate for translation into clinical practice, especially for SH-HCC.

This study unveils a previously unknown double-negative feedback loop between SOAT1 and CPT1A in HCC, highlighting their critical roles in maintaining lipid homeostasis. The synergistic anti-cancer efficacy observed with the combination of avasimibe and etomoxir suggests a promising new therapeutic strategy for HCC, particularly SH-HCC. Future research could focus on optimizing the drug combination, exploring potential biomarkers for patient selection, and conducting clinical trials to evaluate the efficacy and safety of this combination therapy in humans.

The study mainly focused on the SOAT1 and CPT1A pathways in HCC. Other potential pathways involved in lipid metabolism were not extensively investigated. Further research is needed to fully understand the complex interactions among various lipid metabolic pathways in HCC. The in vivo study used a specific HCC model (DEN-induced HCC in mice fed HFD); further studies in other HCC models are necessary to confirm the findings. The sample size of the in vivo studies is relatively small, warranting larger-scale studies for further confirmation.