Rationale: Increased lipid droplet (LD) formation has been linked to
tumor metastasis, stemness, and chemoresistance in various types of
cancer. Here, we revealed that LD formation is critical for the adaptation to
sorafenib in
hepatocellular carcinoma (HCC) cells. We aim to investigate the LD function and its regulatory mechanisms in HCC. Methods: The key
proteins responsible for LD formation were screened by both metabolomics and proteomics in
sorafenib-resistant HCC cells and further validated by immunoblotting and immunofluorescence staining. Biological function of AKR1C3 was evaluated by CRISPR/Cas9-based gene editing. Isotopic tracing analysis with deuterium3-labeled
palmitate or carbon13-labeled
glucose was conducted to investigate
fatty acid (FA) and
glucose carbon flux. Seahorse analysis was performed to assess the glycolytic flux and mitochondrial function. Selective AKR1C3 inhibitors were used to evaluate the effect of AKR1C3 inhibition on HCC
tumor growth and induction of autophagy. Results: We found that long-term
sorafenib treatment impairs
fatty acid oxidation (FAO), leading to LD accumulation in HCC cells. Using multi-omics analysis in cultured HCC cells, we identified that
aldo-keto reductase AKR1C3 is responsible for LD accumulation in HCC. Genetic loss of AKR1C3 fully depletes LD contents, navigating FA flux to
phospholipids,
sphingolipids, and mitochondria. Furthermore, we found that AKR1C3-dependent LD accumulation is required for mitigating
sorafenib-induced mitochondrial lipotoxicity and dysfunction. Pharmacologic inhibition of AKR1C3 activity instantly induces autophagy-dependent LD catabolism, resulting in mitochondrial fission and apoptosis in
sorafenib-resistant HCC clones. Notably, manipulation of AKR1C3 expression is sufficient to drive the metabolic switch between FAO and glycolysis. Conclusions: Our findings revealed that AKR1C3-dependent LD formation is critical for the adaptation to
sorafenib in HCC through regulating
lipid and energy homeostasis. AKR1C3-dependent LD accumulation protects HCC cells from
sorafenib-induced mitochondrial lipotoxicity by regulating lipophagy. Targeting AKR1C3 might be a promising therapeutic strategy for HCC
tumors.