Down-regulation of ACADM-mediated lipotoxicity inhibits invasion and metastasis of estrogen receptor-positive breast cancer cells

doi:10.12122/j.issn.1673-4254.2025.06.06

Abstract

Abstract:

Objective To investigate the effect of downregulation of medium-chain acyl-coenzyme A dehydrogenase (ACADM) on invasion and migration of estrogen receptor-positive breast cancer cells and the underlying mechanism. Methods The Kaplan-Meier Plotter database was used to analyze the ACADM expression levels in breast cancer and normal tissues and their association with patient prognosis. Human breast cancer MCF-7 and T47D cell lines with lentivirus-mediated ACADM knockdown were established, and their in situ tumor formation and metastasis after tail vein injection were evaluated in nude mice. The MCF-7 and T47D cells with ACADM knockdown and their unmodified parental cells were examined with oil-red O staining assay, ROS assay, mitochondrial respiratory chain function assay before and after treatments with ROS scavenger, Elamipretide (a cardiolipin oxidation inhibitor) or SC79 (an AKT activator), and the changes in migration and invasion abilities of the treated cells were analyzed with Transwell invasion assay and Boyden chamber assay. Western blotting was used to detect protein expression levels of related signaling pathways in the treated cells. Results ACADM overexpression was associated with a significantly shorter overall survival of breast cancer patients. In MCF-7 and T47D cells, ACADM knockdown resulted in downregulation of N calnexin, vimentin, p-P13K and p-AKT proteins, increased levels of free fatty acids and reactive oxygen species, lowered activities of mitochondrial respiratory chain complex III and V, and reduced mitochondrial inner phospholipids. ACADM knockdown significantly decreased the invasive capacity of the cells, which were obviously reversed by treatment with ROS scavenger, Elamipretide, and SC79. Conclusion Down-regulation of ACADM inhibits migration and invasion ability of estrogen receptor-positive breast cancer cells by lowering lipotoxicity and impairing mitochondrial function through the ROS/PI3K/AKT pathway.

Key words: breast cancer, medium-chain acyl-coenzyme A dehydrogenase, invasion, lipotoxicity, reactive oxygen species, PI3K-AKT

Jiahao LI, Ruiting XIAN, Rong LI. Down-regulation of ACADM-mediated lipotoxicity inhibits invasion and metastasis of estrogen receptor-positive breast cancer cells[J]. Journal of Southern Medical University, 2025, 45(6): 1163-1173.

Figures/Tables 6

Fig.1 Expression level of ACADM in breast cancer tissues and its effect on malignant behaviors of estrogen receptor (ER)-positive breast cancer cells. A: Expression levels of ACADM detected by Western blotting in normal breast epithelial cells and ER-positive breast cancer cell lines (**P<0.01, ##P<0.01 vs MCF-10A cells). B: Prognosis of breast cancer patients with low and high ACADM expression obtained from the Kaplan-Meier Plotter bioinformatics website. C: Expression of ACADM protein in MCF-7 and T47D cells were detected by Western blotting after down-regulation of ACADM (*P<0.05, **P<0.01 vs NC). D: CCK8 assay for assessing proliferation of MCF-7 cells after down-regulation of ACADM. E: Changes of tumor size of in situ MCF-7 cell xenograft with ACADM knockdown in nude mice. F: HE staining of hepatic metastases in nude mice with tail vein injection MCF-7 cells with or without ACADM knockdown (Original magnification:×200). G: Western blotting for detecting protein expressions in the EMT signaling pathway in MCF-7 cells with ACADM knockdown. *P<0.05, **P<0.01, ***P<0.001 vs NC.

Fig.2 Down-regulation of ACADM-mediated lipotoxicity attenuates invasiveness of MCF-7 and T47D cells. A: Lipid droplets in shACADM MCF7 and T47D cells detected by Oil red O staining (×100). B: Free fatty acid content in shACADM MCF7 and T47D cells detected using a microplate reader (**P<0.01, ***P<0.001 vs NC). C: Ester CoA contents are increased in shACADM MCF-7 and T47D cells (*P<0.05 vs NC). D:Transwell assay for assessing invasiveness of MCF-7 and T47D cells with ACADM knockdown cultured in 4% serum (**P<0.01 vs shACADM). E: Boyden chamber assay for assessing migration ability of MCF-7 and T47D cells with ACADM knockdown cultured in 4% serum (*P<0.05, **P<0.01 vs shACADM).

Fig.3 ACADM knockdown results in increased levels of ROS in breast cancer cells. A: ROS levels in MCF-7 and T47D cells with ACADM knockdown detected by fluorescence microscopy (×100). B: ROS levels in MCF-7 and T47D cells with ACADM knockdown detected by flow cytometry (***P<0.001, ****P<0.0001 vs NC). C: Lipid oxidation (MDA) in MCF-7 and T47D cells with ACADM knockdown detected by colorimetric method (**P<0.01 vs NC). D: ROS content in MCF-7 and T47D cells with ACADM knockdown detected by flow cytometry after treatment with ROS scavenger (****P<0.0001 vs NC; ##P<0.01, ####P<0.0001 vs shACADM). E: Invasion and migration of MCF-7 and T47D cells with ACADM knockdown detected by Transwell assay after treatment with ROS scavengers (**P<0.01, ***P<0.001 vs NC; #P<0.05, ##P<0.01 vs shACADM). F: Invasion and migration of MCF-7 and T47D cells with ACADM knockdown detected by Boyden chamber assay after treatment with ROS scavengers (**P<0.01, ***P<0.001 vs NC; #P<0.05, ###P<0.001 vs shACADM).

Fig.4 Downregulation of ACADM impairs mitochondrial function in breast cancer cells. A: Seahorse technique for detecting oxygen consumption in MCF-7 cells with ACADM knockdown. B: Glycolysis in MCF-7 cells with ACADM knockdown detected using Seahorse technique. C: ATP content in MCF-7 and T47D cells with ACADM knockdown (*P<0.05 vs NC). D: Mitochondrial ROS in MCF-7 and T47D cells with ACADM knockdown detected by confocal fluorescence microscopy (×100). E: Content of mitochondrial ROS in MCF-7 and T47D with ACADM knockdown detected by flow cytometry (***P<0.001, ****P<0.0001 vs NC). F: Activity of mitochondrial respiratory chain complex III in shACADM MCF-7 and T47D cells (*P<0.05 vs NC). G: Activity of mitochondrial respiratory chain complex V in shACADM MCF-7 and T47D cells (*P<0.05, **P<0.01 vs NC).

Fig.5 ACADM knockdown reduces mitochondrial cardiolipin to attenuate invasion and migration of breast cancer cells. A: Hierarchical clustering of lipid species in shACADM MCF-7 and T47D cells. B: Contents of mitochondrial ROS detected by flow cytometry in shACADM MCF-7 and T47D cells treated with Elamipretide (****P<0.0001 vs NC; ###P<0.001, ####P<0.0001 vs shACADM). C: Invasion and migration ability of Elamipretide-treated shACADM MCF-7 and T47D cells detected by Transwell assay (**P<0.01 vs NC; #P<0.05, ##P<0.01 vs shACADM). D: Invasion and migration ability of Elamipretide-treated shACADM MCF-7 and T47D cells detected by Boyden chamber assay (**P<0.01 vs NC; #P<0.05, ##P<0.01 vs shACADM).

Fig.6 ACADM knockdown affects cell invasion capacity by regulating the PI3K/AKT pathway. A: Expressions of PI3K-AKT pathway proteins in shACADM MCF-7 cells and T47D cells detected by Western blotting (*P<0.05, **P<0.01 vs NC). B: Expression of p-AKT protein in shACADM MCF-7 cells and T47D cells with SC79 treatment detected by Western blotting (*P<0.05, **P<0.01 vs NC; #P<0.05, ###P<0.001 vs shACADM). C: Invasion and metastasis ability of shACADM MCF-7 and T47D cells detected by Transwell assay after SC79 treatment (*P<0.05, **P<0.01vs NC; #P<0.05, ##P<0.01 vs shACADM). D: Invasion and migration ability of shACADM MCF-7 and T47D cells detected by Boyden chamber assay after SC79 treatment (*P<0.05 vs NC; #P<0.05, ##P<0.01 vs shACADM). E: ROS levels in shACADM MCF-7 and T47D cells detected by fluorescence microscope after ROS inhibitor treatment (×100). F: Expressions of PI3K-AKT pathway proteins in shACADM MCF-7 cells and T47D cells treated with ROS inhibitor detected by Western blotting (*P<0.05 vs NC; #P<0.05, ##P<0.01 vs shACADM).

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