Aspirin promotes RSL3-induced ferroptosis by suppressing mTOR/SREBP-1/SCD1-mediated lipogenesis in PIK3CA-mutant colorectal cancer

Abstract

Ferroptosis, a new form of regulated cell death triggered by the iron-dependent peroxidation of phospholipids, is associated with cellular metabolism, redox homeostasis, and various signaling pathways related to cancer. Aspirin is a widely used non-steroidal anti-inflammatory drug (NSAID) and has been reported to show therapeutic benefit in cancers harboring oncogenic PIK3CA, which encodes the catalytic p110α subunit of phosphoinositide 3-kinase (PI3K). In this study, we found that aspirin sensitized cancer cells harboring oncogenic activation of PIK3CA to ferroptosis induction. Mechanistically, aspirin inhibited protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling, suppressed downstream sterol regulatory element-binding protein 1 (SREBP-1) expression, and attenuated stearoyl-CoA desaturase-1 (SCD1)-mediated lipogenesis of monounsaturated fatty acids, thus promoting RSL3-induced ferroptosis in colorectal cancer (CRC) cells. Moreover, genetic ablation of SREBP-1 or SCD1 conferred cancer cells greater sensitivity to ferroptosis induction. Conversely, ectopic expression of SREBP-1 or SCD1 restored ferroptosis resistance in CRC cells and abolished the effect of aspirin on RSL3-induced cytotoxicity. Additionally, the synergistic effects of aspirin and RSL3 were confirmed in a xenograft mouse model. The combined use of aspirin and RSL3 resulted in significant tumor suppression. Our work demonstrated that aspirin enhanced the cytotoxic effect of RSL3 in PIK3CA-mutant cancers, and the combination of aspirin and ferroptosis inducer displayed promising therapeutic effects in cancer treatment.

Keywords: Aspirin; Colorectal cancer; Ferroptosis; SCD1; mTOR.

Fig. 1

Aspirin promotes RSL3-induced ferroptosis in PIK3CA-mutant CRC cells. (A) DLD-1 and HCT116 cells were treated with different concentration of RSL3 (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 μM) combined with DMSO or aspirin (2 mM) for 24 h, and cell viability was measured by CCK-8 assays. (B) Violet staining was performed with the indicated concentration of aspirin and RSL3 combination treatment. (C) The changes in cell morphology were observed after treatment with aspirin (2 mM), RSL3 (0.1 μM) or their combination in DLD-1 and HCT116 cells. Scale bar, 100 μm. (D) Cell viability was measured in DLD-1 and HCT116 cells treated with aspirin (2 mM), RSL3 (0.1 μM),or the combination in the presence or absence of Fer-1 for 24 h. (E) Cell viability was measured in DLD-1 and HCT116 cells treated with aspirin (2 mM) and RSL3 (0.1 μM) combination in the presence of with Fer-1 (1 μM), Z-VAD-FMK (10 μM), or 3-MA (1 mM) for 24 h. (F–H) The levels of lipid ROS detected with C11-BODIPY 581/591 (F), MDA detected with the MDA assay kit (G), and ferrous ion detected with FerroOrange dye (H), in DLD-1 and HCT116 cells treated with aspirin (2 mM), RSL3 (0.1 μM) or the combination for 24 h. Scale bar, 50 μm *, P < 0.05; **, P < 0.001; ***, P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Combination treatment with aspirin and RSL3 suppresses the AKT/mTORC1 signaling axis in PIK3CA-mutatnt CRC cells. (A–B) The protein levels of AKT, p-AKT, mTOR, 4E-BP1, p-4E-BP1 were detected in DLD-1 and HCT116 cells treated with aspirin (2 mM), RSL3 (0.1 μM), or their combination for 24 h. (C) DLD-1 and HCT116 cells were pretreated with MHY1485 (2 μM) for 6 h and then exposed to aspirin (2 mM), RSL3 (0.1 μM) or the combination for 24 h. Cell viability was determined by CCK-8 assays. (D) DLD-1 and HCT116 cells were pretreated with rapamycin (50 nM) for 6 h and then exposed to RSL3 (0.1 μM) or Fer-1 (1 μM) for 24 h. Cell viability was determined by CCK-8 assays. (E–G) DLD-1 and HCT116 cells were pretreated with rapamycin (50 nM) or MHY1485 (2 μM) for 6 h and then treated with RSL3 (0.1 μM) for 24h. The levels of lipid ROS detected with C11-BODIPY 581/591(E), MDA detected with the MDA assay kit (F), and ferrous ion detected with FerroOrange dye (G). Scale bar, 50 μm *, P < 0.05; **, P < 0.001; ***, P < 0.001.

Fig. 3

Aspirin sensitizes PIK3CA-mutant CRC cells to RSL3-induced ferroptosis through inhibiting SREBP-1 activity. (A) The protein levels of SREBP-1 and mSREBP-1 were detected by Western blotting in DLD-1 and HCT116 cells treated with aspirin (2 mM), RSL3 (0.1 μM), or the combination for 24 h. (B) The protein levels of SREBP-1 and mSREBP-1 were detected in DLD-1 and HCT116 cells transfected with siNC or siSREBF1. (C) DLD-1 and HCT116 cells transfected with siSREBF1 were treated with RSL3 (0.1 μM) for 24 h and the cell viability was measured by CCK-8 assays. (D) The protein levels of SREBP-1 and mSREBP-1 were detected in DLD-1 and HCT116 cells transfected with vectors expressing SREBP-1a or SREBP-1c. (E) DLD-1 and HCT116 cells overexpressing SREBP-1a or SREBP-1c were treated with RSL3 (0.1 μM) for 24 h and the cell viability was measured. *, P < 0.05; **, P < 0.001; ***, P < 0.001.

Fig. 4

Aspirin sensitizes PIK3CA-mutant CRC cells to ferroptosis by suppressing SCD1 expression. (A) The mRNA levels of ACC, ACLY, FASN, and SCD1 were detected by real time PCR in DLD-1 and HCT116 cells treated with aspirin (2 mM) and RSL3 (0.1 μM) for 24 h. (B) The protein levels of SCD1 were detected in DLD-1 and HCT116 cells treated with aspirin (2 mM), RSL3 (0.1 μM), or the combination for 24 h. (C) The protein levels of SCD1 were detected in DLD-1 and HCT116 cells transfected with siSCD1. (D) DLD-1 and HCT116 cells were transfected with siSCD1 and then treated with RSL3 (0.1 μM) for 24 h. Cell viability was measured by CCK-8 assays. (E–G) DLD-1 and HCT116 cells were transfected with siSCD1 and then treated with RSL3 (0.1 μM) for 24 h. The levels of lipid ROS detected with C11-BODIPY 581/591(E), MDA detected with the MDA assay kit (F), and ferrous ion detected by FerroOrange dye (G). Scale bar, 50 μm *, P < 0.05; **, P < 0.001; ***, P < 0.001.

Fig. 5

Ectopic expression of SCD1 renders PIK3CA-mutant CRC cells resistant to ferroptosis induction. (A) The protein levels of SCD1 were detected in DLD-1 and HCT116 cells transfected with SCD1 overexpression plasmids. (B) DLD-1 and HCT116 cells with SCD1 overexpression were treated with RSL3 (0.1 μM) for 24 h. Cell viability was measured by CCK-8 assays. (C–E) The levels of lipid ROS detected by C11-BODIPY 581/591 (C), MDA detected by the MDA assay kit (D), and ferrous ion detected by FerroOrange dye (E), in RSL3 (0.1 μM)-treated DLD-1 and HCT116 cells with SCD1 overexpression. Scale bar, 50 μm. (F) DLD-1 and HCT116 cells transfected with siSREBF1 were treated with RSL3 (0.1 μM) and SCD1 overexpression plasmid. Cell viability was measured by CCK-8 assays. (G) DLD-1 and HCT116 cells were treated with RSL3 (0.1 μM), OA (100 μM), SA (100 μM), POA (100 μM) or PA (100 μM), with simultaneous treatments of Aspirin (2 mM) for 24 h. The cell viability was measured by CCK-8 assays. *, P < 0.05; **, P < 0.001; ***, P < 0.001.

Fig. 6

The antitumor efficacy of combined aspirin and RSL3 therapy in a human CRC xenograft model. DLD-1 cells were injected subcutaneously into the right flank of nude mice. Six days after inoculation, mice received daily intraperitoneal RSL3 (5 mg/kg), aspirin (100 mg/kg), or the combination of the two drugs (n = 6). (A) Tumor images in different groups. (B) Tumor volume was measured every 2 days. (C) Tumor weight in four groups. (D) The levels of intracellular MDA were measured in different treatment groups. (E) The body weight of mice was measured every 2 days after the xenograft mouse model was established. (F) The expressions of Ki67, 4-HNE, SREBP-1, and SCD1 were determined by immunohistochemical staining, and the staining score was calculated. Scale bar, 40 μm *, P < 0.05; **, P < 0.001; ***, P < 0.001.

Fig. 7

Graphical summary of the mechanism by which aspirin promotes RSL3-induced ferroptosis by inhibiting the mTOR/SREBP-1/SCD1 axis.