DDIT4 Downregulation by siRNA Approach Increases the Activity of Proteins Regulating Fatty Acid Metabolism upon Aspirin Treatment in Human Breast Cancer Cells

Abstract

Repositioning of aspirin for a more effective breast cancer (BC) treatment requires identification of predictive biomarkers. However, the molecular mechanism underlying the anticancer activity of aspirin remains fully undefined. Cancer cells enhance de novo fatty acid (FA) synthesis and FA oxidation to maintain a malignant phenotype, and the mechanistic target of rapamycin (mTORC1) is required for lipogenesis. We, therefore, aimed to test if the expression of mTORC1 suppressor DNA damage-inducible transcript (DDIT4) affects the activity of main enzymes in FA metabolism after aspirin treatment. MCF-7 and MDA-MB-468 human BC cell lines were transfected with siRNA to downregulate DDIT4. The expression of carnitine palmitoyltransferase 1 A (CPT1A) and serine 79-phosphorylated acetyl-CoA carboxylase 1 (ACC1) were analyzed by Western Blotting. Aspirin enhanced ACC1 phosphorylation by two-fold in MCF-7 cells and had no effect in MDA-MB-468 cells. Aspirin did not change the expression of CPT1A in either cell line. We have recently reported DDIT4 itself to be upregulated by aspirin. DDIT4 knockdown resulted in 1.5-fold decreased ACC1 phosphorylation (dephosphorylation activates the enzyme), 2-fold increased CPT1A expression in MCF-7 cells, and 2.8-fold reduced phosphorylation of ACC1 following aspirin exposure in MDA-MB-468 cells. Thus, DDIT4 downregulation raised the activity of main lipid metabolism enzymes upon aspirin exposure which is an undesired effect as FA synthesis and oxidation are linked to malignant phenotype. This finding may be clinically relevant as DDIT4 expression has been shown to vary in breast tumors. Our findings justify further, more extensive investigation of the role of DDIT4 in aspirin's effect on fatty acid metabolism in BC cells.

Keywords: ACC1; CPT1A; DDIT4; aspirin; breast cancer.

Figure 1

Aspirin increases acetyl-CoA carboxylase 1 (ACC1) phosphorylation in the MCF-7 cell line. (A) Representative Western blots of ACC1 phosphorylation and carnitine palmitoyltransferase 1A (CPT1A) expression in MCF-7 and MDA-MB-468 cells after 24 h aspirin (ASA) treatment. (B) Densitometric quantifications of p-ACC1 and CPT1A levels normalized to β-actin. The results are expressed as the mean ± SD (n = 3 in all graphs except CPT1A quantification in MCF-7 cells, where n = 4), * p value < 0.05.

Figure 2

DNA damage-inducible transcript 4 (DDIT4) knockdown reduces ACC1 phosphorylation after aspirin treatment in breast cancer (BC) cell lines. (A) Representative Western blots and densitometric quantifications of p-ACC1 levels normalized to β-actin. MCF-7 and MDA-MB-468 cells were transfected with non-targeting (siCT) or DDIT4 siRNA (siDDIT4) for 24 h and treated with vehicle control (C) or 2 mM of aspirin (ASA) for the next 24 h before cell lysis. DDIT4 was probed on a different blot than p-ACC1. The numbers under the bands indicate densitometric quantifications of relative p-ACC1 level to β-actin. The results in the graphs are expressed as the mean ± SD (n = 3). (B) Comparison of p-ACC1 fold change after aspirin treatment between cells transfected with non-targeting siRNA and DDIT4 siRNA. The results are expressed as the mean ± SD (n = 3), * p value < 0.05.

Figure 3

DDIT4 knockdown enhances CPT1A expression post aspirin treatment in MCF-7 cells. (A) Representative Western blots and densitometric quantifications of CPT1A levels normalized to β-actin. MCF-7 and MDA-MB-468 cells were transfected with non-targeting (siCT) or DDIT4 siRNA (siDDIT4) for 24 h and treated with vehicle control (C) or 2 mM of aspirin (ASA) for the next 24 h before cell lysis. DDIT4 was probed on a different blot than CPT1A in MCF-7 cell lysates. The numbers under the bands indicate densitometric quantifications of relative CPT1A level to β-actin. The results in the graphs are expressed as the mean ± SD (n = 3). (B) Comparison of CPT1A fold change after aspirin treatment between cells transfected with non-targeting siRNA and DDIT4 siRNA. The results are expressed as the mean ± SD (n = 3). * p value < 0.05.