siRNA Knockdown of REDD1 Facilitates Aspirin-Mediated Dephosphorylation of mTORC1 Target 4E-BP1 in MDA-MB-468 Human Breast Cancer Cell Line

Figures & data

Table 1 Regulation of Gene Expression After 24-Hour Treatment with Aspirin and Salicylic Acid

Gene	Function	Positively (↑) or Negatively (↓) Regulates mTORC1	MDA-MB-468				MCF-7
			0.5 mM ASA	2 mM ASA	0.5 mM SA	2 mM SA	0.5 mM ASA	2 mM ASA	0.5 mM SA	2 mM SA
IRS1 (insulin receptor substrate 1)	Docking protein upstream of PI3K	↑	0.59	0.48	NC	0.58	NC	NC	NC	NC
DEPTOR (DEP domain containing mTOR interacting protein)	Component of mTORC1 and mTORC2 complexes; directly interacts with and inhibits mTOR	↓	NC	1.62	NC	1.63	NC	NC	NC	NC
DDIT4 (DNA damage-inducible transcript 4)	Inhibits mTORC1 activity in a TSC1/2 -dependent manner	↓	NC	2.58	NC	2.01	NC	1.93	NC	1.91

Notes: Regulation is expressed as fold change (FC) between treatment and control conditions. Changes in gene expression were determined as described under “Materials and methods“. FC is given only for DEGs with adjusted P<0.05. ↑ and ↓ indicate how a particular gene regulates (activates or inhibits, respectively) mTORC1 activity regarding the previously reported biological function of the gene.

Abbreviations: ASA, aspirin (acetylsalicylic acid); SA, salicylic acid; NC, no change.

Figure 1 REDD1 expression in various cell lines. (A) Effect of aspirin and salicylic acid treatment on REDD1 expression in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. Cells were exposed to 2 mM of aspirin (ASA), salicylic acid (SA), or vehicle control (C) for 24 hours. Equal amounts of proteins (50 µg) from vehicle or drug-treated cells were loaded on each lane of SDS-PAGE gel for electrophoresis, followed by transfer onto PVDF membranes, which were then probed with anti-REDD1 antibody. β-actin was used as loading control. Densitometric quantification of REDD1 levels were normalized to β-actin for fold change calculations. Data are expressed as means±SD of at least three independent experiments. *P<0.05 (vs vehicle control) by Student’s t-test. (B) Baseline levels of REDD1 in non-treated cells.

Figure 2 Aspirin and salicylic acid attenuate mTORC1 signaling in breast cancer and non-cancerous breast epithelial cells. Cells were exposed to 2 mM of aspirin (ASA), salicylic acid (SA), or vehicle control (C) for 24 (MDA-MB-468, MCF10A) or 17 (MCF-7, MDA-MB-231) hours. Equal amounts of proteins (50 µg) from vehicle or drug-treated cells were loaded on each lane of SDS-PAGE gel for electrophoresis, followed by transfer onto PVDF membranes, which were then probed with respective antibody. β-actin was used as loading control. (A) Data for MDA-MB-468 cell line are expressed as means±SD of four independent experiments. *P<0.05 (vs vehicle control) by Student’s t-test. (B) One experiment was performed in MCF-7, MDA-MB-231, and MCF10A cell lines.

Figure 3 Different effects of REDD1 silencing on aspirin-mediated expression of phosphorylated 4E-BP1. Cells were transfected with non-targeting (siCT) or REDD1 siRNA (siREDD1) for 24 (MDA-MB-468, MCF-7, MDA-MB-231 cell lines) or 48 hours (MCF10A) and treated with vehicle control (C) or 2 mM of aspirin (ASA) for the next 24 hours before cell lysis. Equal amounts of proteins (50 µg) were loaded on each lane of SDS-PAGE gel for electrophoresis, followed by transfer onto PVDF membranes, which were then probed with respective antibody. β-actin was used as loading control. REDD1 was probed on a different blot than p-4E-BP1. Data are expressed as means±SD from three independent experiments for MDA-MB-468, MCF-7, MDA-MB-231, and from two independent experiments for MCF10A.