Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase, and induces autophagy in colorectal cancer cells

Abstract

Background & aims: Aspirin reduces the incidence of and mortality from colorectal cancer (CRC) by unknown mechanisms. Cancer cells have defects in signaling via the mechanistic target of rapamycin (mTOR), which regulates proliferation. We investigated whether aspirin affects adenosine monophosphate-activated protein kinase (AMPK) and mTOR signaling in CRC cells.

Methods: The effects of aspirin on mTOR signaling, the ribosomal protein S6, S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) were examined in CRC cells by immunoblotting. Phosphorylation of AMPK was measured; the effects of loss of AMPKα on the aspirin-induced effects of mTOR were determined using small interfering RNA (siRNA) in CRC cells and in AMPK(α1/α2-/-) mouse embryonic fibroblasts. LC3 and ULK1 were used as markers of autophagy. We analyzed rectal mucosa samples from patients given 600 mg aspirin, once daily for 1 week.

Results: Aspirin reduced mTOR signaling in CRC cells by inhibiting the mTOR effectors S6K1 and 4E-BP1. Aspirin changed nucleotide ratios and activated AMPK in CRC cells. mTOR was still inhibited by aspirin in CRC cells after siRNA knockdown of AMPKα, indicating AMPK-dependent and AMPK-independent mechanisms of aspirin-induced inhibition of mTOR. Aspirin induced autophagy, a feature of mTOR inhibition. Aspirin and metformin (an activator of AMPK) increased inhibition of mTOR and Akt, as well as autophagy in CRC cells. Rectal mucosal samples from patients given aspirin had reduced phosphorylation of S6K1 and S6.

Conclusions: Aspirin is an inhibitor of mTOR and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism. These could contribute to its protective effects against development of CRC.

Figure 1

Aspirin inhibits mTOR and activates AMPK in CRC cells. Aspirin-treated CRC whole-cell lysates were immunoblotted. Aspirin inhibits phosphorylation of (A) S6K1, S6 ribosomal protein (S6), and (B) 4E-BP1 in CRC cells. (C) Aspirin induces phosphorylation of AMPK and its substrate ACC in CRC cells. Imagequant software (GE Healthcare, Little Chalfont, UK) was used for densitometry, which is presented as the ratio of phosphorylated to total protein corrected for actin. Phenformin-treated CRC cells were used as controls. AMPK was immunoprecipitated from aspirin-treated whole-cell lysates and assayed with the AMARA peptide. (D) Aspirin increases AMPK activity in HCT116 cells (mean of 3 independent experiments).

Figure 2

AMPK depletion, siRNA-mediated or genetic, does not attenuate aspirin-induced mTOR inhibition. RKO cells, transfected with siRNA against (Ai) AMPK α1, (Aii) α2, or control (con) for 48 hours, were probed as noted. (B) Lysates from aspirin-treated RKO cells transfected with AMPKα1 siRNA were immunoblotted. (C) Lysates from aspirin-treated AMPK^{α1/α2−/−} knockout MEFs and parental cells (wild type [WT]) were immunoblotted.

Figure 3

Influence of Akt on AMPK activation and mTOR inhibition and effects on mTORC2. Whole-cell lysates from (Ai) 10-minute or (Aii) 16-hour aspirintreated HCT116 Akt1/2 knockout and parental (wild type [WT]) cells immunoblotted (duplicate experiments presented). (B) Aspirin-treated CRC cells immunoblotted for p-NDRG1.

Figure 4

Dual targeting of AMPK, Akt, and mTOR signaling with aspirin and metformin. (A) Metformin decreases Akt phosphorylation and increases AMPK phosphorylation in RKO cells. RKO cells were treated with metformin alone, metformin and aspirin, aspirin after pretreatment with metformin (10 minutes), or aspirin alone at concentrations indicated for (B) 10 minutes or (C) 16 hours. Representative results are presented in duplicate.

Figure 5

Aspirin induces apoptosis, inhibition of cell proliferation, and autophagy in CRC cells. Aspirin- or TNF-related apoptosis-inducing ligand (TRAIL) (10 ng/mL) treated CRC cells were lysed and whole-cell, cytoplasmic (CE), or nuclear (NE) extracts were immunoblotted. (A) Aspirin increases cleaved caspase-3 in CRC cells. (B) Aspirin decreases proliferating cell nuclear antigen (PCNA) in both cytoplasmic and nuclear CRC cell extracts. Aspirin decreases cytoplasmic HuR and cyclin A in CRC cells. (C) Aspirin increases LC3-II in HCT116 cells pretreated with bafilomycin A. (D) Increased endogenous LC3 staining in aspirin-, metformin-, or combination-treated RKO cells confirms autophagy at 16 hours. Aspirin induces ULK1 Ser555 phosphorylation in RKO cells but not in AMPK^{α1/α2−/−} knockout MEFs. DAPI, 4′,6-diamidino-2-phenylindole. (E) Aspirin decreases ULK1 Ser757 phosphorylation in HCT and RKO cells. (F) Aspirin increases LC3-II in both AMPK^{α1/α2−/−} MEFs and HCT116 Akt1/2 knockout and respective parental cells.

Figure 6

Aspirin activates AMPK and inhibits mTOR in vivo. Aspirin activates AMPK and ACC phosphorylation in (Ai) liver and (Aii) colon from mice treated with aspirin or phenformin for 21 days (aspirin, 400 mg/kg; phenformin [PHEN], 300 mg/kg body weight). (Bi) Basal untreated total S6 levels in normal rectal mucosa of 3 patients. (Bii & Biii) Aspirin-treated patients (600 mg once-daily [OD], 7 days) show decreased S6 and S6K1 phosphorylation in normal rectal mucosa. Respective graphs show mean densitometry values of phosphorylated:total protein, corrected for actin ± standard error. P, 5mM phenformin; S, serum shock.

Figure 7

Aspirin targets multiple components of the AMPK/mTOR signaling pathway in colorectal cancer. Aspirin increases phosphorylation of AMPK and ULK1. Aspirin inhibits mTOR signaling as evidenced by decreased phosphorylation of S6K1, S6, and 4E-BP1. Hence, aspirin is targeting multiple cellular pathways involved in mRNA stability, cell cycle, autophagy, protein translation, and ribosome biogenesis.