Aspirin Inhibits mTOR Signaling, Activates AMP-Activated Protein Kinase, and Induces Autophagy in Colorectal Cancer Cells

doi:10.1053/j.gastro.2012.02.050

Gastroenterology

Volume 142, Issue 7, June 2012, Pages 1504-1515.e3

https://doi.org/10.1053/j.gastro.2012.02.050 Get rights and content

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.

Section snippets

Antibodies and Reagents

Details are provided in Supplementary Table 1.

Cell Line Culture and Treatment

CRC cell lines (RKO, SW480, and HCT116) are available from the American Type Culture Collection (Middlesex, UK). Professor Bert Vogelstein kindly provided HCT116 Akt1/Akt2 knockout cells.¹⁹ Dr Benoit Viollet kindly provided AMPK α1/α2 knockout mouse embryonic fibroblasts (MEFs).²⁰ Cells grown as monolayers in respective media supplemented with 10% fetal calf serum and 1% penicillin/streptomycin were treated at 60%–70% confluence.

Immunoblotting

Cells were lysed in

Aspirin Inhibits mTOR Signaling and Induces AMPK in CRC Cells

We investigated aspirin's effects on the mTORC1 target proteins S6K1, its substrate S6 ribosomal protein (S6), and 4E-BP1 in 3 CRC cell lines: RKO, SW480, and HCT116. These cell lines represent CRC as a whole and differ in their mutation profile with respect to mTOR pathway genes (Supplementary Table 2).

We used 5 mmol/L aspirin for stimulation having previously observed apoptosis with this concentration in CRC cells.²⁴ There was a striking decrease in S6K1 phosphorylation at 10 minutes and an

Discussion

Here, we show that aspirin inhibits mTOR signaling in CRC cells, as evidenced by inhibition of phosphorylation of S6K1, 4E-BP1, and S6. We show that aspirin activates AMPK in CRC cells. Furthermore, we show that aspirin induces autophagy in CRC cells, a response characteristic of mTOR inhibition. Our results support the concept that aspirin affects multiple components of the AMPK/mTOR signaling pathway.

mTORC1 plays a key role in protein synthesis regulation via its effectors S6K1 and 4E-BP1.

Acknowledgments

The authors thank Professor Grahame Hardie (Dundee), who kindly provided the AMPKα1 antibody, discussion, and expertise; Dr Kevin Green for performing nucleotide assays; Dr Beniot Viollet (INSERM), who kindly provided AMPK^{α1/α2−/−} knockout mouse embryonic fibroblasts; Professor Bert Vogelstein (Johns Hopkins) for providing HCT116 Akt1/Akt2 knockout cells; Dr Masashi and Masako Narita (Cambridge) for advice about endogenous LC-3 staining; and Professor Nick Hastie (MRC HGU, Edinburgh), who

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: Conflicts of interest The authors disclose no conflicts.

: Funding This work was funded by a clinician scientist fellowship to FVND from Cancer Research UK (C26031/A11378) and additional funding from a Centre grant to MGD from CORE Charity.

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Original ResearchBasic and Translational—Alimentary TractAspirin Inhibits mTOR Signaling, Activates AMP-Activated Protein Kinase, and Induces Autophagy in Colorectal Cancer Cells

Background & Aims

Methods

Results

Conclusions

Section snippets

Antibodies and Reagents

Cell Line Culture and Treatment

Immunoblotting

Aspirin Inhibits mTOR Signaling and Induces AMPK in CRC Cells

Discussion

Acknowledgments

Lancet

Cell

J Am Coll Surg

Cancer Cell

Biochim Biophys Acta

Cell Metab

J Biol Chem

Cell Metab

Exp Cell Res

Cell

Cell

Mol Cell

Curr Opin Cell Biol

Biochem Biophys Res Commun

Cancer Cell

Eur J Cancer

Am J Surg

Cancer Cell

Effect of aspirin and NSAIDs on risk and survival from colorectal cancer

Gut

mTOR signaling at a glance

J Cell Sci

Characterization of Rictor phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1

Mol Cell Biol

Rictor is a novel target of p70 S6 kinase-1

Oncogene

Colorectal cancer: mutations in a signalling pathway

Nature

Alterations in PTEN and PIK3CA in colorectal cancers in the EPIC Norfolk study: associations with clinicopathological and dietary factors

BMC Cancer

LKB1-dependent signaling pathways

Annu Rev Biochem

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice

Biochem J

Original Research
Basic and Translational—Alimentary Tract
Aspirin Inhibits mTOR Signaling, Activates AMP-Activated Protein Kinase, and Induces Autophagy in Colorectal Cancer Cells