Metformin blocks MYC protein synthesis in colorectal cancer via mTOR-4EBP-eIF4E and MNK1-eIF4G-eIF4E signaling

Abstract

The antidiabetic drug metformin has been associated with reduced colorectal cancer (CRC) risk and improved prognosis of CRC patients. However, the detailed mechanisms underlying such beneficial effects remain unknown. In this study, we aimed to evaluate metformin activity in CRC models and unveil the underlying molecular mechanisms. We showed that metformin inhibits CRC cell proliferation by arresting cells in the G1 phase of the cell cycle and dramatically reduces colony formation of CRC cells. We discovered that metformin causes a robust reduction of MYC protein level. Through the use of luciferase assay and coincubation with either protein synthesis or proteasome inhibitors, we demonstrated that regulation of MYC by metformin is independent of the proteasome and 3' UTR-mediated regulation, but depends on protein synthesis. Data from polysome profiling and ribopuromycylation assays showed that metformin induced widespread inhibition of protein synthesis. Repression of protein synthesis by metformin preferentially affects cell cycle-associated proteins, by altering signaling through the mTOR-4EBP-eIF4E and MNK1-eIF4G-eIF4E axes. The inhibition of MYC protein synthesis may underlie metformin's beneficial effects on CRC risk and prognosis.

Keywords: MYC; mTOR; cell cycle; colorectal cancer; metformin; protein synthesis.

Figure 1

Metformin suppresses CRC cell growth and colony formation. (A) Metformin inhibits HCT116 and HT29 cell growth in a dose‐dependent manner. (B) Metformin arrests the HCT116 and HT29 cells at G1 phase. (C) Metformin at 2 mm completely abrogates the colony formation ability of HCT116 and HT29 cells. (D) Metformin reduces colony formation of CRC cells in a dose‐dependent fashion. The proliferation data are presented as the means ± SD of values obtained in 3 independent experiments. Student's t‐test was used to assess significance relative to vehicle control. *P < 0.05.

Figure 2

Metformin reduces MYC protein expression in CRC cells. (A) Metformin increases Wnt activity as evaluated by TOPFLASH luciferase reporter. (B) Metformin dramatically reduces MYC protein expression without affecting the MYC RNA level. (C) Metformin promotes glycolysis, as shown by acidic medium, and change of cell morphology into spindle‐like shape. (D) Metformin reduces MYC protein expression in both standard culture condition and glucose‐free condition. (E) Metformin diminishes MYC protein expression in colony formation cells. The luciferase data in (A) are 2 independent experiments (exp1 and exp2) in quadruplicate, and data represent means ± SD. The qRT‐PCR data in (B) are presented as the means ± SD (HCT116, n = 2; HT29, n = 4). Student's t‐test was used to assess significance. *P < 0.05.

Figure 3

Metformin blocks MYC protein synthesis in standard culture conditions. (A,B) Metformin‐induced MYC reduction is neither rescued by the proteasome inhibitor MG132, nor accelerated by the protein synthesis inhibitor CHX in HCT116 cells (A) and in HT29 cells (B). In A and B, cells were treated with metformin for 24 h (10 mm), in the presence or absence of MG132 (10 μM). (C,D) Metformin does not enhance the MYC phosphorylation at threonine 58 in 24 h treatment (C) or in colony formation samples (D). (E) Metformin does not reduce luciferase activity of MYC 3′UTR reporter, at the condition of causing MYC protein level changes. Cells were treated with metformin (5 mm, 10 mm) for 24 h, and culture medium was analyzed using the Secrete‐Pair™ Dual Luminescence Assay Kit.

Figure 4

Metformin blocks MYC protein synthesis in glucose‐free media. (A,B) Metformin‐induced MYC reduction is neither rescued by the proteasome inhibitor MG132, nor accelerated by the protein synthesis inhibitor CHX in HCT116 cells (A) and in HT29 cells (B). In A and B, cells were treated with metformin for 24 h (2 mm), in the presence or absence of MG132 (10 μM). (C) Metformin does not enhance the MYC phosphorylation at threonine 58. (D) Metformin reduces the overexpressed MYC protein in HT29 cells established with lentiviral MYC plasmid lacking 5′UTR and 3′UTR. (E) Metformin reduces MYC protein in COLO320 cells that express high level of endogenous MYC due to genomic amplification.

Figure 5

Metformin induces widespread inhibition of protein synthesis. (A) Metformin abrogates the expression of p‐4EBP1 and p‐eIF4E at 24 h, and the effect is partially rescued by coincubation of cells with the mTOR activator MHY1485, in HCT116 cells. (B) The AMPK activator AICAR reduces MYC expression, similar as metformin in HCT116 cells. (C) Metformin blocks protein synthesis, as shown by reduced FITC signal, in HT29 cells stained with OPP, a puromycin analog that incorporated only to newly synthesized proteins. (D) Left panel shows the rational of using ribopuromycylation (puromycin labeling) to monitor protein synthesis, after which the newly synthesized protein can be detected by antibody against puromycin. Metformin strongly blocks protein synthesis, shown by reduced puromycin labeling. AICAR induces a similar yet weaker effect. HT29 cells were treated with metformin (2.5 mm) or AICAR (0.5 mm) for 24 h, and then, the cells were incubated with puromycin (10 μg/mL) for 20 min. Total proteins were isolated and detected by western blot using puromycin antibody. (E) Metformin causes an increase of mRNA association with polysomes and a reduction of mRNA association with monosomes.

Figure 6

Metformin preferably represses the protein synthesis of cell cycle‐related genes. (A) Venn diagram of proteins that are downregulated by metformin (2 mm for 24 h) in four CRC cell lines identified with reverse‐phase protein array (RPPA). (B) Validation of the overlapping downregulated proteins by western blot. (C) Enrich analysis of the 16 downregulated proteins by RPPA revealed an enrichment of proteins that regulate cell cycle. (D) Metformin reduces the expression of p‐MNK1, concordant with the downregulated MNK1 by metformin in RPPA analysis. (E) A schematic diagram presenting the proposed mechanism of metformin effects.