Poster Presentation 31st Annual Lorne Proteomics Symposium 2026

Repurposing metformin as a chemopreventative in colorectal cancer: phosphoproteomics of cell signalling. (#140)

Vo Van Anh Pham 1 , Matthew McKay 1 , Mark Molloy 1
  1. The University of Sydney - Kolling Institute, St Leonards, NSW, Australia

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second deadliest cancer in Australia. Metformin, a first line anti-diabetic drug, shows epidemiological evidence in reducing CRC risk in non-diabetics. The mechanism of metformin’s anticancer activity in the gut is not well understood. We investigated this by using phosphoproteomics to map the signalling pathways that contribute to metformin actions.

CRC cell lines with different mutational profiles were treated with 2.5mM metformin under low-glucose conditions. Samples were harvested in triplicate at 0, 6, and 24 hours, alongside negative controls. Phosphopeptides were enriched with TiO2 beads and analysed by DIA on an Orbitrap HF-X, with Spectronaut v18.5 used for peptide identification. Kinase-Substrate Enrichment Analysis (KSEA) identified differentially regulated phosphosites, which were mapped to kinase-substrate relationships using NetworKIN and PhosphoSitePlus databases.

The ratio of phosphorylated Serine (S): Threonine (T): Tyrosine (Y) is 90:9.9:0.1. 1,165 phosphopeptides were found differentially expressed between conditions. KSEA results showed Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2) upregulation throughout 24 hours—a known mitotic regulator upstream of AMPK activation, metformin's key stimulating pathway1. Sustained downregulation of Protein Kinase A (PRKACA) was observed, inhibiting cyclic AMP/PKA signalling and promoting AMPK activation2. Cyclin Dependent Kinases (CDKs) were downregulated, coupled with upregulation of DNA-Dependent Protein Kinase (PRKDC), an upstream CDKs inhibitor. These findings are consistent with previously reported experiments, where CRC cells were treated with metformin at 10mM concentration1. Calcium/Calmodulin Dependent Protein Kinase II Alpha (CAMK2A), a known upstream activator of AMPK, was also found upregulated. AMPKβ1 was found phosphorylated at Ser108, likely by CAMK2A, which has been previously reported to be activated under low-glucose condition 3,4.

This analysis reveals that metformin treatment at a therapeutic concentration can activate AMPK through multiple convergent pathways, providing insights into metformin actions and its chemopreventive potential in CRC.

  1. Salovska, B., et al. Phosphoproteomic analysis of metformin signaling in colorectal cancer cells elucidates mechanism of action and potential therapeutic opportunities. Clin Transl Med 13, e1179 (2023).
  2. Aw, D.K.L., Sinha, R.A., Xie, S.Y. & Yen, P.M. Differential AMPK phosphorylation by glucagon and metformin regulates insulin signaling in human hepatic cells. Biochemical and Biophysical Research Communications 447, 569-573 (2014).
  3. Raney, M.A. & Turcotte, L.P. Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle. J Appl Physiol (1985) 104, 1366-1373 (2008).
  4. Dite, T.A., et al. The autophagy initiator ULK1 sensitizes AMPK to allosteric drugs. Nature Communications 8, 571 (2017).