Deep Phosphoproteomic Elucidation of Metformin-Signaling in Heterogenous Colorectal Cancer Cells

The biguanide drug metformin is a safe and widely prescribed drug for type 2 diabetes. Interestingly, hundreds of clinical trials were set to evaluate the potential role of metformin in the prevention and treatment of cancer including colorectal cancer (CRC). To interrogate cell signaling events and networks in CRC and explore the druggability of the metformin-rewired phosphorylation network, we performed a proteomic and phosphoproteomic analysis on a panel of 12 molecularly heterogeneous CRC cell lines. Using in-depth data-independent analysis mass spectrometry (DIA-MS), we profiled a total of 10,142 proteins and 56,080 phosphosites (P-sites) in CRC cells treated with metformin for 30 minutes and 24 hours. Our results indicate that metformin does not directly trigger or inhibit any immediate phosphorylation events. Instead, it primarily remodels cell signaling in the long-term. Strikingly, the phosphorylation response to metformin was highly heterogeneous in the CRC panel, uncovering four groups of metformin responsivity. We further performed a network analysis to systematically estimate kinase/phosphatase activities and reconstruct signaling cascades in each cell line. We created a “MetScore” which catalogs the most consistently perturbed P-sites among CRC cells for future studies. Finally, we leveraged the metformin P-site signature to identify pharmacodynamic interactions revealing a number of candidate metformin-interacting drugs. Together, we provide a data resource using state-of-the-art phosphoproteomics to understand the metformin-induced cell signaling for potential cancer therapeutics. ### Competing Interest Statement JSR has received funding from GSK and Sanofi and fees from Astex and Travere Therapeutics. * AMPK : 5’ AMP-activated protein kinase CAMKK2 : calcium/calmodulin-dependent protein kinase kinase 2/beta CDKs : cyclin-dependent kinases CRC : colorectal cancer DIA-MS : data-independent acquisition mass spectrometry FDR : False discovery rate LC : liquid chromatography LKB1/STK11 : serine/threonine-protein kinase STK11 MAPK3K7/TAK1 : mitogen-activated protein kinase kinase kinase 7 MAPKs : mitogen-activated protein kinases MoA : mechanisms of action NES : normalized enrichment score PER2 : Period circadian protein homolog 2 PGRMC2 : Progesterone receptor membrane component 2 PKN : prior knowledge network mTOR : serine/threonine-protein kinase mTOR MS : mass spectrometry P-site : phosphorylation site T2D : type 2 diabetes