Phosphoproteomic analysis of metformin signaling in colorectal cancer cells elucidates mechanism of action and potential therapeutic opportunities

Abstract

Background The biguanide drug metformin is a safe and widely prescribed drug for type 2 diabetes. Interestingly, hundreds of clinical trials have been set to evaluate the potential role of metformin in the prevention and treatment of cancer including colorectal cancer (CRC). However, the “metformin signaling” remains controversial. Aims and Methods To interrogate cell signaling induced by metformin in CRC and explore the druggability of the metformin‐rewired phosphorylation network, we performed integrative analysis of phosphoproteomics, bioinformatics, and cell proliferation assays on a panel of 12 molecularly heterogeneous CRC cell lines. Using the high‐resolute data‐independent analysis mass spectrometry (DIA‐MS), we monitored a total of 10,142 proteins and 56,080 phosphosites (P‐sites) in CRC cells upon a short‐ and a long‐term metformin treatment. Results and Conclusions We found that metformin tended to primarily remodel cell signaling in the long‐term and only minimally regulated the total proteome expression levels. Strikingly, the phosphorylation signaling response to metformin was highly heterogeneous in the CRC panel, based on a network analysis inferring kinase/phosphatase activities and cell signaling reconstruction. A “MetScore” was determined to assign the metformin relevance of each P‐site, revealing new and robust phosphorylation nodes and pathways in metformin signaling. Finally, we leveraged the metformin P‐site signature to identify pharmacodynamic interactions and confirmed a number of candidate metformin‐interacting drugs, including navitoclax, a BCL‐2/BCL‐xL inhibitor. Together, we provide a comprehensive phosphoproteomic resource to explore the metformin‐induced cell signaling for potential cancer therapeutics. This resource can be accessed at https://yslproteomics.shinyapps.io/Metformin/ .

Elevated basal AMP-activated protein kinase activity sensitizes colorectal cancer cells to growth inhibition by metformin

“…A very comprehensive study of metformin-regulated signalling in 10 different colorectal cancer cell lines has identified that the activity of more than 80 kinases and phosphatases form part of diverse signalling pathways that are regulated by metformin, across the 10 cell lines [ 24 ]. Direct changes to phosphorylation were demonstrated for more than 40 kinases and phosphatases in all cell lines ( figure 5 a , [ 24 ]).…”

“…A very comprehensive study of metformin-regulated signalling in 10 different colorectal cancer cell lines has identified that the activity of more than 80 kinases and phosphatases form part of diverse signalling pathways that are regulated by metformin, across the 10 cell lines [ 24 ]. Direct changes to phosphorylation were demonstrated for more than 40 kinases and phosphatases in all cell lines ( figure 5 a , [ 24 ]). Similarly, several studies have investigated AMPK-dependent phosphorylation and over 180 AMPK-dependent substrates have been identified, including more than 15 kinases and phosphatases ( figure 5 a , [ 24 , 37 – 40 ]).…”

“…Direct changes to phosphorylation were demonstrated for more than 40 kinases and phosphatases in all cell lines ( figure 5 a , [ 24 ]). Similarly, several studies have investigated AMPK-dependent phosphorylation and over 180 AMPK-dependent substrates have been identified, including more than 15 kinases and phosphatases ( figure 5 a , [ 24 , 37 – 40 ]). Hence, both metformin and AMPK-dependent signalling are very complex.…”

“…( a ) A larger number of signalling pathways are regulated by metformin and AMPK. A comparison of proteins reported to encompass AMPK and/or metformin (from 10 CRC cell lines) regulated phosphorylation [ 24 , 37 – 40 ]. Kinases and phosphatases are listed specifically.…”

“…At the molecular level, metformin, in part, inhibits the mitochondrial electron transport chain complex 1, to prevent the production of ATP from AMP and ADP [ 22 ], causing AMP/ATP and ADP/ATP ratios to increase leading, in part, to AMPK activation. In addition, a range of AMPK-independent metformin effects have been reported including miRNA modulation, reduced reactive oxygen species (ROS) generation, suppression of angiogenesis, independent modulation mTORC1 activity, regulation of inflammation and glucose uptake [ 23 , 24 ].…”