Accumulation of mutations in cells changes the properties of proteins in vivo, resulting in dysregulation or altered protein-protein interactions (PPIs). Such altered interactions transform the complex signaling pathways and cell metabolism, eventually causing disease. Therefore, observing the changes in PPIs is an important first step in understanding the state of a disease. We are investigating changes in PPI patterns of various oncoproteins using the single molecule co-immunoprecipitation (single co-IP) technique we developed [1][2][3].
Single-molecule fluorescence technologies (single-molecule pull-down and co-IP) have proven to be an ideal platform for studying the cellular signaling complexes and PPI-networks. However, the throughput was still not enough to profile complex PPI networks. A multidisciplinary team of researchers has developed the method for predicting the responses to a targeted therapy for cancers by profiling protein-protein interactions (PPIs) via single-molecule technology.
We had developed a high-throughput single-molecule imaging chip and an automated imaging system. The imaging chip enables 40 different PPI pairs on a single chip with low sample consumption. The highly automated imaging system integrates robotic controlled microscope stage, auto-focusing capability and image analysis for PPI complex detection. The method is able to assess 100 different PPIs per hour from various cancers, which is equivalent to performing 100 different conventional co-IPs and the following western blots in an hour.
We applied the developed single-molecule co-IP imaging to study PPI networks of epidermal growth factor receptor (EGFR). It is believed that the activating mutations in EGFR gene leads to hyperactivation of EGFR signaling pathways. Receptor tyrosine kinases (RTKs) including EGFR are known to activate signaling pathways through phosphorylating tyrosine residues as binding sites for PPIs. Therefore, the measurement of specific phosphotyrosine levels on EGFR has been the standard for gauging the strength of cell signaling[4].
We revealed that the activity of signaling pathway largely depends on the formation of protein complexes, rather than on specific phosphorylation levels. We also showed that a mutation occurred on EGFR gene changes the components of signaling complexes, which leads to unique PPI outcomes of mutant EGFR.
We had further showed that the responses to a EGFR-targeted treatment is strongly dependent on the formation of EGFR PPI complexes from cell lines, patient-derived xenografts and patient specimens. In particular, we identified tumors responding to EGFR-specific inhibitors from three lung adenocarcinoma samples harboring EGFR-inhibitor sensitive mutations as well as five lung squamous carcinoma samples with no actionable mutations.
Finally, tumor tissues from two patients were analyzed by single-molecule PPI profiling and meaningful counts for EGFR PPI complexes were observed from one of them, who had showed a partial response to gefitinib treatment for one and half year.
This investigation from clinical specimens proved that single-molecule PPI profiling technology is ready-to-be used as the diagnostic tool. Our technology will help to make a decision for predicting clinical outcomes to a targeted treatment even though patients carry no actionable mutations on target genes.
[1] Lee, Nat. Commun., 2013
[2] Lee et al., Nat. Protoc., 2013
[3] Yoo et al., J. Am. Chem. Soc., 2016
[4] Lee et al., Nat. Biomed. Eng., 2018