A research team led by Merck scientists has taken what could represent a first step toward using phosphoproteins as biomarkers for cancer drug development.
Examining prostate cancer cells treated with three investigational drugs targeting the phosphatidylinositol 3-kinase, or PI3K, signaling pathway, the researchers identified 375 nonredundant serine-threonine phosphopeptides within the pathway, of which 71 were regulated by at least one drug and 11 were regulated by all three drugs.
The results, published in a study in this week's Science Translational Medicine, suggest that biomarker panels comprising some of the drug-responsive phosphoproteins could be used as tools for studying and evaluating the effectiveness of PI3K pathway inhibitors, Ronald Hendrickson, director of proteomics at Merck Research Laboratories and one of the study's authors, told ProteoMonitor.
Kinases — the enzyme class responsible for protein phosphorylation — are central to a variety of cellular processes and have been implicated in a range of cancers. They've became a primary target for cancer drug research, making an understanding of phosphorylation within cancer pathways a key to drug development.
"There are many kinase inhibitors being developed across the whole [pharmaceutical] industry," including Merck, Hendrickson said.
The PI3K pathway, specifically, is commonly activated in human tumors, an event that typically coincides with increased activity of AKT, a kinase that promotes tumor cell survival and metastasis. As such, AKT inhibitors are considered a promising class of cancer drugs.
Better techniques are needed, however, to measure and predict tumor sensitivity to these inhibitors. Measuring AKT activity directly is difficult because existing antibodies for phospho-AKTThr308 — the phosphorylation site that directly correlates with AKT activity — have low sensitivity and stability for immunohistochemistry. This makes alternative phosphoprotein biomarkers for AKT activity desirable.
More broadly, proteomic approaches to studying phosphorylation levels of proteins within pathways activated in cancer patients could enable finer characterization of these pathways, allowing for the development of more target-specific drugs.
"In theory, these drug-related phosphorylation sites could be useful biomarker tools for PI3K pathway inhibitors," Hendrickson said. "You can imagine a future state where you might have a panel of phospho-specific antibodies to measure these activated signaling pathways."
Proven biomarkers "are really critical to the goal of advancing personalized medicine," he added. "The struggle is really to match the right drug to the right patient. That's a significant challenge in oncology."
In the study, the researchers treated SILAC-labeled PC-3 cancer cells with three drug compounds: a PDK1 inhibitor, an AKT inhibitor, and the PI3K-mTOR inhibitor PI-103. They then enriched for phosphopeptides using phospho-motif-specific antibodies and sequenced them via electron disassociation mass spectrometry on a Thermo Electron triple quadrupole-ETD instrument.
This analysis yielded 375 non-redundant phosphopeptides tied to the PI3K pathway, a number Hendrickson said was somewhat higher than the researchers had expected, particularly given "that the PIK3 pathway has been so well studied to date."
Seventy-one of these phosphopeptides showed a greater-than-1.4-fold change in phosphorylation in response to treatment by at least one of the three drug compounds; 32 of the peptides showed a greater-than-1.4-fold change in phosphorylation in response to treatment by at least two of the three compounds; and 11 showed a greater-than-1.4-fold change in phosphorylation in response to treatment by all three.
By analyzing these relative responses to the drug treatments, the researchers were able to reconstruct signaling modules correlating with the established nodes of the PI3K pathway, discovering that most of the modulated proteins mapped to nodes located two hops downstream from the inhibited kinases.
To investigate the potential feasibility of using these phosphopeptides as biomarkers for PIK3 pathway inhibition, the scientists then moved to an experiment designed to demonstrate "proof-of-concept for clinical translation," Hendrickson said.
"Merck selected one biomarker, generated a high-quality antibody reagent against that phosphopeptide, and then showed that this biomarker positively correlates with PIK3 pathway activation and predicts AKT inhibitor sensitivity," he said.
The team selected phospho-PRAS40Thr246, which had been the phosphoprotein most highly regulated by the AKT and PI-103 inhibitors and is a direct substrate for AKT. They developed a custom antibody against it and tested its correlation with AKT activation in 67 breast cancer and 96 lung cancer cell lines using reverse-phase protein microarrays and found strong positive correlation in both cases.
They also examined the relationship between phospho-PRAS40 and AKT activation in mouse prostate tumors, again finding a strong positive correlation. Immunohistochemistry performed on both proteins also demonstrated that phospho-PRAS40 exhibited greater subcellular localization and stability during tissue processing than AKT, further suggesting its usefulness as a biomarker.
Phospho-PRAS40 "predicts the sensitivity of cancer cells to inhibitors of AKT," Hendrickson said, summarizing the findings. "The implications of this research are far-reaching and potentially include finding tailored treatments for cancer patients based upon simple drug- and tumor-specific biomarker patterns."
He noted that the technique is potentially applicable to other cancer pathways and types of phosphorylation, as well.
"It's a general technique, and it highlights a general approach for the identification of uniquely regulated but simple phosphopeptides that are altered with a specific targeted agent," he said. "What we've published is a relatively small experiment. We'll have to see how this generalizes to other pathways."