Temporal Effects of Combined Birinapant and Paclitaxel on Pancreatic Cancer Cells Investigated via Large-Scale, Ion-Current-Based Quantitative Proteomics (IonStar)

Abstract

SummaryDespite decades of effort, pancreatic adenocarcinoma (PDAC) remains an intractable clinical challenge.An insufficient understanding of mechanisms underlying tumor cell responses to chemotherapy contributes significantly to the lack of effective treatment regimens. Here, paclitaxel, a first-line chemotherapeutic agent, was observed to interact synergistically with birinapant, a Second Mitochondrial-derived Activator of Caspases mimetic. Therefore, we investigated molecular-level drug interaction mechanisms using comprehensive, reproducible, and well-controlled ion-current-based MS1 quantification (IonStar). By analyzing 40 biological samples in a single batch, we compared temporal proteomic responses of PDAC cells treated with birinapant and paclitaxel, alone and combined. Using stringent criteria (e.g. strict false-discovery-rate FDR control, 2 peptides/protein), we quantified 4069 unique proteins confidently (99.8% without any missing data), and 541 proteins were significantly altered in the three treatment groups, with a FDR of <1%. Interestingly, most of these proteins were altered only by combined birinapant/paclitaxel, and these predominantly represented three biological processes: mitochondrial function, cell growth and apoptosis, and cell cycle arrest. Proteins responsible for activation of oxidative phosphorylation, fatty acid β-oxidation, and inactivation of aerobic glycolysis were altered largely by combined birinapant/paclitaxel compared to single drugs, suggesting the Warburg effect, which is critical for survival and proliferation of cancer cells, was alleviated by the combination treatment. Metabolic profiling was performed to confirm substantially greater suppression of the Warburg effect by the combined agents compared to either drug alone. Immunoassays confirmed proteomic data revealing changes in apoptosis/survival signaling pathways, such as inhibition of PI3K/AKT, JAK/STAT, and MAPK/ERK signal transduction, as well as induction of G2/M arrest, and showed the drug combination induced much more apoptosis than did single agents. Overall, this in-depth, large-scale proteomics study provided novel insights into molecular mechanisms underlying synergy of combined birinapant/paclitaxel, and describes a proteomics/informatics pipeline that can be applied broadly to the development of cancer drug combination regimens.4

Pharmacodynamic Drug–Drug Interactions

“…Blue dots predominate over the red, suggesting an overall synergistic interaction that was confirmed quantitatively by model analysis. This figure is reproduced with permission of the American Society for Biochemistry and Molecular Biology, from Wang et al wileyonlinelibrary.com]…”

“…Traditional chemotherapeutic agents that are not molecularly targeted to specific effector pathways tend to be promiscuous; they often exhibit nonspecific pharmacological effects, which further complicates the prediction of PD DDIs based on first principles. However, ‘omics technologies combined with bioinformatics hold the potential for providing new insights into mechanisms of drug action, and PD DDI studies at a detailed molecular level can reveal new targets or pathways underlying the effects of established drugs …”

Glucose Drives Growth Factor–Independent Esophageal Cancer Proliferation via Phosphohistidine–Focal Adhesion Kinase Signaling

“…The peptide fragments derived from purified human FAK were subjected to HCD MS/MS analysis as previously described 29, 48, 49. The nano-flow reverse phase LC included a Spark Endurance autosampler (Emmen, Holland) and an ultra-high pressure Dionex (Sunnyvale, CA) ultimate Nano-2D Ultra capillary/nano-LC system.…”

Signal-Targeted Therapies and Resistance Mechanisms in Pancreatic Cancer: Future Developments Reside in Proteomics

“…Alteration of other post-translational modifications other than tyrosine phosphorylation was not analysed. Large scale proteomics analysing the temporal effects of paclitaxel on pancreatic cancer cells, highlighted the role of proteins involved in mitochondrial function, survival (PI3K pathway) and cell cycle arrest in a key resistance mechanism [ 56 ]. Proteomics has identified that several negative feedback mechanisms are relieved upon mTORC1 inhibition, thus explaining the disappointing results from clinical trials on this target [ 38 , 39 , 57 , 59 ].…”