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Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
Published on: November 7, 2019
Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer
Qingxiang Lin1, Shichen Shen2, Zhicheng Qian3
1Department of Cell Stress Biology, Roswell Park Comprehensive Cancer Center, Buffalo, New York, USA; Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York, USA; Center of Excellence in Bioinformatics & Life Science, University at Buffalo, State University of New York, Buffalo, New York, USA.
Abstract:
Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current-based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and 'drug response' mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.
Insights
Pancreatic cancer cells develop gemcitabine resistance (GemR) through altered metabolism and increased invasiveness. Targeting key proteins like ribonucleoside-diphosphate reductase large subunit may overcome GemR in pancreatic adenocarcinoma (PDAC).
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Pancreatic adenocarcinoma (PDAC) is a lethal cancer with limited treatment options.
- Gemcitabine (Gem) is a standard chemotherapy, but resistance (GemR) frequently develops, reducing patient survival.
- Understanding the molecular basis of GemR is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the molecular mechanisms underlying gemcitabine resistance in pancreatic cancer.
- To identify key proteins and pathways altered in Gem-resistant PDAC cells.
- To explore potential therapeutic vulnerabilities in Gem-resistant PDAC.
Main Methods:
- Development of highly gemcitabine-resistant (GemR) clones from gemcitabine-sensitive MIAPaCa-2 cells.
- Global quantitative differential proteomics using an MS1 workflow to analyze ~6000 proteins.
- Comparison of protein expression profiles between sensitive and resistant cell lines (MIA-GR8, PANC-1).
Main Results:
- GemR cells exhibited altered cellular metabolism, increased proliferation, migration, and invasiveness.
- S100 calcium binding protein A4 was significantly downregulated; proteins involved in energy production were reduced.
- Upregulation of mesenchymal markers and increased cellular invasiveness were observed.
- Key enzymes in gemcitabine metabolism were altered, decreasing intracellular gemcitabine utilization.
- Ribonucleoside-diphosphate reductase large subunit was significantly upregulated in GemR cells.
Conclusions:
- Adaptive changes in cellular response systems, including drug metabolism and DNA repair, contribute to gemcitabine resistance complexity in PDAC.
- Downregulation of ribonucleoside-diphosphate reductase large subunit correlates with increased gemcitabine sensitivity, reduced proliferation, and migration.
- These findings suggest potential therapeutic targets to overcome gemcitabine resistance in PDAC patients.
