Category: Professional Posters
Purpose: Of the four primary subtypes of breast cancer, approximately 10-15% of breast cancer is classified as triple-negative breast cancer (TNBC). TNBC is the most aggressive and difficult to treat due to its lack of three receptors that are common chemotherapy targets, making prevention crucial for TNBC. With the rise of proteomics in recent years, statins have been extensively studied for prevention in patients with a high risk of developing breast cancer. The purpose of this study was to analyze the changes in protein expression in TNBC cells treated with lovastatin to determine potential mechanisms for the antiproliferative effect of statins.
Methods: Two sets of TNBC treated with two different concentrations of lovastatin were cultured and collected. The cells were then lysed open using a sonication and a TFE solution and protein concentration was determined using Micro-BCA Assay. A “short-stack” sample clean-up was performed using 100 ug of each sample into a 4-15% Tris-Bis gel for approximately 2 cm, followed by an in-gel digestion using trypsin. The resulting peptides were extracted from the gel using 60% acetonitrile/0.1% formic acid. Samples were then dried of all liquid followed by fractionation using a strong cation exchange (SCX) Stage Tip using resulting in 11 fractions. Each sample fraction was resuspended in 0.1% formic acid and analyzed by LC-MS analysis on a Thermo LTQ mass spectrometer to be using data-dependent analysis. Proteins were identified from the LC-MS data using the MyriMatch search algorithm and the human complement of the UniprotKB database. The data were filtered for confident identifications using IDPicker 3.0. Identified proteins were then further investigated for function utilizing the UniProt database. Once the biological functions of the proteins were determined, potential hypotheses as to how lovastatin causes cancer cell apoptosis could be postulated.
Results: A total of 260 proteins were confidently identified from the samples. The upregulation of heat shock proteins indicate that the lovastatin-treated cells were under stress. An explanation for this stress could include the upregulation of peroxiredoxin leading to increased reactive oxygen species (ROS) within the cells and eventual apoptosis. While, a downregulation of proteins associated with replication and transcription such as ribosomal protein L11 and basic transcription factor 3 also offer explanations for the cell’s apoptosis. Other possible cellular processes that appear to be disturbed by lovastatin are cell adhesion, migration, and division evident by the alteration in proteins associated with actin filaments. A decrease in keratin proteins with an increase in filamin proteins leads to a dysregulation of the actin cytoskeleton and actin activity causing the cell shape to be disformed and the cell to lose ability to migrate and divide leading to cell death. Finally, cell viability was disturbed as seen by the upregulation of 14-3-3 protein beta/alpha which is a negative regulator of MAP kinase activation. The inhibition of MAP kinase activation blocks multiple downstream signaling pathways leading again to cell death.
Conclusion: Upon reviewing the changes in protein expression, it is postulated that lovastatin causes it’s antiproliferative effect through a variety of potential mechanisms. Some of these mechanisms include increasing the amount of ROS, altering replication and transcription, altering actin formation, and blocking the activation of MAP kinase. Further research is needed to look more into these potential pathways and confirm the exact mechanism that lovastatin causes cell its antiproliferative effect. However, for this future research proteomics should be utilized because of the depth of information of potential mechanisms that can be found by examining protein expression, as seen in this study.