Abstract 4121: Unveiling discordant views of a novel drug's influence on cell motility from proteomics and immunofluorescence perspectives
Authors: Chen Weining , Woods Nicholas , Law Henry Chun Hin , Qiao Fangfang , Jagadesan Sankarasubramanian , Guda Chittibabu , Bergan Raymond 
Published: March 2024

Abstract
Introduction: Metastasis is a leading cause of death in patients with solid tumors. Our group has discovered KBU2046, a selective inhibitor of cell motility (Nature Communications 2018). We conducted a time-course immunofluorescent staining to investigate the impact of KBU2046 on the cell adhesion and motility components of human prostate cancer cells. Our objective was to compare our findings with our proteomic analysis results in order to gain insights into the novel molecular mechanisms of KBU2046 in modulating cell motility.
Experimental Procedures: Proteomics analysis. We treated PC3 cells with KBU2046 or vehicle (N=3/group) and quantified protein expression using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). In separate experiments (N=4), we performed LC-MS/MS after Tandem Mass Tag (TMT) labeling on isolated membrane fractions. We used bioinformatics tools, including Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway Analysis (IPA), and protein-protein interaction (PPI) network analysis, to characterize significantly differentially expressed proteins upon KBU2046 treatment and tried to verify by Western blots. Immunofluorescence (IF) staining. Following treatment of PC3, 1532CPTX, and 1532NPTX cells with KBU2046 or vehicle at various intervals, cells were stained for β1, β5 integrin, actin, and DAPI. Images were acquired by a Zeiss LSM 800 Confocal Laser Scanning Microscope.
Results: The proteomics analysis demonstrated that KBU2046 was having a major impact on cell motility processes, consistent with our prior functional studies. Despite conducting Western blot analysis, we could not confirm the candidate proteins linked to cell adhesion. Using IF, we examined changes in cell adhesion proteins over time in response to KBU2046 treatment. The time-course IF investigation revealed that KBU2046 affects adhesion proteins kinetically by altering their subcellular localization over time rather than altering their total expression level. This finding corroborates KBU2046's known effects on cell motility, demonstrating that a kinetic-based analysis yields important new information not directly represented in a single time point screen, although functionally related.
Conclusions: Our study underscores the importance of integrating proteomic and imaging methodologies to procure a holistic comprehension of the molecular mechanisms that govern cell behavior, especially those that relate to highly dynamic processes. Through these methodologies, we have obtained a more profound understanding of how cells regulate motility and how that process can be therapeutically modulated.
Citation Format: Weining Chen, Nicholas Woods, Henry Chun Hin Law, Fangfang Qiao, Sankarasubramanian Jagadesan, Chittibabu Guda, Raymond Bergan. Unveiling discordant views of a novel drug's influence on cell motility from proteomics and immunofluorescence perspectives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4121.