Chemical proteomic strategies for deciphering neocarzilin´s mode of action in cancer cells
Final Report Abstract
Natural products address a plethora of different proteins with essential functions. The deconvolution of their targets represents a unique opportunity for unravelling new anti-cancer strategies. We here apply chemical proteomics to identify the targets of the natural product neocarzilin A (NCA) within living cancer cells. NCA is a potent anti-cancer compound which was identified decades ago but still lacks functional characterization. In preliminary studies we synthetically equipped the natural product with an alkyne tag and treated proteomes to identify its cellular target(s). Interestingly, only one protein, the vesicle amine transport protein (VAT-1) with an essential role in cell migration, has been identified as prominent hit. Within this project period we were able to introduce a refined chemical probe that mimics the structural and stereochemical properties of NCA, and identify bone marrow stromal antigen 2 (BST-2) as a key target contributing to the anti-proliferative activity of NCA in cancer cells. This mechanism was validated using BST-2 knockout (KO) HeLa cells, which exhibited reduced sensitivity to NCA treatment. Conversely, reintroduction of BST-2 in KO cells restored the NCA-induced proliferation inhibition to levels similar to wild-type (WT) HeLa cells. Proteome-wide mass spectrometry analysis of NCA-treated WT and KO cancer cells highlighted alterations in specific pathways and revealed decreased BST-2 levels following NCA exposure. Further investigation into the modulation of BST-2 levels using proteasome and lysosome inhibitors indicated that NCA induces lysosomal degradation of BST-2. As BST-2 facilitates the release of epidermal growth factor receptor (EGFR) from lipid rafts, the observed reduction in BST-2 levels resulted in diminished proliferation as indicated by impaired STAT3 phosphorylation. Additionally, fluorescence microscopy showed increased colocalization of EGFR with lipid rafts in the presence of NCA, supporting these findings. Albeit we were able to crystallise and determine the structure of Vat-1 in a new space group, our attempts to obtain complex crystals of Vat-1 and NCA and its derivatives were not successful.
Publications
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Neocarzilin A Is a Potent Inhibitor of Cancer Cell Motility Targeting VAT-1 Controlled Pathways. ACS Central Science, 5(7), 1170-1178.
Gleissner, Carolin M.-L.; Pyka, Carolin L.; Heydenreuter, Wolfgang; Gronauer, Thomas F.; Atzberger, Carina; Korotkov, Vadim S.; Cheng, Weiting; Hacker, Stephan M.; Vollmar, Angelika M.; Braig, Simone & Sieber, Stephan A.
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Neocarzilin Inhibits Cancer Cell Proliferation via BST-2 Degradation, Resulting in Lipid Raft-Trapped EGFR. JACS Au, 4(5), 1833-1840.
Braun, Josef; Hu, Yudong; Jauch, Adrian T.; Gronauer, Thomas F.; Mergner, Julia; Bach, Nina C.; Traube, Franziska R.; Zahler, Stefan & Sieber, Stephan A.
