Abstract 3916: Cyanobacterial natural product Sec61α inhibitors induce broad suppression of the glioma cell secretome
Authors: Mattos Daphne R. , Serrill Jeffrey D. , Gafken Philip R. , McPhail Kerry L. , Vogel Walter K. , Ishmael Jane E. 
Published: June 2022

Abstract
Coibamide A is a cyclopeptide natural product with potent antiproliferative activity against cultured human cancer cells and subcutaneous U87-MG glioblastoma tumors in mice. We have recently determined that coibamide A binds to the Sec61 translocon channel at a site that is close, but not identical, to the binding site of the peptide/polyketide natural product apratoxin A. As the majority of secreted proteins in mammalian cells are trafficked through the conventional secretory pathway via the Sec61 translocon, this raised the possibility that coibamide A and apratoxin A block import and secretion of different Sec61 client proteins. In the present study, we analyzed and compared the secretome of human U87-MG glioblastoma cells in the presence and absence of both marine natural products.
U87-MG glioblastoma cells were treated with coibamide A (5 nM), apratoxin A (5 nM), or vehicle for 24 h. The growth media was then replaced with serum-free media for a further 24 h. Tryptic proteomic samples were generated from cell-free conditioned media and analyzed on a LC-coupled Orbitrap Fusion mass spectrometer. The high-resolution Orbitrap mass analyzer was used to both identify precursor ions for tandem mass (MS2) analysis in the ion trap mass analyzer and to quantitate those ions (MS1). Secreted proteins were identified from peptide–spectrum matches to the human protein database using a decoy database to assign an FDR and quantified from their MS1 ion intensities.
Comparison of mass spectrometric data against the human secretome database revealed 175 proteins in the U87-MG cell secretome that were identified with high confidence. Differences between coibamide A and apratoxin A treatment were not significant, however both Sec61 inhibitors induced significant suppression of the U87-MG secretome relative to vehicle-treated cells. Treatment with coibamide A or apratoxin A resulted in the reduction of 75% of quantifiable secretome (131 proteins). Of the remaining 44 proteins, 34 are directed to intracellular organelles or contain a motif associated with retention in the endoplasmic reticulum and 10 were not suppressed with treatment but were hypothesized to be Sec61 clients.
Malignant glioblastoma cells secrete proteins into the extracellular space and use complex intercellular signaling mechanisms for a survival advantage. Our results indicate that broad-acting inhibitors of Sec61-dependent co-translational translocation block the progression of a significant percentage of the U87-MG glioblastoma cell secretome including several proteins that have previously been detected as biomarkers in the cerebrospinal fluid, blood, or urine of brain tumor patients. Taken together, our work illustrates the feasibility of inducing a reversible suppression of numerous (undrugged) extracellular signals by targeting Sec61-dependent protein biogenesis in the secretory pathway with drug-like molecules.
Citation Format: Daphne R. Mattos, Jeffrey D. Serrill, Philip R. Gafken, Kerry L. McPhail, Walter K. Vogel, Jane E. Ishmael. Cyanobacterial natural product Sec61α inhibitors induce broad suppression of the glioma cell secretome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3916.