Proteomic analyses of brain tumor cell lines amidst the unfolded protein response

Abstract

Brain tumors such as high grade gliomas are among the deadliest forms of human cancers. The tumor environment is subject to a number of cellular stressors such as hypoxia and glucose deprivation. The persistence of the stressors activates the unfolded proteins response (UPR) and results in global alterations in transcriptional and translational activity of the cell. Although the UPR is known to effect tumorigenesis in some epithelial cancers, relatively little is known about the role of the UPR in brain tumors. Here, we evaluated the changes at the molecular level under homeostatic and stress conditions in two glioma cell lines of differing tumor grade. Using mass spectrometry analysis, we identified proteins unique to each condition (unstressed/stressed) and within each cell line (U87MG and UPN933). Comparing the two, we find differences between both the conditions and cell lines indicating a unique profile for each. Finally, we used our proteomic data to identify the predominant pathways within these cells under unstressed and stressed conditions. Numerous predominant pathways are the same in both cell lines, but there are differences in biological and molecular classifications of the identified proteins, including signaling mechanisms, following UPR induction; we see that relatively minimal proteomic alterations can lead to signaling changes that ultimately promote cell survival.

HSP90 inhibitors in the context of heat shock and the unfolded protein response: effects on a primary canine pulmonary adenocarcinoma cell line*

“…Debris was pelleted (16,000 x g, 30 min, 4°C), and lysates were quantified by BCA assay (Pierce ThermoFisher). Cell lysates were prepared as described, and Western blots were run as described [22, 23]. Antibodies for heat shock proteins included anti- HSP110 mAb (cat # 610511, BD Biosciences, Franklin Lakes, NJ, USA), used at 1:500 dilution; anti-HSP90 mAb (cat # 610419, BD Biosciences), used at 1:500 dilution; anti-HSP70/HSP72 mAb (clone C92F3A-5, ADI-SPA-810, Enzo, Farmingdale, NY, USA), used at 1:1000 dilution; anti-HSC70/HSP73 (ADI-SPA-757, Enzo), used at 1:1000 dilution; anti-HSP60 Ab (D307, cat # 4870, Cell Signaling, Danvers, MA, USA), used 1 1:500 dilution; anti-HspBP1 mAb (clone MAB-10201, Orbigen, San Diego, CA, USA), used at 1:500 dilution; anti-aB crystallin mAb (clone 1B6.1-3G4, ADI-SPA-222, Enzo), used at 1:500 dilution; anti-HSF1 Ab (H-311, cat # sc-9144, Santa Cruz Biotechnology, Santa Cruz, CA, USA) used at 1:200 dilution; anti-GRP75/Mortalin mAb (clone 30A5, ADI-SPA-225, Enzo) used at 1:500 dilution; anti-TRAPd mAb (clone C-6, cat # sc-376706), used at 1:500 dilution.…”

“…Antibodies for heat shock proteins included anti- HSP110 mAb (cat # 610511, BD Biosciences, Franklin Lakes, NJ, USA), used at 1:500 dilution; anti-HSP90 mAb (cat # 610419, BD Biosciences), used at 1:500 dilution; anti-HSP70/HSP72 mAb (clone C92F3A-5, ADI-SPA-810, Enzo, Farmingdale, NY, USA), used at 1:1000 dilution; anti-HSC70/HSP73 (ADI-SPA-757, Enzo), used at 1:1000 dilution; anti-HSP60 Ab (D307, cat # 4870, Cell Signaling, Danvers, MA, USA), used 1 1:500 dilution; anti-HspBP1 mAb (clone MAB-10201, Orbigen, San Diego, CA, USA), used at 1:500 dilution; anti-aB crystallin mAb (clone 1B6.1-3G4, ADI-SPA-222, Enzo), used at 1:500 dilution; anti-HSF1 Ab (H-311, cat # sc-9144, Santa Cruz Biotechnology, Santa Cruz, CA, USA) used at 1:200 dilution; anti-GRP75/Mortalin mAb (clone 30A5, ADI-SPA-225, Enzo) used at 1:500 dilution; anti-TRAPd mAb (clone C-6, cat # sc-376706), used at 1:500 dilution. Antibodies for ER chaperones and the UPR, including AKT, pAKT, SAPK/JNK, pSAPK/JNK, were used as described previously[22]. Antibody to PDI, PERK, and ERO1L were from Cell Signaling (ER Stress Antibody Sampler Kit #9956), as was a rabbit mAb to ATF4 (D4B8, #11815), all used at 1:500 dilutions.…”

“…Anti-EGFR mAb was from Novus (clone 29-1; Littleton, CO, USA) used at 1:500 dilution. Anti-actin and anti-tubulin antibodies used for control staining were from Sigma [22, 23]. …”

“…Cells were lysed in buffers provided in the kit, and arrays were probed according to the manufacturer instructions. Arrays were developed as described [22, 24]. …”

“…After 24 hours, cells were lysed and lysates were used to probe an R&D Apoptosis Antibody Array Kit (Catalog # ARY009) (Minneapolis, MN, USA). The array was developed as described [22, 24]. …”