Proteomics and phosphoproteomics profiling of the co-formulation of type I and II interferons, HeberFERON, in the glioblastoma-derived cell line U-87 MG

Abstract

HeberFERON is a co-formulation of Interferon (IFN)-alpha2b and IFN-gamma; in synergic proportions, with a demonstrated effect on skin cancer and other solid tumors. It has antiproliferative effects over glioblastoma multiform (GBM) clones and cell lines in culture, including U-87 MG. Omics studies in U-87 MG showed distinctive expression patterns compared to individual IFNs. Kinase signaling pathways dysregulation can also contribute to HeberFERON effects. Here, we report the first label-free quantitative proteomic and phosphoproteomic analyses to evaluate changes induced by HeberFERON after 72h incubation of U-87 MG cell line. LC-MS/MS analysis identified 7627 proteins with a fold change >2 (p<0.05); 122 and 211 were down- and up-regulated by HeberFERON, respectively. We identified 23549 peptides (5692 proteins) and 8900 phosphopeptides, 412 of these phosphopeptides (359 proteins) were differentially modified with fold change >2 (p<0.05). Proteomic enrichment analysis showed IFN signaling and its control, together to direct and indirect antiviral mechanisms were the main modulated processes. Enrichment analysis of phosphoproteome pointed to the cell cycle, cytoskeleton organization, translation and RNA splicing, autophagy, and DNA repair as biological processes represented. There is a high interconnection of phosphoproteins in a molecular network, where mTOR occupies a centric hub. HeberFERON regulates many phosphosites newly reported or with no clear association to kinases. Of interest is phosphosites increasing phosphorylation were mainly modified by CDK and ERK kinases, thus new cascades regulations can be determining the antiproliferation outcome. Our results contribute to a better mechanistic understanding of HeberFERON in the context of GBM.

An Atlas of Protein Phosphorylation Dynamics During Interferon Signaling

“…This led to the identification of >700 total IFNregulated phosphorylation sites in >500 proteins, vastly increasing our current understanding of factors associated with these pathways. The results we present differentiate themselves from other phosphoproteomic efforts in this area (for example (Vázquez-Blomquist et al, 2022;Viengkhou et al, 2021)) due to the short IFN stimulation periods that we used to capture rapid and transient events relevant to the known timings of JAK/STAT signaling, as well as the comprehensive nature of our approach using all three IFN types. Furthermore, the work adds a complementary additional layer of knowledge to recent studies that have investigated the common and unique specificities of different IFNs with regards to their antiviral activities, or their transcriptome and proteome re-wiring capabilities (Guo et al, 2020;Lum et al, 2024;Matos et al, 2019;Megger et al, 2017;Schuhenn et al, 2022;Yan et al, 2004).…”