HeberFERON distinctively targets Cell Cycle in the glioblastoma-derived cell line U-87MG

Abstract

Background HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, that have shown its clinical superiority over individual interferons in basal cell carcinomas. In Glioblastoma (GBM), HeberFERON has shown promising preclinical and clinical results. This motivated us to design a microarray experiment aimed to identify the molecular mechanisms involved into the distinctive effect of HeberFERON compared with individual interferons. Methods Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive patterns of HeberFERON action. Validation of most distinctive genes was performed by qPCR. Cell Cycle analysis of cell treated by HeberFERON for 24h, 48h and 72h was carried out by flow cytometry. Results The three treatments show different behavior based on the gene expression profiles. Enrichment analysis identified several Mitotic Cell Cycle related events, in particular from Prometaphase to Anaphase, that are exclusively targeted by HeberFERON. FOXM1 transcription factor network which is involved in several Cell Cycle phases and is highly expressed in GBMs is significantly down regulated by HeberFERON. Flow cytometry experiments corroborated the action of HeberFERON over Cell Cycle in a dose and time dependent manner with a clear cellular arrest since 24h post-treatment. Despite the fact that p53 was not down-regulated by HeberFERON several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relation of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain HeberFERON distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes as well as its reduced MDM2 mediated ubiquitination and export from nucleus to cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON over the proliferation of U-87MG. Conclusions We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to individual interferon treatments, where Cell Cycle related events showed the highest relevance.

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

“…Increased phosphorylation of phosphosites unreported in the APC/C component ANAPC1_S223 and the kinesin KIF23_S422 [41] occurred with HeberFERON. The decreased of CENPA, INCENP, APC/C-CDC20 and KIF23 gene expressions with a significantly fold change respect to individual IFNs was observed in a microarray experiment, reinforcing their roles in the G2/M arrest observed [39]. Three phosphosites (S584, S2527, and S2707) with no biological function described in MKI67 , increased phosphorylation [43]; only S584 is predicted to be a CDK1 substrate by NetworKIN.…”

“…INCENP ensures the correct chromosome alignment and segregation, as a scaffold protein regulating the chromosomal passenger complex localization and it is required for chromatin-induced microtubule stabilization and spindle assembly [41]. gene expressions with a significantly fold change respect to individual IFNs was observed in a microarray experiment, reinforcing their roles in the G2/M arrest observed [39]. Three phosphosites (S584, S2527, and S2707) with no biological function described in MKI67, increased phosphorylation [43]; only S584 is predicted to be a CDK1 substrate by NetworKIN.…”

“…Changes in phosphorylation of key proteins in the cell cycle support this is a very important biological process targeted by HeberFERON [39]. The histone H3 variant centromere protein A ( CENPA ) specified accurate segregation of sister chromatids during mitosis [40].…”

Proteomics and Phospho-Proteomics Profiling of the Co-Formulation of Type I and II Interferons, HeberFERON, in the Glioblastoma-Derived Cell Line U-87 MG

“…Cell signaling is the biochemical process by which cells respond to perturbations in their environment, extracellular stimuli, or intracellular cues where the engagement of IFNs to their receptors would be one of those perturbations. Treatment of U-87 MG with HeberFERON causes an antiproliferative effect with a decrease in cell numbers in the treated culture [ 12 ]. Changes in overall quantities of transcripts after 72 h of treatment have already been studied using a microarray chip showing that the cell cycle is one of the most affected processes by the co-formulation [ 12 ].…”

“…Treatment of U-87 MG with HeberFERON causes an antiproliferative effect with a decrease in cell numbers in the treated culture [ 12 ]. Changes in overall quantities of transcripts after 72 h of treatment have already been studied using a microarray chip showing that the cell cycle is one of the most affected processes by the co-formulation [ 12 ]. Protein quantities and protein modification changes are also important ways to understand the biological effect of HeberFERON.…”