Glioblastoma-Specific Protein Interaction Network Identifies PP1A and CSK21 as Connecting Molecules between Cell Cycle–Associated Genes

Abstract

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 α (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis.

Nuclear Protein Phosphatase 1 α (PP1A) Expression is Associated with Poor Prognosis in p53 Expressing Glioblastomas

“…PP1A however, in all its complexity, acting as a molecule associated with both pro-apoptotic and pro-tumorigenic roles [6][7][8], has come to light only recently in GBM, with our previous demonstration of PP1A as a novel connecting molecule in GBMs in the p53 subnetwork [2]. Further the recent literature on specificity of nuclear PP1A expression in GBMs [2] and its potential as a targetable molecule [18][19][20] made it imperative for us to understand the prognostic relevance of this molecule in GBM.…”

“…In GBMs, PP1A has been demonstrated by us to be overexpressed both at mRNA and protein levels, the nuclear expression of PP1A was shown to be highly specific for GBMs [2]. The identification of PP1A was predominantly as a connecting molecule in the p53 containing subnetwork of GBMs.…”

“…3,3′-Diaminobenzidine (SigmaAldrich) was used as the chromogenic substrate. GBM tumors that showed PP1A and p53 overexpression by IHC respectively in our previous studies [2,14] served as positive controls. A negative control (slide in which the primary antibody is omitted) was included with each batch of staining.…”

“…Tumor tissues were thus categorised into p53 positive GBM and p53 negative GBM. PP1A demonstrated both cytoplasmic and nuclear staining, however, only the strong nuclear staining was considered for further analysis, in view of our previous demonstration of high specificity of nuclear expression of PP1A in GBM [2]. In each slide >1000 cells were counted and the percentage of cells with strong nuclear staining was depicted as the labelling index (LI).…”

“…Handling such large data arising from these involves use of computational biology techniques. One such previous study from our group employing bioinformatics methods, explored GBM specific protein interaction networks and had identified Protein Phosphatase 1 α (PP1A) as a novel connecting molecule between cell cycle associated genes [2].…”