Integrated proteomics identified novel activation of dynein IC2-GR-COX-1 signaling in neurofibromatosis type I (NF1) disease model cells.

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up- or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural abnormalities are, in part, caused by up-regulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1.

Glioma initiating cells form a differentiation niche via the induction of extracellular matrices and integrin αV.

Glioma initiating cells (GICs) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC maintenance/differentiation, we established GIC clones having the potential to differentiate into malignant gliomas, and subjected to DNA microarray/iTRAQ based integrated proteomics. 21,857 mRNAs and 8,471 proteins were identified and integrated into a gene/protein expression analysis chart. Gene Ontology analysis revealed that the expression of cell adhesion molecules, including integrin subfamilies, such as α2 and αV, and extracellular matrices (ECMs), such as collagen IV (COL4), laminin α2 (LAMA2), and fibronectin 1 (FN), was significantly upregulated during serum-induced GIC differentiation. This differentiation process, accompanied by the upregulation of MAPK as well as glioma specific proteins in GICs, was dramatically accelerated in these ECM (especially FN)-coated dishes. Integrin αV blocking antibody and RGD peptide significantly suppressed early events in GIC differentiation, suggesting that the coupling of ECMs to integrin αV is necessary for GIC differentiation. In addition, the expression of integrin αV and its strong ligand FN was prominently increased in glioblastomas developed from mouse intracranial GIC xenografts. Interestingly, during the initial phase of GIC differentiation, the RGD treatment significantly inhibited GIC proliferation and raised their sensitivity against anti-cancer drug temozolomide (TMZ). We also found that combination treatments of TMZ and RGD inhibit glioma progression and lead the longer survival of mouse intracranial GIC xenograft model. These results indicate that GICs induce/secrete ECMs to develop microenvironments with serum factors, namely differentiation niches that further stimulate GIC differentiation and proliferation via the integrin recognition motif RGD. A combination of RGD treatment with TMZ could have the higher inhibitory potential against the glioma recurrence that may be regulated by the GICs in the differentiation niche. This study provides a new perspective for developing therapeutic strategies against the early onset of GIC-associated glioma.