Abstract
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.
Highlights
Malignant glioma is the most common and lethal primary brain tumor [1]
It is suggested that glioma initiating cells (GICs) reside in a microenvironment referred to as the niche, which is composed of stem cells, neighboring supportive cells, extracellular matrix (ECM), and other factors required for stem cell renewal [4], and the manipulation of GIC maintenance/differentiation could be applicable for the clinical treatment of malignant glioma
The astrocyte/glioma marker GFAP and the malignancy marker CD44 dramatically expressed upon serum stimulation with higher levels, but those of the neuron marker Tuj1 were not (Fig. 1B c– e, Fig. 1C, and Fig. S1B), demonstrating that the GIC clones had both the characteristics of neural stem cell (NSC) and the capacity to differentiate into glioma cells, and that they were capable of long-term self-renewal, differentiation, and tumorigenesis
Summary
Malignant glioma is the most common and lethal primary brain tumor [1]. Recently, it was proposed that glioma development is initiated and maintained by glioma initiating cells (GICs), a population of cells capable of extensive self-renewal, multi-lineage differentiation, and promotion of glioblastoma multiform (GBM) development, in immunodeficient mice [2]. It is suggested that GICs are responsible for the therapeutic resistance and recurrence of GBM [3], and considered the most effective therapeutic target for the treatment of malignant gliomas. It is suggested that GICs reside in a microenvironment referred to as the niche, which is composed of stem cells, neighboring supportive cells, extracellular matrix (ECM), and other factors required for stem cell renewal [4], and the manipulation of GIC maintenance/differentiation could be applicable for the clinical treatment of malignant glioma. We previously established a concise proteomic strategy consisting of sequential MS-based, in silico, and cell biological analyses to study the mechanisms of neural differentiation using neural stem cell (NSC) models [7], and demonstrated that this approach was effective for elucidating the functions of proteins involved in cellular biological processes
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