Introduction: Glioblastoma (GB) is the most common primary malignant brain tumor in adults accounting for 12% to 15% of all intracranial neoplasms and 60% to 75% of astrocytic tumors. It corresponds to WHO grade IV glioma, typically affects subcortical white matter of cerebral hemispheres and less than a half of patients survive more than 1 year.
Since the isolation of tumor stem cells (TSC) from GB, numerous works have been focusing on this subpopulation of cells instead of the tumor bulk. It has been demonstrated that TSC derived from glioblastomas are closer to the genotype and phenotype of tumors they come from than serum-cultured cell lines since serum causes irreversible differentiation of traditional GB cell lines. Moreover, TSC bear remarkable similarity to normal neural stem cells (NSC). NSC renewal and progenitor differentiation are regulated by the niche or microenvironment surrounding them. Certain signals involved classically in embryonic development like Notch, BMP, Noggin, Eph/ephrins and Hedgehog-Gli (Hh-Gli) seem to be important in maintaining these neurogenic niches and might play an important role in brain tumors.
Gli1, the final effector of Hh-Gli pathway, was originally isolated from a human glioma cell line. A number of groups have some doubts regarding the role of Gli in glioma biology. This assumption may arise from the use of cerebellum samples (with high Gli expression) as control in cDNA amplification experiments. Recent works, however, have confirmed the general presence of an active Hh-Gli pathway in GB by different means. GB-derived neurospheres of GB expressing activation of Hh-Gli, treated with cyclopamine (Hh-Gli inhibitor) showed 40% to 60% reduction in growth and no new neurospheres formation when the inhibitor was retired.
The aim of the present study is to analyze the expression of Hh-Gli intermediates in a series of human GB and TSC derived from some of them. We have compared the levels of transcript with controls of white and grey matter and found important correlations with clinical characteristics of the patients.
Materials and Methods: We studied 43 astrocytomas (39 high-grade and 4 low-grade) and 12 controls: 4 white matter, 4 grey matter and 4 hippocampus (from adult epileptic patients with mesial temporal sclerosis), one culture of NSC from white matter and NSC from hippocampus.
After the histological diagnosis, TSC were cultured. We designed primers of the intermediates of the Hh-Gli pathway: Patch1, Smoothened (Smo), Gli1, Gli2, Sufu, and the stem cell marker CD133. The transcription of these genes in tumours, TSC and controls was quantified with real-time PCR. We demonstrated the multipotentiality of the TSC with differentiation assays in vitro and oncogenicity in athymic mice. Demographic, clinical and radiologic characteristics were obtained from the patient's clinical history. All the data were statistically analyzed with SPSS software.
Results: 26 of 43 tumors showed disregulation of some of the intermediates of Hh-Gli. Some of the intermediates: Gli1, Gli2 and Smo were significantly upregulated in tumors compared to controls. The analysis of 3 lineages of TSC showed an increase of effectors of Hh-Gli with serial passages in vitro. Furthermore, several important clinical characteristics were related to Hh-Gli: Sufu, a tumor suppressor, was upregulated in disseminated tumors across white matter fibers at diagnosis and Smo with an increased tumor size, both factors involved in patient's prognosis.
Conclusions: This study demonstrates that most glial neoplasms show disregulation of Hh-Gli in tumors and TSC and opens an important investigation line towards drug discovery with Hh-Gli inhibitors.
Citation Information: Clin Cancer Res 2010;16(14 Suppl):B9.