Faculty Bibliography

Glioblastoma (GBM), a WHO-defined Grade IV astrocytoma, is the most common and aggressive CNS malignancy. Despite current treatment modalities, the survival time remains dismal. The main cause of mortality in patients with this disease is reoccurrence of the malignancy, which is attributed to treatment-resistant cancer stem cells within and surrounding the primary tumor. Inclusion of novel therapies, such as immuno- and DNA-based therapy, may provide better means of treating GBM. Furthermore, manipulation of recently discovered non-coding microRNAs, some of which regulate tumor growth through the development and maintenance of GBM stem cells, could provide new prospective therapies. Studies conducted by The Cancer Genome Atlas (TCGA) also demonstrate the role of molecular pathways, specifically the activated PI3K/AKT/mTOR pathway, in GBM tumorigenesis. Inhibition of the aforementioned pathway may provide a more direct and targeted method to GBM treatment. The combination of these treatment modalities may provide an innovative therapeutic approach for the management of GBM.

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, portends a poor prognosis despite current treatment modalities. Recurrence of tumor growth is attributed to the presence of treatment-resistant cancer stem cells (CSCs). The targeting of these CSCs is therefore essential in the treatment of this disease. Mechanistic target of rapamycin (mTOR) forms two multiprotein complexes, mTORC1 and mTORC2, which regulate proliferation and migration, respectively. Aberrant function of mTOR has been shown to be present in GBM CSCs. All-trans retinoic acid (ATRA), a derivative of retinol, causes differentiation of CSCs as well as normal neural progenitor cells. The purpose of this investigation was to delineate the role of mTOR in CSC maintenance, and to establish the mechanism of targeting GBM CSCs using differentiating agents along with inhibitors of the mTOR pathways. The results demonstrated that ATRA caused differentiation of CSCs, as demonstrated by the loss of the stem cell marker Nestin. These observations were confirmed by western blotting, which demonstrated a time-dependent decrease in Nestin expression following ATRA treatment. This effect occurred despite combination with mTOR (rapamycin), PI3K (LY294002) and MEK1/2 (U0126) inhibitors. Expression of activated extracellular signal-regulated kinase 1/2 (pERK1/2) was enhanced following treatment with ATRA, independent of mTOR pathway inhibitors. Proliferation of CSCs, determined by neurosphere diameter, was decreased following treatment with ATRA alone and in combination with rapamycin. The motility of GBM cells was mitigated by treatment with ATRA, rapamycin and LY29002 alone. However, combination treatment augmented the inhibitory effect on migration suggesting synergism. These findings indicate that ATRA-induced differentiation is mediated via the ERK1/2 pathway, and underscores the significance of including differentiating agents along with inhibitors of mTOR pathways in the treatment of GBM.

Medulloblastoma is the most common malignant brain tumor in children. Recent studies have implicated sonic hedgehog (SHH) and insulin growth factor (IGF) as important mediators in deregulated pathways, which directly inactivate tuberous sclerosis complex, leading to activation of the serine/threonine kinase, mammalian target of rapamycin (mTOR). mTOR consists of two catalytic subunits of biochemically distinct complexes called mTORC1 and mTORC2. This study aims to further elucidate the role of the mTOR pathway, in the development of medulloblastoma, and assess the use of mTOR inhibitors as novel therapeutic agents. Medulloblastoma cells treated with mTORC1 inhibitor, rapamycin, down-regulated pERK expression initially; however ERK activation was evident upon prolonged treatment. Phosphorylation of mTORC1 substrate, p70S6K at thr389 was reduced by rapamycin and pretreatment with rapamycin abrogated platelet-derived growth factor (PDGF)-induced activation of S6K, as well as that of mTORC2 substrate pAKT(Ser473). Activation of AKT was decreased at 1, 3, and 6 h of treatment, but extended treatment with rapamycin increased expression of pAKT(Ser473). Expression of cyclic dependent kinase inhibitor, P27, decreased following PDGF and increased following rapamycin treatment, suggesting their respective impact on cell proliferation via cell cycle control. Cell proliferation was increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of medulloblastoma cells, while it was suppressed following treatment with rapamycin or U0126 (MEK1/2 inhibitor). pp242, a novel combined mTORC1/2 inhibitor, and rapamycin limited proliferation by reducing the S-Phase entry as assessed by EdU incorporation, while PDGF increased EdU incorporation. pp242 reduced the number of cells entering the S-phase to a greater extent than did rapamycin. Migration of medulloblastoma cells towards fibronectin was suppressed in a time-dependent manner after rapamycin treatment. These results indicate that the mTOR pathway is involved in the pathogenesis of medulloblastoma, and that targeting this pathway may provide a strategy for therapy of medulloblastoma.