Targeting cancer stem cells in glioblastoma multiforme using mTOR inhibitors and the differentiating agent all-trans retinoic acid

Abstract

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.

Cellular and molecular effects of the mTOR inhibitor everolimus

“…Furthermore, they showed that this activation was mediated via the S6K1/PI3K/Ras pathway, and was independent of the upregulation of mTORC2, Akt or its downstream components. This is supported further by studies that have reported the involvement of MEK1/2 (MAPK/ERK kinase 1/2), ERK1/2 and mTOR pathways in a mutual negative-feedback loop mediated via S6K1, in which each pathway can inhibit or activate the other, affecting cellular proliferation or differentiation [178]. Furthermore, Friedman et al [178] demonstrated that utilizing other agents such as PI3K (LY294002) and MEK1/2 (U0126) inhibitors in combination with inhibitors of mTOR (rapamycin) had a synergistic effect in preventing cell migration of glioblastoma cells.…”

“…This is supported further by studies that have reported the involvement of MEK1/2 (MAPK/ERK kinase 1/2), ERK1/2 and mTOR pathways in a mutual negative-feedback loop mediated via S6K1, in which each pathway can inhibit or activate the other, affecting cellular proliferation or differentiation [178]. Furthermore, Friedman et al [178] demonstrated that utilizing other agents such as PI3K (LY294002) and MEK1/2 (U0126) inhibitors in combination with inhibitors of mTOR (rapamycin) had a synergistic effect in preventing cell migration of glioblastoma cells. This echoes a previous study by Bresccia et al [179] who reported that dual inhibition with PI3K and MAPK inhibitors was superior in efficacy to inhibition of a single pathway alone.…”

Combination Treatment with All-Trans Retinoic Acid Prevents Cisplatin-Induced Enrichment of CD133+ Tumor-Initiating Cells and Reveals Heterogeneity of Cancer Stem Cell Compartment in Lung Cancer

“…ATRA has been reported to be effective in the cure and prevention of many types of cancer (reviewed in 30 ), however, its differentiating role against solid tumors CSC’s compartment is still poorly explored. 31–33 Using in vitro cell fate tracing systems and in vivo models of patient-derived xenografts (PDXs), 34 we show here that ATRA is able to interfere with the dynamics of the CSC compartment and prevents chemotherapy-related TICs increase.…”

Immunohistochemical Detection of Cancer Stem Cell Related Markers CD44 and CD133 in Metastatic Colorectal Cancer Patients

“…Several treatment regimens specifically targeting CSCs are now emerging to overcome this problem. One of these approaches is forced differentiation of CSCs combined with targeted therapy [10]. …”