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. 2005 Apr;7(4):356-68.
doi: 10.1593/neo.04595.

mTOR promotes survival and astrocytic characteristics induced by Pten/AKT signaling in glioblastoma

Xiaoyi Hu  1 , Pier Paolo Pandolfi, Yi Li, Jason A Koutcher, Marc Rosenblum, Eric C Holland
Affiliations

mTOR promotes survival and astrocytic characteristics induced by Pten/AKT signaling in glioblastoma

Xiaoyi Hu et al. Neoplasia. 2005 Apr.

Abstract

Combined activation of Ras and AKT leads to the formation of astrocytic glioblastoma multiforme (GBM) in mice. In human GBMs, AKT is not mutated but is activated in approximately 70% of these tumors, in association with loss of PTEN and/or activation of receptor tyrosine kinases. Mechanistic justification for the therapeutic blockade of targets downstream of AKT, such as mTOR, in these cancers requires demonstration that the oncogenic effect of PTEN loss is through elevated AKT activity. We demonstrate here that loss of Pten is similar to AKT activation in the context of glioma formation in mice. We further delineate the role of mTOR activity downstream of AKT in the maintenance of AKT+KRas-induced GBMs. Blockade of mTOR results in regional apoptosis in these tumors and conversion in the character of surviving tumor cells from astrocytoma to oligodendroglioma. These data suggest that mTOR activity is required for the survival of some cells within these GBMs, and mTOR appears required for the maintenance of astrocytic character in the surviving cells. Furthermore, our study provides the first example of conversion between two distinct tumor types usually thought of as belonging to specific lineages, and provides evidence for signal transduction-mediated transdifferentiation between glioma subtypes.

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Figures

Figure 1
Figure 1
Combined RCAS/tv-a and Cre/Lox systems result in Pten deletion and consequent Akt, but not MAPK pathway activation. (A) Schematic representation of the location of PCR primers to detect the wild-type (wt), floxed, and recombined Pten allele. (B) PCR analysis indicates that RCAS-Cre resulted in Pten deletion in some of the neural progenitors (lane 1, upper band) from Ntv-a LP mice, but not in other types of primary brain cells or uninfected neural progenitors (lane 1, lower band). DNA of RCAS-LacZ-infected primary Ntv-a LP brain cells (lane 2), tail DNA from Ntv-a LP+/- (lane 3), and dH2O (lane 4) were used as controls. (C) Western blot analysis shows that Cre decreased Pten protein level and consequently activated the Akt pathway, but not the MAPK pathway, in neuronal progenitors from Ntv-a LP mice. GAPDH was used as a loading control. Supplementary Figure 1. Low-power views (100x) of GFAP/Olig2 staining in the untreated and 7-day-treated mouse GBMs. In contrast to the IHC of GFAP in the untreated tumors (A), the pattern of GFAP staining in the 7-day-treated tumors was similar to that in oligodendrogliomas, and GFAP-positive cells were reactive astrocytes. In the untreated tumors, Olig2 staining was very heterogeneous. There was no Olig2-positive cells in approximately 90% of microscopic views (B), and (C) represents a view with maximum Olig2-positive cells. (E) and (F) show that almost all surviving tumor cells in two treated tumors were Olig2-positive, and that Olig-2 negative cells were reactive with astrocytes and vascular cells. The asterisks in (F) indicate areas of necrosis.
Figure 2
Figure 2
Histologic analysis of GBMs induced by Cre+KRas from Ntv-a LP mice (A) H&E-stained sections illustrates increased cell density, pseudopalisading necrosis (asterisk), microvascular proliferation, and nuclear polymorphism (arrow) (original magnification of pictures on the upper panel and lower left panel, 40x; original magnification of the picture on the lower right panel, 400x). (B) (40x) IHC shows that Pten was absent in the tumor cells but present in the normal brain cells and endothelial cells within the tumors (higher magnification not shown). The tumor cells were positive for P-Erk and PCNA, and some of them were positive for GFAP and nestin. (C) (50x) IHC shows that some GBMs stained strongly positive for GFAP—one of the characteristics of human GBMs.
Figure 3
Figure 3
Loss of Pten is similar to activated Akt in gliomagenesis. (A) Percent GBM-free mice curves indicate that Cre+KRas and Akt+KRas induced GBMs from Ntv-a LP (nestin-expressing neuronal progenitors) with no significant difference in tumor incidence (62.2% vs 48.6%; P = .61). (B) (10x) H&E comparison of the GBMs induced by Cre+KRas and Akt+KRas from Ntv-a LP mice shows similar tumor morphology. (C) (10x) In Ntv-a wt background, Akt+KRas resulted in large (L), medium (M), and small (S) GBMs; whereas in Ntv-a LP background, Cre+KRas led to only one medium-sized GBM and most GBMs were small. (D) Tumor incidence was higher in Ntv-a LP mice compared to Ntv-a wt mice (P < .01). (E) Percentage of large-sized and medium-sized tumors was higher in Ntv-a wt mice.
Figure 4
Figure 4
mTOR activity correlates with activated Akt in Akt+KRas-induced GBMs, and mTOR blockade by CCI-779 decreases P-S6 in the surviving tumor cells. (10x) IHC for HA (representing P-Akt) (A and B) and P-S6 (E and F) on adjacent sections from Akt+KRas-induced GBMs suggests that mTOR activity as measured by P-S6 correlates with activated Akt. (C and G) (10x) IHC of HA and P-S6 on adjacent sections from the Akt+KRas-induced GBM treated with CCI-779 for 48 hours. (D and H) (10x) IHC of HA and P-S6 on adjacent sections from the Akt+KRas-induced GBMs treated with CCI-779 for 7 days.
Figure 5
Figure 5
mTOR blockade induces apoptotic death in GBM cells expressing activated Akt. (A) MRI with contrast enhancement images shows loss of enhancement in a mice bearing GBM after 7-day CCI-779 treatment. (B) (40x) H&E-stained sections demonstrate that in contrast to psudopalisading necrosis in the untreated GBMs (left panel), 48-hour CCI-779 treatment resulted in apoptotic cell death in the tumor (right panel). The asterisk indicates one representative area of pseudopalisading necrosis. (C) (10x) TUNEL assay shows that CCI-779 treatment led to regional apoptotic cell death (brown color). (D) (400x) Higher magnification of the TUNEL assay results from a mouse GBM treated with CCI-779 for 7 days.
Figure 6
Figure 6
mTOR blockade converts surviving tumor cells to an oligodendroglioma-like morphology (A) (400x) Histology comparison of untreated (upper panel) and 7-day-treated GBMs (lower panel) induced by Akt+KRas. Untreated GBM cells showed fibrillary cytology; they were positive for GFAP but most of them were negative for Olig2, as indicated by H&E and IHC. In contrast, surviving tumor cells acquired oligodendroglioma morphology with round nuclei and cleared cytoplasm; GFAP-positive cells were reactive astrocytes; and most of the tumor cells were positive for Olig2. (B) (400x) IHC of Ki-67 in the untreated (left panel) and 7-day-treated GBMs shows that mTOR blockade inhibited proliferation of the surviving tumor cells. (C) Percentage of Olig2-positive cells in 7-day-treated GBMs was much higher than that in the untreated tumors (P < .01). Each symbol represents one tumor, and “-” represents the mean value for each tumor group. (D) mTOR inhibition decreases GFAP protein level in primary astrocytes. Selected primary astrocytes from Gtv-a wt mice were treated with 10 BM LY294002 (LY) or 10 nM rapamycin (R) for 6 days. After 6 days, cells treated with inhibitors were cultured in medium without inhibitors for an additional 6 days (+/-LY, +/-R). The control cells were mocked-treated for 6 days (lane 1) or 12 days (lane 4). Western blot analysis shows that LY294002 inhibited P-Akt and P-S6, and that rapamycin inhibited P-S6, but the inhibitors did not affect P-Erk significantly. LY294002 and rapamycin inhibited GFAP protein to a similar level, whereas astrocytes began to express GFAP after removal of the inhibitors.
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