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. 2009 Mar 6;4(3):226-35.
doi: 10.1016/j.stem.2009.01.007.

PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells

Anne-Marie Bleau  1 , Dolores Hambardzumyan, Tatsuya Ozawa, Elena I Fomchenko, Jason T Huse, Cameron W Brennan, Eric C Holland
Affiliations

PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells

Anne-Marie Bleau et al. Cell Stem Cell. .

Abstract

In normal brain, the side population (SP) phenotype is generated by ABC transporter activity and identifies stem cell and endothelial cell subpopulations by dye exclusion. By drug efflux, the ABCG2 transporter provides chemoresistance in stem cells and contributes to the blood brain barrier (BBB) when active in endothelial cells. We investigated the SP phenotype of mouse and human gliomas. In glioma endothelial cells, ABC transporter function is impaired, corresponding to disruption of the BBB in these tumors. By contrast, the SP phenotype is increased in nonendothelial cells that form neurospheres and are highly tumorigenic. In this cell population, Akt, but not its downstream target mTOR, regulates ABCG2 activity, and loss of PTEN increases the SP. This Akt-induced ABCG2 activation results from its transport to the plasma membrane. Temozolomide, the standard treatment of gliomas, although not an ABCG2 substrate, increases the SP in glioma cells, especially in cells missing PTEN.

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Figures

Figure 1
Figure 1. ABCG2 localizes in vasculature and progenitor cells of PDGF-induced gliomas and identifies side population cells
(A) ABCG2 (red) localizes in blood vessels of gliomas, where it is co-expressed with Von Willebrand factor (vWF) (green); DAPI counterstained in blue. (B) ABCG2 (red), vWF (purple) and nestin (green) shows that some nestin immunoreactive cells are positive for ABCG2 in both endothelial cells and tumor progenitor cells. (C) Hoechst 33342 dye exclusion assay on cells isolated from normal brain cortex identifies a SP and low SP fraction, while a uniform SP fraction is detected in PDGF-induced gliomas. Incubation with verapamil abolished the SP and low SP; MP corresponds to main population. (D) Q-RT-PCR confirms high expression levels of ABCG2 in the low SP and SP as compared to the MP in PDGF-induced tumors. (E) Cell surface phenotype of SP cells shows that in normal brain, the majority of low SP cells are positive for CD31 and/or CD133. In tumor samples, the CD31+ and CD133+ cells are spread over the three populations, indicating a lower ability of endothelial cells to exclude the dye. (F) Quantification of double staining for Hoechst and cell surface markers (CD31, CD133, A2B5 and CD11b) in SP cells isolated from mouse gliomas.
Figure 2
Figure 2. SP cells possess stem cell properties and are enriched in neurospheres
(A) Culture of sorted low SP, SP and MP cells in neurosphere medium shows that only SP cells are able to form neurospheres. (B) Total neurosphere cultures prepared from PDGF-induced tumors stain positive for various stem cell markers such as nestin, Oct4, Sox2, Musashi, ABCG2 and GFAP. Bars = 25 and 50 μm. (C) Q-RT-PCR showing relative ABC transporter mRNA levels in neurospheres as compared to the same cells cultured in DMEM supplemented with 10% FCS. Note that only ABCG2 expression increases under stem cell conditions. (D) Hoechst dye exclusion assay on neurospheres reveals a high percentage of SP cells. Incubation with Verapamil partially abolished the SP fraction while Pantoprazole and Fumitremorgin C (FTC) almost totally abolished the SP phenotype. (E) Sorted SP cells isolated from neurospheres are highly resistant to mitoxantrone as compared to the total population, but were sensitized by the co-incubation with FTC.
Figure 3
Figure 3. Loss of PTEN and temozolomide treatment increase the SP fraction; SP cells with PTEN loss are highly tumorigenic
(A) Loss of PTEN in neurospheres strongly increases the SP phenotype, which is partially block by the incubation with the PI3K inhibitor LY294002. Long-term treatment of neurospheres with 100 μM temozolomide induces an increase in the amount of SP cells, which is greatly favored by PTEN deletion. (B) ABCG2 (red) localizes at the cell membrane in untreated cells as compared to the cytoplasmic staining upon treatment with LY294002. (C) Kaplan-Meier survival curve shows that SP cells isolated from neurospheres with PTEN loss possess a higher tumorigenic potential than the MP cells; temozolomide treatment increases the tumorigenicity of SP cells. (D) Q-RT-PCR in neurospheres showing increased nestin expression after treatment with temozolomide; MGMT expression is enriched in SP cells. (E) Western Blot analysis for ABCG2 indicates that LY294002 (LY) and temozolomide (T) don't modulate ABCG2 expression as compared to DMSO treated cells.
Figure 4
Figure 4. U87MG-ABCG2 stable cell line is resistant to mitoxantrone but not temozolomide
(A) U87-ABCG2 cell line presents a 72kDa band accompanied by post-translational modifications that correlates with high ABCG2 activity (B). (C) The SP2 cells (SP cells enriched by two sorting) are highly resistant to mitoxantrone but not temozolomide.
Figure 5
Figure 5. PI3K/Akt pathway regulates ABCG2 activity in U87MG stable cell line and the SP phenotype of human neurospheres
(A) Immunostaining for hABCG2 (red) shows a membrane staining in SP2 cells versus a cytoplasmic staining in MP2 cells; ZO-1 (green) delimitate the membrane. In the right panel, FACS analysis using cell surface hABCG2 antibody confirms high staining intensity in SP2 cells. Blocking of PI3K with LY294002 induced a change in ABCG2 localization, from the cell membrane to the cytoplasm. (B) Mitoxantrone exclusion assay in SP2 cells after incubation with LY294002, Akt inhibitor IV, perifosine and rapamycin. Blocking of the PI3K and Akt pathways abolished the ability of ABCG2 to efflux mitoxantrone. (C) Hoechst staining of human neurosphere culture shows the presence of a SP that was blocked by FTC. Treatment with LY29004 totally abolished the SP fraction while perifosine displayed a stronger effect in blocking the lower SP fraction. (D) H&E of nude mice injected with SP cells isolated from human GBM neuropsheres. (E). Kaplan-Meier survival curve shows that SP cells isolated from human neurospheres are more tumorigenic then MP cells.
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