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Comparative Study
. 2008 Feb 15;22(4):436-48.
doi: 10.1101/gad.1627008.

PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo

Dolores Hambardzumyan  1 , Oren J Becher, Marc K Rosenblum, Pier Paolo Pandolfi, Katia Manova-Todorova, Eric C Holland
Affiliations
Comparative Study

PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo

Dolores Hambardzumyan et al. Genes Dev. .

Abstract

Medulloblastomas are brain tumors that arise in the cerebellum of children and contain stem cells in a perivascular niche thought to give rise to recurrence following radiation. We used several mouse models of medulloblastomas in parallel to better understand how the critical cell types in these tumors respond to therapy. In our models, the proliferating cells in the tumor bulk undergo radiation-induced, p53-dependent apoptotic cell death. Activation of Akt signaling via PTEN loss transforms these cells to a nonproliferating extensive nodularity morphology. By contrast, the nestin-expressing perivascular stem cells survive radiation, activate PI3K/Akt pathway, undergo p53-dependent cell cycle arrest, and re-enter the cell cycle at 72 h. Furthermore, the ability of these cells to induce p53 is dependent on the presence of PTEN. These cellular characteristics are similar to human medulloblastomas. Finally, inhibition of Akt signaling sensitizes cells in the perivascular region to radiation-induced apoptosis.

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Figures

Figure 1.
Figure 1.
Radiation induces apoptosis in TB of medulloblastoma. (A) Whole mount of control nonirradiated medulloblastoma (SHH + N-myc representative pictures). (B) Whole mount of 6-h-post-2-Gy γIR, stained for H&E, cleaved caspase-3 indicating apoptosis, and PCNA indicating proliferation (red arrows indicate tumors). Immunofluorescence detection of PCNA (green), cleaved caspase-3 (red), and DAPI (blue) of untreated (C) and irradiated (D) tumors (SHH + N-myc, n = 2; SHH + Akt, n = 2). As a result of irradiation, PCNA is excluded from the nuclei and colocalizes with cleaved caspase-3 in the cytoplasm of the TB cells (yellow color on the merged image) Bars: C,D, 20 μm.
Figure 2.
Figure 2.
Two distinct medulloblastoma cell populations survive 2 Gy of γIR. H&E staining for cells of the EN (A) and cells of the PVN (B) (red arrow) (SHH + N-myc representative pictures). (C) Cleaved caspase-3 and PCNA staining showing lack of cleaved caspase-3 and proliferation in the surviving cells of the PVN. Surviving PVN cells are adjacent to blood vessels, express nestin, and are cleaved caspase-3-negative (white arrows). (D) High- and low-magnification images of triple immunostaining with CD34 (red), nestin (green), cleaved caspase-3 (magenta), and DAPI counterstaining (blue) show that all surviving PVN cells are nestin-positive. Bars: A–C, 50 μm; D, 20 and 100 μm.
Figure 3.
Figure 3.
Evaluation of cell death and proliferation over time following 2 Gy of γIR in p53+/+ medulloblastomas. Cells surviving 2 Gy of γIR in the PVN re-enter the cell cycle at 72 h. (A) H&E, TUNEL, cleaved caspase-3, PCNA, and pH3 of untreated tumors and tumors treated by 2 Gy of γIR were collected after 4, 6, 12, 48, and 72 h, as indicated. Clusters of cells re-enter the cell cycle at 72 h (white arrow). (B) Triple immunofluorescent staining of PCNA (green), nestin (red), and DAPI (blue) at 72 h after 2 Gy of γIR in PVN shows high proliferation of nestin-positive cells (white arrows). Bars: A, 100 μm; B, 20 μm.
Figure 4.
Figure 4.
Radiation induces elevation of nestin and pS6RP expression in the cells of the PVN. (A) High and low magnification of triple immunofluorescent staining of CD34 (red), nestin (green), pS6RP (magenta), and DAPI (blue) following 6 h of 2 Gy of γIR (SHH + N-myc). Quantification of immunostaining intensity of nestin (B) and pS6RP (C) at the time points indicated following 2 Gy of γIR (SHH + N-myc and SHH + Akt-driven tumors). PVN in human medulloblastomas are very similar to those seen in mice. (D) Triple immunofluorescent merged images (pS6RP [green]/CD31 [red]/DAPI [blue], CD31 [red]]/nestin [green]/DAPI [blue], nestin [green]/pS6RP [red]/DAPI [blue], and nestin [green]/notch receptor [red]/DAPI [blue]) of human medulloblastoma samples showing existence of stem cell niches around blood vessels similar to mouse tumors (n = 2 frozen and 13 paraffin sections, total 15). Bars: A, 20 and 100 μm; D, 20 μm. Dunnett’s Multiple Comparison Test of ANOVA was used. (**) P < 0.01; (no asterisk) not significant.
Figure 5.
Figure 5.
PTEN loss in combination with SHH overexpression leads to formation of medulloblastomas with EN in mice. Whole mount (A) and higher magnification (B) of H&E, PCNA, and PTEN immunoreactivity as indicated (black arrow represents proliferating cells of PVN and red arrow shows endothelial cells, the only PTEN-positive cells in the tumor). (C) Triple immunofluorescent staining of NeuN (red), pS6RP (green), and DAPI (blue) in the PVN. (D) Triple immunofluorescent staining of NeuN (red), nestin (green), and DAPI (blue). Bars: B, 100 μm; C,D, 20 μm.
Figure 6.
Figure 6.
Radiation-induced cell death and cell cycle arrest are p53-dependent. (A) At 4 h, p53 is immunoreactive in the dying cells outside of the PVN; at 6 h, the cells of the PVN express p53 while the cells outside of the PVN have lost p53 expression (black arrow represents apoptotic cells, and yellow arrow represents arresting cells). Regions of EN do not elevate p53 expression following radiation (SHH + N-myc). Whole mounts of H&E, PCNA, and cleaved caspase-3 of control (B) and 6-h-post-γIR (C) tumors in N-tva/p53−/− background (SHH + Akt) (red arrows point to the tumor regions). (D) Quantification of cleaved caspase-3 staining in medulloblastomas untreated and treated with 2 Gy of γIR in different backgrounds with different combinations (n = 4 for groups 1 and 2; n = 6 for groups 3 and 4, which contain spontaneous medulloblastomas in Ptc+/− background and medulloblastomas in Ptc+/− background irradiated at P1; n = 5 for groups 5 and 6). Triple immunofluorescent images of PCNA (green), cleaved caspase-3 (red), and DAPI (blue) of untreated (E) and 6-h-post-γIR (F) tumors in Ntva/p53−/− background showing very little elevation of cleaved caspase-3. Bars: A, 50 μm; E, 20 μm; F, 100 μm. Two-sided t-test was used for D. (***) P < 0.001.
Figure 7.
Figure 7.
Perifosine inhibits Akt phosphorylation and sensitizes the PVN to radiation-induced cell death in SHH + Akt tumors. Perifosine was administered to tumor-bearing mice i.p. at 30 mgper kilogram daily for 3 d (n = 3). A control group was treated with saline (n = 3). At 12 h after the last dose, 2 Gy TBI was administered (Perifosine + 2 Gy, n = 3). Representative immunohistochemistry for cleaved cas-3 (A) and pAkt S473 (B). (C) Quantification of the surviving PVN area; representative photomicrographs of cleaved caspase-3 staining in 2 Gy and Perifosine + 2 Gy treatment groups are on the left. Fifteen 20× pictures were taken, the surviving areas were quantified, and averages were plotted. Bars: A,B, 100 μm,; C, 50 μm. Two-sided t-test was used in C. (*) P < 0.05.
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References

    1. Ahmed N.N., Grimes H.L., Bellacosa A., Chan T.O., Tsichlis P.N. Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc. Natl. Acad. Sci. 1997;94:3627–3632. - PMC - PubMed
    1. Alvarez-Buylla A., Seri B., Doetsch F. Identification of neural stem cells in the adult vertebrate brain. Brain Res. Bull. 2002;57:751–758. - PubMed
    1. Bao S., Wu Q., McLendon R.E., Hao Y., Shi Q., Hjelmeland A.B., Dewhirst M.W., Bigner D.D., Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006;444:756–760. - PubMed
    1. Browd S.R., Kenney A.M., Gottfried O.N., Yoon J.W., Walterhouse D., Pedone C.A., Fults D.W. N-myc can substitute for insulin-like growth factor signaling in a mouse model of sonic hedgehog-induced medulloblastoma. Cancer Res. 2006;66:2666–2672. - PubMed
    1. Calabrese C., Poppleton H., Kocak M., Hogg T.L., Fuller C., Hamner B., Oh E.Y., Gaber M.W., Finklestein D., Allen M., et al. A perivascular niche for brain tumor stem cells. Cancer Cell. 2007;11:69–82. - PubMed

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