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. 2014 Dec 20:2:160.
doi: 10.1186/s40478-014-0160-4.

Silencing BMI1 eliminates tumor formation of pediatric glioma CD133+ cells not by affecting known targets but by down-regulating a novel set of core genes

Patricia A Baxter, Qi Lin, Hua Mao, Mari Kogiso, Xiumei Zhao, Zhigang Liu, Yulun Huang, Horatiu Voicu, Sivashankarappa Gurusiddappa, Jack M Su, Adekunle M Adesina, Laszlo Perlaky, Robert C Dauser, Hon-chiu Eastwood Leung, Karin M Muraszko, Jason A Heth, Xing Fan, Ching C Lau, Tsz-Kwong Man, Murali Chintagumpala, Xiao-Nan Li

Silencing BMI1 eliminates tumor formation of pediatric glioma CD133+ cells not by affecting known targets but by down-regulating a novel set of core genes

Patricia A Baxter et al. Acta Neuropathol Commun. .

Abstract

Clinical outcome of children with malignant glioma remains dismal. Here, we examined the role of over-expressed BMI1, a regulator of stem cell self-renewal, in sustaining tumor formation in pediatric glioma stem cells. Our investigation revealed BMI1 over-expression in 29 of 54 (53.7%) pediatric gliomas, 8 of 8 (100%) patient derived orthotopic xenograft (PDOX) mouse models, and in both CD133+ and CD133- glioma cells. We demonstrated that lentiviral-shRNA mediated silencing of suppressed cell proliferation in vitro in cells derived from 3 independent PDOX models and eliminated tumor-forming capacity of CD133+ and CD133- cells derived from 2 PDOX models in mouse brains. Gene expression profiling showed that most of the molecular targets of BMI1 ablation in CD133+ cells were different from that in CD133- cells. Importantly, we found that silencing BMI1 in CD133+ cells derived from 3 PDOX models did not affect most of the known genes previously associated with the activated BMI1, but modulated a novel set of core genes, including RPS6KA2, ALDH3A2, FMFB, DTL, API5, EIF4G2, KIF5c, LOC650152, C20ORF121, LOC203547, LOC653308, and LOC642489, to mediate the elimination of tumor formation. In summary, we identified the over-expressed BMI1 as a promising therapeutic target for glioma stem cells, and suggest that the signaling pathways associated with activated BMI1 in promoting tumor growth may be different from those induced by silencing BMI1 in blocking tumor formation. These findings highlighted the importance of careful re-analysis of the affected genes following the inhibition of abnormally activated oncogenic pathways to identify determinants that can potentially predict therapeutic efficacy.

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Figures

Figure 1
Figure 1
Expression of BMI1 in pediatric gliomas. (A) Analysis of BMI1 mRNA in pediatric brain astrocytomas (AST), juvenile pilocytic astrocytoma (JPA), ependymoma (EPN) and glioblastoma (GBM) using qRT-PCR. The relative levels of BMI1 were normalized to an internal control GAPDH and expressed as a percentage (mean ± SD). Red dotted lines indicate the levels of normal adult cerebral tissues. (B) BMI1 mRNA expression in the 8 PDOX mouse models. The relative levels of xenograft tumors during serial sub-transplantations from passage I to V (xeno-I to xeno-V) as well as the original patient tumors (Pt tum) (if available) were quantitated with qRT-PCR. (C) Representative immunohistochemical staining showing the GBM xenograft models with low-medium (IC-3704GBM and IC-3752GBM) and high BMI1 protein expressions (arrows) (IC-1406GBM and IC-2305GBM). (D) Western hybridization showing the increased BMI1 protein expression in the 8 GBM xenograft mouse models using beta-actin as loading control. Note that BMI1 protein was not expressed in the normal adult cerebral tissue, and the relative levels of BMI1 protein in the 8 xenograft models appeared to be correlated with their relative mRNA expressions, particularly in those with high mRNA transcripts (1406, 2305, 1128, and 1227). (E) Immunofluorescent staining showing that BMI1 protein was expressed in FACS-purified CD133+ and CD133- xenograft cells derived from ICb-1227AA.
Figure 2
Figure 2
Suppression of neurosphere formation in vitro in pGBM cells by lentivirus-mediated shRNA specific to BMI1. (A) Representative images showing the suppression of neurosphere formation in two pGBM xenograft models (IC-1206GBM and IC-2305GBM). MTT was added 4 hr prior to the microscopic examination. Dark-colored intracellular crystal was indicative of viable cells. (B) Graphs showing the quantitative analysis of cell proliferation (** P < 0.01) in neurospheres derived from 3 independent PDOX models following the silencing of BMI1 with a panel of five lentivirus-mediated gene-specific shRNAs. O.D. indicates optical density. (C) Western hybridization showing the decreased BMI1 protein expression. CD133+ cells were purified from 3 PDOX models and transduced with Lenti-BMI1-693 (sh693) for 48 hr before being harvested for protein extraction. Tumor cells not treated with lentivirus (Control) and transduced with Lentiviruses containing non-target shRNA (Non-target) were included as references.
Figure 3
Figure 3
Elimination of tumor-forming capabilities of CD133+ cells in vivo in mouse brains by Lenti-BMI1-693. (A) Summary of tumor formation efficiency in tumor cells derived from the two pGBM mouse models. For CD133+ cells, each mouse was implanted with 1,500 cells; and for the CD133− cells, 100,000 cells/mouse. (B) Representative images of H&E-stained paraffin sections. Compared with huge intracerebral xenografts in IC-2305GBM cells transduced with non-target Lenti-shRNA, no tumor formation was observed in mice implanted with tumor CD133+ cells in which BMI1 gene expression was silenced by Lenti-BMI1-693.
Figure 4
Figure 4
Gene expression profiling in pGBM cells. FACS-purified CD133+ and CD133− cells were transduced with Lenti-BMI1-693 for 48 hr in vitro and harvested for RNA extraction. Tumor cells receiving no or non-target lenti-shRNA were included as control. (A) Down-regulation of BMI1 mRNA in CD133+ (+) and CD133- (−) cells transduced by Lenti-BMI1-693. For IC-2305GBM, the CD133− cells were not included due to low cell content. (B) List of gene numbers differentially induced by lentin-BMI1-693 in CD133+ and CD133− cells derived from two PDOX models. Only a small fraction of genes were shared. (C) Heatmaps of gene expression showing changes of known genes previously associated with the over-expressed BMI1 (left panel) and the new targets associated with the silencing of BMI1 (right panel). In the known gene panel, those genes originally up-regulated by over-expressed BMI1 are indicated with up-arrows (↑), and those inhibited by the over-expressed BMI1 are unmarked. No reversal of expression levels were observed in these genes. In the new target panel, genes consistently altered by Lenti-BMI1-693 in the CD133+ cells from all 3 models are highlighted in the blue shaded area. Arrows indicate the 4 genes that were validated with qRT-PCR in (D) where the mRNA levels in cells transduced with Lenti-BMI1-693 were normalized with those transduced with Lenti-non-target shRNA and plotted as fold changes (log2).
Figure 5
Figure 5
Genes differentially altered by lenti-BMI1-693 in CD133 + cells in at least 2 of the 3 PDOX derived cells. (A) Heatmap showing the 46 genes in CD133+ cells as compared with the CD133− cells. (B) Graph showing the top signaling pathways affected by the silencing of BMI1 in CD133+ cells.
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