Hedgehog signaling promotes medulloblastoma survival via Bc/II

Abstract

Activation of the Hedgehog (Hh) pathway has been identified in several cancers, including medulloblastoma, but the mechanisms by which this pathway affects tumor survival and growth are incompletely understood. We investigated whether Hedgehog might promote survival of medulloblastoma cells via up-regulation of BclII. We found that mRNA levels of the Hedgehog pathway effector Gli1 were significantly associated with BclII expression in medulloblastoma and that Gli1 and BclII are both present in regions of decreased apoptosis in nodular medulloblastoma. Transient overexpression of Gli1 and Gli2 in medulloblastoma cultures induced a BclII transcriptional reporter and increased BclII protein levels, whereas stable overexpression of Gli1 was associated with increased BclII mRNA. The Hedgehog antagonist cyclopamine blocked expression of the Hh pathway targets PTCH1 and Gli1, lowered BclII levels, and increased apoptosis in DAOY and UW228 medulloblastoma cells. Apoptotic induction caused by cyclopamine could be rescued in part by enforced expression of Gli1 or BclII. Hh pathway blockade also sensitized medulloblastoma to the effects of the proapoptotic agent lovastatin. These data demonstrate that BclII is an important mediator of Hh activity in medulloblastoma and suggest new strategies for combined chemotherapeutic regimens.

Figure 1

Expression of BclII and the Hh targets Gli1 and Gli2 is increased in the internodular regions of nodular/desmoplastic medulloblastoma. a: A cartoon modeling molecular and cellular factors in nodular medulloblastoma. Cells within medulloblastoma nodules have decreased levels of Gli and BclII, and either differentiate into neurocytes or undergo programmed cell death. b: BclII expression is low in nodules (asterisk) and high in internodular regions. c: Medulloblastoma cells invading the cerebellar molecular layer (arrows) are strongly immunopositive for Gli1, whereas nonneoplastic internal granule layer neurons (asterisk) are negative. d and e: Gli1 staining was frequently strong in internodular regions (arrowhead) but weak within nodules (asterisk). f and g: Expression of Gli2 was also higher between (arrows) compared with within (asterisks) nodules. Apoptotic cells with condensed nuclei lacked Gli2 protein (small arrow, inset). h: Cleaved caspase 3 was detected in lymphocyte-like apoptotic cells (small arrows). Original magnifications: ×200 (b, d, f, g); ×100 (c); ×400 (e, h).

Figure 2

Analysis of Gli and BclII mRNA expression. a: Gli1 expression was measured by quantitative RT-PCR in RNA extracted from five nodular, six classic, and seven anaplastic snap-frozen medulloblastomas, as well as the DAOY cell line. b: BclII mRNA levels in these 18 samples are compared with those of Gli1, with each tumor represented by a filled triangle. The subtype of tumors with above-median Gli1 or BclII expression is further designated using an open square, diamond, or circle.

Figure 3

Gli positively up-regulates BclII expression. a: Transient transfection of constructs encoding Gli1 or Gli2 into DAOY or UW228 cell lines significantly induced transcription of a BclII promoter-luciferase reporter as compared with empty vector. b: Stimulation of Hh signaling in cerebellar granule cell precursor cultures using SHH ligand results in a significant increase in BclII mRNA levels measured using quantitative RT-PCR. c: Both Gli1 and BclII mRNA levels are significantly increased in two DAOY subclones (g5, g29) stably expressing Gli1 compared with a vector-transfected clone (v). d: BclII protein expression, normalized to GAPDH, is elevated in DAOY cells stably transfected with Gli1. Mean levels from two experiments are shown above representative bands. *P < 0.05, **P < 0.005 for two-sided t-tests; ns, not significant.

Figure 4

Analysis of Hh targets and BclII expression after cyclopamine treatment. a: Levels of Gli1 and PTCH1, but not Gli2, are significantly reduced by 5 μmol/L cyclopamine compared with vehicle-treated controls. b: BclII luciferase reporter assays after 5 μmol/L cyclopamine exposure reveal progressive inhibition of BclII expression throughout 30 hours. BclII expression was not inhibited in DAOY cells stably expressing Gli1 (Gli1-5). c: Hedgehog pathway blockade in DAOY and UW228 cells by cyclopamine for 48 hours also inhibited BCL2 protein expression normalized to GAPDH. *P < 0.05, ** P < 0.005 for two-sided t-tests; ns, not significant.

Figure 5

Hh pathway blockade induces apoptosis in medulloblastoma. a: Vector-transfected DAOY cell cultures exposed to 10 μmol/L cyclopamine for 48 hours contained numerous refractile or floating cells. Cell death seemed more limited in cultures treated with vehicle, and in cyclopamine-treated DAOY cells stably expressing Gli1 (Gli1-5). b: Quantification of apoptotic cells after cyclopamine treatment confirmed that it significantly induces apoptosis. Enforced Gli1 expression blocked this increase in cell death in the stable DAOY Gli1-5 subclone. c: DAOY cells were transfected with siRNA targeting BclII or scrambled control (C) siRNA, and then treated with vehicle or 5 μmol/L cyclopamine. The addition of cyclopamine to cells already depleted of BclII did not cause significant additional apoptosis. Decreased levels of BclII protein are documented in a Western blot positioned below the first two bars. d: Two subclones stably transfected with a BclII-expressing plasmid were not sensitive to apoptotic induction by cyclopamine. e: Combined treatment with cyclopamine and lovastatin acts synergistically to induce apoptosis in medulloblastoma. *P < 0.05, **P < 0.005 for two-sided t-tests; ns, not significant.