Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma

Abstract

Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog in non-neoplastic stem cells, we investigated whether Hedgehog blockade could target the stem-like population in glioblastoma multiforme (GBM). We found that Gli1, a key Hedgehog pathway target, was highly expressed in 5 of 19 primary GBM and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors, and in GBM-derived neurospheres, suggesting a potential mechanism for pathway activation. Hedgehog pathway blockade by cyclopamine caused a 40%-60% reduction in growth of adherent glioma lines highly expressing Gli1 but not in those lacking evidence of pathway activity. When GBM-derived neurospheres were treated with cyclopamine and then dissociated and seeded in media lacking the inhibitor, no new neurospheres formed, suggesting that the clonogenic cancer stem cells had been depleted. Consistent with this hypothesis, the stem-like fraction in gliomas marked by both aldehyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast, we found that radiation treatment of our GBM neurospheres increased the percentage of these stem-like cells, suggesting that this standard therapy preferentially targets better-differentiated neoplastic cells. Most importantly, viable GBM cells injected intracranially following Hedgehog blockade were no longer able to form tumors in athymic mice, indicating that a cancer stem cell population critical for ongoing growth had been removed. Disclosure of potential conflicts of interest is found at the end of this article.

Figure 1

Hedgehog pathway members are expressed in gliomas. (A): Ptch, Gli1, Smo, and Shh mRNAs were detected in primary glioblastoma multiforme (GBM) samples. (B): Measurement of Gli1 and Shh mRNA using quantitative reverse transcription-polymerase chain reaction in 19 primary GBM samples and seven GBM cell lines disclosed high Gli1 expression in five tumors. (C): Gli1 immunostaining was present in all malignant glioma samples examined, with some showing weak cytoplasmic staining (upper panel) and others showing more abundant cytoplasmic and nuclear protein (lower panel). Endothelial cells (arrow, lower panel) were negative for Gli1 and served as internal controls. Abbreviations: ntc, no template control; Ptch, Patched; Smo, Smoothened.

Figure 2

Hedgehog signaling is required in established glioma cultures. (A): Effect of 48-hour cyclopamine treatment on the activity of a Gli-dependent luciferase reporter in U87-MG. (B): The growth rate of glioma cultures was significantly inhibited by cyclopamine. (C): U87-MG cells treated with cyclopamine formed significantly fewer colonies in agar. (D): Representative wells with U87-MG colonies in soft agar. (E): U87-MG: Gli1 mRNA level (top panel) and total viable cells per hpf (bottom panel) were reduced by specific shRNA targeting lentivirus 488 compared with either empty vector pLKO.1-puro or the nonfunctional Gli1 shRNA 487. Two-sided t test was used throughout. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: hpf, high-power field; ns, not significant; V, vehicle (ethanol) control.

Figure 3

Glioblastoma multiforme (GBM)-derived neurospheres express Hedgehog (Hh) ligands, receptors, and targets. (A): mRNAs for Gli1, Gli2, Smo, and Ptch1 (including the inducible isoform Ptch1b) were all expressed in both long-term (HSR-GBM1) and low-passage (JHH-GBM3) neurosphere cultures. (B): Immunohistochemistry. Gli1 protein was highly expressed in HSR-GBM1 intracranial xenografts. Note the highly infiltrating cells invading the CC. Arrow in inset points to a nucleus positive for Gli1; surrounding cells were largely negative in this region. (C): HSR-GBM1 secretes biologically active Shh-ligand, as evidenced by the induction of Hh reporter activity in Light2 cells when conditioned medium from the GBM neurosphere cultures was applied. Medium isolated from 293T cells engineered to secrete Shh-N was a positive control. (D): Following application of concentrated (1:5 dilution) conditioned media from Shh-N secreting 293T cultures, Gli1 mRNA levels were elevated fourfold in low-density HSR-GBM1 cells. Two-sided t test was used throughout. *, p < .05. Abbreviations: CC, corpus callosum; ns, not significant; ntc, no template control; Ptch, Patched; Smo, Smooth-ened; T, tumor; V, lateral ventricle.

Figure 4

Hedgehog (Hh) pathway inhibition reduces glioma neurosphere initiation and growth. (A): The formation and growth of HSR-glioblastoma multiforme 1 (GBM1) neurospheres was inhibited by cyclopamine in a dose-dependent fashion. (B): Gli1 but not Gli2 siRNAs significantly inhibited growth of HSR-GBM1 neurospheres relative to non-Hh targeting siRNA control (Gli2 siRNA, p = .061). (C, D): Hh pathway blockade affected the ability of cells to initiate sphere formation, as HSR-GBM1 cultures treated for 7 days with cyclopamine (but not vehicle) had a drastically reduced ability to generate new spheres upon transfer into fresh medium lacking cyclopamine. (E): Cyclopamine modestly augmented the effect of radiation treatment on the growth rate of HSR-GBM1 cells. (F, G, H): Cyclopamine treatment for 7 days significantly increased the fraction of GFAP-expressing cells (F, G) and reduced the fraction of nestin-expressing cells (F, H). All images were taken at a magnification of ×60. Two-sided t test was used throughout. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; siRNA, short interfering RNA; V, vehicle.

Figure 5

Cyclopamine depletes stem-like cells from glioblastoma multiforme (GBM) neurospheres and blocks tumor engraftment. (A): When GBM neurospheres were forced to differentiate, mRNA levels of the proliferation marker Ki67 and the neural stem-cell markers CD133 and Nestin decreased, along with the Hedgehog pathway targets Gli1 and Ptch1b. In contrast, markers of glial differentiation (GFAP) and neuronal differentiation (microtubule-associated protein 2) increased. (B, C): The stem-like side population was significantly decreased by cyclopamine (two-sided t test; *, p < .05; **, p < .01). (D, E): The aldehyde dehydrogenase (ALDH)-positive subpopulation of stem/progenitor cells was also depleted by cyclopamine. (F): The side- and ALDH-positive stem/progenitor cell subpopulations were depleted by cyclopamine and increased by radiation when each was given alone. Cotreatment with cyclopamine and radiation depleted side- and ALDH-positive subpopulations. (G, H): HSR-GBM1 cultures pretreated with vehicle always engrafted when either 1,000 or 10,000 viable cells counted at the end of treatment were injected into the athymic mice. In contrast, when the same number of viable cells was injected after 7 days of treatment with 10 μM cyclopamine, no detectable tumors formed (the arrow points to degenerative changes along the injection needle tract). Log-rank analysis of Kaplan-Meier survival curves indicates that the prolongation of survival associated with cyclopamine pretreatment is significant (p < .0001). Abbreviations: ALDH, aldehyde dehydrogenase; Cyc, cyclopamine; DEAB, 4-(diethylamino)benzaldehyde; GFAP, glial fibrillary acidic protein; SP, side population; SSC, side scatter; T, t test; V, vehicle; Veh, vehicle.