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Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells

Abstract

Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers1. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP–BMPR signalling system—which controls the activity of normal brain stem cells2,3—may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.

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Acknowledgements

This work was supported by the Associazione Italiana per la Ricerca sul Cancro, the Plurigenes EEC grant, funding from the Brain Tumours Funders’ Collaborative, from Neurothon Onlus, and by a donation from BMW Italia. We are grateful to R. Galli, J. Laterra, C. L. Solero and F. McBlane for helpful comments and suggestions.

Author information

Correspondence to A. L. Vescovi.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Detailed description of methods used in this study. (DOC 42 kb)

Supplementary Tables

Supplementary Tables 1–9 containing comprehensive data on all cell preparations used. (DOC 183 kb)

Supplementary Legends

This file contains legends for Supplementary Tables and Supplementary Figures. (DOC 63 kb)

Supplementary Figure 1

A quantitative analysis (Real Time PCR (RT-PCR)) of the expression of the BMPR type 1A, 1B, and 2 genes in the different GBM cell preparations investigated in this study – acutely dissociated or briefly cultured (GBSC) cells. (JPG 749 kb)

Supplementary Figure 2

a, Dose response curves showing the ability of BMP4 to inhibit the proliferation of two representative GBM cell preparations in a dose-dependent fashion. b. Cytofluorimetric analysis showing the phosphorylation and nuclear translocation of phospho Smad 1,5,8 as elicited 1.5 hours post BMP4 treatment in cultured GBM cells. (JPG 475 kb)

Supplementary Figure 3

Effects of various BMPs (all at 100 ng/ml) on the growth of GBM cells. (JPG 441 kb)

Supplementary Figure 4

The figure shows the effect of BMP4 on the expansion rate of briefly cultured GBM cells expanding in the presence of mitogens. (JPG 996 kb)

Supplementary Figure 5

The figure shows one example of the dot plot graph concerning the quantitative analysis of the expression of the CD133 antigen. (JPG 632 kb)

Supplementary Figure 6

Untreated GBM cells constitutively express neuronal markers such as MAP2. (JPG 577 kb)

Supplementary Figure 7

BMP4 inhibits the tumorigenicity of GBM cells. (JPG 1564 kb)

Supplementary Figure 8

Ex vivo pre-treatment of GBM cells with BMP4 inhibits ventricular invasion following orthotopic injection. (JPG 486 kb)

Supplementary Figure 9

Phenotype analysis confirmed that, as shown previously, tumours induced by transplanting briefly cultured GBM cells into the brain of scid/bg animals generated astroglial-like cells. (JPG 1573 kb)

Supplementary Figure 10

a. Quantitative RT–PCR analysis of the expression of 13 different BMPs genes in briefly cultured GBM cells. b. When GBM cells, cultured with mitogens, were exposed to a neutralizing antibody that specifically blocks BMP4, the cell growth rate increased significantly as compared to control conditions. (JPG 1073 kb)

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Further reading

Figure 1: Expression and activation of BMP receptors in human GBM cells.
Figure 2: BMP4 depletes the GBM BTIC population in vitro.
Figure 3: BMP4 elicits a pro-differentiation effect on GBM cells.
Figure 4: BMP4 inhibits tumorigenicity of GBM cells.

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