Marie-Pierre Junier

Mesenchymal Transition and PDGFRA Amplification/Mutation Are Key Distinct Oncogenic Events in Pediatric Diffuse Intrinsic Pontine Gliomas

Diffuse intrinsic pontine glioma (DIPG) is one of the most frequent malignant pediatric brain tumor and its prognosis is universaly fatal. No significant improvement has been made in last thirty years over the standard treatment with radiotherapy. To address the paucity of understanding of DIPGs, we have carried out integrated molecular profiling of a large series of samples obtained with stereotactic biopsy at diagnosis. While chromosomal imbalances did not distinguish DIPG and supratentorial tumors on CGHarrays, gene expression profiling revealed clear differences between them, with brainstem gliomas resembling midline/thalamic tumours, indicating a closely-related origin. Two distinct subgroups of DIPG were identified. The first subgroup displayed mesenchymal and pro-angiogenic characteristics, with stem cell markers enrichment consistent with the possibility to grow tumor stem cells from these biopsies. The other subgroup displayed oligodendroglial features, and appeared largely driven by PDGFRA, in particular through amplification and/or novel missense mutations in the extracellular domain. Patients in this later group had a significantly worse outcome with an hazard ratio for early deaths, ie before 10 months, 8 fold greater that the ones in the other subgroup (p = 0.041, Cox regression model). The worse outcome of patients with the oligodendroglial type of tumors was confirmed on a series of 55 paraffin-embedded biopsy samples at diagnosis (median OS of 7.73 versus 12.37 months, p = 0.045, log-rank test). Two distinct transcriptional subclasses of DIPG with specific genomic alterations can be defined at diagnosis by oligodendroglial differentiation or mesenchymal transition, respectively. Classifying these tumors by signal transduction pathway activation and by mutation in pathway member genes may be particularily valuable for the development of targeted therapies.

What role(s) for TGFα in the central nervous system

Abstract Transforming growth factor α (TGFα) is a member of the epidermal growth factor (EGF) family with which it shares the same receptor, the EGF receptor (EGFR or erbB1). Identified since 1985 in the central nervous system (CNS), its functions in this organ have started to be determined during the past decade although numerous questions remain unanswered. TGFα is widely distributed in the nervous system, both glial and neuronal cells contributing to its synthesis. Although astrocytes appear as its main targets, mediating in part TGFα effects on different neuronal populations, results from different studies have raised the possibility for a direct action of this growth factor on neurons. A large array of experimental data have thus pointed to TGFα as a multifunctional factor in the CNS. This review is an attempt to present, in a comprehensive manner, the very diverse works performed in vitro and in vivo which have provided evidences for (i) an intervention of TGFα in the control of developmental events such as neural progenitors proliferation/cell fate choice, neuronal survival/differentiation, and neuronal control of female puberty onset, (ii) its role as a potent regulator of astroglial metabolism including astrocytic reactivity, (iii) its neuroprotective potential, and (iv) its participation to neuropathological processes as exemplified by astroglial neoplasia. In addition, informations regarding the complex modes of TGFα action at the molecular level are provided, and its place within the large EGF family is precised with regard to the potential interactions and substitutions which may take place between TGFα and its kindred.

NG2+/Olig2+ Cells are the Major Cycle‐Related Cell Population of the Adult Human Normal Brain

A persistent cycling cell population in the normal adult human brain is well established. Neural stem cells or neural progenitors have been identified in the subventricular zone and the dentate gyrus subgranular layer (SGL), two areas of persistent neurogenesis. Cycling cells in other human normal brain areas, however, remains to be established. Here, we determined the distribution and identity of these cells in the cortex, the white matter and the hippocampal formation of adult patients with and without chronic temporal lobe epilepsy using immunohistochemistry for the cell cycle markers Ki‐67 (Mib‐1) and minichromosome maintenance protein 2. Rare proliferative neuronal precursors expressing the neuronal antigen neuronal nuclei were restricted to the SGL. In contrast, the oligodendrocyte progenitor cell markers Olig2 and the surface antigen NG2 were expressed by the vast majority of cycling cells scattered throughout the cortex and white matter of both control and epileptic patients. Most of these cycling cells were in early G1 phase, and were significantly more numerous in epileptic than in non‐epileptic patients. These results provide evidence for a persistent gliogenesis in the human cortex and white matter that is enhanced in an epileptic environment.

CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

BackgroundTumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.MethodsWe screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.ResultsA long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.ConclusionsOur results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

Secreted factors from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway.

Glioma stem‐cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem‐cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long‐term glioblastoma stem‐like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR‐dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem‐cell niches.

Differential regulation of NGF receptors in primary sensory neurons by adjuvant-induced arthritis in the rat.

&NA; In the adult brain, neurotrophins play a key role in adaptive processes linked to increased neuronal activity. A growing body of evidence suggests that chronic pain results from long‐term plasticity of central pathways involved in nociception. We have investigated the involvement of nerve growth factor (NGF) in adaptive responses of primary sensory neurons during the course of a long‐lasting inflammatory pain model. The amount and distribution of the NGF receptors p75NTR and TrkA were measured in the dorsal horn and dorsal root ganglia (DRG) of animals subjected to Freunds adjuvant‐induced arthritis (AIA). We observed an increased immunoreactivity of both receptors in the central terminals of primary sensory neurons in the arthritic state. The increases were seen in the same population of afferent terminals in deep dorsal horn laminae. These changes paralleled the variations of clinical and behavioral parameters that characterize the course of the disease. They occurred in NGF‐sensitive, but not GDNF‐sensitive, nerve terminals. However, p75NTR and TrkA protein levels in the DRG (in the cell body of these neurons) showed different response patterns. An immediate rise of p75NTR was seen in parallel with the initial inflammation that developed after administration of Freunds adjuvant in hindpaws. In contrast, increases of the mature (gp140trk) form of TrkA occurred later and seemed to be linked to the development of the long‐lasting inflammatory response. The changes in receptor expression were observed exclusively at lumbar levels, L3–L5, somatotopically appropriate for the inflammation. Together, these results implicate NGF in long‐term mechanisms accompanying chronic inflammatory pain, via the up‐regulation of its high affinity receptor, and offer additional evidence for differential processes underlying short‐ versus long‐lasting inflammatory pain.

Differential Proteomic Analysis of Human Glioblastoma and Neural Stem Cells Reveals HDGF as a Novel Angiogenic Secreted Factor

Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem‐like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio‐chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two‐dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma‐derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC‐conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC‐conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti‐HDGF antibodies. In vivo, GSC‐conditioned medium induced neoangiogenesis, whereas HDGF‐targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high‐grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties. STEM CELLS 2012;30:845–853

Alternative Lengthening of Telomeres in Human Glioma Stem Cells

Cancer stem cells are increasingly recognized as major therapeutic targets. We report here the isolation of glioma stem cells (GSCs) maintaining telomere length through a telomerase‐independent mechanism known as alternative lengthening of telomeres (ALTs). TG20 cells were isolated from a glioblastoma multiforme, which had the ALT phenotype. They have no detectable telomerase activity and extremely long and heterogeneous telomeres colocalizing with promyelocytic leukemia bodies. The cancer stem cell potential of TG20 cells was confirmed based on their expression of neural stem cell markers, their capacity of in vitro long‐term proliferation and to form intracranial tumors in immune‐deficient mice. Interestingly, we found that both in vitro and in vivo TG20 cells were significantly more resistant to ionizing radiation than GSCs with telomerase activity. Analysis of DNA damage foci, DNA double‐strand breaks repair, and chromosome instability suggest that radiation resistance was related to interference of ALT pathway with DNA damage response. Therefore, our data show for the first time that the ALT pathway can confer to cancer stem cells the capacity to sustain long‐term proliferation as telomerase activity and importantly may also affect treatment efficiency. TG20 cells are thus the first cellular model of GSCs displaying ALT and should prove to be useful for the development of specific treatment strategies. STEM CELLS 2011;29:440–451

Clinical Relevance of Tumor Cells with Stem-Like Properties in Pediatric Brain Tumors

Background Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. Methodology/Principal Findings Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. Conclusions/Significance In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

Transforming Growth Factor α Expression as a Response of Murine Motor Neurons to Axonal Injury and Mutation-induced Degeneration

We previously showed that degenerating adult motor neurons of the murine mutant wobbler, a model of spinal muscular atrophy, express Transforming Growth Factor α (TGFα), a growth factor endowed with glio- and neurotrophic activities. Here, we evaluated whether TGFα expression is a general response of adult motor neurons to injury. Synthesis of its precursor (pro-TGFα) was investigated in another model of motoneuronal degeneration, the murine mutant muscle deficient, and in hypoglossal motor neurons following axonal crush and cut. In control conditions, motor neurons were devoid of pro-TGFα immunoreactivity. In the mutant lumbar spinal cord, pro-TGFα immunoreactive motor neurons appeared as soon as the disease developed and pro-TGFα expression persisted until the latest stages of degeneration. Motor neurons and astrocytes of the white matter weakly immunoreactive for the TGFα receptor were also present in both control and mutant lumbar spinal cords. Following hypoglossal nerve crush and cut, motoneuronal pro-TGFα expression was precocious and transient, visible at one day post-injury and lasting for only 3 days, during which time astrocyte-like cells immunoreactive for both TGFα and its receptor appeared within the injured nucleus. Enhanced TGFα mRNA levels following nerve crush showed that activation occurred at the transcriptional level. These results show that upregulation of TGFα is an early and common response of adult murine motor neurons to injury, regardless of its experimental or genetic origin.