Targeting developmental signalling pathways for the treatment of malignant glioma
Role of NUMB in glioma development
We have studied the function of NUMB in glioma development by transducing glioma cell lines with different NUMB isoforms. Despite its previously described central role in several glioma-related signalling pathways, NUMB overexpression could not change the phenotype of experimental glioma in a therapeutically relevant manner.
Introduction: Glioblastoma multiforme is a highly aggressive CNS tumor with dismal prognosis. The cancer stem cell hypothesis claims that there exists a subpopulation of tumor cells with stem-cell properties, possibly undergoing asymmetric cell division for self-renewal. Developmental signalling pathways (Notch, Hedgehog) regulate stem-cell maintenance physiologically and are reportedly active in gliomas.
Aims: Our aim was to investigate the effects of overexpressing the cell fate determinant NUMB which regulates asymmetric cell division and is known to interact with Notch and Hedgehog signalling.
Methods: Adherent glioma cells were lentivirally transduced with NUMB. Intracranial xenografts were analyzed with respect to animal survival, histology and MRI morphology. gliomaspheres Proliferation was quantified in vitro by generation of growth curves.Differentiation characteristics were determined by morphology, realtime PCR and immunocytochemistry.
Results: NUMB isoforms are expressed in patient material, glioma cell lines and CSC enriched gliomaspheres.NUMB2/4 isoforms were predominant in all samples. When overexpressed in U87, no signicant alteration of proliferation was observed. Growth rates remained constant and no signs of cell cycle arrest could be observed in transduced cells. However, numb4 enhanced growth of U87 cells cultured as spheroids compared to controls. Neither NUMB2 nor NUMB4 could induce expression of glial or neuronal markers, and tumorigenicity in vivo was not significantly impaired, although NUMB4 seemed to even enhance tumor growth. This has to be verified in a larger set of experiments. Histomorphology of NUMB4 xenografts also appeared to be different. Based on analysis of microarray data from studies comparing adherent and neurosphere cell lines, expression of NUMB was expected to be lower in gliomaspheres. Experimentally determined protein levels, however, were only slightly lower than those in U87. By overexpression, none of the human NUMB isoforms could induce changes in morphology that could be interpretated as loss of stem-like phenotype either.Functional analysis of relevant signalling pathways revealed reduction of Notch related transcription factor HES1 by NUMB in adherent glioma cell lines, while inhibition of Hedgehog signalling and stabilization of p53 could not be recapitulated in U87.
influence of numb isoforms on glioblastoma growth
Numb plays a crucial role in the developing central and peripheral nervous system.
The effects of Numb are known to be mediated by several pathways that have also been linked to glioma pathogenesis. Notch and Hedgehog signalling seem to be downregulated by Numb. Recently, a role for Numb in stabilizing p53 has been suggested.
Malignant gliomas, the largest group of primary intracerebral tumours, are one of the most difficult-to-cure cancers. The outcome has not improved significantly in the past years, despite considerable advances in our understanding of the molecular pathogenesis and the improvement of surgical techniques, radio- and chemotherapy. For glioblastoma, the most malignant form of glioma, the median survival time is generally less than one year. Thus novel effective therapies are urgently needed.
The goal of this project is to interfere with cellular signalling pathways that account for self-renewal and differentiation, and therapeutically drive the differentiation of cancer cells (into less harmful ones not capable of self-renewal). Two major pathways involved in the differentiation and proliferation of neural stem cells are the notch and wnt signalling pathways. In glioma, both pathways are upregulated. Thus, it is feasible that they control the self-renewal and proliferation of cancer or cancer stem-like cells.
We have cloned all 4 human numb isoforms into lentiviral vectors. Numb1-4 were overexpressed by lentiviral transduction in different glioma cell lines. Initially, we found decreased viability and signs of apoptosis in cell lines expressing Numb1 and Numb3 isoforms. Preliminary results also showed increased expression of the neuronal marker beta-Tubulin III in cells surviving lentiviral transduction. However, vector sequencing showed mutations in precisely these isoforms. Recent experiments with a non-mutant Numb3 vector suggested similar results.
For Numb4 transduced glioma cell lines, we found – in contrast to the initial hypothesis – increased proliferation and increased chemoresistance to temozolomide, the standard chemotherapeutic drug for GBM nowadays. These changes might be due to increased Hedgehog signalling. Upon implantation of numb2 and numb4 overexpressing cell lines into the brains of nude rats, numb4 tumors grew significantly faster compared to rats harbouring the parental cell line.
Further in vivo experiments and functional studies in vitro regarding proliferation and involvement of signalling pathways are needed to corroborate these findings.