More than 41,000 people are diagnosed with primary brain tumors each year in the U.S. [1
], and glioblastomas (GBMs) are the most common malignant brain tumors in adults [2
]. Even with advances in surgery, imaging, chemotherapy, and radiation therapy over the past three decades, more than 70% of GBM patients still die within 2 years of diagnosis [1
]. New strategies to treat this deadly disease are desperately needed [1
]. In this study, we focus on the potential therapeutic role of NOTCH pathway inhibition, and the requirement for ongoing NOTCH signaling in stem-like tumor cells.
There is emerging evidence that a small population of cancer stem cells (CSCs) within neoplasms is responsible for long-term tumor propagation, with initial studies focused on leukemia [4
]. Several groups have subsequently demonstrated that stem-like cells exist in brain tumors, including GBMs [4
]. CSC markers such as CD133 and side population have been used to prospectively isolate a small fraction of cells in human and rodent brain tumors with a significantly increased potential to generate tumor neurospheres and xenografts [5
]. Tumor-derived neurospheres also have a genetic profile that remains closer to that of the tumor from which they are isolated than traditional adherent GBM cell lines maintained in high serum [12
]. In addition, GBMs propagated as neurospheres maintain stem-like subpopulations, and more accurately replicate the infiltrating growth patterns seen in primary tumors [9
]. GBM neurosphere cultures thus provide an improved pathological model, and may also facilitate the discovery of new therapeutic reagents that can target the stem-like or xenograft-initiating cells required for long-term tumor growth.
locus was first described by Morgan in a strain of Drosophila with notched wing blades [13
]. The gene was subsequently cloned as a cell surface receptor [14
], playing a key role in the development of many different cell types and tissues, including neurons in the central nervous system [15
]. NOTCH signaling is initiated when transmembrane ligands on one cell bind NOTCH receptors on an adjacent cell and cause the γ
-secretase-mediated proteolytic release of the NOTCH intracellular domain (NICD) [19
]. NICD then translocates into the nucleus where it interacts with the transcriptional cofactor CBF1 and activates targets such as the HES and HEY genes, which modulate neuronal and glial differentiation [21
]. In vertebrates, at least four NOTCH receptors (NOTCH 1-4), five ligands (JAG1, 2, DLL1, 3, 4), and multiple effector molecules (HES1-6, HEY1, 2 L
) have been identified [21
]. NOTCH ligands, receptors, and targets have been found in a wide range of neoplasms, including lung, breast, cervix, head/neck, renal, and pancreas carcinoma, neuroblastoma, myeloma, melanoma, choroid plexus tumor, medulloblastoma, and GBM [20
]. In many of these tumor types, it has been shown that increased NOTCH activity promotes tumor growth, whereas NOTCH pathway blockade inhibits proliferation and/or survival. Inhibition of NOTCH signaling is therefore a promising therapeutic avenue in a wide range of cancers.
We and others have demonstrated that the NOTCH signaling pathway plays an important role in the pathogenesis of medulloblastoma and GBM [28
], and that these malignant brain tumors contain stem-like cancer cells with higher NOTCH activity [9
]. It has recently been reported that common chemotherapeutic drugs, including temozolomide, carboplatin, paclitaxel (Taxol), and etoposide (VP16), as well as traditional radiation therapy, predominantly targeted the CD133-negative population, and spared or enriched the CD133-positive population [37
]. We have also observed an increase in the stem-like subpopulation in GBM-derived neurosphere lines following radiation therapy [39
]. Thus, conventional chemotherapies and radiation therapies appear to effectively remove only better-differentiated cells, while leaving many GBM CSCs alive. The NOTCH pathway represents a possible target in stem-like glioma cells, as several groups have shown that GBM CSCs express NOTCH family genes, and that tumor-derived neurospheres have elevated NOTCH activity [9
]. It is known that NOTCH regulates normal neural stem cell self-renewal and differentiation [40
], and that CSCs share many characteristics with their normal cognates, including the signaling pathways that regulate self-renewal. In the present study, we used both established and low-passage GBM cultures as a model to examine the effects of NOTCH pathway blockade on CSCs and tumor growth.