|A new common target for the treatment of invasive cancers?|
‘Rai is a new regulator of neural progenitor migration and glioblastoma invasion’
September 2012 — Glioblastoma multiforme (GBM) is the most common form of brain tumor yet carries an unfortunately poor prognosis owing to its aggressive nature and the high infiltrative capacity of Glioblastoma cells. Its invasive nature makes it difficult to contain or remove surgically, and also a difficult target for localized radiation and chemotherapy treatment. Such tumors are initiated by a specific type of cancer stem cells (GBM initiating cells) which share some characteristics of neural stem cells (NSCs) including extensive self-renewal and migration. Understanding the invasive nature of these cells is of obvious importance for the development of suitable treatments for Glioblastoma patients. Recent work published in Stem Cells by Ortensi et al.1 from various centres in Milan has studied the role of Rai, a member of the Shc-like adaptor proteins, in the regulation of cancer cells. Rai is of particular interest in cancer as, although not normally present in glia, is expressed in high grade gliomas. Their work has uncovered that Rai is a key promoter of migratory activity and, importantly, that its knockdown can reduce cell invasion as well as the downregulation of pro-invasive genes.
The authors studied ‘fresh’ tumor samples in addition to generating primary neurosphere cultures from tumor tissues collected from GBM patients undergoing surgery. They found the expression of Rai in all samples, which showed intense expression at the tumor/host interface and was upregulated in all GBM-derived neurospheres. Interestingly, the differentiation of neurospheres led to a decrease in Rai expression, indicating that its expression may be confined to undifferentiated GBM cells. They then looked to determine the role of Rai in gliomagenesis. First, they achieved Rai silencing in GBM neurosphere cultures using RAI sequence-specific short hairpin RNA (shRNA), then injected either silenced or naïve cells into the nucleus caudatus of immunodeficient mice. This part of the basal ganglia has multiple roles including learning, memory, emotion and language comprehension. Fascinatingly, its involvement in the emotional response to visual beauty has dubbed it one of the ‘neural correlates of romantic love’.2 What they found was that Rai interference significantly reduced the tumorigenic potential of grafted cells, prolonging time before the onset of neurological symptoms and increasing survival compared to naïve cells, while incomplete knockdown of Rai restored the invasive nature of the cells. Further, histological examination postmortem revealed that naïve GBM neurospheres had markedly infiltrated surrounding brain tissues, while silenced cells remained more confined to the site of transplantation and formed a smaller tumor mass overall. There was also a significant reduction in the number of disseminated doublecortin+ cells surrounding Rai silenced grafts, a marker of migrating neuroblasts and infiltrating GBM cells. Importantly, Rai interference was maintained in silenced cells following transplantation, with the rare exception of a small number of cells at the tumor edge. They also confirm the importance of Rai for cell migration in vitro via 2D and 3D Matrigel infiltration, wound healing migration and sphere dispersion assays, all of which confirmed a limited migratory capacity of cells following Rai interference.
Ortensi et al. also found that the cells of the rostral migratory stream (RMS) and subventricular zone (SVZ) in the adult brain are Rai+, indicating a functional role for Rai in primitive migratory cells. By BrdU-labeling of Rai knockout (Rai-/-) mice they then show that neural progenitor migration from the SVZ to the olfactory bulb via the RMS is significantly reduced (by 60%) compared with wild type mice. The migration of progenitor cells from Rai-/- explants in vitro was also significantly impaired. In addition, Rai-silencing caused a reduction in the level of proteins and metalloproteases associated with migration and tumor invasion, including MMP2, MMP3, ADAMs and ADAMTS. The authors found that the Wnt/β-catenin pathway was downregulated accompanied by high levels of β-catenin phosphorylation in Rai-/- neurospheres and also that the β-catenin target Neurogenin2, a regulator of migration and differentiation, was downregulated, although this pathway was not found to be altered in Rai-silenced neurospheres. Interestingly, an activation effect of Rai on the NFĸB pathway was found. In addition, reduced expression of Notch1 was observed in Rai-silenced neurospheres and reactivation of Notch signalling reverted the migratory defect. The effects of Rai interference did not appear to be mediated via the PI3K/Akt pathway however, and therefore the function of Rai in GBM seems independent of Akt signalling. These results reveal the complex interplay of Rai with various signalling pathways in GBM.
To determine whether these varying results arose from different molecular classes of GBM (which have recently been identified by gene expression studies) Ortensi et al. then performed meta-analysis of Rai expression using four independent microarray data sets, which revealed the significant enrichment of Rai in the pro-neural tumor subtype. This subtype is associated with Notch activation, in line with their results following Rai-silencing. Further, classification of each GBM patient sample confirmed the elevated expression of Rai in those belonging to the pro-neural subtype.
These results indicate Rai to be a new regulator of cell migration and invasion which has implications not only for cancer research but in enhancing our understanding of normal cell migratory behaviour. It is fascinating that while elevated Rai had a greater association with pro-neural tumor subtypes, the loss of Rai not only disabled the migratory and invasive capacity of pro-neural GBM cells, but was effective in all GBM subtypes tested in this study, meaning that this might represent a novel common target for cancer treatment. Undoubtedly this will facilitate further research into the potential of this target to prevent cancer metastasis and improve the prognosis of this devastating condition as well as other forms of cancer.
1 Ortensi B, Osti D, Pellegatta S, Pisati F, Brescia P, Fornasari L, Levi D, Gaetani P, Colombo P, Ferri A, Nicolis S, Finocchiaro G, Pelicci G. Rai is a new regulator of neural progenitor migration and glioblastoma invasion. Stem Cells. 2012;30(5):817-32.
2 Ishizu T and Zeki S. Warrant, Eric James. ed. Toward A Brain-Based Theory of Beauty. PLoS ONE 2011;6(7):1–10.
STEM CELLS correspondent Carla B. Maellough reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.