This study indicates that ASPM is tightly associated with the malignant progression of gliomas and that its knockdown produces extensive death of glioma cells in vitro.
As previously described [
7], we found that ASPM expression was strongly correlated with the grade of gliomas. In recurrent tumors, ASPM expression increased not only in patients who progressed to a higher grade, but also in recurrent glioblastomas, suggesting that a progressive increase in ASPM mRNA expression is a marker for the inexorable malignant progression and aggressiveness of gliomas, continuing even when the tumors have already reached their most anaplastic histological stage (Grade IV). Experimental evidence supports this finding since we found a progressive increase in ASPM mRNA expression in serial passages of gliomaspheres
in vitro and in mouse glioma xenografts
in vivo. To explain such a tight correlation between ASPM expression and the malignant progression of tumors, an attractive hypothesis is that symmetric division of CSC-expressing ASPM leads to progressive proliferative expansion of this undifferentiated compartment. Although we found that ASPM was also expressed at the protein level in all glioma grades and overexpressed in Grade IV gliomas, it is worth noting that the results were less striking. This finding has been previously noted and led to the suggestion that ASPM could be in part regulated at the translational level [
7]. However, the dramatic inhibition of gliomasphere proliferation obtained with shRNA-mediated ASPM knockdown highlights the potential importance of this gene in malignant gliomas. Sphere formation is a key behavior of neural and brain tumor stem cells and is used to test stem cell capacity for self-renewal. A previous study also showed that siRNA-mediated inhibition of ASPM resulted in cell proliferation inhibition, thought to be mediated by a G1-phase cell cycle arrest [
6]. The effect that we observed here by stable transduction of shRNA expression in CSC-enriched cells appears even more drastic, possibly because of a more efficient and longer lasting inhibition of
ASPM. Whereas siRNA transfection results in a transient effect, lentiviral-mediated expression of shRNA allows the transgene to integrate into the genome of the host cell, resulting in long-term expression. Production of secondary spheres was also inhibited after ASPM knockdown, a finding compatible with a sustained inhibition of CSC proliferation. Interestingly, stable inhibition of ASPM not only prevented further growth but also produced rapid and extensive cellular apoptotic death in our model. This finding may indicate that, following ASPM inhibition, CSC underwent active maturation and differentiation but could not survive in our experimental setting (serum-free medium without appropriate growth factors). However, experiments by Horvath et
al. conducted in a similar serum-free medium resulted in growth inhibition and not massive cell death. On the other hand, the function of
ASPM in neoplastic cells, particularly gliomas, has not been completely elucidated. In particular, the links between ASPM and key molecular alterations of malignant gliomas, such as EGFR amplification or mutation (EGFRvIII) (an almost constant feature in gliomaspheres - 80% in our experience), remain to be investigated. Nevertheless, some data suggest that these alterations are closely related. ASPM mRNA expression was increased in an EGFRvIII-expressing U87 glioblatoma cell line as compared to an isogenic U87 line, while ASPM expression was inhibited following the addition of erlotinib (an EGFR tyrosine kinase inhibitor) to the EGFRvIII-expressing U87 line. This observation led Horvath et
al to suggest that ASPM (and genes from the same module) is involved downstream of the mutant EGFR. Thus, inhibition of ASPM could prove lethal by severely hampering one of the key signaling pathways of glioblastomas.
Because ASPM is absent or expressed at very low levels in normal brain, the considerable differential expression levels between GBM and normal parenchyma and the extreme in vitro sensitivity of tumor cells to ASPM knockdown support continued research to specifically target ASPM from a therapeutic perspective, focusing on in vivo studies.