Current treatment for malignant glioma is only palliative. Recent studies have suggested that the resistance of BTSCs to current therapies is, at least in part, related to the failures of current treatment.12,13
Therefore, the development of novel therapies to effectively target BTSCs is imperative. One potential avenue of treatment is to take advantage of the similarities between BTSCs and their somatic counterparts, NSCs.37
However, therapies that target key pathways for the survival of both cell types might have unintended effects of killing normal NSCs. Therefore, a worthy treatment would be to target genes and signaling pathways that are required for BTSCs but not for normal NSCs.
MELK is expressed by several organ-specific stem cells, including NSCs.24,38
However, our previous study has suggested that NSCs do not require MELK for their survival, while their self-renewal and proliferation depends on MELK.24
In contrast, knockdown of MELK in stemlike GBM cells resulted in apoptosis.27
These data support our hypothesis that MELK is one attractive target for BTSC.
MELK has also been strongly associated with cancers. We and others27,28,29
have demonstrated that MELK is highly expressed in malignant gliomas and is also related to patient outcome, such that patients with higher levels of MELK mRNA have shorter survival periods than those with lower levels of MELK expression.27
Some studies have shown that MELK plays a functional role in other cancer cells.26,40,41
For example, MELK knockdown decreased in vitro proliferation and anchorage-independent growth of cell lines derived from pancreatic and breast cancer, as well as the in vivo growth of transformed fibroblasts in a subcutaneous xenograft model.25
Lin et al.35
provided further evidence that the action of MELK on cancer cell growth is associated with resistance to apoptosis through the inhibition of a pro-apoptotic function of Bcl-G. In our prior study, we found that MELK is expressed in stemlike GBM cells and knockdown of MELK by siRNA results in apoptosis.27
These findings led us to hypothesize that upregulated MELK expression is a hallmark of survival for cancer stem cells, and targeting MELK may kill BTSCs while sparing normal NSCs. Thus, we sought inhibitors of a MELK-mediated pathway in BTSCs.
Toward the goal of developing novel GBM therapies directed at a MELK-mediated pathway, we analyzed the effect of small molecules on MELK expression in GBM spheres and identified siomycin A as a compound that suppresses MELK expression in GBM spheres. Previously, others demonstrated that siomycin A treatment leads to apoptosis of transformed, but not normal fibroblasts.15
In agreement with their study and the phenotype of MELK knockdown of normal stem/progenitor cells24
and stemlike GBM cells,27
our data suggested that siomycin A treatment significantly reduces cell survival and self-renewal (Fig. ) of stemlike GBM cells, with little toxicity to normal neural stem/progenitor cells (Figs. and ). This cytotoxicity of siomycin A on stemlike GBM cells was coupled to a significant anti-invasive effect in organotypic brain cultures (Fig. ), resulting in a marked reduction of tumor progression in vivo (Fig. ).
However, several open questions still remain. For example, it is unlikely that MELK is the only downstream target of siomycin A. Therefore, the action of siomycin A on other pathways may result in unanticipated toxicity. In addition, ex vivo siomycin A treatment inhibited but did not completely prevent tumor formation in one of the lines (GBM1600 in Fig. ). It is entirely possible that there are siomycin A–resistant stemlike GBM cells. Our in vitro experiments may target only one type of BTSC in GBM, while there may be multiple different kinds of cells with tumor-initiating potential, both among tumors derived from different patients and within a single tumor. Lastly, the lack of demonstrable effect on neurosphere formation of normal stem/progenitor cells or immortalized astrocytes does not completely rule out a more subtle effect on them, a possibility that will need to be explored. Future studies are required to answer these questions.
Our studies demonstrate that siomycin A acts, at least in part, via a MELK-mediated pathway, but they do not clearly define the link between MELK and FOXM1b in BTSC. A previous study15
suggested that the action of siomycin A on FOXM1b has two distinct mechanisms: (1) regulation of the abundance of FOXM1b mRNA and protein and (2) direct inhibition of phosphorylation of the FOXM1b protein. We found that overexpression of MELK, but not FOXM1b, overrode the inhibitory effect of siomycin A on sphere formation derived from GBM cells (Fig. and Supplemental Fig. 4
). If direct inhibition of phosphorylation is the predominant mechanism of siomycin A on FOXM1b in GBM stem cells, then exogenously expressed FOXM1b may not rescue the phenotype of siomycin A–treated stemlike GBM cells. In addition, MELK and FOXM1b may play different roles in BTSCs and their derivatives. In fact, it is not clear yet whether FOXM1b is required for proliferation and/or survival of BTSCs in GBM.42,45
Given that GBM is resistant to the current therapies and BTSCs in GBM may contribute to, at least in part, the therapy resistance, identifying novel therapies that efficiently eradicate BTSCs are crucial.
Our findings indicate siomycin A as a potent inhibitor for a MELK-mediated pathway in stemlike GBM cells. Treatment with siomycin A significantly inhibits survival, proliferation, self-renewal, and invasion of stemlike GBM cells in vitro and significantly reduces tumor growth in vivo. Furthermore, siomycin A treatment has little or no effect on survival or growth of nonstem cells in GBM or normal neural stem/progenitor cells in vitro. These results may provide a rationale to design novel therapeutic strategies that effectively and selectively target BTSCs in GBM with less toxicity to somatic cells. Future work will elucidate the mechanism of siomycin A action as well as the role of MELK and FOXM1b in BTSC.