The finding of EDN3 overexpression in GSC through the expression microarray analysis led to our novel investigation of the EDN system in cultured GSCs. In normal NCC cultures, EDN3/EDNRB signaling prevents the premature differentiation of crest-derived precursors (22
). In vivo, EDN3/EDNRB signaling is required for NCC migration during ENS development (22
). Mutations in EDN3 or EDNRB can lead to the abnormal development of ENS and melanocytes, implying that the loss of function in these two genes may interfere with tumor development by EDN3/EDNRB signaling-dependent GSC. Indeed, blockade of EDN3/EDNRB signaling of GSC leads to the loss of tumorigenic potential in our GSC model. We found that cultured GSC express both neural and mesenchymal signatures characterizing NCC-like cells and therefore hypothesized that autocrine EDN3/EDNRB signaling may be a survival/maintenance factor for GSC. In fact, when GSC undergo serum-induced proliferative differentiation, a significant loss of EDN3 production accompanied by co-downregulation of a series of transcripts related to NCC, RGC and NSC was observed. For instance, SOX2 is a marker of NSC and is essential for maintaining the pluripotent, self-renewal, and undifferentiated phenotypes of embryonic stem cells. Moreover, silencing of SOX2 in GSC can cause the loss of tumorigenicity (45
). FABP7, a glial-specific marker, is a direct target of Notch signaling in RGC, and the involvement of Notch signaling in the maintenance of the tumorigenic potential of GSC has been reported (46
). Thus, autocrine EDN3/EDNRB signaling may prevent GSC from differentiating prematurely.
Direct evidence of the differentiation-inhibiting effect of EDN3 was demonstrated in clonogenic cultures of ENS progenitors supplemented with EDN3, by which neuronal and glial differentiation pathways were blocked and the multipotent state of progenitors was maintained (44
). These observations thus support the view that autocrine EDN3 maintains the undifferentiated state of GSC. Moreover, EDN3 and EDN1 mRNAs are altered reciprocally in GSC in response to differentiation, indicating that EDN3 and EDN1 genes can be epigenetically regulated, and that increased EDN1 may be associated with the onset of tumorigenesis. Indeed, we detected an increased expression of EDN1, but not EDN3, mRNA in patient glioblastoma tumors (compared to GSC), suggesting that EDN3 may play a tumor suppressor-like role to maintain the quiescence and tumorigenic potential of GSC. In agreement with this, a frequent loss of EDN3 expression in breast tumors due to epigenetic inactivation has previously been suggested (29
). Likewise, there were decreased levels of EDN3 and increased levels of EDN1 and EDN2 mRNA in cancerous cervical epithelial cells compared with normal cervical epithelial cells (28
). Thus, the reduction of EDN3 levels (e.g. induced by exogenous cues) may implicate the initiation of proliferative differentiation from quiescent GSC towards more differentiated progeny, by which the proliferative progeny gradually overpopulate a tumor. It appears that sole EDN3/EDNRB signaling alone cannot grant tumorigenic potential to GSC, yet the removal of EDN3/EDNRB signaling contributed undifferentiated, anti-apoptotic, and migratory properties to GSC, which may weaken or eliminate their tumorigenic capacity. Autocrine EDN3 in EDNRB+ GSC, may therefore, play a role in anti-developmental, anti-differentiating, antiapoptotic and anti-tumorigenic functions, thereby help in maintaining a continuous GSC pool.
We found that loss of EDN3 production in GSC through serum-induced differentiation did not cause cell apoptosis, while directly blocking EDN3/EDNRB signaling causes severe GSC apoptosis. These observations seem to support the notion that autocrine EDN3/EDNRB signaling only provides survival benefits when GSC are maintained in SGF condition. A previous study has shown that stimulation of the EDNRB in astrocyte induces cAMP response element-binding protein (CREB) and c-fos expression via multiple MAPK signaling pathways, including the extracellular regulated kinase (ERK) 2, c-Jun N-terminal kinase 1 (JNK1), and p38 kinase (38
). EDN3 stimulation also rapidly increased phosphorylation of ERK2 in neural progenitor cells (39
) and activated IkappaB and MAPK in the human colonic epithelial cells (40
). Likewise, EDN3 treatment resulted in the activation of MAPK-p90 ribosomal S6 kinase-CREB and cAMP-protein kinase A-CREB pathways in melanocyte culture (41
) and induction of ERK1/2 and focal adhesion kinase (FAK) phosphorylation in malenoma cells (43
). One important note is that the activation of EDNRB by EDN3 also leads to loss of expression of the cell adhesion molecule E-cadherin and associated catenin proteins, while increasing Snail and N-cadherin expression (43
). Thus, the expression profiles of BQ788-treated GSC supports the notion that these pathways are the downstream effector pathways of EDN3/EDNRB signaling pathway that contribute GSC survival and migration (Supplementary Table 2
). The usage of an inducible knockdown of EDNRB would strengthen the role of EDNRB signaling in GSC. Moreover, since ex-vivo treatment with BQ788 may already damage the capabilities of self-renewal and proliferative differentiation in GSC, inducible knockdown of EDNRB or EDN3 using viral-delivered hairpin RNA would allow more in depth functional and in vivo studies.
It has been shown that EDN3 first stimulates expression of EDNRB, then when under prolonged exposure to EDN3, EDNRB expression decreases (12
). This may explain why EDNRB are expressed at lower levels in the sphere cultures, which consistently produce EDN3. We found that EDN3 alone is not a potent mitogen for GSC and similar finding was also reported (44
). Interestingly, it was shown that EDNRB antagonists reduced the viability and proliferation of glioma cells, which do not express EDN3 (48
), implying the possibility of blocking EDN1/EDNRB signaling. The decreased glioma cell viability by EDNRB antagonists independent of their cognate receptor was also reported (49
). We speculate that low levels of EDNRB mRNA being expressed in cultured CD133+ GSC used for injection, but not in some tumor xenografts, could be due to tumor initiation from CD133+ GSC being accompanied by activation/upregulation of EDNRA signaling and downregulation of EDNRB signaling in our model system. Alternatively, it is possible that EDNRB may be only expressed by a small subset of quiescent GSC that express the tumor-suppressive phenotype (e.g. CD133+/EDNRB+ cells) for maintaining GSC pool in vivo, which can be retrieved and enriched by SGF culturing. On the other hand, CD133+ cells sorted from xenografts may mostly contain activated GSC (e.g. CD133+/EDNRB-/EDNRA+ cells) that have entered the pathway to tumorigenesis. Interestingly, some tumor xenografts express high levels of EDN3 and EDNRA mRNA, but low levels of EDN1 and EDN2 mRNA, with a negligible level of EDNRB mRNA, suggesting that the growth of EDN3+ tumor may not be EDNRB, but EDNRA signaling-dependent. Promoter hypermethylation of the EDNRB gene in various human malignancies has been reported and suggested that EDNRB may be a candidate tumor-suppressor gene (50
Lastly, our genome-wide expression analysis has provided some molecular explanation for the requirement of EDN3/EDNRB signaling in maintaining GSC sphere cultures. Apparently, EDN3/EDNRB blockade mostly impacts cell structure, cell movement, self-renewal/cell division, and cell survival, rendering GSC non-tumorigenic. We previously demonstrated that CD133+ cells, not CD133− cells, sorted from same sphere cultures contain enriched tumorigenic cells yet they express tumor suppressor phenotype (8
). Thus, true GSC may be quiescent prior to undergoing asymmetric cell division to simultaneously self-
renew and generate more differentiated progeny. It appears that differentiated progeny are active-growing cells that make up the most population in tumor spheres and are likely the effector progeny which can undergo proliferative differentiation to produce more differentiated progeny that expresses hyperproliferative and hyperangiogenic phenotype, leading to tumor formation in animals. Therefore, identifying essential genes (e.g. EDN3) which are shared by both GSC and their immediate differentiated progeny/daughter cells would enhance its clinical value since both populations can be targeted. Thus, treatment with BQ788 not only abolishes the self-renewal capacity of quiescent GSC but also prevent differentiated progeny from populating tumor spheres and forming tumor in animals.
Depletion of EDN3+ cells will likely prevent either CD133+ or CD133− GSC from regenerating a tumor. Futures studies should determine whether addition of EDNRB antagonist can sensitize cells to radio-chemotherapy in treatment of established glioblastoma tumors in animals. Our data support the view that the prevention of GSC-mediated tumor recurrence may need to focus on targeting active stem cell pathways in GSC (such as EDN3/EDNRB pathway), not proliferative pathways. Obviously, the cure for brain cancer requires eliminating both GSC and non-GSC populations; therefore, it is important to evaluate the synergistic benefits of incorporating GSC-targeted therapies into conventional cancer treatments.