TLX may promote survival and
TLX may promote survival and prevent apoptosis in NB 10cl in ml synthesis . Here, we ask whether the survival-promoting effects of TLX are mediated by Apoptosis Signal-regulating Kinase-1 (ASK1, MAP3K5). ASK1 is a member of the Mitogen-Activated Protein (MAP) kinase family and it preferentially activates the JNK and p38 MAPK pathways, resulting cell death in NB cells , , , , , . Emerging evidence demonstrates that ASK1 is involved in the pathogenesis of inflammatory, cardiovascular, and neurodegenerative diseases , , .
The pro-apoptotic role of ASK1 has been extensively studied , , , , , , , but what remains to be explored is its role in pediatric cancer cells. Here, we show that TLX interacts with ASK1 in stabilizing TLX and enhancing HIF-1α expression in NB cells. Pharmacological inhibition of Akt activity, as well as siRNA knockdown of ASK1 expression, resulted in down-regulation of HIF-1α and VEGF-A in NB cells. These results suggest that post-translational regulation mediated by TLX and its association with ASK1 is an important mechanism for HIF-1α expression in NB cells. These findings support a novel role for ASK1 in maintaining TLX, which may act as a survival factor for NB cells.
Materials and methods
Discussion Until now, there has been little evidence that ASK1 possesses a pro-survival potential in cancer cells. TLX is known to be essential in neural stem cell renewal, while ASK1 is a pro-apoptotic kinase. Our study shows that both TLX and ASK1 are expressed higher in the SP cells of established NB lines and primary NB lines, both cell lines were shown to possess a higher capacity of self-renewal than non-SP cells (Fig. S1) . Thus, we examined whether ASK1 has other roles in NB and found that increasing ASK1 enhances TLX and its downstream HIF-1α, whereas ASK1 knockdown diminishes apoptosis of IMR32 in hypoxia. We found that the pro-apoptotic pASK1T838 stabilized TLX, which contributed to the maintenance of survival for primary NB cells. The C-terminals of both TLX and ASK1 are required for their interaction and the subsequent amplification of downstream signaling events. Under normal conditions, ASK1 forms a homo-oligomer through its C-terminal coiled-coil domain . Upon oxidative stress, the C-terminal structure is altered to expose its trans-autophosphorylation site, Thr-838. However, ASK1 activation is a prerequisite for the ubiquitination of the C-terminal ASK1 by Roquin-2 E3 ubiquitin ligase, followed by the interaction of de-ubiquitinating enzyme USP9X . Thus, it is possible that the C-terminus of TLX, which also binds with the ubiquitin ligases, such as VHL  and perhaps also Smurf1 (targets the TGF-β-receptor I  might interact also with Roquin-2 or other ubiquitin ligases in order to sustain ASK1 in an active form. Our study revealed a new mechanism of ASK1-TLX stabilization, driving the enhancement of HIF-1α and VEGF-A, which contributes to tumor angiogenesis. Inhibition of Akt activation prevented TLX-mediated amplification of HIF-1α and VEGF, indicating its dependency on Akt phosphorylation. Akt1 is known to suppress ASK1 by inhibiting phosphorylation of ASK1 at ASK1S966 . Thus, increase of TLX will activate Akt to be followed by ASK1 inhibition, rendering destabilization of TLX acting as a feedback. On the other hand, stabilization of TLX could be a trigger activating the angiogenic responses and repressing transcription of PTEN . This will maintain activated Akt as a feed-forward mechanism. Another intriguing finding of this study is the discrepancy in ASK1 modifications between the established NB lines, the primary NB lines, and the xenografts derived from the primary NB cells. The SP fraction of cells in the established lines, which are thought to be enriched for tumorigenic cells, showed high levels of expression of pASK1S966 than pASK1T838. In contrast, primary NB cells expressed pASK1T838 and low levels of pASK1S966. Established NB lines might have been selected to express high levels of TLX for continued proliferation, and by repressing PTEN, TLX will maintain pAkt, which causes an increase in inhibitory pASK1S966 during extended culture periods. Primary NB cells are not selected to the same degree and are likely to be sensitive to oxidative stress, which activates pASK1T838 and stabilizes TLX. Almost all NB153 tumor spheres cells expressed high levels of TLX and pASK1T838, but no pASK1S966. The tumor spheres consist of enriched multipotent cells, and oxidative stress might be higher . This is valid even though they are derived from the same patient, as in the NB128 xenograft, which expressed low levels of pASK1T838, as well as pASK1S966. This might be due to the fact that primary NB xenografts grow in more heterogeneous conditions when compared with sphere cultures.