To identify novel drug candidates for the common childhood tumor IH, we screened chemical libraries for compounds that would inhibit the vasculogenic stem cell, HemSCs, isolated from IH tumor tissue. We identified Rapamycin, an mTOR inhibitor, based on its relative selectivity for HemSCs over BM-MSCs, a normal human stem cell with a similar phenotype. Rapamycin also suppressed self-renewal and modified the differentiative status of the HemSCs. Finally, Rapamycin prevented HemSCs, either alone or combined with cbEPCs, from forming blood vessels in vivo and increased regression of already formed vessels. Our data suggest that the primary action of Rapamycin is to cause the HemSCs to lose their stem cell properties; this represents an entirely unique mechanism for blocking human post-natal vasculogenesis. We base this on the experiments in which pre-treatment of HemSCs for defined periods of time diminished self-renewal in vitro and inhibited vasculogenesis in vivo. Furthermore, inhibiting proliferation per se
with Roscovitine was not sufficient to block vasculogenesis. An alternative mechanism to Rapamycin's anti-vasculogenic effect in vivo might be the inhibition of the interaction between HemSC and cbEPCs. Evidence for the mTOR pathway was shown by the activation of the upstream, positive modulator AKT in proliferating IH tumor specimens. Finally, we show that Rapamycin exerts non-overlapping, inhibitory effects on HemSCs and ECs compared to corticosteroids (See schematic in Supplemental Fig. 8
The role of phosphatase and tensin homologue (PTEN) in controlling the homeostasis and self renewal of stem/progenitor cells in multiple tissues is well established (Hill and Wu, 2009
) (Dubrovska et al., 2009
; Groszer et al., 2001
; Inoue-Narita et al., 2008
). However, the contribution of pathways downstream of PTEN, and especially the AKT/mTOR axis, is less defined. Both over-activation and suppression of mTOR activity has been shown to affect selfrenewal of stem cells. When human embryonic stem cells (hESCs) are cultivated under self-renewal conditions, mTOR inhibition suffices to disrupt pluripotency and trigger mesoderm and endoderm activities (Zhou et al., 2009
). In hematopoeitic stem cells (HSC), over-activation of the mTOR pathway by conditional deletion of PTEN or tuberous sclerosis protein 1, or by constitutive activation of AKT, (Kharas et al., 2010
) led to loss of HSC quiescence and reduced long-term HSC function. Rapamycin was shown to rescue HSC function (Yilmaz et al., 2006
) (Chen et al., 2008
; Zhang et al., 2006
). The effects of rapamycin treatment on IH-derived stem cells suggest that tightly-controlled mTOR activity is also essential for post-natal, tissue-resident stem cells.
IH appears in the first weeks of life and with higher incidence in low birth weight pre-term infants (Amir et al., 1986
). Following the proliferative phase, there is spontaneous regression leading to a fibrofatty residuum. This unique life cycle suggests that this tumor derives from immature progenitor cells, perhaps of neural crest origin (Haggstrom et al., 2006
) (Wu et al., 2008
), that have not accomplished their full differentiation plan. If that is the case, an intervention that will push these cells to lose their “stemness”, perhaps using Rapamycin or another modulator of mTOR activity, is an appealing strategy to prevent growth and/or induce early involution.
Taken together, our findings suggest two potential advantages of mTOR inhibitors for the treatment of non-responsive IH. First, our finding that corticosteroids and Rapamycin act by distinct mechanisms to suppress the vasculogenic potential of HemSCs suggests that mTOR inhibitors can be used as “steroid sparing” agents, reducing the required dose of corticosteroid. Moreover, due to the induction of HemSCs differentiation by Rapamycin, the treatment period might be shortened. Rapamycin has adverse effects, but in most cases, these effects are dose or concentration-dependent(Sindhi et al., 2005
). Second, in some patients, the permanent fibrofatty residuum after tumor involution can be disfiguring. We found that Rapamycin blocks HemSCs adipogenic differentiation, in accordance with previous reports demonstrating a role for intact mTOR activity for adipogenic differentiation (Zhang et al., 2009
) It might then be the case that treating proliferating IH with Rapamycin would prevent this sequela and be advantageous over a natural involution.