The data presented here indicate that a stress response gene (p66Shc) and a stem cell regulatory gene (Notch-3) share in an interplay that controls stem cell renewal and hypoxia response. These results are consistent with the tenet that stem cells are harbored in vivo in a low-oxygen environment [1, , , , , , , , –10], the niche, and with the consequent hypothesis that self-renewal potential of stem cells is strictly linked to the capacity of these cells to survey in a hypoxic environment. In particular, as far as the role of p66Shc/Notch-3 interplay in stem cell survival is concerned, we here show that Notch-3 upregulates its own ligand, Jagged-1, in an ERK-dependent manner. This finding is in agreement with the recent reports indicating that in addition to its established capability to induce RBP-Jκ-dependent transcription of HES-like genes, the activated Notch-3 promotes ERK phosphorylation [15, 25]. In this regard, we show that the administration of an antibody that halts the ligand/receptor Jagged-1/Notch-3 interaction reduces the self-renewal of MS. Consequently, we suggest that these data indicate that the p66Shc/Notch-3/Jagged-1 axis may be crucial for stem/progenitor cell self-renewal and survival and that Notch-3 activity acts throughout a canonical ligand-receptor interaction and activation in the absence of hypoxia . However, in the presence of hypoxic stress, we show that such a pathway is flanked by a p66Shc/Notch-3-dependent hypoxia survival response, which relies on the upregulation of CA-IX gene expression. Interestingly, similarly to what was observed for Jagged-1, the Notch-3-dependent upregulation of CA-IX gene expression is mediated by ERK activation. This finding is in line with a recent report indicating that CA-IX expression is modulated by an ERK1/2-dependent pathway, which functions in a manner parallel to, but independent from, the HIF-1α-dependent upregulation of CA-IX gene . Intriguingly, a protein-protein interaction between Notch and HIF-1α proteins has recently been shown to be capable of modulating gene transcription in stem/progenitor cells . In this regard, although we could not demonstrate such a protein-protein complex in the present investigation (data not shown), the available data, together with the results presented here, support the notion that Notch and hypoxia regulation are intimately connected at multiple levels (i.e., HIF-1α regulation  and ERK activation [this investigation]). On the basis of the data reported above, we speculate that these results are consistent with the hypothesis that stem cells are endowed with a genetic program aimed at promoting self-renewal and survival in a hypoxic environment. In turn, low oxygen tension is expected to set the stem cell niche makeup in vivo [10, 12]. Moreover, according to the stem cell hypothesis of cancer, it may be conceived that the dysregulation of such an integrated capacity to survive in a hypoxic environment and to self-renew may confer a growth advantage on cancer (stem) cells. In this regard, there are reports indicating that upregulation of Notch-3, Jagged-1, and CA-IX genes in breast cancer tissues are associated with a poor prognosis [27, 28]. Our results suggest that these genes may be part of a common pathway aimed at promoting survival of cancer (stem) cells. As for p66Shc expression in breast cancer, it has previously been shown that the gene is highly expressed in breast cancer cells with metastatic potential but not in less aggressive ones . In fact, p66Shc has been characterized, so far, for its capacity to induce cell death in the presence of oxidative stress by means of the serine 36 residue phosphorylation  (supplemental online Fig. 1). In this investigation, we found that an S36A p66Shc mutant protein is a better inducer of Notch-3 and CA-IX gene expression than wild-type p66Shc protein in the presence of hypoxia. Because the phosphorylation at the serine 36 residue depends upon oxidative stress, our data also suggest that oxidative stress may inhibit the capacity of p66Shc to upregulate stem cell/hypoxia survival. Intriguingly, it has been reported that Rac-1, a potent activator of p66Shc-dependent oxidative stress , is crucial for maintaining epidermal tissue stem cell survival and self-renewal . Hence, similarly to (or in cooperation with) Rac-1, p66Shc may operate as a double-edged sword: on one hand, playing a prosurvival role in a low-oxygen environment (such as the niche), and on the other, inducing cell death in a pro-oxidant environment. Intriguingly, p66Shc−/− mice experience an advantage for survival late in life when tissue oxidative stress level is increased . This scenario fits in with the theory of antagonistic pleiotropy , which predicts that genes playing detrimental roles late in life are an unforeseen by-product of evolution, due to the selective pressure on such genes to play a vital role in basic functions. In this regard, stem cell survival may be the vital function for which p66shc has been evolutionary selected. In conclusion, our results provide evidence that p66Shc (a major modulator of mammalian aging ), Jagged-1/Notch-3 (two members of an evolutionary-conserved stem cell regulatory pathway ), and CA-IX (a hypoxia-survival gene ) share in a molecular machinery that coordinates stem cell self-renewal and survival in hypoxic conditions. This notion is expected to contribute to the better comprehension of the role of aging in the intricate relationship between cancer and stem cells.