Studies indicate that label-retaining cells co-purify with tissue-specific adult stem cells, suggesting a physiologic role for quiescence in preservation of self-renewal within regenerative hierarchies that govern development, stasis, aging and cancer. Central to this model is the functional asymmetry of stem cell division that yields mitotic siblings with distinct fates. While one sibling forfeits self-renewing capacity and enters a stage of transient amplification, a second retains it and enters a state of quiescence. Doing so enables retention of proliferative capacity and evasion of the negative effects of excessive cell division, including telomeric erosion, accumulation of reactive oxygen species and increased risk of mutation. Quiescence is also a potent blockade to differentiation,11
suggesting a role in developmental potency. Despite the critical role of quiescence, the molecular and genomic events associated with entry into and maintenance of quiescence are incompletely understood. Work presented here describes a regulatory relationship between ΔNp63α and Notch3 that governs quiescence and demonstrates for the first time that ΔNp63α promotes quiescence and suggests a mechanism by which ΔNp63α promotes stem cell longevity.
While the hypotheses surrounding the role of adult stem cells in cancer initiation and etiology remain controversial and unproven, there is abundant evidence indicating that diverse tumors possess a subpopulation of cells that are uniquely tumorigenic and able to self-renew. Multiple studies have demonstrated that this subpopulation displays broad-spectrum resistance to cytotoxic chemotherapeutics and ionizing radiation,45–47
thereby implicating this subpopulation in cancer recurrence. Other studies demonstrate a correlation between label retention and chemoresistance in cancer models, suggesting that cellular quiescence may confer resistance to therapeutics that target proliferating cells.23
Consistent with this are studies indicating that subversion of quiescence in leukemic stem cells renders these cells sensitive to chemotherapeutics.48
Therefore, targeting genetic pathways governing stem cell quiescence in the setting of adjuvant therapeutics represents a promising strategy to reduce cancer recurrence. Here, we present data indicating that disruption of Notch signaling subverts quiescence in a cell culture model with features of MaSCs. Additionally, we demonstrate that Notch signaling may mediate a cellular response to diverse quiescence-inducing stimuli, including ΔNp63α activity, suggesting a fundamental role in cellular quiescence that may apply to multiple cancer stem cell models.
Cellular and developmental context are critical determinants of Notch signaling output that may account for the diverse cellular responses to perturbations in Notch signaling.27–30
This diversity is best illustrated by abundant and compelling evidence that Notch signaling can be either oncogenic or tumor suppressive in distinct cellular contexts. A model that may account for this variability holds that Notch signaling instructs mutually exclusive cell fates upon Notch donor and recipient cells via lateral inhibition;42
however, the molecular determinants of context specificity are largely unidentified. Within the context of governance of stem cell quiescence, our study suggests that ΔNp63α may be one such determinant of Notch signaling output. Additionally, it is unclear whether the four Notch family members mediate identical Notch signaling outputs. In this manuscript, we reported that while only Notch3 was responsive to ΔNp63α, both Notch1 and Notch2 were expressed at higher levels. This may suggest that effects of DAPT are due to inhibition of signaling from multiple Notch receptors. In this manuscript we present data indicating that shRNA-mediated ablation of Notch3 was sufficient to prevent quiescence following growth factor reduction. Our data indicate that Notch signaling is anti-proliferative and promotes expression of genes associated with quiescence and support a model in which activation of Notch by ΔNp63α represents a mechanism by which stem cell quiescence is maintained. Consistent with this model is the remarkable finding that the mammary glands of MMTV-Notch1ICD
undergo a developmental blockade that disrupts lobulo-alveolar development in pregnant mice.36
This is in contrast to several other MMTV-based breast cancer models characterized by precocious lobulo-alveolar development and is consistent with a model in which Notch signaling suppresses activation of the mammary regenerative hierarchy.
Two recent studies have also implicated p53 in the governance of stem cell activity. One identified necdin as a gene that is regulated by p53 in the absence of any cellular or genotoxic stress and showed that necdin was necessary to maintain the ratio of long-term hematopoetic stem cells to short-term hematopoetic stem cells.49
A second study showed that p53-/-
mammary epithelial cells had greater mammosphere-forming capacity and greater mammary regenerative capacity than mammary epithelial cells from wild-type counterparts.50
Importantly this study showed that the increased regenerative activity that resulted from p53 ablation was neutralized by DAPT, suggesting strongly that Notch signaling was activated in response to p53 suppression. This study also showed that long-term BrdU retention was compromised in p53-/-
mice. Collectively, these studies indicate that a complex relationship between ΔNp63 isoforms and p53 may underlie the governance of quiescence vs. activation in adult stem cell populations.
Our conclusion that ΔNp63α promotes cellular quiescence coupled to studies indicating that it is required to avoid cellular senescence40,51,52
suggests a dynamic model in which ΔNp63α balances quiescence and senescence to preserve long-term replicative capacity and a prolonged life span. While there is abundant evidence that p53 is a potent inducer of cellular senescence, more recent studies have indicated that it can also promote quiescence and, in so doing, prevent senescence. Importantly, this study demonstrated that the ability of p53 to induce quiescence is the result of p53-mediated suppression of senescence.53
While the cellular and molecular mechanisms underlying this paradox are incompletely understood, another recent study has implicated the status of the mTOR signaling pathway in the p53-mediated outcome.54
Additionally, ΔNp63α has been shown to be a transcriptional target of p53.55
This coupled to the fact that ΔNp63α is expressed in a highly cell-type dependent manner suggests a model in which cells that are capable of p53-dependent regulation of ΔNp63α may be prone to quiescence, while those in which p53 is present but ΔNp63α expression is repressed may be prone to senescence.