Inhibition of oxidative metabolism leads to p53 genetic inactivation and transformation in neural stem cells

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis.Alterations of mitochondrial metabolism are found in several cancers (1). This can occur through inactivation of components of the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) (1–5). In particular, high-grade gliomas (HGGs) display mutations in the TCA enzymes isocitrate dehydrogenase IDH1 and IDH2 (5). Notably, gliomas also present mutations in mitochondrial DNA (mtDNA) and alterations of the ETC, but whether these are early or late events in cancer pathogenesis remains to be determined (6–14). Finally, p53, which has emerged as an important regulator of mitochondrial metabolism and cellular redox control (15–17), is often found mutated or functionally inactivated in HGG. Its inactivation in neural progenitor/stem cells (NPCs), which act as HGG cells of origin, contributes to gliomagenesis (18–22). In particular, deletion of a significant portion of the p53 DNA binding domain induces the accumulation of cooperative oncogenic events, thus leading to HGG (21). However, it remains to be determined whether p53 metabolic functions contribute to suppression of neoplastic transformation in the nervous system. Although these studies suggest an involvement of altered mitochondria metabolism in brain tumorigenesis, direct evidence of its role as a driver or contributing factor in pathogenesis of HGG and other human cancers is missing. More generally, the role of mitochondrial dysfunction in regulation of tumor suppressive control remains only partially investigated.Here, we studied the effect of oxidative metabolism inhibition in normal NPCs. Our findings show that inhibition of respiration via knockdown (KD) of the complex I subunit NDUFA10 or by reducing mtDNA copy number results in p53 genetic loss, via a mechanism involving generation of reactive oxygen species (ROS) and ROS-mediated oxidative damage. In turn, this causes a glycolytic switch, a marked growth advantage, and tumor formation upon transplantation in the mouse brain. Overall, this study reveals that, in NPCs, the relationship between p53 and mitochondrial metabolism is bidirectional, with p53 being activator of mitochondrial metabolism as well as target for genetic inactivation upon inhibition of respiratory chain activity.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions

DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.Adult stem cells (SCs) are thought to be resistant to DNA damage (DD)-induced apoptosis or senescence owing to the activation of unique pro-survival and DD repair (DDR) responses (1–3). Genetic alterations that decrease DNA repair activities lead to increased DD and reduced self-renewal in SCs, suggesting that DDR is critical to preservation of SC function (1, 4, 5). DDR decreases during physiological aging, a phenomenon correlated with the accumulation of endogenous DD and decreased self-renewal in aged SCs (6–9).In differentiated cells, DD triggers a checkpoint response that leads to apoptosis or senescence and depends on activation of the tumor suppressor p53 (10). This is considered a powerful tumor-suppressor mechanism, as demonstrated by the finding that p53 is invariably inactivated in spontaneous tumors (11). After irradiation, p53 is up-regulated in populations enriched for hematopoietic, hair follicle bulge, and colon SCs (5, 12–15). Whether this is critical for activation of the DDR response and maintenance of self-renewal, why p53 induction does not result in SC apoptosis or senescence, and how tumor suppression is executed in SCs remain unclear, however. Indirect evidence indicates that the cell cycle inhibitor p21, a downstream effector of p53, might be involved in DD processing in SCs. In the absence of p21, SCs exhaust prematurely (16) and after a low radiation dose display reduced reconstitution capacity (17). Here we report our studies on the role of p53 and p21 in DD processing of highly purified hematopoietic SCs (HSCs) and mammary SCs (MaSCs).     

Regulation of p53 is critical for vertebrate limb regeneration

Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration.Unlike mammals, which exhibit limited regenerative abilities, the urodele amphibians—or salamanders—are capable of regenerating an extraordinary range of body structures, including ocular tissues, tail, sections of the heart, parts of the nervous system, and entire limbs (1). In salamanders, such as the newt and axolotl, limb regeneration depends on the formation of a blastema, a mound of progenitor cells of restricted potential that arises after amputation (2–4). Following a period of proliferation, blastema cells redifferentiate and restore the structures of the limb.Extensive evidence indicates that limb regeneration depends on reprogramming of cells in mature limb tissues. Upon amputation, muscle, cartilage, and connective tissue cells underneath the injury site lose their differentiated characteristics and re-enter the cell cycle to give rise to the blastema (5–8). This mechanism has also been observed during zebrafish heart and fin regeneration (9, 10). In contrast, reversals of the differentiated state are rarely observed in mammalian tissues, which led to the suggestion that inability to undergo dedifferentiation could contribute to the failure of regeneration in mammals (11). Despite their significance, the mechanisms underlying regulation of the differentiated state during vertebrate regeneration remain poorly understood.Recently, the tumor suppressor p53, whose best-characterized functions are in the maintenance of genome stability (12), has been implicated in the suppression of artificial cell reprogramming to pluripotency (13–17) and the promotion of differentiation pathways in mammals (18). In addition, it has been observed that inhibiting p53 disrupts limb regrowth in salamanders (19), although its role in this context has remained unknown. It is possible that p53 could play a role in the regulation of dedifferentiation and redifferentiation events intrinsic to vertebrate regeneration. Our results demonstrate that the regulation of p53 activity is critical for limb regeneration by controlling key cell fate decisions throughout this process.