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.     

Altered cofactor regulation with disease-associated p97/VCP mutations

Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget’s disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (k_cat/K_m) of p97 by 11-fold. Whereas both p37 and p47 decrease the K_m of ATP in p97, p37 increases the k_cat of p97. In contrast, regulation by p47 is biphasic, with decreased k_cat at low levels but increased k_cat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69–92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis.The p97 AAA [ATPase associated with diverse cellular activities; also called VCP (valosin-containing protein)] participates in key steps in ubiquitin-dependent protein quality control (1), autophagy (2, 3), membrane remodeling (4, 5), and numerous other fundamentally important cellular functions. p97 directs proteins to two major degradation systems: the proteasome and autophagy pathways. This key role of p97 underscores its importance in protein homeostasis and strongly implicates p97 in neurodegenerative diseases (6). Dominantly inherited missense mutations in p97 were initially associated with a rare degenerative disorder termed inclusion body myopathy with Paget’s disease of bone and frontotemporal dementia (IBMPFD) (7, 8). Subsequently, it has been found that mutations in p97 cause a wider spectrum of degenerative diseases that includes amyotrophic lateral sclerosis (9–12) and parkinsonism (13). Thus, the acronym IBMPFD is insufficient. Moreover, mutations in the heterogeneous nuclear ribonucleoproteins hnRNPA2B1 and hnRNPA1 also cause a syndrome similar to conditions associated with VCP mutations (14). To account for the varied phenotypes and genetic etiologies of IBMPFD, it has been suggested that the term multisystem proteinopathy type 1 (MSP1) (14) be used to describe the phenotypic syndrome associated with mutations in p97. Disease mutants of p97 contribute to pathology by disrupting autophagosome (2, 3) and endosome maturation, which leads to vacuolation, weakness, and muscle atrophy (15, 16). However, the molecular disease mechanisms associated with mutant p97 are still being investigated, with the eventual hope of developing therapeutic agents for these serious illnesses.p97 assembles into a homohexamer, as revealed by X-ray crystallography (17, 18). A single monomer is composed of an N domain, which interacts with p97 cofactor proteins, and two ATPase domains, D1 and D2. The D1 and D2 domains form stacked hexameric rings, whereas the N domains extend outward, coplanar with the D1 ring. Most of the disease mutations occur in the N domain, whereas some occur in the D1 or the N–D1 linker region (see SI Appendix, Fig. S1A for examples). All p97 disease mutants tested thus far can form stable hexamers (19, 20) and exhibit increased D2 ATPase activity (19–23). Disease mutations lead to increased proteolytic susceptibility of the D2 ring (19). Structural and biochemical studies suggest that disease mutations alter N-domain and D1 conformations (20, 23, 24) and cause defects in interdomain communication between neighboring subunits.A major role of the N domain is to recruit cofactors (25, 26), such as the Npl4 (nuclear protein localization homolog 4) and Ufd1 (ubiquitin fusion degradation 1) heterodimers (27), and an array of 13 UBX (ubiquitin regulatory X) domain cofactors (28). By recruiting certain cofactor proteins, the N domain may link the mechanochemical activity of ATP hydrolysis to the unfolding or disassembly of substrate proteins. p47, the first p97 UBX cofactor discovered, is required for p97-mediated membrane fusion (29). Binding of p47 (also called NSFL1 cofactor p47 or UBX domain-containing protein 2C) to the N domain of p97 significantly reduces the diameter of the p97 ring (29) and inhibits wild-type p97 ATPase activity (30). Although actively studied, the physiological functions of p97–cofactor complexes and their mechanisms are largely unknown.X-ray crystallography of p97 has revealed that the N domain of p97 is conformationally flexible (17, 18), adopting two primary conformations. In the up conformation, the N domain extends above the D1 ring, whereas in the down conformation, the N domain lies coplanar with the D1 ring. The conformation is nucleotide-dependent, that is, determined by the binding state of the D1 domain (17). It has been proposed that the flexibility of the N domain is crucial to ATP hydrolysis, because modifying the N domain reduces ATPase activity. Specifically, reducing N-domain mobility inhibits wild-type p97 ATPase activity (20). Moreover, removing the N domain (1∼209) altogether was shown to block the enhanced ATPase activity of a disease mutant (20).In wild-type p97, the N domains exist in a tightly regulated, heterogeneous arrangement of up and down conformations. In contrast, disease mutants exhibit dysregulated N-domain conformations (12, 17, 24). Crystal structures of two disease mutants showed all six N domains of the complex in the up conformation, a behavior that has been observed in only disease mutants (23). A recent study found that this uniform arrangement is a secondary effect of reduced ADP binding by the D1 domain, whose state controls N-domain conformation (23).Altered conformation of the N domain in p97 disease mutants is further supported by atypical p97 cofactor binding in cells (31–33). Decreased binding to a UBX cofactor, UBXD1, is observed in 293T cells expressing p97 disease mutants and leads to a blockade of caveolin 1 trafficking (33). Intriguingly, disease mutants can coimmunoprecipitate more p47 and Npl4/Ufd1 heterodimers than WT p97, suggesting elevated binding affinities for p47 and Npl4/Ufd1 in mutant cells (31, 33). However, the consequences of altered binding to cofactors in cells that express mutant p97 have not been investigated biochemically. To provide a mechanistic understanding of cofactor-regulated ATPase activity, we analyzed the effect of p37 and p47 on the ATPase activity of WT and disease mutants of p97 in this study.     

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.     

F-box protein FBXO31 directs degradation of MDM2 to facilitate p53-mediated growth arrest following genotoxic stress

The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage.One of the most fundamental and extensively studied anticancer mechanisms is the large increase in the levels of the tumor suppressor p53 that occurs following DNA damage (reviewed in ref. 1). The increased p53 then mediates growth arrest and/or apoptosis. The importance of this anticancer mechanism is highlighted by the mutation or functional inactivation of the p53 gene in more than 50% of human cancers (2, 3).In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that interacts directly with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome (reviewed in ref. 4). Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Originally it was proposed that MDM2 degradation was caused by auto-ubiquitination; however, subsequent experiments showed that the E3 ubiquitin ligase activity of MDM2 is not required for its degradation (5).We originally identified the F-box protein FBXO31 in an RNAi screen as one of 17 factors required for oncogenic BRAF to induce senescence in primary human cells (6). F-box proteins are best known for their role as the substrate-recognition components of the SKP1/CUL1/F-box protein (SCF) class of E3 ubiquitin ligases (7). The F-box motif is responsible for the ability of F-box proteins to interact with the SCF complex and to promote ubiquitination of their targets (8).One of the other genes we isolated in our original RNAi screen was p53 (6), raising the possibility that FBXO31 and p53 function in a common pathway(s). Consistent with this idea, both FBXO31 and p53 can induce growth arrest (9, 10), and we have found that after DNA damage there is a posttranslational increase of FBXO31 levels, as there is for p53 (9). These considerations prompted us to ask whether there was a functional relationship between FBXO31 and p53.