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.     

Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are neuromuscular diseases, caused by accumulation of CUG and CCUG RNAs in toxic aggregates. Here we report that the increased stability of the mutant RNAs in both types of DM is caused by deficiency of RNA helicase p68. We have identified p68 by studying CCUG-binding proteins associated with degradation of the mutant CCUG repeats. Protein levels of p68 are reduced in DM1 and DM2 biopsied skeletal muscle. Delivery of p68 in DM1/2 cells causes degradation of the mutant RNAs, whereas delivery of p68 in skeletal muscle of DM1 mouse model reduces skeletal muscle myopathy and atrophy. Our study shows that correction of p68 may reduce toxicity of the mutant RNAs in DM1 and in DM2.Myotonic dystrophy type 1 (DM1) is a neuromuscular disease characterized by myotonia, distal muscle weakness, heart conduction defects, and, in the congenital form, a delay in myogenesis and severe cognitive abnormalities (1). DM1 is caused by expanded CTG repeats within the 3′ untranslated region of the DMPK gene (2). Myotonic dystrophy type 2 (DM2) is a late-onset disease that is caused by expanded CCTG repeats in intron 1 of the ZNF9/CNBP gene (3). Development of therapeutic approaches for DM1 or DM2 is an urgent need. Numerous data suggest that DM1 and DM2 are caused by RNA gain-of-function mechanisms (4–6). Initial studies showed that mutant RNAs mainly affect two RNA-binding proteins, CUG-binding protein 1 (CUGBP1) and muscleblind-like protein 1 (MBNL1) (7–9). CUG repeats elevate protein levels of CUGBP1 by increasing its stability (5). In addition, CUG repeats change signal transduction pathways, such as the glycogen synthase kinase 3β (GSK3β)–cyclin D3 pathway, regulating CUGBP1 activity (5, 10). CUG and CCUG repeats form double-stranded hairpin structures and sequester MBNL1 (9, 11, 12). Several other RNA-binding proteins, such as Staufen1 and two members of the DEAD-box RNA helicases family, DDX5/p68 and DDX6, are also involved in DM1 (13–15).We showed that the mutant CUG and CCUG RNAs are very stable (16), suggesting that the activity of RNA-binding proteins regulating RNA decay is reduced in DM1 and in DM2. In this study, we tested this hypothesis by isolation and analysis of several CCUG-binding proteins. We found that the levels of one of these proteins, p68, are reduced in DM1 and DM2 biopsied muscle and that correction of p68 leads to degradation of the mutant CUG and CCUG RNAs, disintegration of RNA foci, and reduction of DM muscle pathology.     

UbcH7 regulates 53BP1 stability and DSB repair

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.Prompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2–6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7, 8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9–11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12–14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12, 16, 17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12–14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 stabilizes 53BP1, forcing cells to choose NHEJ, but not HR, to repair DSBs, which poses a significant threat to cells treated with DNA damage, especially S-phase genotoxins, such as camptothecin (CPT), a topoisomerase 1 (Top1) inhibitor. The ternary CPT-Top1-DNA complex places a roadblock in the path of advancing DNA replication forks, leading to replication fork collapse and generation of one-ended DSBs. Such one-ended DSBs require HR, but not NHEJ, to repair (8). In contrast, repair of one-ended DSBs by NHEJ leads to radial chromosomes and cell death (12–14). Therefore, stabilization of 53BP1 by UbcH7 depletion increased the sensitivity of cancers cells to CPT and other DNA damaging agents. Our data suggest a novel strategy in enhancing the anticancer effect of radiotherapy or chemotherapy through stabilizing or increasing 53BP1.     

The ATM–p53 pathway suppresses aneuploidy-induced tumorigenesis

The spindle assembly checkpoint (SAC) is essential for proper sister chromatid segregation. Defects in this checkpoint can lead to chromosome missegregation and aneuploidy. An increasing body of evidence suggests that aneuploidy can play a causal role in tumorigenesis. However, mutant mice that are prone to aneuploidy have only mild tumor phenotypes, suggesting that there are limiting factors in the aneuploidy-induced tumorigenesis. Here we provide evidence that p53 is such a limiting factor. We show that aneuploidy activates p53 and that loss of p53 drastically accelerates tumor development in two independent aneuploidy models. The p53 activation depends on the ataxia-telangiectasia mutated (ATM) gene product and increased levels of reactive oxygen species. Thus, the ATM-p53 pathway safeguards not only DNA damage but also aneuploidy.Faithful transmission of genetic materials is of fundamental importance to the survival of all organisms. In eukaryotes, replicated chromosomes are held together as sister chromatids by the cohesin complexes established during the replication and are segregated to daughter cells in mitosis. The timing of the sister chromatid separation is controlled by the spindle assembly checkpoint (SAC), which monitors the status of microtubule attachment at kinetochores. The SAC is activated when kinetochores are not attached (i.e., occupied) by microtubules and/or when there is a lack of tension at sister kinetochores (1, 2), under both of which situations separation of sister chromatids needs to be actively prevented or missegregation of chromosomes would ensue. The activation of SAC leads to the inhibition of the anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase that targets securin and cyclin B1 for destruction (3–6). Both securin and cyclin B1 are recognized and brought to APC/C by the adaptor protein Cdc20. Not surprisingly, APC^Cdc20 is inhibited by the SAC. The inhibition is carried out by two proteins, Mad2 and BubR1 (for more detailed and recent reviews, see refs. 7 and 8). Genetic analyses in budding yeasts unequivocally demonstrated that the spindle assembly checkpoint was essential in maintaining chromosomal stability (9, 10). Studies of engineered mouse strains carrying mutations in SAC components also indicated the importance of the checkpoint in maintaining chromosome stability (11–19), and BUBR1 was found mutated in a rare human disorder, mosaic variegated aneuploidy (20).A hallmark of human cancers is genomic instability including chromosomal instability (CIN). CIN can be numerical changes in whole chromosomes (aneuploidy) or structural alterations such as translocations. Aneuploidy is found in nearly all of the major human tumor types (21), and it was the abnormal chromosome numbers in cancerous cells that prompted Boveri to propose nearly a century ago that cancer was caused by aneuploidy (22). Nearly all SAC-compromised mouse strains develop spontaneous tumors, although the rates vary substantially (for a summary see ref. 23). Together with the finding of BUBR1 mutation in mosaic variegated aneuploidy, a condition that predisposes patients to childhood cancers (20), the tumor results in SAC mutants strongly argue that aneuploidy can induce tumorigenesis. However, the spontaneous tumor development in SAC mutant mice is usually late onset and at relatively low rates, indicating that aneuploidy does not present a serious risk of tumor development. The low risk of aneuploidy-induced tumorigenesis suggests that there are limiting factors. One such factor could be the general unfitness of aneuploid cells when compared with euploid cells, which is true from budding yeasts to mammals (24, 25). This unfitness likely stems from the imbalances in gene dosage that lead to changes in a score of physiological parameters including energy metabolism (24, 25). Mouse embryonic fibroblasts (MEFs) trisomy for chromosome 1, 13, 16, or 19 grew less robustly than the diploid MEFs and showed resistance to transformation (24). These findings support the notion that aneuploidy can be tumor suppressing under certain circumstances (15, 26). However, the fact that most human cancer cells are aneuploid (21) indicates that there must be ways to overcome the unfitness barrier and that once this barrier is overcome, aneuploidy is beneficial to the tumor development.Here we report that p53 is another limiting factor in aneuploidy-induced tumorigenesis. We provide evidence that p53 is activated by aneuploidy and the activation depends on ataxia-telangiectasia mutated (ATM). We further show that aneuploidy resulted in heightened energy metabolism and increased levels of intracellular reactive oxygen species, which caused oxidative DNA damage and ATM activation.