AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

Post-translational modification localizes MYC to the nuclear pore basket to regulate a subset of target genes involved in cellular responses to environmental signals.

The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states.

The ubiquitin-specific protease USP36 SUMOylates EXOSC10 and promotes the nucleolar RNA exosome function in rRNA processing.

The RNA exosome is an essential 3' to 5' exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3' to 5' exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.

The Pancreatic Cancer Early Detection (PRECEDE) Study is a Global Effort to Drive Early Detection: Baseline Imaging Findings in High-Risk Individuals.

BACKGROUND: Pancreatic adenocarcinoma (PC) is a highly lethal malignancy with a survival rate of only 12%. Surveillance is recommended for high-risk individuals (HRIs), but it is not widely adopted. To address this unmet clinical need and drive early diagnosis research, we established the Pancreatic Cancer Early Detection (PRECEDE) Consortium. METHODS: PRECEDE is a multi-institutional international collaboration that has undertaken an observational prospective cohort study. Individuals (aged 18-90 years) are enrolled into 1 of 7 cohorts based on family history and pathogenic germline variant (PGV) status. From April 1, 2020, to November 21, 2022, a total of 3,402 participants were enrolled in 1 of 7 study cohorts, with 1,759 (51.7%) meeting criteria for the highest-risk cohort (Cohort 1). Cohort 1 HRIs underwent germline testing and pancreas imaging by MRI/MR-cholangiopancreatography or endoscopic ultrasound. RESULTS: A total of 1,400 participants in Cohort 1 (79.6%) had completed baseline imaging and were subclassified into 3 groups based on familial PC (FPC; n=670), a PGV and FPC (PGV+/FPC+; n=115), and a PGV with a pedigree that does not meet FPC criteria (PGV+/FPC-; n=615). One HRI was diagnosed with stage IIB PC on study entry, and 35.1% of HRIs harbored pancreatic cysts. Increasing age (odds ratio, 1.05; P<.001) and FPC group assignment (odds ratio, 1.57; P<.001; relative to PGV+/FPC-) were independent predictors of harboring a pancreatic cyst. CONCLUSIONS: PRECEDE provides infrastructure support to increase access to clinical surveillance for HRIs worldwide, while aiming to drive early PC detection advancements through longitudinal standardized clinical data, imaging, and biospecimen captures. Increased cyst prevalence in HRIs with FPC suggests that FPC may infer distinct biological processes. To enable the development of PC surveillance approaches better tailored to risk category, we recommend adoption of subclassification of HRIs into FPC, PGV+/FPC+, and PGV+/FPC- risk groups by surveillance protocols.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.

SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.

Diminished WNT -> ß-catenin -> c-MYC signaling is a barrier for malignant progression of BRAFV600E-induced lung tumors.

Oncogene-induced senescence (OIS) is proposed as a cellular defense mechanism that restrains malignant progression of oncogene-expressing, initiated tumor cells. Consistent with this, expression of BRAF(V600E) in the mouse lung epithelium elicits benign tumors that fail to progress to cancer due to an apparent senescence-like proliferative arrest. Here we demonstrate that nuclear ß-catenin → c-MYC signaling is essential for early stage proliferation of BRAF(V600E)-induced lung tumors and is inactivated in the subsequent senescence-like state. Furthermore, either ß-catenin silencing or pharmacological blockade of Porcupine, an acyl-transferase essential for WNT ligand secretion and activity, significantly inhibited BRAF(V600E)-initiated lung tumorigenesis. Conversely, sustained activity of ß-catenin or c-MYC significantly enhanced BRAF(V600E)-induced lung tumorigenesis and rescued the anti-tumor effects of Porcupine blockade. These data indicate that early stage BRAF(V600E)-induced lung tumors are WNT-dependent and suggest that inactivation of WNT → ß-catenin → c-MYC signaling is a trigger for the senescence-like proliferative arrest that constrains the expansion and malignant progression of BRAF(V600E)-initiated lung tumors. Moreover, these data further suggest that the trigger for OIS in initiated BRAF(V600E)-expressing lung tumor cells is not simply a surfeit of signals from oncogenic BRAF but an insufficiency of WNT → ß-catenin → c-MYC signaling. These data have implications for understanding how genetic abnormalities cooperate to initiate and promote lung carcinogenesis.

Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers.

The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix-loop-helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non-small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.

SUMO protease SENP1 deSUMOylates and stabilizes c-Myc.

Posttranslational modifications play a crucial role in the proper control of c-Myc protein stability and activity. c-Myc can be modified by small ubiquitin-like modifier (SUMO). However, how SUMOylation regulates c-Myc stability and activity remains to be elucidated. The deSUMOylation enzyme, SENP1, has recently been shown to have a prooncogenic role in cancer; however, mechanistic understanding of this is limited. Here we show that SENP1 is a c-Myc deSUMOylating enzyme. SENP1 interacts with and deSUMOylates c-Myc in cells and in vitro. Overexpression of wild-type SENP1, but not its catalytically inactive C603S mutant, markedly stabilizes c-Myc and increases its levels and activity. Knockdown of SENP1 reduces c-Myc levels, induces cell cycle arrest, and drastically suppresses cell proliferation. We further show that c-Myc can be comodified by both ubiquitination and SUMOylation. SENP1-mediated deSUMOylation reduces c-Myc polyubiquitination, suggesting that SUMOylation promotes c-Myc degradation through the proteasome system. Interestingly, SENP1-mediated deSUMOylation promotes the accumulation of monoubiquitinated c-Myc and its phosphorylation at serine 62 and threonine 58. SENP1 is frequently overexpressed, correlating with the high expression of c-Myc, in breast cancer tissues. Together, these results reveal that SENP1 is a crucial c-Myc deSUMOylating enzyme that positively regulates c-Myc's stability and activity.

Modeling differentiation-state transitions linked to therapeutic escape in triple-negative breast cancer.

Drug resistance in breast cancer cell populations has been shown to arise through phenotypic transition of cancer cells to a drug-tolerant state, for example through epithelial-to-mesenchymal transition or transition to a cancer stem cell state. However, many breast tumors are a heterogeneous mixture of cell types with numerous epigenetic states in addition to stem-like and mesenchymal phenotypes, and the dynamic behavior of this heterogeneous mixture in response to drug treatment is not well-understood. Recently, we showed that plasticity between differentiation states, as identified with intracellular markers such as cytokeratins, is linked to resistance to specific targeted therapeutics. Understanding the dynamics of differentiation-state transitions in this context could facilitate the development of more effective treatments for cancers that exhibit phenotypic heterogeneity and plasticity. In this work, we develop computational models of a drug-treated, phenotypically heterogeneous triple-negative breast cancer (TNBC) cell line to elucidate the feasibility of differentiation-state transition as a mechanism for therapeutic escape in this tumor subtype. Specifically, we use modeling to predict the changes in differentiation-state transitions that underlie specific therapy-induced changes in differentiation-state marker expression that we recently observed in the HCC1143 cell line. We report several statistically significant therapy-induced changes in transition rates between basal, luminal, mesenchymal, and non-basal/non-luminal/non-mesenchymal differentiation states in HCC1143 cell populations. Moreover, we validate model predictions on cell division and cell death empirically, and we test our models on an independent data set. Overall, we demonstrate that changes in differentiation-state transition rates induced by targeted therapy can provoke distinct differentiation-state aggregations of drug-resistant cells, which may be fundamental to the design of improved therapeutic regimens for cancers with phenotypic heterogeneity.

Correction: Modeling differentiation-state transitions linked to therapeutic escape in triple-negative breast cancer.

[This corrects the article DOI: 10.1371/journal.pcbi.1006840.].

GRB7 dependent proliferation of basal-like, HER-2 positive human breast cancer cell lines is mediated in part by HER-1 signaling.

GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and overexpressed with HER-2 in human breast cancer. This study addresses the role of GRB7 in HER-2 positive human breast cancers resistant to HER-2 targeted therapy. HCC1954, 21MT1, and JIMT1 are basal like HER-2 positive breast cancer cell lines based on expression profiling. These three cell lines are resistant to trastuzumab and lapatinib treatment. Knockdown of GRB7 protein expression with siRNA transfection as well as lentiviral vector mediated shRNA over-expression decreased the growth of HCC1954, 21MT1, and JIMT1 cells in vitro and the growth of tumor xenografts these cells formed in animal models. When assayed by ki-67 staining and TUNEL assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC1954 cells and increased apoptosis in JIMT1 cells. Phospho-proteome profiling found HER-1 tyrosine phosphorylation was reduced with GRB7 knock down in JIMT1 cells. Immuno-blotting and immuno-precipitation experiments found HER-1 phosphorylation was reduced with GRB7 knock down in all three cell lines. HER-1 knock down via siRNA transient transfection as well as blocking HER-1 function with panitumumab decreased proliferation of all three cell lines in vitro. Our study finds that GRB7 has an essential growth promoting function which is mediated in part by HER-1 activation. The potential of HER-1 targeting in therapy resistant HER-2 positive breast cancer merits further study.

The ubiquitin-specific protease USP36 is a conserved histone H2B deubiquitinase.

Histone H2B monoubiquitination plays a critical role in the regulation of gene transcription. Deregulation of H2B monoubiquitination contributes to human pathologies, such as cancer. Here we report that human USP36 is a novel H2Bub1 deubiquitinase. We show that USP36 interacts with H2B and deubiquitinates H2Bub1 in cells and in vitro. Overexpression of USP36 markedly reduced the levels of H2Bub1 in cells. Using the p21 gene as a model, we demonstrate that depletion of USP36 increases H2Bub1 at the p21 locus, primarily within its gene body. Consistently, knockdown of USP36 induced the expression of p21 and inhibits cell proliferation. Together, our results reveal USP36 as a novel H2B deubiquitinase and shed light on its additional functions in regulating gene expression.

The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc.

c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCF(Fbw7) (a SKP1-cullin-1-F-box complex that contains the F-box and WD repeat domain-containing 7, Fbw7, as the F-box protein) and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) 28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ but not the nucleoplasmic Fbw7α. However, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc's nucleolar degradation pathway.

Aspirin therapy reduces the ability of platelets to promote colon and pancreatic cancer cell proliferation: Implications for the oncoprotein c-MYC.

Aspirin, an anti-inflammatory and antithrombotic drug, has become the focus of intense research as a potential anticancer agent owing to its ability to reduce tumor proliferation in vitro and to prevent tumorigenesis in patients. Studies have found an anticancer effect of aspirin when used in low, antiplatelet doses. However, the mechanisms through which low-dose aspirin works are poorly understood. In this study, we aimed to determine the effect of aspirin on the cross talk between platelets and cancer cells. For our study, we used two colon cancer cell lines isolated from the same donor but characterized by different metastatic potential, SW480 (nonmetastatic) and SW620 (metastatic) cancer cells, and a pancreatic cancer cell line, PANC-1 (nonmetastatic). We found that SW480 and PANC-1 cancer cell proliferation was potentiated by human platelets in a manner dependent on the upregulation and activation of the oncoprotein c-MYC. The ability of platelets to upregulate c-MYC and cancer cell proliferation was reversed by an antiplatelet concentration of aspirin. In conclusion, we show for the first time that inhibition of platelets by aspirin can affect their ability to induce cancer cell proliferation through the modulation of the c-MYC oncoprotein.

ΔN-ASPP2, a novel isoform of the ASPP2 tumor suppressor, promotes cellular survival.

ASPP2 is a tumor suppressor that works, at least in part, through enhancing p53-dependent apoptosis. We now describe a new ASPP2 isoform, ΔN-ASPP2, generated from an internal transcription start site that encodes an N-terminally truncated protein missing a predicted 254 amino acids. ΔN-ASPP2 suppresses p53 target gene transactivation, promoter occupancy, and endogenous p53 target gene expression in response to DNA damage. Moreover, ΔN-ASPP2 promotes progression through the cell cycle, as well as resistance to genotoxic stress-induced growth inhibition and apoptosis. Additionally, we found that ΔN-ASPP2 expression is increased in human breast tumors as compared to adjacent normal breast tissue; in contrast, ASPP2 is suppressed in the majority of these breast tumors. Together, our results provide insight into how this new ASPP2 isoform may play a role in regulating the ASPP2-p53 axis.

Targeting c-MYC by antagonizing PP2A inhibitors in breast cancer.

The transcription factor c-MYC is stabilized and activated by phosphorylation at serine 62 (S62) in breast cancer. Protein phosphatase 2A (PP2A) is a critical negative regulator of c-MYC through its ability to dephosphorylate S62. By inactivating c-MYC and other key signaling pathways, PP2A plays an important tumor suppressor function. Two endogenous inhibitors of PP2A, I2PP2A, Inhibitor-2 of PP2A (SET oncoprotein) and cancerous inhibitor of PP2A (CIP2A), inactivate PP2A and are overexpressed in several tumor types. Here we show that SET is overexpressed in about 50-60% and CIP2A in about 90% of breast cancers. Knockdown of SET or CIP2A reduces the tumorigenic potential of breast cancer cell lines both in vitro and in vivo. Treatment of breast cancer cells in vitro or in vivo with OP449, a novel SET antagonist, also decreases the tumorigenic potential of breast cancer cells and induces apoptosis. We show that this is, at least in part, due to decreased S62 phosphorylation of c-MYC and reduced c-MYC activity and target gene expression. Because of the ubiquitous expression and tumor suppressor activity of PP2A in cells, as well as the critical role of c-MYC in human cancer, we propose that activation of PP2A (here accomplished through antagonizing endogenous inhibitors) could be a novel antitumor strategy to posttranslationally target c-MYC in breast cancer.

Inhibition of 5-lipoxygenase selectively triggers disruption of c-Myc signaling in prostate cancer cells.

Myc is up-regulated in almost all cancer types and is the subject of intense investigation because of its pleiotropic effects controlling a broad spectrum of cell functions. However, despite its recognition as a stand-alone molecular target, development of suitable strategies to block its function is hindered because of its nonenzymatic nature. We reported earlier that arachidonate 5-lipoxygenase (5-Lox) plays an important role in the survival and growth of prostate cancer cells, although details of the underlying mechanisms have yet to be characterized. By whole genome gene expression array, we observed that inhibition of 5-Lox severely down-regulates the expression of c-Myc oncogene in prostate cancer cells. Moreover, inhibition of 5-Lox dramatically decreases the protein level, nuclear accumulation, DNA binding, and transcriptional activities of c-Myc. Both the 5-Lox inhibition-induced down-regulation of c-Myc and induction of apoptosis are mitigated when the cells are treated with 5-oxoeicosatetraenoic acid, a metabolite of 5-Lox, confirming a role of 5-Lox in these processes. c-Myc is a transforming oncogene widely expressed in prostate cancer cells and maintains their transformed phenotype. Interestingly, MK591, a specific 5-Lox inhibitor, strongly affects the viability of Myc-overactivated prostate cancer cells and completely blocks their invasive and soft agar colony-forming abilities, but it spares nontransformed cells where expression of 5-Lox is undetectable. These findings indicate that the oncogenic function of c-Myc in prostate cancer cells is regulated by 5-Lox activity, revealing a novel mechanism of 5-Lox action and suggesting that the oncogenic function of c-Myc can be suppressed by suitable inhibitors of 5-Lox.

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.

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis.

Mnt (Max's next tango) is a Max-interacting transcriptional repressor that can antagonize both the proproliferative and proapoptotic functions of Myc in vitro. To ascertain the physiologically relevant functions of Mnt and to help define the relationship between Mnt and Myc in vivo, we generated a series of mouse strains in which Mnt was deleted in T cells in the absence of endogenous c-Myc or in the presence of ectopic c-Myc. We found that apoptosis caused by loss of Mnt did not require Myc but that ectopic Myc expression dramatically decreased the survival of both Mnt-deficient T cells in vivo and Mnt-deficient MEFs in vitro. Consequently, Myc-driven proliferative expansion of T cells in vitro and thymoma formation in vivo were prevented by the absence of Mnt. Consistent with T-cell models, mouse embryo fibroblasts (MEFs) lacking Mnt were refractory to oncogenic transformation by Myc. Tumor suppression caused by loss of Mnt was linked to increased apoptosis mediated by reactive oxygen species (ROS). Thus, although theoretically and experimentally a Myc antagonist, the dominant physiological role of Mnt appears to be suppression of apoptosis. Our results redefine the physiological relationship between Mnt and Myc and requirements for Myc-driven oncogenesis.