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Summary RNautophagy is a newly described type of selective autophagy whereby cellular RNAs are transported into lysosomes for degradation. This process involves the transmembrane protein SIDT2, which transports double-stranded RNA (dsRNA) across the endolysosomal membrane. We previously demonstrated that SIDT2 is a transcriptional target of p53, but its role in tumorigenesis, if any, is unclear. Unexpectedly, we show here that Sidt2−/− mice with concurrent oncogenic KrasG12D activation develop significantly fewer tumors than littermate controls in a mouse model of lung adenocarcinoma. Consistent with this observation, loss of SIDT2 also leads to enhanced survival and delayed tumor development in an Apcmin/+ mouse model of intestinal cancer. Within the intestine, Apcmin/+;Sidt2−/− mice display accumulation of dsRNA in association with increased phosphorylation of eIF2α and JNK as well as elevated rates of apoptosis. Taken together, our data demonstrate a role for SIDT2, and by extension RNautophagy, in promoting tumor development., Graphical Abstract, Highlights Loss of the SIDT2 double-stranded RNA (dsRNA) transporter •leads to accumulation of dsRNA in tissues•is associated with increased apoptosis•reduces tumor burden in mouse models of lung adenocarcinoma and intestinal cancer, Biological Sciences; Molecular Biology; Cell Biology; Cancer

Using oncogene-expressing cells to interrogate p53 function under physiological oxygen conditions, Valente et al. show that p53 deficiency drives concurrent dysregulation of a range of cellular processes. These findings highlight the pleiotropic effects of p53 inactivation., The mechanisms by which TP53, the most frequently mutated gene in human cancer, suppresses tumorigenesis remain unclear. p53 modulates various cellular processes, such as apoptosis and proliferation, which has led to distinct cellular mechanisms being proposed for p53-mediated tumor suppression in different contexts. Here, we asked whether during tumor suppression p53 might instead regulate a wide range of cellular processes. Analysis of mouse and human oncogene-expressing wild-type and p53-deficient cells in physiological oxygen conditions revealed that p53 loss concurrently impacts numerous distinct cellular processes, including apoptosis, genome stabilization, DNA repair, metabolism, migration, and invasion. Notably, some phenotypes were uncovered only in physiological oxygen. Transcriptomic analysis in this setting highlighted underappreciated functions modulated by p53, including actin dynamics. Collectively, these results suggest that p53 simultaneously governs diverse cellular processes during transformation suppression, an aspect of p53 function that would provide a clear rationale for its frequent inactivation in human cancer.

The p53 gene is mutated in over half of all cancers, reflecting its critical role as a tumor suppressor. Although p53 is a transcriptional activator that induces myriad target genes, those p53-inducible genes most critical for tumor suppression remain elusive. Here, we leveraged p53 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and RNA-seq (RNA sequencing) data sets to identify new p53 target genes, focusing on the noncoding genome. We identify Neat1, a noncoding RNA (ncRNA) constituent of paraspeckles, as a p53 target gene broadly induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1−/− mice, we examined the functional role of Neat1 in the p53 pathway. We found that Neat1 is dispensable for cell cycle arrest and apoptosis in response to genotoxic stress. In sharp contrast, Neat1 plays a crucial role in suppressing transformation in response to oncogenic signals. Neat1 deficiency enhances transformation in oncogene-expressing fibroblasts and promotes the development of premalignant pancreatic intraepithelial neoplasias (PanINs) and cystic lesions in KrasG12D-expressing mice. Neat1 loss provokes global changes in gene expression, suggesting a mechanism by which its deficiency promotes neoplasia. Collectively, these findings identify Neat1 as a p53-regulated large intergenic ncRNA (lincRNA) with a key role in suppressing transformation and cancer initiation, providing fundamental new insight into p53-mediated tumor suppression.

The tumor protein p53 (TP53, best known as p53) transcription factor is a critical tumor suppressor, but those p53-inducible genes most important for tumor suppression have remained unclear. Using unbiased RNA interference and CRISPR (Clustered Regularly Interspersed Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) screens, genetically engineered mouse models, human cancer genome analysis, and integrative eCLIP-sequencing and RNA-sequencing analyses, we reveal a new branch of p53-mediated tumor suppression involving the RNA splicing regulator Zinc finger Matrin-type 3, Zmat3.

Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens invivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

How cancer cells respond to nutrient deprivation remains poorly understood. In certain cancer cells, deprivation of cystine induces a non-apoptotic, iron-dependent form of cell death termed ferroptosis. Recent evidence suggests that ferroptosis sensitivity may be modulated by the stress-responsive transcription factor and canonical tumor suppressor protein p53. Using CRISPR/Cas9 genome editing, small-molecule probes, and high-resolution, time-lapse imaging, we find that stabilization of wild-type p53 delays the onset of ferroptosis in response to cystine deprivation. This delay requires the p53 transcriptional target CDKN1A (encoding p21) and is associated with both slower depletion of intracellular glutathione and a reduced accumulation of toxic lipid-reactive oxygen species (ROS). Thus, the p53-p21 axis may help cancer cells cope with metabolic stress induced by cystine deprivation by delaying the onset of non-apoptotic cell death.

p53 is a crucial tumour suppressor that responds to diverse stress signals by orchestrating specific cellular responses, including transient cell cycle arrest, cellular senescence and apoptosis, which are all processes associated with tumour suppression. However, recent studies have challenged the relative importance of these canonical cellular responses for p53-mediated tumour suppression and have highlighted roles for p53 in modulating other cellular processes, including metabolism, stem cell maintenance, invasion and metastasis, as well as communication within the tumour microenvironment. In this Opinion article, we discuss the roles of classical p53 functions, as well as emerging p53-regulated processes, in tumour suppression.

The mechanisms by which the p53 tumor suppressor acts remain incompletely understood. To gain new insights into p53 biology we used high throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. Chromatin immunoprecipitation sequencing reveals 4785 p53 bound sites in the genome located near 3193 genes involved in diverse biological processes. RNA sequencing analysis shows that only a subset of p53 bound genes is transcriptionally regulated yielding a list of 432 p53 bound and regulated genes. Interestingly we identify a host of autophagy genes as direct p53 target genes. While the autophagy program is regulated predominantly by p53 the p53 family members p63 and p73 contribute to activation of this autophagy gene network. Induction of autophagy genes in response to p53 activation is associated with enhanced autophagy in diverse settings and depends on p53 transcriptional activity. While p53 induced autophagy does not affect cell cycle arrest in response to DNA damage it is important for both robust p53 dependent apoptosis triggered by DNA damage and transformation suppression by p53. Together our data highlight an intimate connection between p53 and autophagy through a vast transcriptional network and indicate that autophagy contributes to p53 dependent apoptosis and cancer suppression. © 2013 by Cold Spring Harbor Laboratory Press.

The link between EBV infection and Burkitt lymphoma (BL) is strong, but the mechanism underlying that link has been elusive. We have developed a mouse model for EBV-associated BL in which LMP2A, an EBV latency protein, and MYC are expressed in B cells. Our model has demonstrated the ability of LMP2A to accelerate tumor onset, increase spleen size, and bypass p53 inactivation. Here we describe the results of total gene expression analysis of tumor and pretumor B cells from our transgenic mouse model. Although we see many phenotypic differences and changes in gene expression in pretumor B cells, the transcriptional profiles of tumor cells from LMP2A/λ-MYC and λ-MYC mice are strikingly similar, with fewer than 20 genes differentially expressed. We evaluated the functional significance of one of the most interesting differentially expressed genes, Egr1, and found that it was not required for acceleration of tumor onset by LMP2A. Our studies demonstrate the remarkable ability of LMP2A to affect the pretumor B-cell phenotype and tumorigenesis without substantially altering gene expression in tumor cells.

The p53 transcription factor is a critical tumor suppressor in humans and mice. Despite this essential function, the molecular pathways through which p53 acts in tumor suppression remain enigmatic. To define the transcriptional programs through which p53 suppresses carcinogenesis, we have taken combined mouse genetic and genomic approaches. We previously generated a panel of p53 knock-in mouse strains expressing mutants in the first (p5325,26), second (p5353,54), or both (p5325,26,53,54) of two amino-terminal transcriptional activation domains (TADs). In terms of transcriptional activity, we have discovered that p5325,26 is severely impaired for transactivation of the majority of canonical p53 target genes (e.g. p21, Puma, Noxa), but retains the ability to activate a set of primarily novel p53 target genes, while p5325,26,53,54 lacks transactivation activity altogether. The p5353,54 mutant, in contrast, is uncompromised for transactivation of p53 target genes. The p5325,26,53,54 mutant is completely defective in tumor suppression, underscoring the importance of transcriptional activation for p53-mediated tumor suppression. Intriguingly, the p5325,26 mutant retains full activity in suppressing a variety of cancers, indicating that efficient transactivation of most canonical p53 target genes is dispensable for tumor suppression. As p5325,26 activates only a subset of p53-inducible genes, yet retains tumor-suppressor activity, it has helped pinpoint a small set of novel, direct p53-inducible tumor suppression-associated target genes (TSAGs) whose functions we are currently interrogating through genetic screens. Additionally, we have made the surprising discovery that the p5353,54 mutant suppresses pancreatic cancer more efficiently than wild-type p53, and that this capacity correlates with the ability of p5353,54 to hyperactivate a subset of p53 target genes. Indeed, our analysis of these genes has uncovered Ptpn14, a direct p53 target gene critical for mediating p53 function in suppressing pancreatic cancer cell growth. Ptpn14 encodes a protein tyrosine phosphatase that negatively regulates the oncoprotein Yap in both human and mouse cells. These studies thus reveal a p53-Ptpn14-Yap axis in pancreatic cancer. Together, these strategies will help to elaborate the transcriptional networks fundamental for p53 function in tumor suppression. Citation Format: Stephano Spano Mello, Kathryn Bieging-Rolett, Alyssa Kaiser, Elizabeth Joy Valente, Nitin Raj, Jacob McClendon, Brittany Maria Flowers, David Warren Morgens, Michael Cory Bassik, Laura Donatella Attardi. Deconstructing p53 pathways in tumor suppression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr IA07.

The p53 tumor suppressor is a stress sensor, driving cell cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19(Arf) tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19(Arf)-independent. Accordingly, p19(Arf)-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19(Arf) is required for replicative senescence, a condition associated with an activated DNA damage response, as p19(Arf)-/- MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19(Arf) is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19(Arf) has a specific role. We therefore examined the role of p19(Arf) in cellular responses to chronic, low-dose DNA-damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5 G y γ-irradiation daily or 150 μM hydroxyurea, a replication stress inducer. In contrast to their response to acute DNA damage, p19(Arf)-/- MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19(Arf) in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19(Arf)-/- MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19(Arf) in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA-damaging agent treatment is insufficient to promote senescence in p19(Arf)-/- MEFs, suggesting that the role of p19(Arf) in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19(Arf) for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19(Arf) serves as a tumor suppressor. Moreover, our study helps reconcile reports suggesting crucial roles for both p19(Arf) and DNA damage-signaling pathways in tumor suppression.

ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa , leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

Burkitt’s lymphoma (BL) is characterized by translocation of the MYC gene to an immunoglobulin locus. Transgenic mouse models have been used to study the molecular changes that are necessary to bypass tumor suppression in the presence of translocated MYC. Inactivation of the p53 pathway is a major step to tumor formation in mouse models that is also seen in human disease. Human BL is often highly associated with Epstein-Barr virus (EBV). The EBV latency protein latent membrane protein 2A (LMP2A) is known to promote B cell survival by affecting levels of pro-survival factors. Using LMP2A transgenic mouse models, we have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work uncovers a contribution of EBV to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL.

The activation of the p53 protein by cellular stress signals is fundamental for tumor suppression but also promotes pathological states, such as provoking the side effects of genotoxic cancer therapies. To better understand the mechanisms of p53 action in different contexts, we have leveraged both mouse genetic and genomic approaches. First, we have used mouse genetics to define transcriptional programs involved in p53 function in different in vivo settings, specifically by generating a panel of p53 transcriptional activation domain mutant knock-in mouse strains. These include strains expressing p53 mutants in the first (p5325,26), second (p5353,54), or both transactivation domains (p5325,26,53,54). We have observed that p5325,26 is severely compromised for transactivation of most classical p53 target genes, but retains the ability to activate a subset of p53 targets, while p5325,26,53,54 lacks transactivation activity completely. Interestingly, although unable to induce apoptosis or cell cycle arrest in response to acute DNA damage signals, p5325,26 retains full activity in suppressing cancers of a wide range of types, indicating that robust transactivation of most canonical p53 targets is dispensable for tumor suppression. Importantly, as p5325,26 activates only a subset of p53-dependent genes, yet retains tumor suppressor activity, it has helped to define a small set of novel p53-inducible tumor suppression-associated genes, which we are currently analyzing in detail. Second, we have utilized genomic approaches to better understand p53 function. Using ChIP-sequencing and RNA-sequencing to analyze transcriptional programs in acute DNA damage-treated mouse embryo fibroblasts, our studies have revealed an extensive p53-regulated autophagy program that contributes to p53 responses. Together, these approaches will help better define the transcriptional networks important for p53 action in different settings. Citation Format: Colleen A. Brady, Daniela Kenzelmann Broz, Dadi Jiang, Stephano Spano Mello, Kathryn Bieging, Thomas M. Johnson, Leslie A. Jarvis, Margaret M. Kozak, Shashwati Basak, Laura D. Attardi. Deconstructing p53 pathways in vivo. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA4.