Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

p53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation-driven pancreatic cancer.

Oncogenic KRAS activation, inflammation and p53 mutation are key drivers of pancreatic cancer (PC) development. Here we report iASPP, an inhibitor of p53, as a paradoxical suppressor of inflammation and oncogenic KRASG12D-driven PC tumorigenesis. iASPP suppresses PC onset driven by KRASG12D alone or KRASG12D in combination with mutant p53R172H. iASPP deletion limits acinar-to-ductal metaplasia (ADM) in vitro but accelerates inflammation and KRASG12D-induced ADM, pancreatitis and PC tumorigenesis in vivo. KRASG12D/iASPPΔ8/Δ8 tumours are well-differentiated classical PCs and their derivative cell lines form subcutaneous tumours in syngeneic and nude mice. Transcriptomically, either iASPP deletion or p53 mutation in the KRASG12D background altered the expression of an extensively overlapping gene set, comprised primarily of NF-κB and AP1-regulated inflammatory genes. All these identify iASPP as a suppressor of inflammation and a p53-independent oncosuppressor of PC tumorigenesis.

Regulation of immunological tolerance by the p53-inhibitor iASPP.

Maintenance of immunological homeostasis between tolerance and autoimmunity is essential for the prevention of human diseases ranging from autoimmune disease to cancer. Accumulating evidence suggests that p53 can mitigate phagocytosis-induced adjuvanticity thereby promoting immunological tolerance following programmed cell death. Here we identify Inhibitor of Apoptosis Stimulating p53 Protein (iASPP), a negative regulator of p53 transcriptional activity, as a regulator of immunological tolerance. iASPP-deficiency promoted lung adenocarcinoma and pancreatic cancer tumorigenesis, while iASPP-deficient mice were less susceptible to autoimmune disease. Immune responses to iASPP-deficient tumors exhibited hallmarks of immunosuppression, including activated regulatory T cells and exhausted CD8+ T cells. Interestingly, iASPP-deficient tumor cells and tumor-infiltrating myeloid cells, CD4+, and γδ T cells expressed elevated levels of PD-1H, a recently identified transcriptional target of p53 that promotes tolerogenic phagocytosis. Identification of an iASPP/p53 axis of immune homeostasis provides a therapeutic opportunity for both autoimmune disease and cancer.

Defect in Ser312 phosphorylation of Tp53 dysregulates lipid metabolism for fatty accumulation and fatty liver susceptibility: Revealed by lipidomics.

The Tp53 gene is a well-known tumour suppressor, mutation of which (e.g. prevention of Ser312 phosphorylation) induces deletion or expression of an inactive p53 protein to increase the susceptibility of tumour occurance. However, the role of Tp53 gene in maintaining metabolic homeostasis for regulating physio-pathological activities is still not well-understood. This study aimed to use the lipidomics study as a systematic approach to understand the relationship between the phenotypic effects of Tp53 mutation on lipid-related endogenous metabolites. Plasma and liver samples from mice carrying a Tp53 Ser312 to Ala mutation and wild type mice were collected, lipids were extracted by liquid-liquid extraction method and analyzed by the RPLC-LTQ-FTMS for the lipidomics study. Our results indicated that defect in Ser312 phosphorylation of Tp53 leads the lipid disturbance (e.g. triacylglycerols) for fatty accumulation and fatty liver susceptibility, which is with preference of females. Histological observation by staining with haematoxylin and eosin further validated our lipidomics findings. To our conclusion, fatty liver occurrence may have different phenotypes, one of which is strongly linked with the Tp53 mutation and is susceptible in females. Lipidomics as a technique to detect a great number of endogenous compounds provides precise metabolic information that may further help improve personalized diagnosis of Chronic hepatic diseases.

ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis.

During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.

Cell autonomous role of iASPP deficiency in causing cardiocutaneous disorders.

Desmosome components are frequently mutated in cardiac and cutaneous disorders in animals and humans and enhanced inflammation is a common feature of these diseases. Previous studies showed that inhibitor of Apoptosis Stimulating p53 Protein (iASPP) regulates desmosome integrity at cell-cell junctions and transcription in the nucleus, and its deficiency causes cardiocutaneous disorder in mice, cattle, and humans. As iASPP is a ubiquitously expressed shuttling protein with multiple functions, a key question is whether the observed cardiocutaneous phenotypes are caused by loss of a cell autonomous role of iASPP in cardiomyocytes and keratinocytes specifically or by a loss of iASPP in other cell types such as immune cells. To address this, we developed cardiomyocyte-specific and keratinocyte-specific iASPP-deficient mouse models and show that the cell-type specific loss of iASPP in cardiomyocytes or keratinocytes is sufficient to induce cardiac or cutaneous disorders, respectively. Additionally, keratinocyte-specific iASPP-deficient mice have delayed eyelid development and wound healing. In keratinocytes, junctional iASPP is critical for stabilizing desmosomes and iASPP deficiency results in increased and disorganized cell migration, as well as impaired cell adhesion, consistent with delayed wound healing. The identification of a cell autonomous role of iASPP deficiency in causing cardiocutaneous syndrome, impaired eyelid development and wound healing suggests that variants in the iASPP gene also may contribute to polygenic heart and skin diseases.

Caspase cleavage of iASPP potentiates its ability to inhibit p53 and NF-κB.

An intriguing biological question relating to cell signaling is how the inflammatory mediator NF-kB and the tumour suppressor protein p53 can be induced by similar triggers, like DNA damage or infection, yet have seemingly opposing or sometimes cooperative biological functions. For example, the NF-κB subunit RelA/p65 has been shown to inhibit apoptosis, whereas p53 induces apoptosis. One potential explanation may be their co-regulation by common cellular factors: inhibitor of Apoptosis Stimulating p53 Protein (iASPP) is one such common regulator of both RelA/p65 and p53. Here we show that iASPP is a novel substrate of caspases in response to apoptotic stimuli. Caspase cleaves the N-terminal region of iASPP at SSLD294 resulting in a prominent 80kDa fragment of iASPP. This caspase cleavage site is conserved in various species from zebrafish to Homo sapiens. The 80kDa fragment of iASPP translocates from the cytoplasm to the nucleus via the RaDAR nuclear import pathway, independent of p53. The 80kDa iASPP fragment can bind and inhibit p53 or RelA/p65 more efficiently than full-length iASPP. Overall, these data reveal a potential novel regulation of p53 and RelA/p65 activities in response to apoptotic stimuli.

Phosphorylation of ASPP2 by RAS/MAPK pathway is critical for its full pro-apoptotic function.

We reported recently that apoptosis-stimulating protein of p53 (ASPP) 2, an activator of p53, co-operates with oncogenic RAS to enhance the transcription and apoptotic function of p53. However, the detailed mechanism remains unknown. Here we show that ASPP2 is a novel substrate of mitogen-activated protein kinase (MAPK). Phosphorylation of ASPP2 by MAPK is required for RAS-induced increased binding to p53 and increased transactivation of pro-apoptotic genes. In contrast, an ASPP2 phosphorylation mutant exhibits reduced p53 binding and fails to enhance transactivation and apoptosis. Thus phosphorylation of ASPP2 by RAS/MAPK pathway provides a novel link between RAS and p53 in regulating apoptosis.

Requirement for phosphorylation of P53 at Ser312 in suppression of chemical carcinogenesis.

The p53 tumour suppressor is activated in response to a wide variety of genotoxic stresses, frequently via post-translational modification. Using a knock in mouse model with a Ser312 to Ala mutation, we show here that phosphorylation of p53 on Ser312 helps to prevent tumour induction by the alkylating agent MNU, which predominantly caused T cell lymphomas. This is consistent with our previous observation that p53(312A/A) mice are more susceptible to X-ray induced tumourigenesis. Phosphorylation on Ser312 aids p53's interaction with E2F1, and enhances p53-mediated apoptosis. Loss of E2F1 alone does not affect tumour susceptibility to MNU, but its absence partially rescues tumour formation in p53(312A/A) mice, thus reflecting the oncogenic properties of E2F1. Our data confirms the participation of Ser312 phosphorylation in tumour suppression by p53.

Phosphorylation of Ser312 contributes to tumor suppression by p53 in vivo.

The tumor suppressor p53 is a master sensor of stress, and posttranslational modifications are key in controlling its stability and transcriptional activities. p53 can be phosphorylated on at least 23 Ser/Thr residues, the majority of which are phosphorylated by stress-related kinases. An exception is Ser315 in human p53 (Ser312 in mouse), which is predominantly phosphorylated by cell cycle-related kinases. To understand the biological importance of Ser312 phosphorylation in vivo, we generated p53Ser312Ala knock-in mice. We show here that, although Ser312 is not essential for mouse life span under normal physiological conditions, Ser312Ala mutation dampens p53's activity during embryonic development. This is evident from its partial rescue of embryonic lethality caused by Mdm4 deletion. In agreement with the notion that Ser312 mutation weakens p53 function, Ser312Ala mice are also more susceptible to tumorigenesis following a sublethal ionizing radiation dose. Importantly, in the cohort studied, Ser312 mutation predisposes mice to develop thymic lymphomas and liver tumors, partly due to p53Ser312Ala's inability to fully induce a set of p53 target genes including p21 and cyclin G1. Thus, we demonstrate that phosphorylation of Ser312 is required for p53 to function fully as a tumor suppressor in vivo.

RPLC-ion-trap-FTMS method for lipid profiling of plasma: method validation and application to p53 mutant mouse model.

A reversed-phase liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometric method was developed for the profiling of lipids in human and mouse plasma. With the use of a fused-core C 8 column and a binary gradient, more than 160 lipids belonging to eight different classes were detected in a single LC-MS run. The method was fully validated and the analytical characteristics such as linearity ( R (2), 0.994-1.000), limit of detection (0.08-1.28 microg/mL plasma), repeatability (RSD, 2.7-7.9%) and intermediate precision (RSD, 2.7-15.6%) were satisfactory. The method was successfully applied to p53 mutant mice plasma for studying some phenotypic effects of p53 expression.

A complex barcode underlies the heterogeneous response of p53 to stress.

The tumour suppressor p53 is activated following stress and initiates a heterogeneous response in a cell-, tissue- and stress-dependent manner. This heterogeneity is reflected in the different physiological outcomes that follow p53 activation. One mechanism that may contribute to this variability is the promoter selectivity of p53 target genes. p53 is at the hub of numerous signalling pathways that are triggered in response to particular stresses, all of which can leave their mark on p53 by way of post-translational modifications and interactions with cofactors. The precise combination of these marks, much like the bars in a barcode, dictates the behaviour of p53 in any given situation.

ASPP2: a gene that controls life and death in vivo.

The fundamental role of apoptosis in tumor prevention and the important role of p53 in this process are now universally recognized. Recently, several families of p53-binding proteins have been shown to influence p53's decision to direct the cells either into the apoptotic pathway or in cell cycle arrest. Among them, the ASPP family specifically regulate p53-dependent apoptosis. Its member ASPP2 was discovered more than 10 years ago as a binding partner of p53 and its role as a positive regulator of p53 mediated apoptosis has been clearly established in vitro. However, its physiological importance in vivo has just emerged through the generation and characterisation of the ASPP2-deficient mice. We now know that ASPP2 is a haploinsufficient tumor suppressor and an important activator of p53 during mouse development and tumor suppression in vivo. ASPP2 might be a novel target for future cancer therapy.

ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth.

ASPP2 stimulates the apoptotic function of the p53 family in vivo. We show here that ASPP2-/- pups died before weaning. This postnatal lethality was significantly enhanced in p53+/- background and both deletions are synthetic lethal. ASPP2+/- mice developed spontaneous tumors. The tumor onset was accelerated by gamma-irradiation or in p53+/- background. Tumors derived from ASPP2+/- mice retained wild-type ASPP2 allele even though some of them lost p53. These provide the first genetic evidence that ASPP2 is a haploinsufficient tumor suppressor that shares overlapping function(s) with p53 in mouse development and tumor suppression.

The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization.

ASPP1 and ASPP2 are both proteins that interact with p53 and enhance its ability to induce apoptosis by selectively elevating the expression of proapoptotic p53-responsive genes. iASPP(RAI) is a third member of the family that is the most conserved inhibitor of p53-mediated apoptosis. Here, we have described iASPP, a longer form of iASPP(RAI), which at 828 amino acids is more than twice the size of iASPP(RAI). Using two antibodies that recognize both iASPP and iASPP(RAI), we report that this longer form of iASPP is the predominant form of the molecule expressed in cells. Like iASPP(RAI), iASPP also binds to p53 and inhibits apoptosis induced by p53 overexpression. However, whereas iASPP(RAI) is predominantly nuclear, the N-terminus of iASPP is entirely cytoplasmic, and the longer iASPP is located in both the cytoplasm and the nucleus. The effect upon subcellular localization of the longer N-terminus of iASPP means that this new, longer form of the molecule may be subject to greater regulation and provides another layer in the control of p53-induced apoptosis.

To die or not to die: how does p53 decide?

p53 is frequently mutated in cancer and as a result is one of the most intensely studied tumour suppressors. Analysis of the primitive forms of p53 found in Caenorhabditis elegans and Drosophila, alongside studies using transgenic mouse models, indicate that the induction of apoptosis is both the most conserved function of p53 and vital for tumour suppression. p53-mediated apoptosis occurs through a combination of mechanisms which include pathways that are both dependent and independent of alterations in gene expression. In response to genotoxic insult, these pathways probably act together, thereby amplifying the apoptotic signal. However, the picture is complicated because the p53 activity is determined by stress type and individual cellular characteristics. The numerous p53 responsive genes that have been identified also provide further means of controlling the actions of p53. The recent discoveries of proteins that interact with p53 and specifically regulate the ability of p53 to trigger apoptosis have provided further mechanistic insights into the role of p53 in inducing cell death. Understanding the molecular basis of the proapoptotic action of p53 can assist in our quest to reintroduce or reactivate p53 in human tumours.

The ASPP family: deciding between life and death after DNA damage.

It is well established that p53 is a primary target for mutation in human cancer. p53 carries out the important task of ensuring that damaged DNA is not passed on during cell division, a duty that it performs by either inhibiting the cell cycle or inducing apoptosis. However, it is unclear how this decision is made. The recent identification of the ASPP family of proteins, which act to direct the cell away from cell cycle arrest and towards death following p53 upregulation, may explain how this dilemma is resolved. Furthermore, the observation that ASPP2 is in fact the full length form of the previously identified 53BP2/Bbp protein has clarified the ambiguous data that has been generated in relation to this molecule. The further characterisation of these proteins will enable us to gain further insights into the response of the cell to DNA damage and the progression of the cell towards malignancy.