Alice Carrier

Pancreatic cancer is a disease with an extremely poor prognosis. Tumor protein 53-induced nuclear protein 1 ( TP53INP1 ) is a proapoptotic stress-induced p53 target gene. In this article, we show by immunohistochemical analysis that TP53INP1 expression is dramatically reduced in pancreatic ductal adenocarcinoma (PDAC) and this decrease occurs early during pancreatic cancer development. TP53INP1 reexpression in the pancreatic cancer-derived cell line MiaPaCa2 strongly reduced its capacity to form s.c., i.p., and intrapancreatic tumors in nude mice. This anti-tumoral capacity is, at least in part, due to the induction of caspase 3-mediated apoptosis. In addition, TP53INP1 −/− mouse embryonic fibroblasts (MEFs) transformed with a retrovirus expressing E1A/ras V12 oncoproteins developed bigger tumors than TP53INP1 +/+ transformed MEFs or TP53INP1 −/− transformed MEFs with restored TP53INP1 expression. Finally, TP53INP1 expression is repressed by the oncogenic micro RNA miR-155, which is overexpressed in PDAC cells. TP53INP1 is a previously unknown miR-155 target presenting anti-tumoral activity.

Chemokines are key regulators of migration in lymphoid tissues. In the thymus, maturing thymocytes move from the outer capsule to the inner medulla and thereby interact with different types of stromal cells that control their maturation and selection. In the process of searching for molecules specifically expressed at different stages of mouse thymic differentiation, we have characterized the cDNA coding for the thymus-expressed chemokine (TECK) and its receptor CCR9. The TECK receptor gene was isolated and shown to be localized on the mouse chromosome 9F1-F4. Thymic dendritic cells have been initially thought to be a prevalent source of TECK. In contrast, our results indicate that thymic epithelial cells constitute the predominant source of TECK. Consistent with the latter distribution, the TECK receptor is highly expressed by double-positive thymocytes, and TECK can chemoattract both double-positive and single-positive thymocytes. The TECK transcript is also abundantly expressed in the epithelial cells lining the small intestine. In conclusion, the interplay of TECK and its receptor CCR9 is likely to have a significant role in the recruitment of developing thymocytes to discrete compartments of the thymus.

Abstract CC chemokine receptor (CCR) 9, the receptor for the CC-chemokine CCL25/thymus-expressed chemokine (TECK), is mainly expressed by thymocytes and by intraepithelial (IEL) and lamina propria lymphocytes of the small intestine. To study the biologic role of CCR9, a mouse strain was generated in which the CCR9 gene was deleted. In spite of the high level of CCR9 found in double- and single-positive thymocytes and of the expression of its corresponding ligand on thymic stromal cells, CCR9 deletion had no major effect on intrathymic T-cell development. It was noted that there was only a one-day lag in the appearance of double-positive cells during fetal ontogeny in CCR9−/− thymi. When tested in chemotaxis assay, thymocytes isolated from CCR9−/− mice failed to respond to TECK/CCL25. Taken together, these results suggest that in thymocytes, CCR9 is the only physiologic receptor for TECK/CCL25, and that it is dispensable for proper T-cell development. Bone marrow pre-pro–B cells migrate in response to TECK/CCL25, but more mature B cells do not. Consistent with this observation, it was shown that there are fewer pre-pro–B cells in CCR9−/−mice than in wild-type mice. However, this diminution does not appear to have a detectable effect on the generation of a normal complement of mature B cells. Finally, it was shown that in the small intestine of CCR9-deficient mice, the intraepithelial T-cell–to–epithelial cell ratio is decreased, an observation that can be accounted for by a marked diminution of the T-cell receptor γδ+ compartment.

Abstract p53 exerts its tumor suppressor function mainly through transcriptional induction of target genes involved in several processes, including cell cycle checkpoints, apoptosis, and regulation of cell redox status. p53 antioxidant function is dependent on its transcriptional activity and proceeds by sequential induction of antioxidant and proapoptotic targets. However, none of the thus far renowned p53 targets have proved able to abolish on their own the intracellular reactive oxygen species (ROS) accumulation caused by p53 deficiency, therefore pointing to the existence of other prominent and yet unknown p53 antioxidant targets. Here, we show that TP53INP1 represents such a target. Indeed, TP53INP1 transcript induction on oxidative stress is strictly dependent on p53. Mouse embryonic fibroblasts (MEF) and splenocytes derived from TP53INP1-deficient (inp1−/−) mice accumulate intracellular ROS, whereas overexpression of TP53INP1 in p53-deficient MEFs rescues ROS levels to those of p53-proficient cells, indicating that TP53INP1 antioxidant function is p53 independent. Furthermore, accumulation of ROS in inp1−/− cells on oxidant challenge is associated with decreased expression of p53 targets p21/Cdkn1a, Sesn2, TAp73, Puma, and Bax. Mutation of p53 Ser58 (equivalent to human p53 Ser46) abrogates transcription of these genes, indicating that TP53INP1-mediated p53 Ser58 phosphorylation is implicated in this process. In addition, TP53INP1 deficiency results in an antioxidant (N-acetylcysteine)-sensitive acceleration of cell proliferation. Finally, TP53INP1 deficiency increases oxidative stress–related lymphoma incidence and decreases survival of p53+/− mice. In conclusion, our data show that TP53INP1 is a major actor of p53-driven oxidative stress response that possesses both a p53-independent intracellular ROS regulatory function and a p53-dependent transcription regulatory function. [Cancer Res 2009;69(1):219–26]

TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy.

Abstract Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid β-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria–endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.

The worldwide epidemic of obesity is a major public health concern. Obesity is a major risk factor for noncommunicable diseases such as type 2 diabetes and cardiovascular diseases, clustered in the so-called metabolic syndrome (MS). Other main chronic illnesses are promoted by excessive body weight, including cancer and neurodegenerative pathologies, both affecting a number of people worldwide. In recent years, the primary role of an excess of reactive oxygen species (oxidative stress) resulting from altered redox control in the etiology of all of these pathologies has been unveiled. Interestingly, it appears that oxidative stress is both the cause and the consequence of obesity and associated disorders. This Forum features reviews that recapitulate the current knowledge on the link between oxidative stress and MS in the physiopathology of different biological systems. Antioxid. Redox Signal. 26, 429–431.

Mitochondrial respiration (oxidative phosphorylation, OXPHOS) is an emerging target in currently refractory cancers such as pancreatic ductal adenocarcinoma (PDAC). However, the variability of energetic metabolic adaptations between PDAC patients has not been assessed in functional investigations. In this work, we demonstrate that OXPHOS rates are highly heterogeneous between patient tumors, and that high OXPHOS tumors are enriched in mitochondrial respiratory complex I at protein and mRNA levels. Therefore, we treated PDAC cells with phenformin (complex I inhibitor) in combination with standard chemotherapy (gemcitabine), showing that this treatment is synergistic specifically in high OXPHOS cells. Furthermore, phenformin cooperates with gemcitabine in high OXPHOS tumors in two orthotopic mouse models (xenografts and syngeneic allografts). In conclusion, this work proposes a strategy to identify PDAC patients likely to respond to the targeting of mitochondrial energetic metabolism in combination with chemotherapy, and that phenformin should be clinically tested in appropriate PDAC patient subpopulations.

The <i>TP53INP1</i> gene encodes two protein isoforms, TP53INP1α and TP53INP1β, located into the nucleus. Their synthesis is increased during cellular stress by p53-mediated activation of transcription. Overexpression of these isoforms induces apoptosis, suggesting an involvement of TP53INP1s in p53-mediated cell death. It was recently shown that p53-dependent apoptosis is promoted by homeodomain-interacting protein kinase-2 (HIPK2), which is known to bind p53 and induce its phosphorylation in promyelocytic leukemia protein nuclear bodies (PML-NBs). In this work we show that TP53INP1s localize with p53, PML-IV, and HIPK2 into the PML-NBs. In addition, we show that TP53INP1s interact physically with HIPK2 and p53. In agreement with these results we demonstrate that TP53INP1s, in association with HIPK2, regulate p53 transcriptional activity on <i>p21</i>, <i>mdm2</i>, <i>pig3</i>, and <i>bax</i> promoters. Furthermore, TP53INP1s overexpression induces G₁ arrest and increases p53-mediated apoptosis. Although a TP53INP1s and HIPK2 additive effect was observed on apoptosis, G₁ arrest was weaker when HIPK2 was transfected together with TP53INP1. These results indicate that TP53INP1s and HIPK2 could be partners in regulating p53 activity.

TP53INP1 is an alternatively spliced gene encoding two nuclear protein isoforms (TP53INP1α and TP53INP1β), whose transcription is activated by p53. When overexpressed, both isoforms induce cell cycle arrest in G1 and enhance p53-mediated apoptosis. TP53INP1s also interact with the p53 gene and regulate p53 transcriptional activity. We report here that TP53INP1 expression is induced during experimental acute pancreatitis in p53−/− mice and in cisplatin-treated p53−/− mouse embryo fibroblasts (MEFs). We demonstrate that ectopic expression of p73, a p53 homologue, leads to TP53INP1 induction in p53-deficient cells. In turn, TP53INP1s alters the transactivation capacity of p73 on several p53-target genes, including TP53INP1 itself, demonstrating a functional association between p73 and TP53INP1s. Also, when overexpressed in p53-deficient cells, TP53INP1s inhibit cell growth and promote cell death as assessed by cell cycle analysis and colony formation assays. Finally, we show that TP53INP1s potentiate the capacity of p73 to inhibit cell growth, that effect being prevented when the p53 mutant R175H is expressed or when p73 expression is blocked by a siRNA. These results suggest that TP53INP1s are functionally associated with p73 to regulate cell cycle progression and apoptosis, independently from p53.

Thymus-specific serine protease (TSSP) was initially reported as a putative protease specifically expressed in the endosomal compartment of cortical thymic epithelial cells (cTEC). As such, TSSP is potentially involved in the presentation of the self-peptides that are bound to MHC class II molecules expressed at the cTEC surface and are involved in the positive selection of CD4(+) thymocytes. We tested this hypothesis by generating mutant mice deprived of Prss16, the gene encoding TSSP. TSSP-deficient mice produced normal numbers of T cells, despite a decrease in the percentage of cTEC expressing high surface levels of MHC class II. By using sensitive transgenic models expressing MHC class II-restricted TCR transgenes (Marilyn and OT-II), we showed that the absence of TSSP markedly impaired the selection of Marilyn and OT-II CD4(+) T cells. In contrast, selection of CD8(+) T cells expressing an MHC class I-restricted TCR transgene (OT-I) was unaffected. Therefore, TSSP is involved in the positive selection of some CD4(+) T lymphocytes and likely constitutes the first serine protease to play a function in the intrathymic presentation of self-peptides bound to MHC class II complexes.

Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role in astroglial cell death occurring in diverse neuropathological conditions. Numerous studies indicate that neuroglobin (Ngb) promotes neuron survival, but nothing is known regarding the action of Ngb in astroglial cell survival. Thus, the purpose of this study was to investigate the potential glioprotective effect of Ngb on hydrogen peroxide (H2 O2 )-induced oxidative stress and apoptosis in cultured mouse astrocytes. Incubation of cells with subnanomolar concentrations of Ngb (10-14 -10-10 M) was found to prevent both H2 O2 -evoked reduction in surviving cells number and accumulation of reactive oxygen species in a concentration-dependent manner. Furthermore, Ngb treatment abolishes H2 O2 -induced increase in mitochondrial oxygen consumption rates. Concomitantly, Ngb treatment rescues H2 O2 -associated reduced expression of endogenous antioxidant enzymes (superoxide dismutases and catalase) and prevents the stimulation of the expression of pro-inflammatory genes (inducible nitric oxide synthase, cyclooxygenase-2, and interleukin (IL) IL-6 and IL-33). Moreover, Ngb blocks the stimulation of Bax (pro-apoptotic) and the inhibition of Bcl-2 (anti-apoptotic) gene expression induced by H2 O2 , which in turn abolishes caspase 3 activation. The protective effect of Ngb upon H2 O2 induced activation of caspase 3 activity and cell death can be accounted for by activation of protein kinase A and mitogen-activated protein kinase transduction cascade. Finally, we demonstrate that Ngb increases Akt phosphorylation and prevents H2 O2 -provoked inhibition of ERK and Akt phosphorylation. Taken together, these data demonstrate for the first time that Ngb is a glioprotective agent that prevents H2 O2 -induced oxidative stress and apoptotic astroglial cell death. Protection of astrocytes from oxidative insult may thus contribute to the neuroprotective effect of Ngb.

Tumor protein 53 induced nuclear protein 1 (TP53INP1) is a p53 target gene that induces cell growth arrest and apoptosis by modulating p53 transcriptional activity. TP53INP1 interacts physically with p53 and is a major player in the p53-driven oxidative stress response. Previously, we demonstrated that TP53INP1 is downregulated in an early stage of pancreatic cancerogenesis and when restored is able to suppress pancreatic tumor development. TP53INP1 downregulation in pancreas is associated with an oncogenic microRNA miR-155. In the present work, we studied the effects of TP53INP1 on cell migration. We found that TP53INP1 inactivation correlates with increased cell migration both in vivo and in vitro. The impact of TP53INP1 expression on cell migration was studied in different cellular contexts: mouse embryonic fibroblast and different pancreatic cancer cell lines. Its expression decreases cell migration by the transcriptional downregulation of secreted protein acidic and rich in cysteine (SPARC). SPARC is a matrix cellular protein, which governs diverse cellular functions and has a pivotal role in regulating cell-matrix interactions, cellular proliferation and migration. SPARC was also showed to be upregulated in normal pancreas and in pancreatic intraepithelial neoplasia lesions in a pancreatic adenocarcinoma mouse model only in the TP53INP1-deficient animals. This novel TP53INP1 activity on the regulation of SPARC expression could explain in part its tumor suppressor function in pancreatic adenocarcinoma by modulating cellular spreading during the metastatic process.

Abstract It was already described that genetic inhibition of NUPR1 induces tumor growth arrest. In this paper we studied the metabolism changes after NUPR1 downregulation in pancreatic cancer cells, which results in a significant decrease of OXPHOS activity with a concomitant lower ATP production which precedes the necrotic cell death. We demonstrated that NUPR1 downregulation induces a mitochondrial failure with a loss of the mitochondrial membrane potential, a strong increase in ROS production and a concomitant relocalization of mitochondria to the vicinity of the endoplasmic reticulum (ER). In addition, the transcriptomic analysis of NUPR1-deficient cells shows a decrease in the expression of some ER stress response-associated genes. Indeed, in ER stressors-treated cells with thapsigargin, brefeldin A or tunicamycin, a greater increase in necrosis and decrease of ATP content was observed in NUPR1-defficent cells. Finally, in vivo experiments, using acute pancreatitis which induces ER stress as well as NUPR1 activation, we observed that NUPR1 expression protects acinar cells from necrosis in mice. Importantly, we also report that the cell death observed after knocking-down NUPR1 expression is completely reversed by incubation with Necrostatin-1, but not by inhibiting caspase activity with Z-VAD-FMK. Altogether, these data enable us to describe a model in which inactivation of NUPR1 in pancreatic cancer cells results in an ER stress that induces a mitochondrial malfunction, a deficient ATP production and, as consequence, the cell death mediated by a programmed necrosis.

Cancer cells reprogram their metabolism to meet bioenergetics and biosynthetic demands. The first observation of metabolic reprogramming in cancer cells was made a century ago ("Warburg effect" or aerobic glycolysis), leading to the classical view that cancer metabolism relies on a glycolytic phenotype. There is now accumulating evidence that most cancers also rely on mitochondria to satisfy their metabolic needs. Indeed, the current view of cancer metabolism places mitochondria as key actors in all facets of cancer progression. Importantly, mitochondrial metabolism has become a very promising target in cancer therapy, including for refractory cancers such as Pancreatic Ductal AdenoCarcinoma (PDAC). In particular, mitochondrial oxidative phosphorylation (OXPHOS) is an important target in cancer therapy. Other therapeutic strategies include the targeting of glutamine and fatty acids metabolism, as well as the inhibition of the TriCarboxylic Acid (TCA) cycle intermediates. A better knowledge of how pancreatic cancer cells regulate mitochondrial metabolism will allow the identification of metabolic vulnerabilities and thus novel and more efficient therapeutic options for the benefit of each patient.

Metabolic reprogramming is a feature of cancers for which recent research has been particularly active, providing numerous insights into the mechanisms involved. It occurs across the entire cancer process, from development to resistance to therapies. Established tumors exhibit dependencies for metabolic pathways, constituting vulnerabilities that can be targeted in the clinic. This knowledge is of particular importance for cancers that are refractory to any therapeutic approach, such as Pancreatic Ductal Adenocarcinoma (PDAC). One of the metabolic pathways dysregulated in PDAC is autophagy, a survival process that feeds the tumor with recycled intracellular components, through both cell-autonomous (in tumor cells) and nonautonomous (from the local and distant environment) mechanisms. Autophagy is elevated in established PDAC tumors, contributing to aberrant proliferation and growth even in a nutrient-poor context. Critical elements link autophagy to PDAC including genetic alterations, mitochondrial metabolism, the tumor microenvironment (TME), and the immune system. Moreover, high autophagic activity in PDAC is markedly related to resistance to current therapies. In this context, combining autophagy inhibition with standard chemotherapy, and/or drugs targeting other vulnerabilities such as metabolic pathways or the immune response, is an ongoing clinical strategy for which there is still much to do through translational and multidisciplinary research.

AbstractTumor protein 53-induced nuclear protein 1 (TP53INP1) is an antiproliferative and proapoptotic protein involved in cell stress response. To address its physiological roles in colorectal cancer and colitis, we generated and tested the susceptibility of Trp53inp1-deficient mice to the development of colorectal tumors induced by injection of the carcinogen azoxymethane followed by dextran sulfate sodium (DSS)-induced chronic colitis. Trp53inp1-deficient mice showed an increased incidence and multiplicity of tumors compared to those of wild-type (WT) mice. Furthermore, acute colitis induced by DSS treatment was more severe in Trp53inp1-deficient mice than in WT mice. Treatment with the antioxidant N-acetylcysteine prevented colitis and colitis-associated tumorigenesis more efficiently in WT mice than in Trp53inp1-deficient mice, suggesting a higher oxidative load in the latter. Consistently, we demonstrated by electron spin resonance and spin trapping that colons derived from deficient mice produced more free radicals than those of the WT during colitis and that the basal blood level of the antioxidant ascorbate was decreased in Trp53inp1-deficient mice. Collectively, these results indicate that the oxidative load is higher in Trp53inp1-deficient mice than in WT mice, generating a more-severe DSS-induced colitis, which favors development of colorectal tumors in Trp53inp1-deficient mice. Therefore, TP53INP1 is a potential target for the prevention of colorectal cancer in patients with inflammatory bowel disease. We thank Anne Gillet for blastocyst injection and chimera breeding, Gilles Warcollier and the animal house staff for mouse care, Patricia Spoto for help in genotyping, Marie-Noëlle Lavaud for histological analyses, Patrice Berthézène for statistical analyses, and Jean-Charles Dagorn and Mathias Chamaillard for discussions.This work was supported by INSERM, CNRS, the Association pour la Recherche sur le Cancer (B.M.), and Plate-forme RIO-MNG (B.M.). J.G. was supported by doctoral fellowships from the Ministère de l'Education Nationale de la Recherche et de la Technologie, the Association pour la Recherche sur le Cancer, and the Société Française du Cancer. C.C. was supported by a postdoctoral fellowship from the Association pour la Recherche sur le Cancer, and M.G. was supported by a postdoctoral fellowship from INSERM.

In the recent years, we have provided evidence that Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) is a key stress protein with antioxidant-associated tumor suppressive function. The TP53INP1 gene, which is highly conserved in mammals, is over-expressed during stress responses including inflammation. This gene encodes two protein isoforms with nuclear or cytoplasmic subcellular localization depending on the context. TP53INP1 contributes to stress responses, thus preventing stress-induced dysfunctions leading to pathologies such as cancer. Two major mechanisms by which TP53INP1 functions have been unveiled. First, in the nucleus, TP53INP1 was shown to regulate the transcriptional activity of p53 and p73 by direct interaction, and to mediate the antioxidant activity of p53. Second, independently of p53, TP53INP1 contributes to autophagy and more particularly mitophagy through direct interaction with molecular actors of autophagy. TP53INP1 is thus required for the homeostasis of the mitochondrial compartment, and is therefore involved in the regulation of energetic metabolism. Finally, the antioxidant function of TP53INP1 stems from the control of mitochondrial reactive oxygen species production. In conclusion, TP53INP1 is a multifaceted protein endowed with multiple functions, including metabolic regulation, as is its main functional partner p53.

Cell metabolism is reprogrammed in cancer cells to meet their high bioenergetics and biosynthetic demands. This metabolic reprogramming is accompanied by alterations in redox metabolism, characterized by accumulation of reactive oxygen species (ROS). Elevated production of ROS, mostly by mitochondrial respiration, is counteracted by higher production of antioxidant defenses (mainly glutathione and antioxidant enzymes). Cancer cells are adapted to a high concentration of ROS, which contributes to tumorigenesis, metastasis formation, resistance to therapy and relapse. Frequent genetic alterations observed in pancreatic ductal adenocarcinoma (PDAC) affect KRAS and p53 proteins, which have a role in ROS production and control, respectively. These observations led to the proposal of the use of antioxidants to prevent PDAC development and relapse. In this review, we focus on the therapeutic strategies to further increase ROS level to induce PDAC cell death. Combining the promotion of ROS production and inhibition of antioxidant capacity is a promising avenue for pancreatic cancer therapy in the clinic.

Abstract Identifying critical factors involved in the metastatic progression of hepatocellular carcinoma (HCC) may offer important therapeutic opportunities. Here, we report that the proapoptotic stress response factor TP53INP1 is often selectively downregulated in advanced stage IV and metastatic human HCC tumors. Mechanistic investigations revealed that TP53INP1 downregulation in early-stage HCC cells promoted metastasis via DUSP10 phosphatase-mediated activation of the ERK pathway. The DUSP10 promoter included putative binding sites for p73 directly implicated in modulation by TP53INP1. Overall, our findings show how TP53INP1 plays a critical role in limiting the progression of early-stage HCC, with implications for developing new therapeutic strategies to attack metastatic HCC. Cancer Res; 77(17); 4602–12. ©2017 AACR.

The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox-driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1-deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS-identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as a molecular regulator of redox-driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research.

Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro-metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kα-selective inhibition prevented macro-metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the produced lipid second messenger PIP3 , with a selective decrease of C36:2 PI-3,4,5-P3 . Tumoural PI3Kα inactivation prevented the accumulation of pro-tumoural CD206-positive macrophages in the tumour-adjacent tissue. Tumour cell-intrinsic PI3Kα promotes pro-metastatic features that could be pharmacologically targeted to delay macro-metastatic evolution.

Thymus-specific serine protease (TSSP) is a novel protease that may contribute to the generation of the peptide repertoire presented by MHC class II molecules in the thymus. Although TSSP deficiency has no quantitative impact on the development of CD4 T cells expressing a polyclonal T cell receptor (TCR) repertoire, the development of CD4 T cells expressing the OTII and Marilyn transgenic TCRs is impaired in TSSP-deficient mice. In this study, we assess the role of TSSP in shaping the functional endogenous polyclonal CD4 T cell repertoire by analyzing the response of TSSP-deficient mice to several protein antigens (Ags). Although TSSP-deficient mice responded normally to most of the Ags tested, they responded poorly to hen egg lysozyme (HEL). The impaired CD4 T cell response of TSSP-deficient mice to HEL correlated with significant alteration of the dominant TCR-β chain repertoire expressed by HEL-specific CD4 T cells, suggesting that TSSP is necessary for the intrathymic development of cells expressing these TCRs. Thus, TSSP contributes to the diversification of the functional endogenous CD4 T cell TCR repertoire in the thymus.

Tumor protein p53-induced nuclear protein 1 (TP53INP1) is involved in cell stress response. Its expression is lost at the pancreatic intraepithelial neoplasia 1b (PanIN1b)/PanIN2 stage of pancreatic carcinogenesis. Our objective was to determine whether TP53INP1 loss of expression contributes to pancreatic cancer formation in a conditional KrasG12D mouse model. We generated Kras-INP1KO mice using LSL-Kras(G12D/+);Pdx1-Cre(+/-) mice (Kras mice) and TP53INP1(-/-) mice. Analysis of pancreases during ageing shows that in the presence of activated Kras, TP53INP1 loss of expression accelerated PanIN formation and increased pancreatic injury and the number of high-grade lesions as compared with what occurs in Kras mice. Moreover, cystic lesions resembling intraductal papillary mucinous neoplasm (IPMN) were observed as early as 2 months of age. Remarkably, TP53INP1 is down-regulated in human IPMN. Activation of the small GTPase Rac1 shows that more oxidative stress is generated in Kras-INP1KO than in Kras mice pancreas despite elevated levels of the Nrf2 antioxidant regulator. We firmly establish the link between Kras-INP1KO pancreatic phenotype and oxidative stress with rescue of the phenotype by the antioxidant action of N-acetylcysteine. Our data provide in vivo functional demonstration that TP53INP1 deficiency accelerates progression of pancreatic cancer, underlining its role in the occurrence of IPMN and highlighting the importance of TP53INP1 in the control of oxidative status during development of pancreatic cancer.

Type 1 diabetes is a chronic autoimmune disease in which genetic predispositions affect the immune system, leading to a loss of T cell tolerance to β cells and consequent T cell-mediated destruction of insulin-producing islet cells. Genetic studies have suggested that PRSS16 is linked to a diabetes susceptibility locus of the extended HLA class I region in humans. PRSS16 encodes what we believe to be a novel protease, thymus-specific serine protease (TSSP), which shows predominant expression in thymic epithelial cells and is suspected to have a restricted role in the class II presentation pathway. Consistently, Tssp is necessary for the intrathymic selection of few class II-restricted T cell receptor specificities in B6 mice. To directly assess the role of Tssp in autoimmune diabetes, we generated Tssp-deficient (Tssp°) NOD mice. While remaining immunocompetent, Tssp° NOD mice were protected from diabetes and severe insulitis. Diabetes resistance of Tssp° NOD mice was a property of the CD4 T cell compartment that is acquired during thymic selection and correlated with an impaired selection of CD4 T cells specific for islet antigens. Hence, in the NOD mouse, Tssp is a critical regulator of diabetes development through the selection of the autoreactive CD4 T cell repertoire.

Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription.Paradoxically, N-Myc induces p53 gene expression.Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death.Here we aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis.TP53INP1 was found to be one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets.HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival.Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells.These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma.

We have designed an expression vector permitting the production in the periplasm of Escherichia coli of a fusion protein comprising a dimer of bacterial alkaline phosphatase (PhoA) and two Fab or scFv fragments of a monoclonal antibody directed against human IgG. Each hybrid protein expressed both high specificity for the antigen and full PhoA activity. We show that crude periplasmic extracts containing these conjugates can be used as such in enzyme immunoassays for the detection of human IgG, as exemplified in the case of anti-hepatitis B immunoglobulin.

The p53-transcriptional target TP53INP1 is a potent stress-response protein promoting p53 activity. We previously showed that ectopic overexpression of TP53INP1 facilitates cell cycle arrest as well as cell death. Here we report a study investigating cell death in mice deficient for TP53INP1. Surprisingly, we found enhanced stress-induced apoptosis in TP53INP1-deficient cells. This observation is underpinned in different cell types in vivo (thymocytes) and in vitro (thymocytes and MEFs), following different types of injury inducing either p53-dependent or -independent cell death. Nevertheless, absence of TP53INP1 is unable to overcome impaired cell death of p53-deficient thymocytes. Stress-induced ROS production is enhanced in the absence of TP53INP1, and antioxidant NAC complementation abolishes increased sensitivity to apoptosis of TP53INP1-deficient cells. Furthermore, antioxidant defenses are defective in TP53INP1-deficient mice in correlation with ROS dysregulation. Finally, we show that autophagy is reduced in TP53INP1-deficient cells both at the basal level and upon stress. Altogether, these data show that impaired ROS regulation in TP53INP1-deficient cells is responsible for their sensitivity to induced apoptosis. In addition, they suggest that this sensitivity could rely on a defect of autophagy. Therefore, these data emphasize the role of TP53INP1 in protection against cell injury. Antioxid. Redox Signal. 15, 1639–1653.

Abstract Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo , we further revealed that the chronic administration of low-doses of paclitaxel - i.e . metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors.

TP53INP1 is a stress-induced protein, which acts as a dual positive regulator of transcription and of autophagy and whose deficiency has been linked with cancer and metabolic syndrome. Here, we addressed the unexplored role of TP53INP1 and of its Drosophila homolog dDOR in the maintenance of neuronal homeostasis under chronic stress, focusing on dopamine (DA) neurons under normal ageing- and Parkinson's disease (PD)-related context. Trp53inp1-/- mice displayed additional loss of DA neurons in the substantia nigra compared to wild-type (WT) mice, both with ageing and in a PD model based on targeted overexpression of α-synuclein. Nigral Trp53inp1 expression of WT mice was not significantly modified with ageing but was markedly increased in the PD model. Trp53inp2 expression showed similar evolution and did not differ between WT and Trp53inp1-/- mice. In Drosophila, pan-neuronal dDOR overexpression improved survival under paraquat exposure and mitigated the progressive locomotor decline and the loss of DA neurons caused by the human α-synuclein A30P variant. dDOR overexpression in DA neurons also rescued the locomotor deficit in flies with RNAi-induced downregulation of dPINK1 or dParkin. Live imaging, confocal and electron microscopy in fat bodies, neurons, and indirect flight muscles showed that dDOR acts as a positive regulator of basal autophagy and mitophagy independently of the PINK1-mediated pathway. Analyses in a mammalian cell model confirmed that modulating TP53INP1 levels does not impact mitochondrial stress-induced PINK1/Parkin-dependent mitophagy. These data provide the first evidence for a neuroprotective role of TP53INP1/dDOR and highlight its involvement in the regulation of autophagy and mitophagy in neurons.

Pancreatic Ductal AdenoCarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. The objective of this work was to investigate the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative study combining functional, morphological and omics-based evaluation of gastrocnemius muscle from genetically-engineered mice developing autochthonous pancreatic tumor and cachexia (KIC GEMM). KIC cachectic PDAC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber’s size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. While the number of mitochondria per cell is not altered, mitochondrial mass is decreased by a factor of 2 and the mitochondrial DNA by a factor of 3, suggesting a defect in mitochondrial genome homeostasis. Muscle atrophy is associated with strong mitochondrial metabolic defects that are not limited to carbohydrate, protein and redox metabolism, but concern also lipid and nucleic acid metabolism. This work provides a framework to guide towards the most relevant targets in the clinic to limit PDAC-induced cachexia, in particular mitochondrial fatty acid metabolism.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the human cancers with the poorest prognosis. Interestingly, we found that mitochondrial respiration in primary human PDAC cells depends mainly on the fatty acid oxidation (FAO) to meet basic energy requirements. Therefore, we treated PDAC cells with perhexiline, a well-recognized FAO inhibitor used in cardiac diseases. Some PDAC cells respond efficiently to perhexiline, which acts synergistically with chemotherapy (gemcitabine) in vitro and in two xenografts in vivo. Importantly, perhexiline in combination with gemcitabine induces complete tumor regression in one PDAC xenograft. Mechanistically, this co-treatment causes energy and oxidative stress promoting apoptosis but does not exert inhibition of FAO. Yet, our molecular analysis indicates that the carnitine palmitoyltransferase 1C (CPT1C) isoform is a key player in the response to perhexiline and that patients with high CPT1C expression have better prognosis. Our study reveals that repurposing perhexiline in combination with chemotherapy is a promising approach to treat PDAC.

Pancreatic Ductal AdenoCarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. The objective of this work was to investigate the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative study combining functional, morphological and omics-based evaluation of gastrocnemius muscle from genetically-engineered mice developing autochthonous pancreatic tumor and cachexia (KIC GEMM). KIC cachectic PDAC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber’s size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. While the number of mitochondria per cell is not altered, mitochondrial mass is decreased by a factor of 2 and the mitochondrial DNA by a factor of 3, suggesting a defect in mitochondrial genome homeostasis. Muscle atrophy is associated with strong mitochondrial metabolic defects that are not limited to carbohydrate, protein and redox metabolism, but concern also lipid and nucleic acid metabolism. This work provides a framework to guide towards the most relevant targets in the clinic to limit PDAC-induced cachexia, in particular mitochondrial fatty acid metabolism.

Pancreatic Ductal AdenoCarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. The objective of this work was to investigate the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative study combining functional, morphological and omics-based evaluation of gastrocnemius muscle from genetically-engineered mice developing autochthonous pancreatic tumor and cachexia (KIC GEMM).KIC cachectic PDAC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber’s size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. While the number of mitochondria per cell is not altered, mitochondrial mass is decreased by a factor of 2 and the mitochondrial DNA by a factor of 3, suggesting a defect in mitochondrial genome homeostasis.Muscle atrophy is associated with strong mitochondrial metabolic defects that are not limited to carbohydrate, protein and redox metabolism, but concern also lipid and nucleic acid metabolism.This work provides a framework to guide towards the most relevant targets in the clinic to limit PDAC-induced cachexia, in particular mitochondrial fatty acid metabolism.

In cancer, immune cells can play conflicting roles, either protective, by elimination of tumor cells during immune surveillance, or detrimental, by promoting carcinogenesis during inflammation. We report here that the thymus-specific serine protease (TSSP), which is involved in CD4(+) T cell maturation in the thymus, exerts a tumor suppressor activity. Mice genetically deficient for TSSP are highly prone to spontaneous cancer development. The absence of TSSP also increases the rate of induced colitis-associated colorectal (CAC) tumor formation, through exacerbated colon inflammation. Adoptive transfer of T cells in various combinations (CD4(+) and CD8(+) from wild-type and/or knockout mice) into T cell-deficient mice showed that the TSSP-deficient CD4(+) T cell compartment promotes tumor development, associated with high levels of the cytokine IL-17A. Inhibition of IL-17A during CAC tumor formation prevents the increased carcinogenesis and colic immune disequilibrium observed in TSSP-deficient mice. Therefore, our data demonstrate that antitumoral immune surveillance requires thymic TSSP-driven production of CD4(+) T cells contributing to inflammatory balance.

Significance Reactive oxygen species (ROS) play a role in signaling in immune cells, in particular in B cell activation and terminal differentiation in secondary lymphoid organs. Nevertheless, their role in B cell development in the bone marrow (BM) still remains poorly explored. TP53INP1 is a target of the tumor suppressor p53, which mediates its antioxidant activity. We report the surprising observation that chronic oxidative stress in TP53INP1-deficient mice rescues B lymphopoiesis in the BM during aging. ROS sustain IL-7R signaling in aged TP53INP1-deficient BM through maintenance of active STAT5 transcription factor, driving the expression of the B lineage Pax5 transcription factor. This work suggests that antioxidants cannot be in favor of antibody-producing cell development.

Doxorubicin (Dox), a widely used antitumor anthracycline antibiotic, plays an undisputed key role in the treatment of many neoplasic diseases. In this study, the protective role of resveratrol against Dox-induced erythrocytes and plasma toxicity has been evaluated in rats. Animals were treated with resveratrol (25 mg/kg b.w.) by intraperitoneal injection during 8 days. At the 4(th) day of treatment, rats were intraperitoneally injected with a single dose of Dox (20 mg/kg b.w.). At the end of the treatment, blood samples were collected following standard procedure and processed for oxidative stress parameters (malondialdehyde (MDA), carbonyl protein, free iron, calcium and H(2)O(2) levels), transaminases and antioxidant enzymes as catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Data showed that Dox drastically increased erythrocytes and plasma MDA, free iron, H(2)O(2) and carbonyl proteins but decreased calcium levels and also decreased erythrocytes CAT, POD and SOD activity. Besides, Dox decreased plasma CAT and SOD but unexpectedly increased POD activity. Dox also increased plasma ALT and AST levels and decreased them into erythrocytes. Co-treatment with resveratrol counteracted almost all Dox's effects. In conclusion, Dox induced a pro-oxidative stress into erythrocytes and resveratrol exerted real antioxidant properties which can be attributed, at least in part, to free iron and calcium modulation.

Pancreatic adenocarcinoma (PA) incidence is rising worldwide, especially in France. The evolution of known risk factors such as tobacco smoking, obesity, type 2 diabetes, chronic pancreatitis, or constitutional mutations is not sufficient to explain this trend. Pesticides are known risk factors in other malignancies. Previous studies have outlined pesticides' influence in PA, such as dichlorodiphenyltrichloroethane as plausible risk factors. The general population is directly or indirectly exposed to pesticides through air, food or water. Some of these chemicals may accumulate in the body all along lifetime and may harm carriers. The toxic mixing effects of these chemicals are not well documented. Several hypotheses have been put forward to explain how pesticides can induce indirect (fatty pancreas, induced diabetes) or direct (oxidative stress, cell damage) carcinogenesis in pancreatic cells through inflammation. A strong corpus exists acknowledging pesticides as a PA risk factor. However, published studies do not provide a sufficient level of evidence to prove causality and current prospective case-control studies are still ongoing.

Garlic is a commonly used spice in folk medicine that can exert adverse health effects when given at a high dose. Grape seed and skin extract (GSSE) exhibits a variety of beneficial effects even at a high dose. In the present study we evaluated the toxicity of high-dose garlic treatment on liver and the protective effect of GSSE. Rats were intraperitoneally administered either with garlic extract (5 g·(kg body weight) –1 ) or GSSE (500 mg·(kg body weight) –1 ) or a combination of garlic and GSSE at the same doses daily for 1 month. Plasma and hepatic levels of cholesterol, triacylglycerol, and transaminases and liver antioxidant status were evaluated. Data showed that a high garlic dose induced liver toxicity and a pro-oxidative status characterized by increased malondialdehyde and decreased antioxidant enzyme activities as catalase, peroxidase, and superoxide dismutase. Garlic increased intracellular H 2 O 2 but decreased free iron and Ca 2+ . GSSE alone or in co-treatment with garlic had the reverse effect and counteracted almost all garlic-induced deleterious impacts to near control levels. In conclusion, a high garlic dose induced a pro-oxidative state characterized by the Fenton reaction between H 2 O 2 and free iron, inducing Ca 2+ depletion, while GSSE exerted antioxidant properties and Ca 2+ repletion.

High garlic dose could exert adverse health properties and grape seed and skin extract (GSSE) exhibit a variety of beneficial effects, even at high dose. In the present study we evaluated the toxic effect of high garlic dose treatment on antioxidant status of the blood compartment and the protective effect of GSSE. Rats were intraperitoneally (i.p.) administered either with garlic extract (5 g/kg bw) or GSSE (500 mg/kg bw) or a combination of garlic and GSSE at the same doses daily during one month. Plasma parameters and erythrocytes antioxidant status were evaluated. Data confirmed that high garlic dose induced anemia and a pro-oxidative state into erythrocytes characterized by increased malondialdehyde (MDA), carbonyl protein and antioxidant enzyme activities as catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Garlic also elevated intracellular hydrogen peroxide (H(2)O(2)) and free iron whereas GSSE treatment counteracted almost all garlic deleterious effects. In conclusion, high garlic dose induced a pro-oxidative state into erythrocytes via the Fenton reaction between H(2)O(2) and free iron, and GSSE exerted antioxidant properties.

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is mainly due to its rapidly acquired resistance to all conventional treatments. Despite drug-specific mechanisms of resistance, none explains how these cells resist the stress induced by any kind of anticancer treatment. Activation of stress-response pathways relies on the post-translational modifications (PTMs) of involved proteins. Among all PTMs, those mediated by the ubiquitin family of proteins play a central role. Our aim was to identify alterations of ubiquitination, neddylation, and sumoylation associated with the multiresistant phenotype and demonstrate their implications in the survival of PDAC cells undergoing treatment. This approach pointed at an alteration of promyelocytic leukemia (PML) protein sumoylation associated with both gemcitabine and oxaliplatin resistance. We could show that this alteration of PML sumoylation is part of a general mechanism of drug resistance, which in addition involves the abnormal activation of NF-κB and cAMP response element binding pathways. Importantly, using patient-derived tumors and cell lines, we identified a correlation between the levels of PML expression and sumoylation and the sensitivity of tumors to anticancer treatments.-Swayden, M., Alzeeb, G., Masoud, R., Berthois, Y., Audebert, S., Camoin, L., Hannouche, L., Vachon, H., Gayet, O., Bigonnet, M., Roques, J., Silvy, F., Carrier, A., Dusetti, N., Iovanna, J. L., Soubeyran, P. PML hyposumoylation is responsible for the resistance of pancreatic cancer.

Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) plays an important role during cell stress response in synergy with the potent "genome-keeper" p53. In human, the gene encoding TP53INP1 is expressed at very high level in some pathological situations, such as inflammation and prostate cancer (PC). TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse, making TP53INP1 a relevant specific target for molecular therapy of Castration Resistant (CR) PC. In that context, detection of TP53INP1 in patient biological fluids is a promising diagnostic avenue. We report here successful development of a new Enzyme-Linked Immunosorbent Assay (ELISA) detecting TP53INP1, taking advantage of molecular tools (monoclonal antibodies (mAbs) and recombinant proteins) generated in the laboratory during the course of basic functional investigations devoted to TP53INP1. The ELISA principle is based on a sandwich immunoenzymatic system, TP53INP1 protein being trapped by a first specific mAb coated on microplate then recognized by a second specific mAb. This new assay allows specific detection of TP53INP1 in serum of several PC patients. This breakthrough paves the way towards investigation of a large cohort of patients and assessment of clinical applications of TP53INP1 dosage.

Targeting metabolic reprogramming has proven successful in oncology, but this field requires better identification of drugs that inhibit mitochondrial metabolism in cancer cells. Recent work from Dr Wolf's group reveals that the primary target of the antitumor compound SMIP004-7 is mitochondrial complex I (NDUFS2 subunit), inhibition of which promotes anticancer immune surveillance.

Abstract Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in the biosynthesis of catecholamines. We describe here the isolation of the chicken TH gene and the analysis of 3 kb of its 5′ flanking region. The chicken TH transcription unit spans 19 kb. The 60‐bp proximal promoter contains a TATA box and a cyclic AMP response element (CRE) sequence. The 5′ flanking region contains several AP1‐, AP2‐, and octamer‐like sequences as well as a glucocorticoid response element at position ‐1.4 kb. A construct containing the 3‐kb 5′ flanking DNA fused to the chloramphenicol acetyltransferase (CAT) gene was transiently transfected into PC12 cells, and the effect of various effectors was tested. Only forskolin increased the CAT activity, likely owing to the presence of the CRE sequence. Constructs prepared by progressively deleting the 5′ flanking DNA were transfected into PC12 and QT6 (quail transformed fibroblasts) cells. In both cell types, the transcriptional activity increased with deletion of the 5′ flanking region. These results show that the 60‐bp region containing the TATA box and the CRE is sufficient to act as a constitutive promoter for the chicken TH gene and that this region appears to be negatively controlled by upstream sequences.

In this study, the protective role of grape seed and skin extract (GSSE) against high garlic dose-induced renal toxicity has been evaluated. Rats were intraperitoneally injected with garlic (5 g/kg bw) or GSSE (500 mg/kg bw) or a combination of garlic and GSSE at the same doses daily for one month. Renal oxidative stress markers and antioxidant status were evaluated. We also measured plasma creatinine and urea. Data showed that high garlic dose induced renal toxicity by increasing creatinine and urea and a pro-oxidative status characterized by increased malondialdehyde, carbonyl protein, calcium and H2O2, but decreased free iron. Unexpectedly garlic increased catalase but decreased peroxidase and superoxide dismutase activities. GSSE co-treatment counteracted almost all garlic-induced deleterious effects. In conclusion, high garlic dose induced a pro-oxidative state characterized by the Fenton reaction between H2O2 and free iron, inducing Ca2+ depletion, while GSSE exerted antioxidant properties and Ca2+ repletion.

The gene for tyrosine hydroxylase, the first and rate-limiting enzyme in the biosynthetic pathway of catecholamine neurotransmitters, has been localized in situ to chromosome 6 in the chicken. It is the first DNA marker to be reported for this telocentric macrochromosome. Use of a 45-kb biotinylated chicken-specific cosmid probe and a sensitive fluorescent detection system proved to be highly efficient, with over 90% of metaphases showing positive hybridization signals on one or (usually) both chromosome 6 homologs, in physically mapping this single-gene locus.

In this study, the protective role of grape seed and skin extract (GSSE) against doxorubicin-induced blood toxicity has been evaluated in rats. Rats were treated with the extract for 8 days and injected with doxorubicin (20 mg/kg) at the 4th day. At the end of the treatment, blood samples were collected for oxidative stress parameters determination and antioxidant enzymes. Doxorubicin increased erythrocytes and plasma malondialdehyde, free iron, H2O2 and carbonylation, decreased calcium and also decreased erythrocytes catalase, peroxidase and superoxide dismutase (specially the Fe isoform). Doxorubicin also decreased plasma catalase and superoxide dismutase (Cu/Zn and Fe isoforms) but increased peroxidase. Doxorubicin increased plasma alanine aminotransferase and aspartate aminotransferase but decreased them within erythrocytes. GSSE co-treatment counteracted almost all deleterious effects induced by doxorubicin. In conclusion, doxorubicin induced a pro-oxidative stress into rat erythrocytes and plasma and GSSE exerted antioxidant properties which can be attributed to free iron and calcium modulation.

The maturation of a specific subset of CD4+ T lymphocytes in the thymus is dependent on cortical thymic epithelial cells expressing the protease thymus-specific serine protease (TSSP, also known as PRSS16). Recently, we unveiled the involvement of TSSP in tumor suppression through its effect on the CD4+ T compartment.

Ulva rigida exhibits antioxidant activity that protects against oxidative stress. In the present investigation, we conducted experiments in HeLa cells exposed to hydrogen peroxide (H2O2) and found that the U. rigida ethanolic precipitate inhibited H2O2 apoptosis. This precipitate prevented the H2O2 stress-induced decline in the mitochondrial membrane potential (MMP). P21 Bax expression was decreased in H2O2-treated cells compared with untreated cells. Conversely, p21 Bax was consistently detected in cells that were co-treated with H2O2 and the U. rigida ethanolic precipitate. Bcl-xL expression increased in cells co-treated with H2O2 and the U. rigida ethanolic precipitate compared with H2O2-treated cells. Based on the obtained results, H2O2-induced apoptosis was inhibited by the U. rigida ethanol precipitate early in the apoptotic process through the upregulation of Bcl-xL, the prevention of full-length Bax cleavage molecule and the subsequent inhibition of MMP loss, which is a crucial step in the apoptotic cascade. Practical Applications Increasing evidence from both experimental and clinical studies has suggested that oxidative stress plays a major role in aging and pathogenesis. Currently, the search for safe and efficacious medicinal plants that possess antioxidant activity has attracted particular interest. In this context, this is the first report on the antioxidant activity of Ulva rigida in a cell line: U. rigida protected the cells from oxidative stress-induced apoptosis. The U. rigida ethanol precipitate inhibited apoptosis via the upregulation of Bcl-xL, the prevention of full-length Bax cleavage to its short and potent form, and the subsequent inhibition of the loss of the mitochondrial membrane potential. Herein, we report the mechanism by which U. rigida protects against oxidative stress-induced apoptosis. This may provide knowledge to enable the development of novel therapeutic strategies using U. rigida compounds to treat diseases that are attributed to oxidative disorders.

The chicken nerve growth factor (βNGF) gene has been mapped by fluorescent in situ hybridization to a pair of microchromosomes, confirming previous reports of the existence of a single gene locus. A 39-kb genomic fragment cloned in a cosmid vector and including the 5′ end of the βNGF locus was biotinylated for nonradioactive detection of the gene. This report adds to the increasing evidence proving microchromosomal localization of highly conserved and biologically fundamental genes. The implications of such genes belonging to very small linkage groups for the transmission of alleles from generation to generation together with the relevance of nonisotopic in situ hybridization for avian gene mapping are considered.

Chemokines are key regulators of migration in lymphoid tissues. In the thymus, maturing thymocytes move from the outer capsule to the inner medulla and thereby interact with different types of stromal cells that control their maturation and selection. In the process of searching for molecules specifically expressed at different stages of mouse thymic differentiation, we have characterized the cDNA coding for the thymus-expressed chemokine (TECK) and its receptor CCR9. The TECK receptor gene was isolated and shown to be localized on the mouse chromosome 9F1-F4. Thymic dendritic cells have been initially thought to be a prevalent source of TECK. In contrast, our results indicate that thymic epithelial cells constitute the predominant source of TECK. Consistent with the latter distribution, the TECK receptor is highly expressed by double-positive thymocytes, and TECK can chemoattract both double-positive and single-positive thymocytes. The TECK transcript is also abundantly expressed in the epithelial cells lining the small intestine. In conclusion, the interplay of TECK and its receptor CCR9 is likely to have a significant role in the recruitment of developing thymocytes to discrete compartments of the thymus.

Cancer is a complex pathology characterized by aberrant cell proliferation, resistance to induced cell death, and tumoral cell capacity to leave initial tissue and form distant tumors (metastasis). In addition, cancer cells favor angiogenesis which is necessary for tumor survival, progression and dissemination. Genetic events leading to genome instability enable those cell deregulations, in particular gain of oncogenes and loss of tumor suppressors functions observed in all cancer cells. The tumor protein p53 is encoded by the tumor suppressor gene TP53 which is mutated in more than fifty percent of human tumors, these mutations leading to loss of its tumor suppressive function.

Physical mapping of the human immunoglobulin λ locus (IGL) on chromosome 22q11 has shown the existence of at least 52 variable region gene segments. These Vλ genes are associated with EcoRI fragments detectable in Southern blots of genomic DNA samples. The current physical map of the IGL locus includes a unique Vλ8 gene (IGL8a, accession no. Z73650) in a 3.7 kb EcoRI fragment. However, our Southern blot-EcoRI-restriction fragment length polymorphism studies on the Brazilian population using a specific probe for the Vλ8 gene (pVL8 probe) have revealed the presence of two additional fragments bearing Vλ8 sequences (8.0 kilobase (kb) at 100% frequency and 6.0 kb at 10% frequency). We have used human/rodent somatic cell hybrid DNAs to locate these new Vλ8 genes outside the major locus on chromosome 22q11.2. Polymerase chain reactions using specific primers for the IGLV8a gene on the somatic hybrid panel showed that chromosome 8 (besides 22q11) also comprises Vλ8 sequences. This finding represents evidence for the dispersion of the human IGLV8 gene family outside the major locus (orphan genes).

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.

We have characterized 11 overlapping yeast artificial chromosomes (YACs) in the 1p13 region, 8 of them containing the human nerve growth factor (NGF) gene (HGMW-approved symbol NGFB). Sequence-tagged sites (STSs) corresponding to YAC extremities have been designed and used for chromosome assignment on a panel of monochromosomic somatic cell hybrids to check for YAC chimerism, in parallel with analyses by fluorescence in situ hybridization. Determination of end STS content and restriction mapping of the YACs led to the construction of a 3-Mb YAC contig. Four microsatellite markers from the Généthon collection and seven genes known to map to the 1p13 region have been ordered on the contig around the NGF gene. A new gene transcript from the Genexpress catalog has been localized on the contig. This work provides an integrated physical, genetic, and genic map of this chromosome 1 region. It constitutes a basis for determining the structure of the NGF gene and for further characterizing its genic environment.

DNA-dependent protein kinase (DNA-PK), a member of phosphatidylinositol-kinase family, is a key protein in mammalian DNA double-strand break (DSB) repair that helps to maintain genomic integrity. DNA-PK also plays a central role in immune cell development and protects telomerase during cellular aging. Epigenetic deregulation due to endogenous and exogenous factors may affect the normal function of DNA-PK, which in turn could impair DNA repair and contribute to genomic instability. Recent studies implicate a role for epigenetics in the regulation of DNA-PK expression in normal and cancer cells, which may impact cancer progression and metastasis as well as provide opportunities for treatment and use of DNA-PK as a novel cancer biomarker. In addition, several small molecules and biological agents have been recently identified that can inhibit DNA-PK function or expression, and thus hold promise for cancer treatments. This review discusses the impact of epigenetic alterations and the expression of DNA-PK in relation to the DNA repair mechanisms with a focus on its differential levels in normal and cancer cells.

&lt;div&gt;Abstract&lt;p&gt;Identifying critical factors involved in the metastatic progression of hepatocellular carcinoma (HCC) may offer important therapeutic opportunities. Here, we report that the proapoptotic stress response factor TP53INP1 is often selectively downregulated in advanced stage IV and metastatic human HCC tumors. Mechanistic investigations revealed that TP53INP1 downregulation in early-stage HCC cells promoted metastasis via DUSP10 phosphatase-mediated activation of the ERK pathway. The DUSP10 promoter included putative binding sites for p73 directly implicated in modulation by TP53INP1. Overall, our findings show how TP53INP1 plays a critical role in limiting the progression of early-stage HCC, with implications for developing new therapeutic strategies to attack metastatic HCC. &lt;i&gt;Cancer Res; 77(17); 4602–12. ©2017 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Supplementary Figures 1-6 from Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

Supplementary Methods from Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

Supplementary Figure Legends 1-6 from Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

&lt;p&gt;Supplementary Tables 1-3 contains primer sequences for qPCR, luciferase reporter and ChIP assays. Figure Legends for Supplementary Figures 1-4 are also included.&lt;/p&gt;

&lt;p&gt;Effects of TP53INP1 shRNAs on migration and invasion was rescued by TP53INP1 overexpression, ruling out potential off-target effects of the knockdown shRNAs.&lt;/p&gt;

&lt;p&gt;pERK1/2, DUSP10 and p73 immunohistochemical staining of xenograft tumors generated from HCC cells with and without TP53INP1 stably repressed.&lt;/p&gt;

&lt;p&gt;Mitomycin C studies show TP53INP1-mediated migration and invasion is not a result of the cells' altered ability to proliferate.&lt;/p&gt;

&lt;p&gt;Western Blot assay to show abolishment of pERK1/2 upon addition of ERK inhibitor U0126.&lt;/p&gt;

Supplementary Figures 1-6 from Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

Supplementary Methods from Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

Supplementary Figure Legends 1-6 from Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

&lt;p&gt;Western Blot assay to show abolishment of pERK1/2 upon addition of ERK inhibitor U0126.&lt;/p&gt;

&lt;p&gt;Supplementary Tables 1-3 contains primer sequences for qPCR, luciferase reporter and ChIP assays. Figure Legends for Supplementary Figures 1-4 are also included.&lt;/p&gt;

&lt;p&gt;Effects of TP53INP1 shRNAs on migration and invasion was rescued by TP53INP1 overexpression, ruling out potential off-target effects of the knockdown shRNAs.&lt;/p&gt;

&lt;p&gt;Mitomycin C studies show TP53INP1-mediated migration and invasion is not a result of the cells' altered ability to proliferate.&lt;/p&gt;

&lt;p&gt;pERK1/2, DUSP10 and p73 immunohistochemical staining of xenograft tumors generated from HCC cells with and without TP53INP1 stably repressed.&lt;/p&gt;

Summary Objective Increased consumption of meat is an epidemiologically validated risk condition for pancreatic cancer development, but the underlying mechanisms and whether it is related to induction of epithelial cell plasticity are unknown. Design Experimental protocol to test the influence of high consumption of meat was compared to pancreatic inflammation experimental models. To determine the molecular drivers promoting pancreatic cell plasticity, we compared transcriptomics data sets from human samples of pancreatic inflammation and pancreatic cancer (PDAC) prone to plasticity and validated in vivo, ex vivo and in vitro the main identified target. Results Meat-enriched diet promoted plasticity of pancreatic acinar cells, that transdifferentiated in duct-like cells, and presented PI3K activation. We identified a selective PI3K activation gene signature enriched with plasticity. In this signature, PHGDH , which encodes an enzyme responsible for amino acid serine synthesis, was differentially expressed. High level of PHGDH in acinar cells was necessary for the proliferative action of PI3Kα sustained by an increased maximal mitochondrial capacity and decreased cyclin-dependent inhibitor p27 level. PHGDH level was decreased in transdifferentiated acinar cells. In this context, active PI3Kα promoted cell plasticity but decreased the number of cycling cells. Both epithelial-restricted genetic inactivation of PI3Kα and full PI3Kα inhibition by pharmacological dosage reduced inflammation-induced tissue damage, while a pharmacological PI3Kα activator promoted PanIN precancer lesion development. Conclusion Meat-enriched diet promoted plasticity. Blockage of plasticity by PI3Kα inhibition provoked an increased rate of acinar cell proliferation that had a beneficial impact on the tissue microenvironment less prone to precancer lesion development. What is already known on this topic It is now well accepted that inflammatory conditions predispose to pancreatic tumour development; increased consumption of red and processed meat is an epidemiologically validated risk condition, but the underlying mechanisms are unknown. What this study adds We identify PI3K activation as a common molecular pathway activated by increased consumption of red and processed meat and by inflammatory condition to promote pancreatic plasticity and precancer lesion development. How this study might affect research, practice or policy As we show that treatments with the clinically available PI3Kα inhibitor block pancreatic plasticity under inflammatory stress while maintaining pancreas mass and limiting inflammatory reaction damage, they may represent an efficient and safe preventive interception drug in patients at risk of developing pancreatic cancer. PI3K pro-cancer action is exacerbated by the loss of serine synthesis enzyme; hence, diets that alter amino acid synthesis should be tightly controlled in those patients.

&lt;div&gt;Abstract&lt;p&gt;p53 exerts its tumor suppressor function mainly through transcriptional induction of target genes involved in several processes, including cell cycle checkpoints, apoptosis, and regulation of cell redox status. p53 antioxidant function is dependent on its transcriptional activity and proceeds by sequential induction of antioxidant and proapoptotic targets. However, none of the thus far renowned p53 targets have proved able to abolish on their own the intracellular reactive oxygen species (ROS) accumulation caused by p53 deficiency, therefore pointing to the existence of other prominent and yet unknown p53 antioxidant targets. Here, we show that TP53INP1 represents such a target. Indeed, TP53INP1 transcript induction on oxidative stress is strictly dependent on p53. Mouse embryonic fibroblasts (MEF) and splenocytes derived from TP53INP1-deficient (inp1&lt;sup&gt;−/−&lt;/sup&gt;) mice accumulate intracellular ROS, whereas overexpression of TP53INP1 in p53-deficient MEFs rescues ROS levels to those of p53-proficient cells, indicating that TP53INP1 antioxidant function is p53 independent. Furthermore, accumulation of ROS in inp1&lt;sup&gt;−/−&lt;/sup&gt; cells on oxidant challenge is associated with decreased expression of p53 targets p21/&lt;i&gt;Cdkn1a, Sesn2, TAp73, Puma&lt;/i&gt;, and &lt;i&gt;Bax&lt;/i&gt;. Mutation of p53 Ser&lt;sup&gt;58&lt;/sup&gt; (equivalent to human p53 Ser&lt;sup&gt;46&lt;/sup&gt;) abrogates transcription of these genes, indicating that TP53INP1-mediated p53 Ser&lt;sup&gt;58&lt;/sup&gt; phosphorylation is implicated in this process. In addition, TP53INP1 deficiency results in an antioxidant (&lt;i&gt;N&lt;/i&gt;-acetylcysteine)-sensitive acceleration of cell proliferation. Finally, TP53INP1 deficiency increases oxidative stress–related lymphoma incidence and decreases survival of p53&lt;sup&gt;+/−&lt;/sup&gt; mice. In conclusion, our data show that TP53INP1 is a major actor of p53-driven oxidative stress response that possesses both a p53-independent intracellular ROS regulatory function and a p53-dependent transcription regulatory function. [Cancer Res 2009;69(1):219–26]&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;p53 exerts its tumor suppressor function mainly through transcriptional induction of target genes involved in several processes, including cell cycle checkpoints, apoptosis, and regulation of cell redox status. p53 antioxidant function is dependent on its transcriptional activity and proceeds by sequential induction of antioxidant and proapoptotic targets. However, none of the thus far renowned p53 targets have proved able to abolish on their own the intracellular reactive oxygen species (ROS) accumulation caused by p53 deficiency, therefore pointing to the existence of other prominent and yet unknown p53 antioxidant targets. Here, we show that TP53INP1 represents such a target. Indeed, TP53INP1 transcript induction on oxidative stress is strictly dependent on p53. Mouse embryonic fibroblasts (MEF) and splenocytes derived from TP53INP1-deficient (inp1&lt;sup&gt;−/−&lt;/sup&gt;) mice accumulate intracellular ROS, whereas overexpression of TP53INP1 in p53-deficient MEFs rescues ROS levels to those of p53-proficient cells, indicating that TP53INP1 antioxidant function is p53 independent. Furthermore, accumulation of ROS in inp1&lt;sup&gt;−/−&lt;/sup&gt; cells on oxidant challenge is associated with decreased expression of p53 targets p21/&lt;i&gt;Cdkn1a, Sesn2, TAp73, Puma&lt;/i&gt;, and &lt;i&gt;Bax&lt;/i&gt;. Mutation of p53 Ser&lt;sup&gt;58&lt;/sup&gt; (equivalent to human p53 Ser&lt;sup&gt;46&lt;/sup&gt;) abrogates transcription of these genes, indicating that TP53INP1-mediated p53 Ser&lt;sup&gt;58&lt;/sup&gt; phosphorylation is implicated in this process. In addition, TP53INP1 deficiency results in an antioxidant (&lt;i&gt;N&lt;/i&gt;-acetylcysteine)-sensitive acceleration of cell proliferation. Finally, TP53INP1 deficiency increases oxidative stress–related lymphoma incidence and decreases survival of p53&lt;sup&gt;+/−&lt;/sup&gt; mice. In conclusion, our data show that TP53INP1 is a major actor of p53-driven oxidative stress response that possesses both a p53-independent intracellular ROS regulatory function and a p53-dependent transcription regulatory function. [Cancer Res 2009;69(1):219–26]&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Identifying critical factors involved in the metastatic progression of hepatocellular carcinoma (HCC) may offer important therapeutic opportunities. Here, we report that the proapoptotic stress response factor TP53INP1 is often selectively downregulated in advanced stage IV and metastatic human HCC tumors. Mechanistic investigations revealed that TP53INP1 downregulation in early-stage HCC cells promoted metastasis via DUSP10 phosphatase-mediated activation of the ERK pathway. The DUSP10 promoter included putative binding sites for p73 directly implicated in modulation by TP53INP1. Overall, our findings show how TP53INP1 plays a critical role in limiting the progression of early-stage HCC, with implications for developing new therapeutic strategies to attack metastatic HCC. &lt;i&gt;Cancer Res; 77(17); 4602–12. ©2017 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

A spin-label (P-OPC) composed of the nitroxide-containing ring proxyl linked at the C1 position of the intercalating fluorescent chromophore oxazolopyridocarbazole (OPC) has been synthesized. The spin-labeled OPC was found to interact with DNA and polynucleotides according to an external minor groove binding mode with association constant values Kapp ranging from 10(5) to 10(6) M-1. External binding was obvious from the inability of P-OPC to increase the length of sonicated DNA upon binding, the low unwinding angle (9.6 degrees) of circular PM2 DNA, and the low energy transfer from DNA bases to bound chromophore. Binding of P-OPC to DNA or polynucleotide results in a strong immobilization of the proxyl moiety, resulting in the appearance of an asymmetric and broad ESR spectrum with a maximal hyperfine splitting of 56.5 G. In the equilibrium conditions, the occurrence of superimposed ESR spectra related to the P-OPC fraction undergoing rapid motion and to the P-OPC fraction immobilized allows the estimation of the concentrations of free and DNA-bound spin-label. The external mode of binding to DNA as well as the characteristics of the ESR spectra make P-OPC suitable for the determination of DNA binding parameters of nonintercalating ligands using competition experiments. The measurement of the binding constants of distamycin A to poly[d(A-T)] and poly[d(G-C)] is taken as an example.

Abstract The recurrence and metastasis of hepatocellular carcinoma (HCC) portends a poor prognosis and represents important clinical challenges. There is a great need to identify critical factors involved in HCC metastasis that will facilitate the development of new therapeutic strategies. We have previously found that the initiation, growth and self-renewal of CD133+ liver tumors to be fine-tuned by a balance of miR-130b overexpression and tumor protein 53 inducible nuclear protein 1 (TP53INP1) down-regulation, suggesting that TP53INP1 is a critical effector driving hepatocarcinogenesis. In this study, we aimed to further investigate the role of TP53INP1 in HCC metastasis. We showed TP53INP1 to be frequently down-regulated in advanced stage IV and metastatic human HCC tumors as compared with early stage (I-III) and primary tumors. Functional studies in immortalized normal liver cell line MIHA and HCC cell line MHCC-97L found TP53INP1 suppression in HCC to promote metastasis in vitro and in vivo. To elucidate the downstream signaling mechanism by which TP53INP1 regulates HCC metastasis, a Proteome Profiler Human Phospho-Kinase Array was utilized. Phospho-ERK was found to be consistently up-regulated in HCC cell lines with TP53INP1 knocked down, with its involvement in TP53INP1-mediated metastasis subsequently validated by rescue experiments using an ERK inhibitor U0126 or shERK1/2 knockdown approach. ERK1/2 is known to be negatively regulated by a family of dual-specificity MAPK phosphatase called DUSP/MKP. Screening of a panel of DUSP/MKP family members by qPCR identified DUSP10 to be commonly down-regulated in both MIHA and MHCC-97L cells with TP53INP1 suppressed as compared to controls. The importance of DUSP10 inactivation on ERK in TP53INP1-mediated HCC was substantiated by rescue experiments whereby DUSP10 ectopic expression in TP53INP1 suppressed cells reversed up-regulated phospho-ERK expression and enhanced HCC aggressiveness. Subsequent analysis of the DUSP10 promoter region by open-access database revealed four putative binding sites for p73, of which transcriptional activity has previously been found to be modulated by TP53INP1. Chromatin immunoprecipitation and luciferase reporter assays collectively demonstrated that TP53INP1 is vital for p73 transcriptional activity of DUSP10. Taken together, TP53INP1 down-regulation promotes HCC metastasis through a p73-dependent DUSP10/ERK signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Kai Yu Ng, Lok Hei Chan, Stella Chai, Man Tong, Xin Yuan Guan, Nikki P Lee, Yun Fei Yuan, Dan Xie, Terence K Lee, Alice Carrier, Nelson J Dusetti, Stephanie Ma. TP53INP1 negatively regulates ERK1/2 via p73-dependent DUSP10 expression to promote metastasis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4842. doi:10.1158/1538-7445.AM2017-4842

Tumor protein 53-induced nuclear protein 1 (TP53INP1) deficiency leads to oxidative stress-associated obesity and insulin resistance. Although skeletal muscle has a predominant role in the development of metabolic syndrome, the bioenergetics and functional consequences of TP53INP1 deficiency upon this tissue remain undocumented. To clarify this issue, gastrocnemius muscle mechanical performance, energy metabolism, and anatomy were investigated in TP53INP1-deficient and wild-type mice using a multidisciplinary approach implementing noninvasive multimodal-NMR techniques. TP53INP1 deficiency increased body adiposity but did not affect cytosolic oxidative stress, lipid content, and mitochondrial pool and capacity in myocyte. During a fatiguing bout of exercise, the in vivo oxidative ATP synthesis capacity was dramatically reduced in TP53INP1-deficient mice despite ADP level (the main in vivo stimulator of mitochondrial respiration) did not differ between both genotypes. Moreover, TP53INP1 deficiency did not alter fatigue resistance but paradoxically increased the contractile force, whereas there were no differences for muscle fiber-type distribution and calcium homeostasis between both genotypes. In addition, muscle proton efflux was decreased in TP53INP1-deficient mice, thereby indicating a reduced blood supply. In conclusion, TP53INP1 plays a role in muscle function and bioenergetics through oxidative capacity impairment possibly as the consequence of abnormal mitochondrial respiration regulation and/or defective blood supply.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a paradigmatic model of micrometastatic disease, characterized by both relapse of surgery and failure of long-term disease control with chemotherapies in resected, locally advanced unresectable and metastatic patients. Molecular characterization of large cohorts of PDAC patients failed to identify actionable pathways associated with mutations except for oncogenic Kras, found in more than 80% of all patients. Class I PI3K being a key regulator of both cell survival and cell migration, we questioned if nonmutated PI3K activity downstream oncogenic Kras is a clinically relevant target for this pathology. To achieve this goal, we devised a novel preclinical protocol in a genetically engineered mouse model (GEMM) including a noninvasive monitoring using circulating cell-free DNA (cfDNA) level as a novel biomarker of micrometastatic disease. We previously showed that, downstream Kras, nonmutant ubiquitous PI3Kalpha is crucial for PDAC development. Among all the mRNA expression-based hallmarks of biologic pathways, we now show that PI3K/Akt/mTOR hallmark is significantly increased in PDAC patients with advanced metastatic disease. High scoring of PI3Kalpha activation, based on expression levels of PI3Kalpha-regulated curated genes, is correlated to a poor prognosis, and is found in metastatic PDAC cell lines and in patient-derived ex vivo cultures of locally invasive PDAC cancer cells. When testing global or isoform-selective inhibitors in GEMM and human cell lines, we show that biologically efficient PI3K inhibitors annihilate PI3Kalpha strongly, but not necessarily specifically, among the four class I PI3K isoforms, resulting in Akt2 inactivation. Similar results are obtained with siRNA and in PIK3CA oncogenic cell lines. PI3Kalpha controls pancreatic tumor cell line motility and migration. In a GEMM model of PDAC, mice with high level of cfDNA present a shorter survival and develop a metastatic disease. Genetic or pharmacologic inhibition of PI3Kalpha in these mice decreases tumor volume, metastasis, ascitic fluid development, cfDNA quantity and M2 macrophage infiltration. Here, we used GEMMs to define a cfDNA-positive aggressive subtype of PDAC and identify a pharmacologically actionable prometastatic feature driven by PI3Kalpha. We propose PI3K as a therapeutic strategy to suppress PDAC progression and metastasis by scavenging peritumoral CD206-positive macrophages. Our data demonstrate that PI3K agents could be efficient on micrometastatic disease (measured by cfDNA), resulting in delay on macrometastatic dissemination. This abstract is also being presented as Poster B04. Citation Format: Benoit Thibault, Fernanda Ramos-Delgado, Elvire Pons-Tostivint, Nicole Therville, Gabriella Reyes-Castellanos, Marie Tosolini, Jean-Pierre Delord, Dieter Saur, Alice Carrier, Pierre Cordelier, Céline Basset, Julie Guillermet-Guibert. Pancreatic ductal adenocarcinoma requires PI3Kalpha activity to accelerate circulating DNA-positive metastatic disease [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR07.

Abstract Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. We aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis.Gene and protein expression was analysed by real-time RT-PCR and immunoblot. Cell survival/death was examined by flow cytometry study of Annexin V-staining. The prognostic value of TP53INP1 expression in tumor tissues was investigated in three independent cohorts of neuroblastoma patients. TP53INP1 was one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma. Citation Format: Jeyran Shahbazi, Christopher J. Scarlett, Murray Norris, Bing Liu, Michelle Haber, Andrew E. Tee, Alice Carrier, Andrew V. Biankin, Wendy B. London, Glenn M. Marshall, Richard Lock, Tao Liu. Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5138. doi:10.1158/1538-7445.AM2014-5138

Review on TP53INP1 (tumor protein p53 inducible nuclear protein 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Ulva rigida (Chlorophyta) has been suggested to contain cytoprotective agents against oxidative stress because of its antioxidant activity. However the antioxidant mechanisms of U. rigida have not yet been explored. In the present study, the protective effect of U. rigida against oxidative stress was evaluated, and the underlying molecular mechanisms were investigated in HeLa cervical carcinoma cells. Quantification of apoptosis by flow cytometry indicated that U. rigida extract inhibited H2O2-induced apoptosis in HeLa cells. In addition, using DiOC6(3) localization as a measurment of mitochondrial integrity, we demonstrated that U. rigida extract prevented the decline of membrane mitochondrial potential (ΔΨ) induced by H2O2 stress. Western blot analysis revealed that the level of the pro-apoptotic protein p21 Bax decreased in H2O2 treated cells compared to the untreated cells, while it was consistely detected in cells co-treated with H2O2 and U. rigida extract. These can be associated to a cleavage, following H2O2 stress, of the full-length form of p21 Bax into a p18 Bax form accepted to be more potent in disrupting mitochondrial integrity. This study provided a possible explanation for the antioxidant activity of U. rigida, and implied an application of this alga for the therapy of diseases involving oxidative stress.

Les connaissances de la biologie du cancer du pancréas ont grandement bénéficié des études des altérations génétiques, épigénétiques et de l'expression moléculaire dans les modèles expérimentaux, mais aussi dans les lésions pré-cancéreuses et les tissus tumoraux. Les gènes suppresseurs de tumeur p16, TP53 et DPC4/smad4 sont altérés alors que l'oncogène KRAS est activé. Cette activation par mutation ponctuelle sur le codon 12 est l'élément majeur et apparaît précocement dans la carcinogenèse pancréatique. À un stade plus tardif de développement tumoral, on observe une augmentation de l'activité télomérase, une surexpression des facteurs de croissance et angiogénique et/ou de leur récepteurs (EGF, Nerve Growth Factor, gastrine, VEGF, FGF, PDGF), des facteurs d'invasion (métalloprotéinases, activateur tissulaire du plasminogène) et des microARN. Le microenvironnement joue aussi un rôle important dans le caractère invasif et métastatique avec une inter-relation forte entre cellules tumorales et cellules étoilées pancréatiques ainsi que la matrice extracellulaire. Ce microenvironnement est responsable de fibrose, hypoxie et hypovascularisation autant de freins à la biodisponibilité des drogues anti-tumorales. Le ciblage du microenvironnement fait partie des nouvelles stratégies thérapeutiques en association avec les chimiothérapies.The understanding of the biology of pancreatic carcinoma has greatly benefited from studies of genetic/epigenetic alterations and molecular expression in experimental models as well as precancerous and cancerous tissues by mean of molecular amplification and large-scale transcriptoma analysis. P16, TP53, DPC4/Smad4 tumor suppressor pathways are genetically inactivated in the majority of pancreatic carcinomas, whereas oncogenic k-ras is activated. The activating point mutation of the KRAS oncogene on codon 12 is the major event and occurs early in pancreatic carcinogenesis. At a late stage of tumor development, an increase of telomerase activity, an over expression of growth factors and/or their receptors (EGF, Nerve Growth Factor, gastrin), of pro-angiogenic factors (VEGF, FGF, PDGF), of invasiveness factors (metalloproteinases, tissue plasminogen activators) occurs. The microenvironment plays also a key role in the invasive and metastatic process of pancreatic carcinoma with a strong relationship between cancerous cells and pancreatic stellate cells as well as extracellular matrix. This microenvironment strongly participates to the tumor fibrosis, hypoxia and hypovascularization inducing an inaccessibility of drugs. Nowadays, the targeting of microenvironment takes a special place in the new therapeutic strategies of pancreatic cancer in combination with chemotherapy.

Abstract Genetic inhibition of NUPR1 induces tumor growth arrest. Inactivation of NUPR1 expression in pancreatic cancer cells results in lower ATP production, higher consumption of glucose with a significant switch from OXPHOS to glycolysis followed by necrotic cell death. Importantly, induction of necrosis is independent of the caspase activity. We demonstrated that NUPR1 inactivation triggers a massive release of Ca 2+ from the endoplasmic reticulum (ER) to the cytosol and a strong increase in ROS production by mitochondria with a concomitant relocalization of mitochondria to the vicinity of the ER. In addition, transcriptomic analysis of NUPR1-deficient cells shows the induction of an ER stress which is associated to a decrease in the expression of some ER stress response-associated genes. Indeed, during ER stress induced by the treatment with thapsigargin, brefeldin A or tunicamycin, an increase in the mitochondrial malfunction with higher induction of necrosis was observed in NUPR1-defficent cells. Finally, activation of NUPR1 during acute pancreatitis protects acinar cells of necrosis in mice. Altogether, these data enable us to describe a model in which inactivation of NUPR1 in pancreatic cancer cells results in an ER stress that induces a mitochondrial malfunction, a deficient ATP production and, as consequence, the cell death by necrosis. Highlights NUPR1 expression promotes pancreatic cancer development and progression NUPR1-depletion is a promising therapeutic strategy to be used for treating cancers NUPR1-depletion induces ER stress, mitochondrial malfunction and a significant switch from OXPHOS to glycolysis followed by necrotic cell death Inactivation of NUPR1 antagonizes cell growth by coupling a defective ER-stress response and a caspase-independent necrosis.

The antitumor activity of exercise by means of enhanced immune activation is documented, but better identification of the underlying mechanisms is required to develop new therapeutic strategies. Recent work from the Dr Bar-Sagi group reveals that exercise engages IL-15 signaling and pharmacological activation of the IL-15/IL-15R axis mimics the exercise-driven immune cell-mediated cytotoxicity in pancreatic cancer.

ABSTRACT Aims Pancreatic Ductal AdenoCarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. The objective of this work was to investigate the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative study combining functional, morphological and omics-based evaluation of gastrocnemius muscle from genetically-engineered mice developing autochthonous pancreatic tumor and cachexia (KIC GEMM). Results KIC cachectic PDAC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fiber’s size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. While the number of mitochondria per cell is not altered, mitochondrial mass is decreased by a factor of 2 and the mitochondrial DNA by a factor of 3, suggesting a defect in mitochondrial genome homeostasis. Innovation Muscle atrophy is associated with strong mitochondrial metabolic defects that are not limited to carbohydrate, protein and redox metabolism, but concern also lipid and nucleic acid metabolism. Conclusion This work provides a framework to guide towards the most relevant targets in the clinic to limit PDAC-induced cachexia, in particular mitochondrial fatty acid metabolism.

Pancreatic Ductal Adenocarcinoma (PDAC) remains a major unresolved disease because of its remarkable therapeutic resistance. Even patients who respond to initial therapy experience relapse in most cases. The mechanisms underlying therapy-acquired resistance supporting relapse are poorly understood. In this study, we aimed to determine the metabolic features of PDAC during relapse, specifically adaptations of mitochondrial and redox metabolism. We used preclinical PDAC mouse models (patient-derived xenografts and murine syngeneic allografts) that present complete regression under initial chemotherapeutic treatment but relapse after a certain time. Relapsed tumors were analyzed ex vivo by flow cytometry to measure mitochondrial and redox characteristics. Molecular mechanisms were investigated by quantification of ATP and antioxidants levels, RT-qPCR and bulk RNA-sequencing. Our findings show that mitochondrial metabolism is reprogrammed during relapse, with increased mitochondrial mass, ATP levels, mitochondrial superoxide anions, and total ROS levels, in relapsed compared to control tumors in both models; mitochondrial membrane potential is increased in the xenografts model only. This mitochondrial metabolic reprogramming occurs during treatment-induced regression and at relapse onset. At the molecular level, antioxidant defenses are increased in relapsed tumors and during treatment. These data suggest that treatment-induced oxidative stress may cause the appearance of treatment-adapted cells, known as drug-tolerant persister (DTP) cells. Finally, the combined treatment of arsenic trioxide (ROS inducer) and buthionine sulfoximine (glutathione synthesis inhibitor) is able to completely prevent relapse in PDAC xenografts. In conclusion, targeting redox metabolism via ROS production and antioxidant inhibition is a very promising approach to prevent relapse in PDAC patients.