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

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]

Using a bioinformatic approach, we identified a TP53INP1-related gene encoding a protein with 30% identity with tumor protein 53-induced nuclear protein 1 (TP53INP1), which was named TP53INP2. TP53INP1 and TP53INP2 sequences were found in several species ranging from Homo sapiens to Drosophila melanogaster, but orthologues were found neither in earlier eukaryotes nor in prokaryotes. To gain insight into the function of the TP53INP2 protein, we carried out a yeast two-hybrid screening that showed that TP53INP2 binds to the LC3-related proteins GABARAP and GABARAP-like2, and then we demonstrated by coimmunoprecipitation that TP53INP2 interacts with these proteins, as well as with LC3 and with the autophagosome transmembrane protein VMP1. TP53INP2 translocates from the nucleus to the autophagosome structures after activation of autophagy by rapamycin or starvation. Also, we showed that TP53INP2 expression is necessary for autophagosome development because its small interfering RNA-mediated knockdown strongly decreases sensitivity of mammalian cells to autophagy. Finally, we found that interactions between TP53INP2 and LC3 or the LC3-related proteins GABARAP and GABARAP-like2 require autophagy and are modulated by wortmannin as judged by bioluminescence resonance energy transfer assays. We suggest that TP53INP2 is a scaffold protein that recruits LC3 and/or LC3-related proteins to the autophagosome membrane by interacting with the transmembrane protein VMP1. It is concluded that TP53INP2 is a novel gene involved in the autophagy of mammalian cells.

Tumor 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.

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.

Les polypes dentelés colorectaux constituent un groupe hétérogène de lésions comprenant, entre autres, les polypes hyperplasiques (PH) et les adénomes dentelés sessiles (ADS) dont la distinction n’est pas toujours aisée. Il est toutefois important pour le pathologiste de reconnaître les ADS car ils semblent être les précurseurs des adénocarcinomes dentelés impliqués dans la voie « dentelée » de la carcinogenèse colique.Nous avons sélectionné 102 biopsies de polypes dentelés colorectaux et évalué les paramètres suivants : localisation, taille, nombre de fragments par polype, aspect superficiel ou tangentiel des biopsies, type de résection, localisation de l’aspect dentelé, branchement, horizontalisation, dilatation et hernie à travers la musculaire muqueuse des cryptes, type cellulaire, papilles épithéliales, éosinophilie cytoplasmique et dysplasie.Quatre-vingt et un polypes correspondaient à des PH (79 %), sept à des ADS (7 %) dont un présentait des foyers de dysplasie, cinq à des adénomes dentelés traditionnels (ADT) (5 %) et trois à des polypes mixtes (PH et adénome tubuleux : 3 %). Seuls six polypes dentelés (6 %) n’ont pu être classés. Les critères majeurs permettant de distinguer l’ADS ont été l’aspect dentelé de toute la hauteur des cryptes et la rareté des cellules indifférenciées à la base des cryptes. De plus, les ADS ont été significativement plus souvent localisés au côlon droit et de plus grande taille que les PH (médiane : 11 mm versus 4 mm).L’ADS peut être distingué du PH en pratique quotidienne. La présence de dysplasie dans l’ADS renforce l’hypothèse que ce polype a un potentiel carcinogénétique et mérite d’être suivi cliniquement au même titre qu’un adénome traditionnel.Serrated polyps of the colorectum are a heterogenous group of mucosal lesions including hyperplastic polyps (HP) and sessile serrated adenomas (SSA), but their morphologic distinction is not always straightforward. However, it is important for the pathologist to identify SSA because recent data show that they might be the precursors of serrated adenocarcinomas which are probably involved in the serrated pathway.We selected 102 serrated colorectal polyps resected by colonoscopy and evaluated the following parameters: location, size, number of biopsies per polyp, superficial or tangential biopsies, type of resection, location of the serrated feature, branching, horizontalisation, dilatation and herniation of crypts through the muscularis mucosae, cellular type, epithelial tufts, cytoplasmic eosinophilia and dysplasia.There were 81 HP (79%), seven SSA (7%) of which one showed foci of dysplasia, five traditional serrated adenomas (5%) and three mixed polyps (HP and tubulous adenoma: 3%). Only six serrated polyps could not be classified. The main architectural criterion for diagnosing SSA was a serrated pattern throughout the crypt axis and the rarity of undifferentiated cells at the base of the crypts. Moreover, clinical characteristics were also helpful, since sessile serrated adenomas were significantly more often located in the right colon and larger (median: 11 mm versus 4 mm) than HP.SSA can be distinguished morphologically from HP in a daily practice. The presence of foci of dysplasia in one case of SSA supports the hypothesis that these polyps have a carcinogenetic potential and should have the same clinical follow-up as traditional adenomas.

AIMOverexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1 and p53 expression in gastric cancer.METHODSTP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed.RESULTSTP53INP1 was expressed in 98% (139/142 cases) of non-cancerous gastric tissues and was down-expressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%). Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3% vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients). P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues. A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed, loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P=0.0006).CONCLUSIONTP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1 is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may reflect the malignant grade of gastric cancer and is regarded as an adverse prognostic factor.

TP53INP1, formerly called either TEAP (thymus-expressed acidic protein) or SIP (stress-induced protein), was identified by microarray technology in mouse. It is a novel gene plausibly involved in the function of thymus (Carrier et al., 1999) and is induced by stress during the acute phase of pancreatitis (Tomasini et al., 2001). The human TP53INP1 counterpart (also called p53DINP1: p53-dependent damage-inducible nuclear protein 1, Okamura et al., 2001) and SIP (Tomasini et al., in press) is a proapoptotic gene induced through TP53 activation. This gene is a stress-induced gene with potential antitumoral properties. We report here the localization of TP53INP1 gene to human chromosome 8q22, in a region that shows conserved synteny with region A1–A2 of the murine chromosome 4 where the mouse gene was mapped (Carrier et al., 2000).

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.

CONTEXTThe tumor protein p53-induced nuclear protein 1 (TP53INP1) gene was found using DNA microarray technology as an overexpressed gene in acute pancreatitis. However, expression of TP53INP1 in chronic pancreatitis has not been previously reported.OBJECTIVEThis study investigated TP53INP1 gene expression and its relationship with p53 and apoptosis in spontaneous chronic pancreatitis in the Wistar-Bonn/Kobori rat.METHODSNinety four-week-old male Wistar-Bonn/Kobori rats were fed a special breeding diet until sacrifice. Camostat mesilate (n=30) or a herbal medicine (Saiko-keishi-to; n=30) were mixed with the diet, while the other 30 rats were untreated. The rats were sacrificed every 4 weeks for 20 weeks, and the pancreas was examined. In addition, 6 four-week-old male Wistar-Bonn/Kobori rats were sacrificed and studied as starting reference. Finally, Wistar rats (n=36) were studied as controls.MAIN OUTCOME MEASURETP53INP1 mRNA expression was determined by reverse transcription-polymerase chain reaction using semi-quantitative analysis, direct sequencing and in situ hybridization.RESULTSTP53INP1 mRNA was strongly expressed at 12 weeks when chronic pancreatitis developed, with a second peak at 20 weeks. The expression kinetics of TP53INP1 mRNA paralleled acinar cell apoptosis assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The p53 mRNA expression showed a single peak at 12 weeks. In situ hybridization revealed that TP53INP1 mRNA was expressed mainly in acinar cells. Therapeutic drugs such as camostat mesilate and a herbal medicine Saiko-keishi-to suppressed the TP53INP1 mRNA expression. TP53INP1 mRNA induction in acinar cells was confirmed with in vitro experiments using an arginine-induced rat pancreatic acinar AR4-2J cell injury model.CONCLUSIONSTP53INP1 expression may reflect the acute-phase response and apoptosis of acinar cells in the course of chronic pancreatitis.

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.

Nous rapportons ici la description clinico‐pathologique et moleculaire d‘un syndrome myeloproliferatif (SMP) atypique qui presente la particularite d‘etre associe a des translocations equilibrees touchant la region p12 du bras court du chromosome 8. Le SMP 8p12 est un syndrome proche de la leucemie myeloide chronique (LMC), mais avec des caracteristiques particulieres dont une hyperplasie des lignages myeloide et lymphoide avec une frequence elevee (60 %) de lymphome, et une hypereosinophilie. Ce syndrome evolue vers une transformation leucemique aigue myeloide rapidement fatale. A l‘heure actuelle, seule le greffe de moelle osseuse allogenique semble etre efficace pour eradiquer ou supprimer le clone malin. L‘anomalie chromosomique recurrente detectee dans tous les cas est une translocation equilibree de la region 8p12 qui va fusionner le gene FGFR1 ( fibroblast growth factor receptor 1) avec un autre gene partenaire. A l‘heure actuelle, cinq genes ont deja ete identifies et clones : FOP en 6q27, CEP110 en 9q33, FIM\RAMP\ZNF198 en 13q12, HERV‐K en 19q13.3, et BCR en 22q11. Les proteines de fusion X‐FGFR1 conservent la region N‐terminale des proteines sauvages contenant des motifs de dimerisation liee au domaine catalytique intact de FGFR1. Les proteines de fusion sont delocalisees dans le cytoplasme des cellules, sont activees de facon constitutive par dimerisation grâce aux motifs d‘oligomerisation dans la partie N‐terminale des proteines de fusion et induisent la survie de cellules hematopoietiques cultivees en absence d‘IL‐3 via des mecanismes anti‐apoptotiques. Ainsi, l‘activation constitutive des proteines tyrosine kinases de fusion X‐FGFR1 et, par voie de consequence, l‘activation constitutive de signaux intracellulaires de transduction contribuent a l‘etat neoplasique in vivo du SMP 8p12, un nouveau sous‐type bien caracterise de syndromes myeloproliferatifs chroniques du a l‘activation constitutive de FGFR1. Les voies de transduction ainsi activees et les proteines de fusion bien caracterisees constituent d‘excellentes cibles pour une therapie moleculaire.

Constitutive activation of aberrant fibroblast growth factor receptor 1 (FGFR1) kinase as a consequence of gene fusion such as FOP-FGFR1 associated with t(6; 8)(q27;p11-12) translocation, is the hallmark of an atypical aggressive stem cell myeloproliferative disorder (MPD) in humans. In this study, we show that expression of FOP-FGFR1 in primary bone marrow cells induced by retroviral transduction generates a MPD in mice. Constitutive FOP-FGFR1 kinase activity was both essential and sufficient to cause a chronic myeloproliferative syndrome in the murine bone marrow transplantation model. In contrast to the human disorder, lymphoproliferation and progression to acute phase were not observed. Lymphoid symptoms, however, appeared when onset of the disease was delayed as the result of mutation of FOP-FGFR1 at tyrosine 511, the phospholipase C gamma (PLCgamma) binding site.

Nous rapportons ici la description clinico‐pathologique et moleculaire d‘un syndrome myeloproliferatif (SMP) atypique qui presente la particularite d‘etre associe a des translocations equilibrees touchant la region p12 du bras court du chromosome 8. Le SMP 8p12 est un syndrome proche de la leucemie myeloide chronique (LMC), mais avec des caracteristiques particulieres dont une hyperplasie des lignages myeloide et lymphoide avec une frequence elevee (60 %) de lymphome, et une hypereosinophilie. Ce syndrome evolue vers une transformation leucemique aigue myeloide rapidement fatale. A l‘heure actuelle, seule le greffe de moelle osseuse allogenique semble etre efficace pour eradiquer ou supprimer le clone malin. L‘anomalie chromosomique recurrente detectee dans tous les cas est une translocation equilibree de la region 8p12 qui va fusionner le gene FGFR1 ( fibroblast growth factor receptor 1) avec un autre gene partenaire. A l‘heure actuelle, cinq genes ont deja ete identifies et clones : FOP en 6q27, CEP110 en 9q33, FIM\RAMP\ZNF198 en 13q12, HERV‐K en 19q13.3, et BCR en 22q11. Les proteines de fusion X‐FGFR1 conservent la region N‐terminale des proteines sauvages contenant des motifs de dimerisation liee au domaine catalytique intact de FGFR1. Les proteines de fusion sont delocalisees dans le cytoplasme des cellules, sont activees de facon constitutive par dimerisation grâce aux motifs d‘oligomerisation dans la partie N‐terminale des proteines de fusion et induisent la survie de cellules hematopoietiques cultivees en absence d‘IL‐3 via des mecanismes anti‐apoptotiques. Ainsi, l‘activation constitutive des proteines tyrosine kinases de fusion X‐FGFR1 et, par voie de consequence, l‘activation constitutive de signaux intracellulaires de transduction contribuent a l‘etat neoplasique in vivo du SMP 8p12, un nouveau sous‐type bien caracterise de syndromes myeloproliferatifs chroniques du a l‘activation constitutive de FGFR1. Les voies de transduction ainsi activees et les proteines de fusion bien caracterisees constituent d‘excellentes cibles pour une therapie moleculaire.

A rare atypical myeloproliferative disorder (aMPD) associated with chromosomal translocations involving the short arm of chromosome 8, region p11-p12 has been described. In most patients, the cytogenetic abnormality is a t(8;13)(p12;q12) that fuses fibroblast growth factor receptor 1, the 8p12 key gene, to FIM/ZNF198 gene. Prognosis is poor with frequent evolution to acute myeloid leukaemia within 1 year of diagnosis. We report a new patient with aMPD with a t(8;13) translocation. Complete haematological, cytogenetic and molecular remission was demonstrated 39 months after allogeneic bone marrow transplantation. This is the first report to demonstrate a molecular remission in this disorder.