Nelson Dusetti

Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development

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/rasV12 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.

Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down‐regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis

Defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in colorectal‐cancer research. Our strategy was to characterize genes whose expression is altered during colorectal carcinogenesis. To this end, the phenotype of a colorectal tumour was previously established by partial sequencing of a large number of its transcripts and the genes of interest were selected by differential screening on high‐density filters with mRNA of colorectal cancer and normal adjacent mucosa. Fifty‐one clones were found over‐expressed and 23 were under‐expressed in the colorectal‐cancer tissues of the 5 analyzed patients. Among the latter, clones 6G2 and 32D6 were found of particular interest, since they had significant homology with several homeodomain‐containing genes. The highest degree of similarity was with the murine Cdx1 for 6G2, and with the murine Cdx2 and hamster Cdx3 for 32D6. Using a RT‐PCR approach, complete sequence of both types of homeobox‐containing cDNA was obtained. The amino‐acid sequence of the human Cdx1 is 85%; identical to the mouse protein, and human Cdx2 has 94% identity with the mouse Cdx2 and hamster Cdx3. Tissue‐distribution analysis of Cdx1 and Cdx2 mRNA showed that both transcripts were specifically expressed in small intestine, in colon and rectum. Twelve tissue samples from colorectal adenocarcinomas and the corresponding normal mucosa were analyzed by Northern blot. Expression of the 2 types of mRNA was either reduced or absent in 10 of them. Several colon‐cancer cell lines were also analyzed. Cdx2 mRNA was absent from LS174T cells and Cdx1 mRNA was absent in PF11, TC7 and SW480 cells; none was detected in HT29 cells. It was concluded that decrease in human Cdx1 and/or Cdx2 expression is associated with colorectal tumorigenesis. Int. J. Cancer 74:35‐44.

Strengthened glycolysis under hypoxia supports tumor symbiosis and hexosamine biosynthesis in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma is one of the most intractable and fatal cancer. The decreased blood vessel density displayed by this tumor not only favors its resistance to chemotherapy but also participates in its aggressiveness due to the consequent high degree of hypoxia. It is indeed clear that hypoxia promotes selective pressure on malignant cells that must develop adaptive metabolic responses to reach their energetic and biosynthetic demands. Here, using a well-defined mouse model of pancreatic cancer, we report that hypoxic areas from pancreatic ductal adenocarcinoma are mainly composed of epithelial cells harboring epithelial-mesenchymal transition features and expressing glycolytic markers, two characteristics associated with tumor aggressiveness. We also show that hypoxia increases the “glycolytic” switch of pancreatic cancer cells from oxydative phosphorylation to lactate production and we demonstrate that increased lactate efflux from hypoxic cancer cells favors the growth of normoxic cancer cells. In addition, we show that glutamine metabolization by hypoxic pancreatic tumor cells is necessary for their survival. Metabolized glucose and glutamine converge toward a common pathway, termed hexosamine biosynthetic pathway, which allows O-linked N-acetylglucosamine modifications of proteins. Here, we report that hypoxia increases transcription of hexosamine biosynthetic pathway genes as well as levels of O-glycosylated proteins and that O-linked N-acetylglucosaminylation of proteins is a process required for hypoxic pancreatic cancer cell survival. Our results demonstrate that hypoxia-driven metabolic adaptive processes, such as high glycolytic rate and hexosamine biosynthetic pathway activation, favor hypoxic and normoxic cancer cell survival and correlate with pancreatic ductal adenocarcinoma aggressiveness.

TP53INP1s and homeodomain-interacting protein kinase-2 (HIPK2) are partners in regulating p53 activity.

The TP53INP1 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 p21, mdm2, pig3, and bax 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.

The Pancreatitis-Associated Protein Is Induced by Free Radicals in AR4-2J Cells and Confers Cell Resistance to Apoptosis

BACKGROUND & AIMS Free radicals are involved in the pathogenesis of acute pancreatitis, during which pancreatitis-associated protein (PAP)-I is overexpressed. We explored whether PAP-I expression could be induced by oxidative stress and whether it could affect apoptosis. METHODS AR4-2J cells were exposed to H2O2 or menadione, and PAP-I messenger RNA (mRNA) expression was analyzed by Northern blotting. RESULTS Maximal expression was observed with 0.1 mmol/L H2O2 or with 0.05 mmol/L menadione. Induction was detectable after 12 hours, reached a climax at 18 hours, and then decreased. Pretreatment of the cells with pyrrolidine dithiocarbamate completely abolished PAP-I mRNA induction, suggesting involvement of NFkappaB in the signaling pathway. These findings were confirmed in transient transfection assays using a plasmid containing the PAP-I promoter linked to the chloramphenicol acetyltransferase reporter gene. Then the relationship between PAP-I induction and protection against cell damage during oxidative stress was considered. Constitutive PAP-I expression in AR4-2J cells after transfection with PAP-I complementary DNA conferred significant resistance to apoptosis induced by low doses of H2O2 but not to necrosis induced by high doses of H2O2. CONCLUSIONS These results suggest that during oxidative stress, PAP-I might be part of a mechanism of pancreatic cell protection against apoptosis.

Tumor Protein 53-Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

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 Ser(58) (equivalent to human p53 Ser(46)) abrogates transcription of these genes, indicating that TP53INP1-mediated p53 Ser(58) 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.

Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identification of two functional interleukin-6 response elements in the rat PAP I promoter region.

Expression of the pancreatitis-associated protein I (PAP I), an exocrine pancreatic protein, increases rapidly and strongly in acinar cells during the acute phase of pancreatitis. This is reminiscent of the response to stress of acute phase proteins. We have previously demonstrated that serum factors from rats with acute pancreatitis, but not from healthy rats, could induce endogenous PAP I gene expression in the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C., Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204, 238-243). In the present work, we have evaluated the influence of several mediators of inflammation on rat PAP I gene transcription in these cells. Tumor necrosis factor α induced an increase in PAP I mRNA expression, and interferon caused an even greater increase in PAP I mRNA level. These stimulations were antagonized by dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also ineffective. IL-6 and dexamethasone together induced a marked stimulation of PAP I gene transcription, and this effect was slightly attenuated by IL-1. To analyze the cis-regulatory elements responsible for the induction of transcription, we fused a 1.2-kilobase segment of the rat PAP I promoter to the chloramphenicol acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA was transfected into AR-42J cells. Addition of IL-6 or dexamethasone was ineffective, whereas their mixture increased the CAT activity 12 times. Progressive deletions of the PAP I promoter were then fused to the CAT gene, and the constructs were transfected to AR-42J cells. A 12-fold increase in CAT activity was seen upon IL-6/dexamethasone treatment with constructs containing more than 274 base pairs upstream from the cap site. In that region, two sequences are similar to the canonical IL-6 response element. Site-directed mutagenesis of these regions strongly decreased induction, showing that they were functional. PAP I should therefore be classified among acute phase proteins of class 2, whose expression is increased by IL-6 acting in combination with glucocorticoids.

TP53INP1 is a novel p73 target gene that induces cell cycle arrest and cell death by modulating p73 transcriptional 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.

Colitis and Colitis-Associated Cancer Are Exacerbated in Mice Deficient for Tumor Protein 53-Induced Nuclear Protein 1

ABSTRACT 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.

TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death

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.