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.apoptosis ͉ pancreatic cancer ͉ ponasterone A ͉ tumor suppressor ͉ micro RNA

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 underexpressed 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. Colorectal cancer is the second most common tumour in men and the third in women in the Western countries. Although much is known about the epidemiology, morphology and genetics of colorectal tumorigenesis, our knowledge of the perturbations of gene expression that occur in colorectal tumours remains limited. Such tumours may arise from benign adenomatous polyps, which later progress to adenocarcinomas through several molecular events. They thus provide a very useful paradigm for studying the molecular genetic bases of cancer. The multistep process leading to colorectal tumorigenesis probably involves the loss of function of tumour-suppressor genes, as well as the activation of oncogenes. Several important genes have already been identified, but they do not account for the whole process, and other genes are probably involved.Efforts have been made to characterize these genes. Differential hybridization techniques and screening of substracted libraries allowed the elucidation of some of them (Bartsch et al., 1986;Denis et al., 1993;Yow et al., 1988;Schweinfest et al., 1993;Kondoh et al., 1992; Barnard et al., 1992a, b). We developed an alternative strategy, in which the phenotype of a colorectal tumour was established by partial sequencing of a large number of randomly selected transcripts (Frigerio et al., 1995). This repertory of ESTs should therefore contain most of the differentially expressed genes. Recently, Nguyen et al. (1995) have developed an efficient method of differential screening in which cDNA clones are gridded on high-density colony filters and hybridized with complex probes derived from poly (A) 1 RNA from different cells or tissues. The signals observed are measured, providing a ''hybridization s...

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 GABARAPlike2, 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.

TP53INP1 is an alternatively spliced gene encoding two nuclear protein isoforms (TP53INP1a and TP53INP1b), 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 À/À mice and in cisplatin-treated p53 À/À 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.

We have previously described an inverse relationship between Cdx1 and Cdx2 mRNA levels and the extent of dysplasia and severity of clinical outcome in colorectal carcinoma, suggesting that altered expression of these genes was associated with colorectal carcinogenesis or tumor progression. To investigate further their involvement in the physiopathology of colorectal cancer, HT29 colon carcinoma cells that show very low Cdx expression were transfected with Cdx1 and/or Cdx2 cDNA to elicit their overexpression. Growth rate, tumorigenicity, resistance to apoptosis, and migration potential of the corresponding cells were analyzed. Growth rate of cells overexpressing Cdx2 decreased by half, whereas overexpression of Cdx1 had no effect. However, cells overexpressing both Cdxs had a growth rate reduced to 20% of control. In cells overexpressing Cdx1 or Cdx2, tumorigenicity and resistance to apoptosis induced by serum starvation, ceramide, or staurosporine were not changed compared with control cells; yet phorbol esterstimulated cell migration was decreased by 50%. In cells overexpressing both Cdx1 and Cdx2, tumorigenicity was decreased by 50%, resistance to apoptosis was significantly lowered, and stimulated cell migration was further decreased to 15% of control compared with cells expressing Cdx1 or Cdx2. Finally, cells overexpressing both Cdxs showed strongly decreased Bcl-2 expression, which could account for their increased sensitivity to apoptosis. These findings show that, in HT29 cells, both Cdx1 and Cdx2 genes must be expressed to reduce tumorigenic potential, to increase sensitivity to apoptosis, and to reduce cell migration, suggesting that the two genes control the normal phenotype by independent pathways. This may explain why loss of Cdx1 or Cdx2 expression is associated with tumor development and invasiveness in colorectal tumors.In an effort to characterize the mechanisms involved in colorectal cancer initiation and progression, we have developed a strategy based on the constitution of a large repertoire of transcripts from a colorectal tumor, all characterized by partial sequencing (1). Expression of these expressed sequence tags in normal and cancerous colon was compared, and those most differentially expressed were selected. Genes detected by these means may be causative or instrumental in tumor induction or/and progression. Looking for such genes, we found that the Cdx1 and Cdx2 homeotic genes were concomitantly down-regulated in about 85% of colorectal cancers (2). Such low expression of Cdx1 or Cdx2 in colon carcinoma was verified by immunohistochemistry (3, 4) and by reverse transcription polymerase chain reaction (5) studies. Cdx1 and Cdx2 are interesting candidates that could play a role in colon cancer pathology because Chawengsaksophak et al. (6) recently reported the occurrence of multiple intestinal adenomatous polyps in the proximal colon of Cdx2 ϩ/Ϫ mice, suggesting that lowering Cdx2 levels in intestinal cells would suffice to induce intestinal tumors. Also, Suh and Traber showed that expre...