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Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive malignancy characterized by an excessive resistance to all known anticancer therapies, a still largely elusive phenomenon. To identify original mechanisms, we have explored the role of post-translational modifications (PTMs) mediated by members of the ubiquitin family. Although alterations of these pathways have been reported in different cancers, no methodical search for these kinds of anomalies has been performed so far. Therefore, we studied the ubiquitin-, Nedd8-, and SUMO1-specific proteomes of a pancreatic cancer cell line (MiaPaCa-2) and identified changes induced by gemcitabine, the standard PDAC's chemotherapeutic drug. These PTMs profiles contained both known major substrates of all three modifiers as well as original ones. Gemcitabine treatment altered the PTM profile of proteins involved in various biological functions, some known cancer associated genes, many potentially cancer-associated genes, and several cancer-signaling networks, including canonical and noncanonical WNT and PI3K/Akt/MTOR pathways. Some of these altered PTMs formed groups of functionally and physically associated proteins. Importantly, we could validate the gemcitabine-induced PTMs variations of relevant candidates and we could demonstrate the biological significance of such altered PTMs by studying in detail the sumoylation of SNIP1, one of these new targets.

Background: Pancreatic cancer cells generate metastases because they can survive the stress imposed by the new environment of the host tissue. To mimic this process, pancreatic cancer cells which are not stressed in standard culture conditions are injected into nude mice. Because they develop xenografts, they should have developed adequate stress response. Characterizing that response might provide new strategies to interfere with pancreatic cancer metastasis., Methodology/principal Findings: In the human pancreatic cancer cell lines Panc-1, Mia-PaCa2, Capan-1, Capan-2 and BxPC3, we used Affymetrix DNA microarrays to compare the expressions of 22.000 genes in vitro and in the corresponding xenografts. We identified 228 genes overexpressed in xenografts and characterized the implication of one of them, WSB1, in the control of apoptosis and cell proliferation. WSB1 generates 3 alternatively spliced transcripts encoding distinct protein isoforms. In xenografts and in human pancreatic tumors, global expression of WSB1 mRNA is modestly increased whereas isoform 3 is strongly overexpressed and isoforms 1 and 2 are down-regulated. Treating Mia-PaCa2 cells with stress-inducing agents induced similar changes. Whereas retrovirus-forced expression of WSB1 isoforms 1 and 2 promoted cell growth and sensitized the cells to gemcitabine- and doxorubicin-induced apoptosis, WSB1 isoform 3 expression reduced cell proliferation and enhanced resistance to apoptosis, showing that stress-induced modulation of WSB1 alternative splicing increases resistance to apoptosis of pancreatic cancer cells., Conclusions/significance: Data on WSB1 regulation support the hypothesis that activation of stress-response mechanisms helps cancer cells establishing metastases and suggest relevance to cancer development of other genes overexpressed in xenografts.

p8 is a transcription cofactor whose expression is strongly and rapidly activated in pancreatic acinar cells during the acute phase of pancreatitis. A p8-deficient mouse strain was generated as a tool to investigate its function. Upon induction of acute pancreatitis, myeloperoxidase activity in pancreas and serum concentrations of amylase and lipase were much higher and pancreatic lesions more severe in p8-deficient mice than in wild-type, indicating that p8 expression decreased pancreatic sensitivity to pancreatitis induction. The protective mechanism might involve the pancreatitis-associated protein (PAP I), whose strong induction during pancreatitis is p8-dependent, because administration of anti-PAP I antibodies to rats increased pancreatic inflammation during pancreatitis. In addition, 100 ng/ml PAP I in the culture medium of macrophages prevented their activation by tumor necrosis factor alpha, strongly suggesting that PAP I was an anti-inflammatory factor. Finally, PAP I was able to inhibit NFkappaB activation by tumor necrosis factor alpha, in macrophages and in the AR42J pancreatic acinar cell line. In conclusion, p8 improves pancreatic resistance to inducers of acute pancreatitis by a mechanism implicating the expression of the anti-inflammatory protein PAP I.

The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)-associated protein, which in turn binds the DNA-damage-associated 53BP1 protein to facilitate DNA repair following DNA γ-irradiation. Contrary to the HAT-associated activity, MSL1-dependent DNA-repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1-dependent DNA-repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after γ-irradiation, it is eventually submitted to sustained down-regulation, presumably to allow development of MSL1-associated DNA-repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1-dependent HAT activity to the vicinity of damaged DNA. MSL1-dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery. J. Cell. Physiol. 221: 594–602, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Pancreatitis-associated protein (PAP) is almost absent in normal pancreas, but is strongly induced in acute pancreatitis. PAP mRNA is also expressed in cancer cells, including pancreatic ductal adenocarcinoma. However, the clinicopathological significance of PAP in human pancreatic cancer is not clear. We examined PAP expression in pancreatic tissues from individuals with pancreatic ductal adenocarcinoma using immunohistochemistry. PAP was overexpressed in 79% (30 of 38) of pancreatic ductal adenocarcinoma, 19% (7 of 36) of chronic pancreatitis, and 29% (2 of 7) of mucinous cystadenoma. PAP was found in malignant ductular structures in pancreatic carcinomas as well as in benign proliferating ductules and acinar cells in chronic pancreatitis. It was not expressed in normal pancreas. The incidence of PAP overexpression was significantly higher in pancreatic cancer than in the other pancreatic diseases (P < 0.01). PAP overexpression was significantly correlated with nodal involvement, distant metastasis (P < 0.05), and short survival (P < 0.01) in pancreatic cancer. These results suggest that overexpression of PAP in human pancreatic ductal adenocarcinoma indicates tumor aggressiveness. [ABSTRACT FROM AUTHOR]

Background: Ras is an area of intensive biochemical and genetic studies and characterizing downstream components that relay ras-induced signals is clearly important. We used a systematic approach, based on DNA microarray technology to establish a first catalog of genes whose expression is altered by ras and, as such, potentially involved in the regulation of cell growth and transformation. Results: We used DNA microarrays to analyze gene expression profiles of rasV12/E1A-transformed mouse embryonic fibroblasts. Among the ~12,000 genes and ESTs analyzed, 815 showed altered expression in rasV12/E1A-transformed fibroblasts, compared to control fibroblasts, of which 203 corresponded to ESTs. Among known genes, 202 were up-regulated and 410 were down-regulated. About one half of genes encoding transcription factors, signaling proteins, membrane proteins, channels or apoptosis-related proteins was up-regulated whereas the other half was down-regulated. Interestingly, most of the genes encoding structural proteins, secretory proteins, receptors, extracellular matrix components, and cytosolic proteins were down-regulated whereas genes encoding DNA-associated proteins (involved in DNA replication and reparation) and cell growth-related proteins were up-regulated. These data may explain, at least in part, the behavior of transformed cells in that down-regulation of structural proteins, extracellular matrix components, secretory proteins and receptors is consistent with reversion of the phenotype of transformed cells towards a less differentiated phenotype, and up-regulation of cell growth-related proteins and DNA-associated proteins is consistent with their accelerated growth. Yet, we also found very unexpected results. For example, proteases and inhibitors of proteases as well as all 8 angiogenic factors present on the array were down-regulated in transformed fibroblasts although they are generally up-regulated in cancers. This observation suggests that, in human cancers, proteases, protease inhibitors and angiogenic factors could be regulated through a mechanism disconnected from ras activation. Conclusions: This study established a first catalog of genes whose expression is altered upon fibroblast transformation by rasV12/E1A. This catalog is representative of the genome but not exhaustive, because only one third of expressed genes was examined. In addition, contribution to ras signaling of posttranscriptional and post-translational modifications was not addressed. Yet, the information gathered should be quite useful to future investigations on the molecular mechanisms of oncogenic transformation. [ABSTRACT FROM AUTHOR]

The p8 protein is involved in the cellular stress response of many tissues. Because p8 is overexpressed in many cancers, we investigated whether its expression was required for tumour development. Mouse embryo fibroblasts (MEFs) from p8+/+ and p8-/- animals were transformed with the pBaberasV12/E1A retroviral vector, which expresses both the rasV12 mutated protein and the E1A oncogene. As expected, transformed p8+/+ MEFs could form colonies in soft agar. However, transformed p8-/- MEFs could not. In addition, subcutaneous or intraperitoneal injections of transformed p8-/- MEFs always led to tumour formation in nude mice, but, again, no tumour was observed with transformed p8-/-MEFs. However, restoring p8 expression in transformed p8-/- MEFs before injection led to tumour formation. In the tumours, p8 expression was induced during tumour development It was concluded that p8 expression in transformed MEFs is necessary for tumour formation, suggesting that the stress-response mechanisms governed by p8 are required for tumour establishment. [ABSTRACT FROM AUTHOR]

Investigates the presence of pancreatitis-associated protein (PAP), which is present in small amounts in rat pancreas, in rat intestines. Probing of the cytoplasmic DNA library from rat jejunum; Immunodetection of PAP transcripts in duodenum, jejunum and ileum homogenates; Influence of fasting and feeding on PAP mitochondrial RNA concentration.

Pancreatitis-associated protein I (PAP I) is a pancreatic secretory protein strongly expressed during acute pancreatitis in the rat and human. We hypothesized that its expression was part of a general and coordinated response of the organ against aggression. An opposite pattern of PAP I mRNA expression has recently been described in the mouse. The murine PAP I mRNA was described to be highly expressed in normal pancreas and down-regulated during pancreatitis. The important implications of these unexpected findings led us to investigate the expression of murine PAP I in cerulein-induced pancreatitis. Northern blot analysis demonstrated a very low level of PAP I mRNA in the healthy mouse pancreas and strong overexpression during acute pancreatitis. Western blot analysis confirmed that changes in pancreatic PAP I levels were parallel to those of the mRNA and the protein was localized by immunohistochemistry to the acinar cells. It was concluded that, during the course of acute pancreatitis, the pattern of PAP I expression in the mouse pancreas was comparable to that already observed in the rat and human. Although we have no explanation for the discrepancy between our results and those recently reported, the expression pattern of PAP I in the mouse exocrine pancreas described in the present study suggests that the pancreatic response to aggression might be conserved in mammals. [ABSTRACT FROM AUTHOR]

The multikinase inhibitor sorafenib was the first drug approved by the FDA for treating patients with advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a major challenge for improving the effectiveness of HCC treatment. Previously, we identified several genes modulated after sorafenib treatment of human HCC cells, including the stress-inducible nuclear protein 1 (NUPR1) gene. Multiple studies have shown that NUPR1 regulates autophagy, apoptosis, and chemoresistance. Here, we demonstrate that treatment of HCC cells with sorafenib resulted in the activation of autophagic flux. NUPR1 knock-down (KD) in HCC cells was associated with increased p62 expression, suggesting an impairment of autophagic flux, and with a significant increase of cell sensitivity to sorafenib. In NUPR1 KD cells, reduced levels of NUPR1 were associated with the increased expression of p73 as well as its downstream transcription targets PUMA, NOXA, and p21. Simultaneous silencing of p73 and NUPR1 in HCC cells resulted in increased resistance to sorafenib, as compared to the single KD of either gene. Conversely, pharmacological activation of p73, via the novel p73 small molecule activator NSC59984, determined synergistic anti-tumor effects in sorafenib-treated HCC cells. The combination of NSC59984 and sorafenib, when compared to either treatment alone, synergistically suppressed tumor growth of HCC cells in vivo. Our data suggest that the activation of the p73 pathway achieved by NUPR1 KD potentiates sorafenib-induced anti-tumor effects in HCC cells. Moreover, combined pharmacological therapy with the p73 activator NSC59984 and sorafenib could represent a novel approach for HCC treatment. [ABSTRACT FROM AUTHOR]

Objectives. p8 protein has mitogenic activity and is linked to the development of pancreatic carcinoma. However, little is known about the expression and physiological significance of this protein in other human carcinomas. Methods. In this study, we immunohistochemically investigated p8 expression in thyroid neoplasms as well as in the normal thyroid gland. Results. p8 was expressed in normal follicular cells, but no normal thyroid was regarded as overexpressing p8. On the other hand, 44.3% of papillary carcinoma overexpressed p8 and the incidence was directly linked to the tumor size (p=0.0340) and lymph node metastasis (p=0.0145). In follicular tumors, the incidence of p8 overexpression did not depend on histological type. In anaplastic (undifferentiated) carcinoma, p8 was overexpressed only in 5.0%, which was significantly lower than in papillary (p=0.0006) and follicular carcinomas (p=0.0049). In normal follicules and follicular tumors, p8 was localized mainly in the nucleus except for two adenomas. On the other hand, p8 localization was more cytoplasmic in papillary carcinoma larger than 1.0 cm (p=0.0186) and with a poorly differentiated lesion (p=0.0313). Conclusions. These results suggest that the overexpression and cytoplasmic localization of p8 protein may reflect disease progression of papillary carcinoma, whereas this protein plays little part in thyroid carcinoma after anaplastic transformation. [Copyright &y& Elsevier]