Reduced nuclear protein 1 expression improves insulin sensitivity and protects against diet-induced glucose intolerance through up-regulation of heat shock protein 70.

We recently reported that deletion of the stress-regulated nuclear protein 1 (Nupr1) protected against obesity-associated metabolic alterations due to increased beta cell mass, but complete Nupr1 ablation was not advantageous since it led to insulin resistance on a normal diet. The current study used Nupr1 haplodeficient mice to investigate whether a partial reduction in Nupr1 expression conferred beneficial effects on glucose homeostasis. Islet number, morphology and area, assessed by immunofluorescence and morphometric analyses, were not altered in Nupr1 haplodeficient mice under normal diet conditions and nor was beta cell BrdU incorporation. Glucose and insulin tolerance tests indicated that there were no significant changes in in vivo insulin secretion and glucose clearance in Nupr1 haplodeficient mice, and beta cell function in vitro was normal. However, reduced Nupr1 expression decreased visceral fat deposition and significantly increased insulin sensitivity in vivo. In contrast to wild type animals, high fat diet-fed Nupr1 haplodeficient mice were not hyperinsulinaemic or glucose intolerant, and their sustained insulin sensitivity was demonstrated by appropriate insulin-induced Akt phosphorylation, as determined by Western blotting. At the molecular level, measurements of gene expression levels and promoter activities identified Nupr1-dependent inhibition of heat shock factor-1-induced heat shock protein 70 (Hsp70) expression as a mechanism through which Nupr1 regulates insulin sensitivity. We have shown for the first time that Nupr1 plays a central role in inhibiting Hsp70 expression in tissues regulating glucose homeostasis, and reductions in Nupr1 expression could be used to protect against the metabolic defects associated with obesity-induced insulin resistance.

New strategies and designs in pancreatic cancer research: consensus guidelines report from a European expert panel.

Although the treatment of pancreatic ductal adenocarcinoma (PDAC) remains a huge challenge, it is entering a new era with the development of new strategies and trial designs. Because there is an increasing number of novel therapeutic agents and potential combinations available to test in patients with PDAC, the identification of robust prognostic and predictive markers and of new targets and relevant pathways is a top priority as well as the design of adequate trials incorporating molecular-driven hypothesis. We presently report a consensus strategy for research in pancreatic cancer that was developed by a multidisciplinary panel of experts from different European institutions and collaborative groups involved in pancreatic cancer. The expert panel embraces the concept of exploratory early proof of concept studies, based on the prediction of response to novel agents and combinations, and randomised phase II studies permitting the selection of the best therapeutic approach to go forward into phase III, where the recommended primary end point remains overall survival. Trials should contain as many translational components as possible, relying on standardised tissue and blood processing and robust biobanking, and including dynamic imaging. Attention should not only be paid to the pancreatic cancer cells but also to microenvironmental factors and stem/stellate cells.

Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway.

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Tumor protein p53-induced nuclear protein (TP53INP1) expression in medullary thyroid carcinoma: a molecular guide to the optimal extent of surgery?

BACKGROUND: Medullary thyroid cancer (MTC) is characterized by early regional lymph node metastasis, the presence of which represents a critical obstacle to cure. At present no molecular markers have been successfully integrated into the clinical care of sporadic MTC. The present study was designed to evaluate TP53INP1 expression in MTC and to assess its ability to guide the surgeon to the optimal extent of surgery performed with curative intent. METHODS: Thirty-eight patients with sporadic MTC were evaluated. TP53INP1 immunoexpression was studied on embedded paraffin material and on cytological smears. RESULTS: TP53INP1 was expressed in normal C cells, in C-cell hyperplasia, and in 57.9% of MTC. It was possible to identify two groups of MTC according to the proportion of TP53INP1 expressing tumor cells: group 1 from 0% to <50% and group 2 from 50% to 100% of positive cells. Patients with a decreased expression of TP53INP1 (group 1) had a lower rate of nodal metastasis (18.8% versus 63.4% in group 2; P = 0.009), with only minimal lymph node involvement per N1 patient (2.7% of positive lymph nodes versus 22.9%; P < 0.001) and better outcomes (100% of biochemical cure versus 55.5%; P < 0.001). Patients with distant metastases were only observed in group 2. Cytological samples exhibit similar results to their embedded counterparts. CONCLUSIONS: TP53INP1 immunoexpression appears to be a clinical predictor of lymph node metastasis in MTC. The evaluation of TP53INP1 expression may guide the extent of lymph node dissection in the clinically node-negative neck. These findings require prospective validation.

PAP1 signaling involves MAPK signal transduction.

Pancreatitis-associated protein 1 (PAP1) belongs to the Reg family of secretory proteins. Several important biological roles have been attributed to PAP1 but the signaling pathways activated by this protein remain only partially understood. Here, we describe the intracellular pathways triggered by PAP1 in a pancreatic acinar cell line. Taking advantage of the fact that PAP1 induces its own transcription, we performed ChIP assays to analyze the recruitment of transcriptional factors on its promoter. Our results show that PAP1 increased the transactivation activity of pap1 and the binding on its promoter of the nuclear factors C/EBPbeta, P-CREB, P-ELK1, EGR1, STAT3, and ETS2, which are downstream targets of MAPK signaling. p44/42, p38, and JNK MAPKs activity increased after PAP1 treatment. In addition, pharmacological inhibition of these kinases markedly inhibited the induction of pap1 mRNA. Taken together, these results indicated that the mechanism of PAP1 action involves the activation of the MAPK superfamily.

Human pancreatitis-associated protein. Messenger RNA cloning and expression in pancreatic diseases.

A human pancreatic cDNA library was screened with the cDNA encoding rat "pancreatitis-associated protein" (PAP). The selected clone encoded a secretory protein structurally related to rat PAP. The protein had the same size as rat PAP and showed 71% amino acid identity, the six half-cystines being in identical positions. Domains of the proteins showing homologies with calcium-dependent lectins were also conserved. In addition, expression in pancreas of the genes encoding the human protein and rat PAP showed similar characteristics: both were expressed at very low levels in control tissue and overexpressed during the acute phase of pancreatitis, contrary to most secretory products. The human protein was therefore named human pancreatitis-associated protein (PAP-H). Antibodies raised to a synthetic peptide of PAP-H detected a single band with an M(r) compatible with PAP-H in Western blot analysis of proteins extracted from a pancreas presenting with acute pancreatitis. In that tissue, the protein could be immunolocalized to the apical regions of acinar cells. An immunoassay was also constructed to quantify the protein in serum. Elevated PAP-H levels were observed in patients with acute pancreatitis and in some patients with chronic pancreatitis. Values were close to background in healthy subjects and in patients with other abdominal diseases. These results confirm that PAP-H synthesis increases during inflammation and suggest a possible use of the protein as biological marker of acute pancreatitis.

Resectable pancreatic head adenocarcinoma: Is R0 resection an illusion? Genetic evaluation of venous resection margin affirmed unrecognized disease.

PURPOSE: To assess the K-ras gene mutation in the histologically negative venous margin of a pancreaticoduodenectomy (PD) specimen and its impact on survival. METHOD: From 2007 to 2010, 22 patients underwent R0 PD for resecable pancreatic adenocarcinoma. All specimens were stained and the portal vein (PV) bed was identified by blue ink; a 2mm3 sample (including the blue ink) was cut from a microscopic free-tumor block. DNA was extracted and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation. Twelve specimens (55%) (kras+ group) were identified with a K-ras mutation in the venous margin resection, and 10 specimens (kras- group) did not have K-ras mutation detected in the venous margin resection. RESULTS: The two groups were comparable. Overall 3years survival of patients of kras+ group versus patients of kras- group was 0 and 17% (P=0.03), respectively. Median survival time of patients of kras+ group versus patients of kras- group was 16months vs 25months (P=0.04; 95% confidence interval [1,11-1,88]), respectively. CONCLUSION: Genetic evaluation of venous resection margin affirmed unrecognized disease with strong impact on survival in more than 50% of patients with histologically R0 resection.

The pancreatitis-associated protein VMP1, a key regulator of inducible autophagy, promotes Kras(G12D)-mediated pancreatic cancer initiation.

Both clinical and experimental evidence have firmly established that chronic pancreatitis, in particular in the context of Kras oncogenic mutations, predisposes to pancreatic ductal adenocarcinoma (PDAC). However, the repertoire of molecular mediators of pancreatitis involved in Kras-mediated initiation of pancreatic carcinogenesis remains to be fully defined. In this study we demonstrate a novel role for vacuole membrane protein 1 (VMP1), a pancreatitis-associated protein critical for inducible autophagy, in the regulation of Kras-induced PDAC initiation. Using a newly developed genetically engineered model, we demonstrate that VMP1 increases the ability of Kras to give rise to preneoplastic lesions, pancreatic intraepithelial neoplasias (PanINs). This promoting effect of VMP1 on PanIN formation is due, at least in part, by an increase in cell proliferation combined with a decrease in apoptosis. Using chloroquine, an inhibitor of autophagy, we show that this drug antagonizes the effect of VMP1 on PanIN formation. Thus, we conclude that VMP1-mediated autophagy cooperate with Kras to promote PDAC initiation. These findings are of significant medical relevance, molecules targeting autophagy are currently being tested along chemotherapeutic agents to treat PDAC and other tumors in human trials.

TAp73 loss favors Smad-independent TGF-ß signaling that drives EMT in pancreatic ductal adenocarcinoma.

Advances made in pancreatic cancer therapy have been far from sufficient and have allowed only a slight improvement in global survival of patients with pancreatic ductal adenocarcinoma (PDA). Recent progresses in chemotherapy have offered some hope for an otherwise gloomy outlook, however, only a limited number of patients are eligible because of important cytotoxicity. In this context, enhancing our knowledge on PDA initiation and evolution is crucial to highlight certain weaknesses on which to specifically target therapy. We found that loss of transcriptionally active p73 (TAp73), a p53 family member, impacted PDA development. In two relevant and specific engineered pancreatic cancer mouse models, we observed that TAp73 deficiency reduced survival and enhanced epithelial-to-mesenchymal transition (EMT). Through proteomic analysis of conditioned media from TAp73 wild-type (WT) and deficient pancreatic tumor cells, we identified a secreted protein, biglycan (BGN), which is necessary and sufficient to mediate this pro-EMT effect. Interestingly, BGN is modulated by and modulates the transforming growth factor-ß (TGF-ß) pathway, a key regulator of the EMT process. We further examined this link and revealed that TAp73 impacts the TGF-ß pathway by direct regulation of BGN expression and Sma and Mad-related proteins (SMADs) expression/activity. Absence of TAp73 leads to activation of TGF-ß signaling through a SMAD-independent pathway, favoring oncogenic TGF-ß effects and EMT. Altogether, our data highlight the implication of TAp73 in the aggressiveness of pancreatic carcinogenesis through modulation of the TGF-ß signaling. By suggesting TAp73 as a predictive marker for response to TGF-ß inhibitors, our study could improve the classification of PDA patients with a view to offering combined therapy involving TGF-ß inhibitors.

NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance.

Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits are modest, and as its mechanisms of action remain elusive, a better understanding of its anticancer effects is needed. Based on our previous study results, we investigated here the implication of the nuclear protein 1 (NUPR1) in HCC and its role in sorafenib treatment. NUPR1 is a stress-inducible protein that is overexpressed in various malignancies, but its role in HCC is not yet fully understood. We found that NUPR1 expression was significantly higher in primary human HCC samples than in the normal liver. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibited the cell growth, migration and invasion of HCC cells, both in vitro and in vivo. Moreover, NUPR1 silencing influenced the expression of RELB and IER3 genes. Unsurprisingly, RELB and IER3 knockdown also inhibited HCC cell viability, growth and migration. Using gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as downregulated gene nodes, and several HCC-related oncogenes were also suppressed. We identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1-regulated gene and demonstrated that RUNX2 gene silencing inhibits HCC cell viability, growth, migration and increased cell sensitivity to sorafenib. We propose that the NUPR1/RELB/IER3/RUNX2 pathway has a pivotal role in hepatocarcinogenesis. The identification of the NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management.

Rapid PCR cloning and sequence determination of the rat lithostathine gene.

The rat lithostathine gene was isolated from a genomic library using a rapid screening procedure involving PCR amplification. It was characterized over 2.7 kbp of gene sequence and 2.43 kbp of 5'-flanking sequence. The 5'-end of the coding sequence was determined by primer extension of lithostathine mRNA. The lithosathine sequence spanned over six exons. The promoter region of the gene contained the TATAAA and CCAAT consensus sequences 30 and 107 bp upstream of the cap site, respectively. Furthermore, a tract of (TG)22 repeat, with potential Z-DNA conformation, was found at position-1081.

Cloning, sequencing and expression of the L5, L21, L27a, L28, S5, S9, S10 and S29 human ribosomal protein mRNAs.

During systematic analysis of the mRNAs expressed in a human colorectal carcinoma with the aim of evidencing new makers of the disease (Frigerio et al. (1995), in press), we isolated several clones corresponding to homologs of rat ribosomal protein mRNAs L5, L21, L27a, L28, S5, S9, S10 and S29. Because expression of several mRNAs encoding ribosomal proteins was found strongly altered during colorectal carcinogenesis, sequence of these transcripts, not previously described in human, was completed and their expression analyzed. Northern blot analysis of RNAs extracted from colorectal cancer and ajdacent normal tissue from 6 patients revealed in all of them perturbations of expression in cancer, compared to normal. No correlation could however be made between the level of expression and the severity of the disease. Yet, abnormal patterns with additional larger transcripts were observed in some patients for rpL5, rpL28 and rpS10.

The pancreatitis associated protein III (PAP III), a new member of the PAP gene family.

A third member of the rat pancreatitis associated protein (PAP) gene family is described here. Its messenger RNA was cloned from an intestinal cDNA library and sequenced. The encoded protein, designated PAP III, shows 66% and 63% identity with the rat PAP I and II, respectively. The PAP III gene is constitutively expressed in the small intestine and in the pancreas with acute pancreatitis, but not in the healthy pancreas.

Two transcripts are generated from the pancreatitis associated protein II gene by alternative splicing in the 5' untranslated region.

We have previously reported the coding sequence of the rat PAP II mRNA. We show in this paper the existence in rat pancreas of two forms of PAP II mRNA with identical coding sequence but a different 5'-untranslated region. We demonstrate that this is the result of a differential splicing.

Expression of p8 in human pancreatic cancer.

The p8 gene is a recently identified gene with mitogenic activity. p8 expression is induced in acute pancreatitis, pancreatic development, and regeneration. However, the expression of p8 in pancreatic cancer is not reported. We investigated p8 expression in 72 human pancreatic tissues, including 38 pancreatic cancers (PCs), by immunohistochemistry. p8 was overexpressed (positive cells >25% in 1,000 cells) in 71% (27 of 38) of PCs, but in only 17% (3 of 18) of chronic pancreatitis cases. There was no overexpression in mucinous cystadenoma or in normal pancreas. The p8 overexpression rate in PC was significantly higher than that in other conditions (P < 0.05). Reverse transcription-PCR analysis confirmed p8 mRNA overexpression (tumor/nontumor ratio >2) in 75% (3 of 4) of PCs. p8 was overexpressed also in human pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). These results suggest that p8 is involved in the development of pancreatic cancer, reflecting its mitogenic activity.

Overexpression of p8 is inversely correlated with apoptosis in pancreatic cancer.

A recently identified gene, p8, has cell growth-promoting activity and is strongly induced in acute pancreatitis. In this study, we detected p8 and single-stranded DNA (ssDNA) for apoptosis by immunohistochemistry in human pancreatic cancer. The p8 was overexpressed (>30% per 1000 cancer cells) in 26 of 44 (59%) pancreatic cancers, and apoptosis (ssDNA-positive cells >10% per 1000 cancer cells) was recognized in 18 of 44 (41%) pancreatic cancers. There was a significant inverse correlation between the p8 overexpression and apoptosis (P < 0.05). Moreover, the expression pattern of high p8 and low ssDNA was seen significantly more often in lower age (<65 years), in moderately or poorly differentiated cancers, and in node-positive cases (P < 0.05). The p8 expression and apoptosis were not significantly correlated with survival. These results suggest that p8 overexpression is involved in antiapoptotic activity and the biological characteristics of pancreatic cancer.

Stromal SLIT2 impacts on pancreatic cancer-associated neural remodeling.

Pancreatic ductal adenocarcinoma (PDA) is a critical health issue in the field of cancer, with few therapeutic options. Evidence supports an implication of the intratumoral microenvironment (stroma) on PDA progression. However, its contribution to the role of neuroplastic changes within the pathophysiology and clinical course of PDA, through tumor recurrence and neuropathic pain, remains unknown, neglecting a putative, therapeutic window. Here, we report that the intratumoral microenvironment is a mediator of PDA-associated neural remodeling (PANR), and we highlight factors such as 'SLIT2' (an axon guidance molecule), which is expressed by cancer-associated fibroblasts (CAFs), that impact on neuroplastic changes in human PDA. We showed that 'CAF-secreted SLIT2' increases neurite outgrowth from dorsal root ganglia neurons as well as from Schwann cell migration/proliferation by modulating N-cadherin/ß-catenin signaling. Importantly, SLIT2/ROBO signaling inhibition disrupts this stromal/neural connection. Finally, we revealed that SLIT2 expression and CAFs are correlated with neural remodeling within human and mouse PDA. All together, our data demonstrate the implication of CAFs, through the secretion of axon guidance molecule, in PANR. Furthermore, it provides rationale to investigate the disruption of the stromal/neural compartment connection with SLIT2/ROBO inhibitors for the treatment of pancreatic cancer recurrence and pain.

Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation.

Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis.

Expression of the stress-induced p8 mRNA is transiently activated after culture medium change.

We report here that the mere fact of changing culture medium for fresh medium induced in several cell lines the expression of stress-activated genes including protein kinases p38, JNK and ERK1/2 and the transcription factor C/EBPbeta. As a consequence, p8, a gene induced by stress in several tissues, was strongly up-regulated. Induction did not occur after change for cell-conditioned medium. Induction was however transient, with a peak at 60 min for p38, at 15-30 min for JNK and at 15 min for ERK1/2, at 2-3 hours for C/EBPbeta and at 4-6 hours for p8. Repression of the induction was due to the secretion of thermolabile molecule(s) that progressively conditioned the medium. As low as 25% of conditioned medium added to fresh culture medium was sufficient to abolish the stress response. Taken together, our data indicate that the renewal of culture medium induces a transient cellular stress that may be a source of artifacts in experiments performed shortly after a change of culture medium.

Cdx1 promotes cellular growth of epithelial intestinal cells through induction of the secretory protein PAP I.

Expression of the Cdx1 homeobox gene in epithelial intestinal cells promotes cellular growth and differentiation. Cdx1and the Pancreatitis Associated Protein I (PAP I) are concomitantly expressed in the epithelial cells of the lower part of the intestinal crypts. Because Cdx1 is a transcription factor and PAP I, in other tissues, is a proliferative factor, we looked for a relationship between these two proteins in the intestinal-derived IEC-6 cells. After stable transfection with a Cdx1 expression vector, they produce high levels of the PAP I transcript and protein indicating a functional link between the two genes. Demonstration of Cdx1 binding to the PAP I promoter region and suppression of PAP I induction after deletion of the corresponding sequence indicated that Cdx1 is a transcription factor controlling PAP I gene expression in intestinal cells. By infecting IEC-6 cells with adenoviruses expressing PAP I, we demonstrated that PAP I induces mitosis in these cells. On the other hand, inhibition of the PAP I expression in the IEC-6 Cdxl-expressing cells using an antisense strategy confirmed the requirement of this protein for the effect of Cdx1 on cell growth. Finally, addition of the immunopurified PAP I to the culture medium promotes cell growth of the IEC-6 cells in a dose-dependent manner. Maximal effect was obtained at 1 ng/ml. Taken together these results demonstrate that PAP I is a target of the Cdx1 homeobox gene in intestinal cells which participates in the regulation of intestinal cell growth via an autocrine and/or paracrine mechanism.