Collagen-derived proline promotes pancreatic ductal adenocarcinoma cell survival under nutrient limited conditions.

Tissue architecture contributes to pancreatic ductal adenocarcinoma (PDAC) phenotypes. Cancer cells within PDAC form gland-like structures embedded in a collagen-rich meshwork where nutrients and oxygen are scarce. Altered metabolism is needed for tumour cells to survive in this environment, but the metabolic modifications that allow PDAC cells to endure these conditions are incompletely understood. Here we demonstrate that collagen serves as a proline reservoir for PDAC cells to use as a nutrient source when other fuels are limited. We show PDAC cells are able to take up collagen fragments, which can promote PDAC cell survival under nutrient limited conditions, and that collagen-derived proline contributes to PDAC cell metabolism. Finally, we show that proline oxidase (PRODH1) is required for PDAC cell proliferation in vitro and in vivo. Collectively, our results indicate that PDAC extracellular matrix represents a nutrient reservoir for tumour cells highlighting the metabolic flexibility of this cancer.

Germline copy number variation in the YTHDC2 gene: does it have a role in finding a novel potential molecular target involved in pancreatic adenocarcinoma susceptibility?

OBJECTIVE: The vast majority of pancreatic cancers occurs sporadically. The discovery of frequent variations in germline gene copy number can significantly influence the expression levels of genes that predispose to pancreatic adenocarcinoma. We prospectively investigated whether patients with sporadic pancreatic adenocarcinoma share specific gene copy number variations (CNVs) in their germline DNA. PATIENTS AND METHODS: DNA samples were analyzed from peripheral leukocytes from 72 patients with a diagnosis of sporadic pancreatic adenocarcinoma and from 60 controls using Affymetrix 500K array set. Multiplex ligation-dependent probe amplification (MLPA) assay was performed using a set of self-designed MLPA probes specific for seven target sequences. RESULTS: We identified a CNV-containing DNA region associated with pancreatic cancer risk. This region shows a deletion of 1 allele in 36 of the 72 analyzed patients but in none of the controls. This region is of particular interest since it contains the YTHDC2 gene encoding for a putative DNA/RNA helicase, such protein being frequently involved in cancer susceptibility. Interestingly, 82.6% of Sicilian patients showed germline loss of one allele. CONCLUSIONS: Our results suggest that the YTHDC2 gene could be a potential candidate for pancreatic cancer susceptibility and a useful marker for early detection as well as for the development of possible new therapeutic strategies.

Neonatal screening for cystic fibrosis: comparing the performances of IRT/DNA and IRT/PAP.

BACKGROUND: French health authorities promoted a study on 553,167 newborns comparing the performances of IRT/DNA and IRT/PAP for CF newborn screening. METHODS: In parallel to IRT/DNA, PAP was assayed in newborns with IRT>50 µg/L. Provisional PAP cutoffs at 3.0 µg/L when 50100 were used. Positive newborns were subjected to sweat test. Optimal cutoffs were established by a non-inferiority method. RESULTS: 95 CF newborns were identified (83 classical forms (ClF), including 9 meconium ileus (MI), and 12 atypical (mild) forms (AF) Of them, IRT/DNA identified 85 (73 ClF including 5 MI and 12 AF). PAP cutoffs at 1.8 µg/L when 50< IRT<100 µg/L and 0.6 µg/L when IRT>100 µg/L would identify 82 CF: 77 ClF, including 8 MI, and 5 AF. The number of sweat tests was 314 and 1039 in the IRT/DNA and IRT/PAP strategies, respectively. CONCLUSIONS: Using the optimal cutoffs, the sensitivity of the IRT/PAP strategy would not be inferior to that of IRT/DNA if identification of MF is not required.

Analysis of germline gene copy number variants of patients with sporadic pancreatic adenocarcinoma reveals specific variations.

OBJECTIVES: The rapid fatality of pancreatic cancer is, in large part, the result of diagnosis at an advanced stage in the majority of patients. Identification of individuals at risk of developing pancreatic adenocarcinoma would be useful to improve the prognosis of this disease. There is presently no biological or genetic indicator allowing the detection of patients at risk. Our main goal was to identify copy number variants (CNVs) common to all patients with sporadic pancreatic cancer. METHODS: We analyzed gene CNVs in leukocyte DNA from 31 patients with sporadic pancreatic adenocarcinoma and from 93 matched controls. Genotyping was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix). RESULTS: We identified 431 single nucleotide polymorphism (SNP) probes with abnormal hybridization signal present in the DNA of all 31 patients. Of these SNP probes, 284 corresponded to 3 or more copies and 147 corresponded to 1 or 0 copies. Several cancer-associated genes were amplified in all patients. Conversely, several genes supposed to oppose cancer development were present as single copy. CONCLUSIONS: These data suggest that a set of 431 CNVs could be associated with the disease. This set could be useful for early diagnosis.

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.

Artificial neural networks for assessing the risk factors for urinary calcium stones according to gender and family history of stone.

OBJECTIVE: In a previous work, we evidenced that artificial neural networks (ANNs) were more informative than classical statistical analyses for assessing the risk of idiopathic calcium nephrolithiasis in male stone-formers. MATERIAL AND METHODS: We compared risk factors for idiopathic calcium nephrolithiasis (age, body mass index, calcemia, calcium oxalate supersaturation, and 24-h calciuria, oxaluria, uricosuria, citraturia, urea, and sodium) in four populations: men and women with and without a family history of stone (FHS). A total of 119 males (58 with an FHS, 61 without) and 59 females (30 with an FHS, 29 without) were compared to healthy controls. For each variable, receiver operating characteristic (ROC) curve indices were calculated by means of ANNs. RESULTS: In men without an FHS, the most discriminant variables were 24-h urea (ROC curve index 0.76), supersaturation (ROC curve index 0.72), 24-h calciuria (ROC curve index 0.68), 24-h uricosuria (ROC curve index 0.64), 24-h oxaluria (ROC curve index 0.63), 24-h sodium (ROC curve index 0.62), and calcemia (ROC curve index 0.60). In men with an FHS, only supersaturation (ROC curve index 0.67) was discriminant. In women without FHS, calcemia (ROC curve index 0.67), 24-h calciuria (ROC curve index 0.64), and 24-h uricosuria (ROC curve index 0.62) were discriminant. In women with an FHS, supersaturation (ROC curve index 0.70), 24-h uricosuria (ROC curve index 0.69), 24-h urea (ROC curve index 0.68), and 24-h calciuria (ROC curve index 0.67) were discriminant. CONCLUSIONS: Risk factors for idiopathic calcium nephrolithiasis were roughly the same in men with or without an FHS, and in women with an FHS. In these patients, calcium oxalate supersaturation and 24-h urea were the most discriminant factors. Conversely, in women without an FHS, calcium abnormalities (calcemia, 24-h calciuria) were discriminant and should prompt a search for infraclinical primary hyperparathyroidism or sarcoidosis.

Artificial neural networks for assessing the risk of urinary calcium stone among men.

The pathophysiology of idiopathic calcium oxalate nephrolithiasis involves metabolic abnormalities. Previous studies gave conflicting results about the metabolic factors in stone formers. Artificial neural networks (ANN) are new methods based on computer programming that have outperformed conventional methods in prediction of outcomes in different medical applications. The aim of our study was to compare with ANN the clinical and biochemical parameters implicated in urinary calcium stone between stone formers (SF) and controls (C). We compared 11 clinical and biochemical variables among 119 male idiopathic calcium oxalate SF and 96 C by univariate and multivariate statistical analyses. Univariate analyses included discriminant analysis, logistic regression analysis, and ANN. For multivariate analyses, stepwise discriminant analysis and ANN were performed. Variables included age, body mass index (BMI), family history of nephrolithiasis, supersaturation with respect to calcium oxalate, calcemia, and 24-h urinary calcium, oxalate, uric acid, urea, sodium, and citrate. With univariate and multivariate analyses, ANN were as efficient as classical statistical analyses in discriminating the different parameters. The sensitivity, the specificity, and the percentage of correctly classified patients were similar in all analyses. With ANN, supersaturation (receiver operating characteristic, ROC curves index 0.73) and urea (ROC 0.72) were the most discriminant followed by family history and urinary calcium (ROC 0.67). ROC index was 0.63 for citrate, 0.61 for oxalate and urate, 0.60 for sodium and calcemia, 0.58 for age, and 0.56 for BMI, but these parameters were not statistically different between SF and C. ANN gave additional information since they made it possible to determine the cut-off values of the parameters and their predictive power. Cut-off values for being a stone former were 8.9 for supersaturation and 363 mmol/day for urinary urea with a predictive power of 0.69 and 0.70, respectively. Univariate and multivariate analysis evidenced supersaturation and 24-h urinary urea excretion as the most discriminant parameters between the two populations. In addition to high supersaturation, the negative impact of protein intake was confirmed.

Combining immunoreactive trypsinogen and pancreatitis-associated protein assays, a method of newborn screening for cystic fibrosis that avoids DNA analysis.

OBJECTIVES: To evaluate the performance of a strategy in which, after immunoreactive trypsinogen (IRT) determination, genetic analysis is replaced by a biological test, the pancreatitis-associated protein (PAP) enzyme-linked immunosorbent assay (ELISA). STUDY DESIGN: The French newborn screening program includes cystic fibrosis (CF) screening by the IRT/CFTR mutation strategy. PAP was assayed on screening cards, in parallel with IRT, in all newborns from 5 French regions (n = 204,749). Analysis of PAP values in CF and non-CF newborns with elevated IRT allowed direct comparison between the current strategy and the proposed IRT/PAP strategy. RESULTS: A protocol in which newborns with IRT >50 ng/mL and PAP >1.8 ng/mL and those with IRT >100 ng/mL and PAP >1.0 ng/mL are directly recalled for sweat testing would have the same performance as the IRT/CFTR mutation strategy. CONCLUSIONS: The IRT/PAP strategy is an alternative for CF newborn screening, which avoids the drawbacks of genetic analysis and is cheaper and easier to implement than the current IRT/CFTR mutation strategy.

Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and DNA microarray analysis of livers reveals an aberrant gene expression response.

BACKGROUND: p8 is a DNA-binding protein induced in many tissues in response to LPS treatment. Hence, p8 could be a mediator of LPS-associated effects or, on the contrary, p8 expression may be part of the protective mechanism of the tissues in response to LPS. Finally, p8 expression in response to LPS could also be a simple epiphenomenon. METHODS: To investigate the role of p8 in vivo, we generated p8-deficient mice by gene targeting. Because p8 is a stress protein, we analyzed the response of p8-/- mice to a systemic stress induced by LPS injection. Liver was chosen as model organ to monitor alterations in gene expression. RESULTS: LPS resulted in higher serum TNF-alpha concentration and higher mortality rate in p8-deficient mice than in wild-type. Also, liver and pancreas, but not lung, from p8-/- mice showed increased amounts of MPO and HPO. To gain insight into the molecular bases of such susceptibility, we used high density DNA microarrays consisting of ~6000 genes and ESTs to compare gene regulation in response to LPS in p8+/+ and p8-/- livers. In wild-type, 105 genes and 73 ESTs were up-regulated and 232 genes and 138 ESTs down-regulated. By contrast, 212 genes and 125 ESTs were found up-regulated and 90 genes and 85 ESTs down regulated in p8-/- mice. Among them, only 93 (51 induced and 42 repressed) corresponded to the wild-type pattern, demonstrating that p8 deficiency hinders the normal response to LPS, which may account for the increased sensitivity of p8-/-mice to the endotoxin. CONCLUSIONS: The large number of genes showing abnormal regulation after LPS suggests that p8 is an important regulatory factor involved in many cellular defence pathways.

Gene expression profiling by DNA microarray analysis in mouse embryonic fibroblasts transformed by rasV12 mutated protein and the E1A oncogene.

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 approximately 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 post-transcriptional 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.