Ezequiel Calvo

Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction.

Quantification of mRNA expression levels using real-time reverse transcription PCR (RT-PCR) is increasingly used to validate results of DNA microarrays or GeneChips. It requires an improved method that is more robust and more suitable for high-throughput measurements. In this report, we compare a user non-influent, second derivative method with that of a user influent, fit point method that is widely used in the literature. We also describe the advantage of using a double correction: one correction using the expression levels of a housekeeping gene of an experiment as an internal standard and a second using reference expression levels of the same housekeeping gene in the tissue or cells. The first correction permits one to decrease errors due to sample preparation and handling, while the second correction permits one to avoid the variation of the results with the variability of housekeeping in each tissue, especially in experiments using various treatments. The data indicate that the real-time PCR method is highly efficient with an efficiency coefficient close to the theoretical value of two. The results also show that the second derivative method is more accurate than the fit point method in quantifying low gene expression levels. Using triplicate experiments, we show that measurement variations using our method are low with a mean of variation coefficients of <1%.

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.

Cholesterol uptake disruption, in association with chemotherapy, is a promising combined metabolic therapy for pancreatic adenocarcinoma

Significance Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second deadliest cancer by 2030. Advances in therapeutic treatments are urgently required to fight against this fatal disease. Here, elucidation of the metabolic signature of PDAC has identified the low-density lipoprotein receptor (LDLR), which facilitates cholesterol uptake, as a promising therapeutic target. Blocking of LDLR reduces the proliferative and clonogenic potential of PDAC cells and decreases activation of the ERK1/2 survival pathway. Moreover, LDLR silencing sensitizes PDAC cells to chemotherapeutic drugs and potentiates the tumoral regression promoted by chemotherapy. Finally, Ldlr is highly expressed at all stages of human PDAC and expression is associated with an increased risk of PDAC recurrence. The malignant progression of pancreatic ductal adenocarcinoma (PDAC) is accompanied by a profound desmoplasia, which forces proliferating tumor cells to metabolically adapt to this new microenvironment. We established the PDAC metabolic signature to highlight the main activated tumor metabolic pathways. Comparative transcriptomic analysis identified lipid-related metabolic pathways as being the most highly enriched in PDAC, compared with a normal pancreas. Our study revealed that lipoprotein metabolic processes, in particular cholesterol uptake, are drastically activated in the tumor. This process results in an increase in the amount of cholesterol and an overexpression of the low-density lipoprotein receptor (LDLR) in pancreatic tumor cells. These findings identify LDLR as a novel metabolic target to limit PDAC progression. Here, we demonstrate that shRNA silencing of LDLR, in pancreatic tumor cells, profoundly reduces uptake of cholesterol and alters its distribution, decreases tumor cell proliferation, and limits activation of ERK1/2 survival pathway. Moreover, blocking cholesterol uptake sensitizes cells to chemotherapeutic drugs and potentiates the effect of chemotherapy on PDAC regression. Clinically, high PDAC Ldlr expression is not restricted to a specific tumor stage but is correlated to a higher risk of disease recurrence. This study provides a precise overview of lipid metabolic pathways that are disturbed in PDAC. We also highlight the high dependence of pancreatic cancer cells upon cholesterol uptake, and identify LDLR as a promising metabolic target for combined therapy, to limit PDAC progression and disease patient relapse.

Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis

Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all cancers and shows remarkable resistance to cell stress. Nuclear protein 1 (Nupr1), which mediates stress response in the pancreas, is frequently upregulated in pancreatic cancer. Here, we report that Nupr1 plays an essential role in pancreatic tumorigenesis. In a mouse model of pancreatic cancer with constitutively expressed oncogenic Kras(G12D), we found that loss of Nupr1 protected from the development of pancreatic intraepithelial neoplasias (PanINs). Further, in cultured pancreatic cells, nutrient deprivation activated Nupr1 expression, which we found to be required for cell survival. We found that Nupr1 protected cells from stress-induced death by inhibiting apoptosis through a pathway dependent on transcription factor RelB and immediate early response 3 (IER3). NUPR1, RELB, and IER3 proteins were coexpressed in mouse PanINs from Kras(G12D)-expressing pancreas. Moreover, pancreas-specific deletion of Relb in a Kras(G12D) background resulted in delayed in PanIN development associated with a lack of IER3 expression. Thus, efficient PanIN formation was dependent on the expression of Nupr1 and Relb, with likely involvement of IER3. Finally, in patients with PDAC, expression of NUPR1, RELB, and IER3 was significantly correlated with a poor prognosis. Cumulatively, these results reveal a NUPR1/RELB/IER3 stress-related pathway that is required for oncogenic Kras(G12D)-dependent transformation of the pancreas.

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

BackgroundRas 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.ResultsWe 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.ConclusionsThis 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.

Epididymosomes Convey Different Repertoires of MicroRNAs Throughout the Bovine Epididymis

ABSTRACT Epididymosomes are small membrane vesicles that are secreted by epididymal epithelial cells and are involved in posttesticular sperm maturation. Although their role in protein transfer to the sperm membrane is well documented, we report their capacity to transport microRNAs (miRNAs), which are potent regulators of posttranscriptional gene expression. Using a microperfusion technique combined with a global microarray approach, we demonstrated that epididymosomes from two discrete bovine epididymal regions (caput and cauda) possess distinct miRNA signatures. In addition, we also established that miRNA repertoires contained within epididymosomes differ from those of their parent epithelial cells, suggesting that miRNA populations released from the cells may be selectively sorted. Binding of DilC12-labeled epididymosomes to primary cultured epididymal cells was measured by flow cytometry, and the results indicated that epididymosomes from the median caput and their miRNA content may be incorporated into distal caput epithelial cells. Overall, these findings reveal that distinct miRNA repertoires are released into the intraluminal fluid in a region-specific manner and could be involved in a novel mechanism of intercellular communication throughout the epididymis via epididymosomes.

Transcriptional profiling of the injured sciatic nerve of mice carrying the Wld(S) mutant gene: Identification of genes involved in neuroprotection, neuroinflammation, and nerve regeneration

Wallerian degeneration (WD) involves the fragmentation of axonal segments disconnected from their cell bodies, segmentation of the myelin sheath, and removal of debris by Schwann cells and immune cells. The removal and downregulation of myelin-associated inhibitors of axonal regeneration and synthesis of growth factors by these two cell types are critical responses to successful nerve repair. Here, we analyzed the transcriptome of the sciatic nerve of mice carrying the Wallerian degeneration slow (Wld(S)) mutant gene, a gene that confers axonal protection in the distal stump after injury, therefore causing significant delays in WD, neuroinflammation, and axonal regeneration. Of the thousands of genes analyzed by microarray, 719 transcripts were differentially expressed between Wld(S) and wild-type (wt) mice. Notably, the Nmnat1, a transcript contained within the sequence of the Wld(S) gene, was upregulated by five to eightfold in the sciatic nerve of naive Wld(S) mice compared with wt. The injured sciatic nerve of wt could be further distinguished from the one of Wld(S) mice by the preferential upregulation of genes involved in axonal processes and plasticity (Chl1, Epha5, Gadd45b, Jun, Nav2, Nptx1, Nrcam, Ntm, Sema4f), inflammation and immunity (Arg1, Lgals3, Megf10, Panx1), growth factors/cytokines and their receptors (Clcf1, Fgf5, Gdnf, Gfrα1, Il7r, Lif, Ngfr/p75(NTR), Shh), and cell adhesion and extracellular matrix (Adam8, Gpc1, Mmp9, Tnc). These results will help understand how the nervous and immune systems interact to modulate nerve repair, and identify the molecules that drive these responses.

Role of MicroRNAs in Controlling Gene Expression in Different Segments of the Human Epididymis

Background The molecular mechanisms implicated in regionalized gene expression in the human epididymis have not yet been fully elucidated. Interestingly, more than 200 microRNAs (miRNAs) have been identified in the human epididymis and could be involved in the regulation of mRNA stability and post-transcriptional expression in this organ. Methods Using a miRNA microarray approach, we investigated the correlation between miRNA signatures and gene expression profiles found in three distinct regions (caput, corpus and cauda) of human epididymides from 3 donors. In silico prediction of transcript miRNA targets was performed using TargetScan and Miranda softwares. FHCE1 immortalized epididymal cell lines were cotransfected with mimic microRNAs and plasmid constructs containing the 3′UTR of predicted target genes downstream of the luciferase gene. Results We identified 35 miRNAs differentially expressed in the distinct segments of the epididymis (fold change ≥2, P-value≤0.01). Among these miRNAs, miR-890, miR-892a, miR-892b, miR-891a, miR-891b belonging to the same epididymis-enriched cluster located on the X chromosome, are significantly more expressed in the corpus and cauda regions than in the caput. Interestingly, a strong negative correlation (r = −0,89, P-value≤0.001) was found between the pattern of expression of miR-892b and its potential mRNA target Esrrg (Estrogen Related Receptor Gamma) and with miR-145 and Cldn10 mRNA (r = −0,92, P-value≤0.001). We confirmed that miR-145 and miR-892b inhibit the expression of the luciferase reporter via Cldn10 and Esrrg 3′ UTRs, respectively. Conclusion Our study shows that the expression of miRNAs is segmented along the human epididymis and correlates with the pattern of target gene expression in different regions. Therefore, epididymal miRNAs may be in control of the maintenance of gene expression profile in the epididymis, which dictates segment-specific secretion of proteins and establishes physiological compartments that directly or indirectly affect sperm maturation and fertility.

Intra-uterine growth restriction and the programming of left ventricular remodelling in female rats

Epidemiological studies link intra‐uterine growth restriction (IUGR) with increased incidence of hypertension and cardiac disease in adulthood. Our rat model of IUGR supports this contention and provides evidence for the programming of susceptibility for hypertension in all offspring. Moreover, in the female offspring only, gross anatomical changes (cardiac ventricle to body ratios) and increased left cardiac ventricular atrial natriuretic peptide (ANP) mRNA levels provide evidence for programming of cardiac disease in this gender. The aim of the current study was to measure changes in cardiac tissue that support remodelling that could be implicated in the initiation of hypertrophy. Adult female rats from our IUGR model and age‐ and sex‐matched controls were killed at 12 weeks of age. Left cardiac ventricles were removed and used for monitoring changes in several key genes, Na+,K+‐ATPase β1 protein expression, cardiomyocyte morphology and contractility as well as citrate synthase and aconitase activities. When compared to controls, female offspring of our IUGR rat model exhibit higher expression (mRNA) of ANP and the atrial isoform of the myosin light chain, lower levels of Na+,K+‐ATPase β1 protein, increased cardiomyocyte depth and volume, increased sarcomere length, diminished cardiomyocyte contractility and lower aconitase activity. Female offspring of our IUGR rat model exhibit changes as adults that are consistent with the onset of cardiac remodelling. The decrease in aconitase activity suggests that oxidative stress may be implicated in this response.

Sequence-specific Recruitment of Heterochromatin Protein 1 via Interaction with Krüppel-like Factor 11, a Human Transcription Factor Involved in Tumor Suppression and Metabolic Diseases

Background: Chromatin remodeling mechanisms utilized by Krüppel-like factor proteins remain poorly defined. Results: Krüppel-like factor 11 directly recruits heterochromatin protein 1α to promoters in a sequence-specific manner. Conclusion: Coupling to heterochromatin protein 1 α and its histone methyltransferase underlies Krüppel-like factor-mediated gene expression and tumor suppression. Significance: This data advances our understanding of how chromatin-mediated mechanisms achieve these functions with increased specificity for target genes. Heterochromatin protein 1 (HP1) proteins are “gatekeepers” of epigenetic gene silencing that is mediated by lysine 9 of histone H3 methylation (H3K9me). Current knowledge supports a paradigm whereby HP1 proteins achieve repression by binding to H3K9me marks and interacting to H3K9 histone methyltransferases (HMTs), such as SUV39H1, which methylate this residue on adjacent nucleosomes thereby compacting chromatin and silencing gene expression. However, the mechanism underlying the recruitment of this epigenetic regulator to target gene promoters remains poorly characterized. In the current study, we reveal for the first time a mechanism whereby HP1 is recruited to promoters by a well characterized Krüppel-like transcription factor (KLF), in a sequence-specific manner, to mediate complex biological phenomena. A PXVXL HP1-interacting domain identified at position 487–491 of KLF11 mediates the binding of HP1α and KLF11 in vitro and in cultured cells. KLF11 also recruits HP1α and its histone methyltransferase, SUV39H1, to promoters to limit KLF11-mediated gene activation. Indeed, a KLF11ΔHP1 mutant derepresses KLF11-regulated cancer genes, by inhibiting HP1-SUV39H1 recruitment, decreasing H3K9me3, while increasing activation-associated marks. Biologically, impairment of KLF11-mediated HP1-HMT recruitment abolishes tumor suppression, providing direct evidence that HP1-HMTs act in a sequence-specific manner to achieve this function rather than its well characterized binding to methylated chromatin without intermediary. Collectively, these studies reveal a novel role for HP1 as a cofactor in tumor suppression, expand our mechanistic understanding of a KLF associated to human disease, and outline cellular and biochemical mechanisms underlying this phenomenon, increasing the specificity of targeting HP1-HMT complexes to gene promoters.