Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Targeting REG3ß limits pancreatic ductal adenocarcinoma progression through CTGF downregulation.

The crosstalk between the transformed tumoral cells and their microenvironment is a key aspect for pancreatic ductal adenocarcinoma (PDAC) progression. This molecular dialog is intensively studied because it may result in an efficient therapeutic target. Contrary to this near microenvironment, the stromal portion in direct contact with the transformed cells, a far microenvironment, placed at the periphery of the tumor mass, produces factors signaling tumors. Among these factors, REG3ß, produced by this part of the pancreas, is an important factor in promoting tumor progression. This paper demonstrated that targeting REG3ß protein with specific antibodies limits the PDAC tumor growth in an orthotopic, syngeneic mice model induced by injection of Panc02 cells. Then, we showed that CTGF is over-expressed in response to REG3ß in PDAC-derived cells. Moreover, inactivation of REG3ß by treating tumors with anti-REG3ß antibodies results in a strong decrease of CTGF in PDAC tumors. Lastly, we demonstrated that forced expression of CTGF in xenografted Panc02 cells abolishes the therapeutic effect of the anti-REG3ß antibody treatment. Altogether, these results indicate that the effect of REG3ß in promoting PDAC progression is mediated by CTGF over-activation. Thus, REG3ß is a promising therapeutic target to treat PDAC with an original rationale. In conclusion, we demonstrated that the far microenvironment is essential for PDAC progression by producing active secretory factors, and some of them could be used as therapeutic targets.

Hedgehog signaling pathway regulates gene expression profile of epididymal principal cells through the primary cilium.

Primary cilia (PC) are organelles that sense and respond to dynamic changes of the extracellular milieu through the regulation of target genes. By using the epididymis as a model system, we determined the contribution of primary cilia in the regulation of epithelial cell functions through the transduction of the Hedgehog (Hh) signaling pathway. Both Sonic (SHH) and Indian Hedgehog (IHH) ligands were detected in epididymal epithelial cells by confocal microscopy and found secreted in the extracellular space. Gene expression profiling preformed on ciliated epithelial cells indicated that 153 and 1052 genes were differentially expressed following treatment with the Hh agonist SAG or the Hh antagonist cyclopamine (Cyclo), respectively. Strikingly, gene ontology analysis indicated that genes associated with immune response were the most affected following Hh modulation. The contribution of epididymal PC to canonical Hh pathway transduction was validated by ciliobrevin D treatment, which induced a significant decrease in PC length and a reduction in the expression Hh signaling targets. Such findings bring us closer to a molecular understanding of the subtle immune balance observed in some epithelia, including the epididymis and the intestine, which are organs featuring both tolerance toward autoimmune spermatozoa (or commensal bacteria) and defense against pathogens.

Transcriptome of 17ß-hydroxysteroid dehydrogenase type 2 plays both hormone-dependent and hormone-independent roles in MCF-7 breast cancer cells.

Breast cancer is a major cause of cancer-related death for women in western countries. 17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) play important roles in the last step of sex-hormone activation and the first step of sex-hormone inactivation. 17ß-HSD2 is responsible for oxidizing the sex hormones. We used microarray technology to analyze the effect of 17ß-HSD2 on the MCF-7 cell transcript profile after knocking down 17ß-HSD2. Five hundred forty-two genes were regulated 1.5-fold or higher after treatment with 17ß-HSD2 siRNA. Knocking down 17ß-HSD2 interrupted nucleosome assembly. Pathway-Act-Network analysis showed that the MAPK and apoptosis signaling pathways were most regulated. In the gene-gene interaction network analysis, UGT2B15, which is involved in hormone metabolism, was the most regulated core gene. FOS, GREB1, and CXCL12 were the most regulated genes, and CXCL12 was related to tumor migration. Following 17ß-HSD2 knock-down, the cell viability decreased to 75.9%. The S-phase percentage decreased by 19.4%, the Q2-phase percentage in cell apoptosis testing increased by 1.5 times, and cell migration decreased to 66.0%. These results were consistent with our gene chip analysis and indicated that 17ß-HSD2 plays both hormone-dependent and hormone-independent enzymatic roles. In-depth investigations of this enzyme on the genomic level will help clarify its related molecular mechanisms.

Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient.

In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high-fertility (HF) and four low-fertility (LF) bulls with comparable post-thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation.

Mechanisms Underlying the Regulation of HP1γ by the NGF-PKA Signaling Pathway.

Heterochromatin protein 1 γ (HP1γ) is a well-known chromatin protein, which regulates gene silencing during the execution of processes associated with embryogenesis, organ maturation, and cell differentiation. We find that, in vivo, the levels of HP1γ are downregulated during nervous system development. Similar results are recapitulated in vitro during nerve growth factor (NGF)-induced neuronal cell differentiation in PC12 cells. Mechanistically, our experiments demonstrate that in differentiating PC12 cells, NGF treatment decreases the association of HP1γ to silent heterochromatin, leads to phosphorylation of this protein at S83 via protein kinase A (PKA), and ultimately results in its degradation. Genome-wide experiments, using gain-of-function (overexpression) and loss-of-function (RNAi) paradigms, demonstrate that changing the level of HP1γ impacts on PC12 differentiation, at least in part, through gene networks involved in this process. Hence, inactivation of HP1γ by different post-translational mechanisms, including reduced heterochromatin association, phosphorylation, and degradation, is necessary for neuronal cell differentiation to occur. Indeed, we show that the increase of HP1γ levels has the reverse effect, namely antagonizing neuronal cell differentiation, supporting that this protein acts as a barrier for this process. Thus, these results describe the regulation and participation of HP1γ in a novel membrane-to-nucleus pathway, through NGF-PKA signaling, which is involved in NGF-induced neuronal cell differentiation.

Pancreatic Adenocarcinoma Therapeutic Targets Revealed by Tumor-Stroma Cross-Talk Analyses in Patient-Derived Xenografts.

Preclinical models based on patient-derived xenografts have remarkable specificity in distinguishing transformed human tumor cells from non-transformed murine stromal cells computationally. We obtained 29 pancreatic ductal adenocarcinoma (PDAC) xenografts from either resectable or non-resectable patients (surgery and endoscopic ultrasound-guided fine-needle aspirate, respectively). Extensive multiomic profiling revealed two subtypes with distinct clinical outcomes. These subtypes uncovered specific alterations in DNA methylation and transcription as well as in signaling pathways involved in tumor-stromal cross-talk. The analysis of these pathways indicates therapeutic opportunities for targeting both compartments and their interactions. In particular, we show that inhibiting NPC1L1 with Ezetimibe, a clinically available drug, might be an efficient approach for treating pancreatic cancers. These findings uncover the complex and diverse interplay between PDAC tumors and the stroma and demonstrate the pivotal role of xenografts for drug discovery and relevance to PDAC.

Identification of the principal transcriptional regulators for low-fat and high-fat meal responsive genes in small intestine.

BACKGROUND: High-fat (HF) diet is a well-known cause of obesity. To identify principle transcriptional regulators that could be therapeutic targets of obesity, we investigated transcriptomic modulation in the duodenal mucosa following low-fat (LF) and HF meal ingestion. METHODS: Whereas one group of mice was sacrificed after fasting, the others were fed ad libitum with LF or HF meal, and sacrificed 30 min, 1 h and 3 h after the beginning of the meal. A transcriptome analysis of the duodenal mucosa of the 7 groups was conducted using both microarray and serial analysis of gene expression (SAGE) method followed by an Ingenuity Pathways Analysis (IPA). RESULTS: SAGE and microarray showed that the modulation of a total of 896 transcripts in the duodenal mucosa after LF and/or HF meal, compared to the fasting condition. The IPA identified lipid metabolism, molecular transport, and small molecule biochemistry as top three molecular and cellular functions for the HF-responsive, HF-specific, HF-delay, and LF-HF different genes. Moreover, the top transcriptional regulator for the HF-responsive and HF-specific genes was peroxisome proliferator-activated receptor alpha (PPARα). On the other hand, the LF-responsive and LF-specific genes were related to carbohydrate metabolism, cellular function and maintenance, and cell death/cellular growth and proliferation, and the top transcriptional regulators were forkhead box protein O1 (FOXO1) and cAMP response element binding protein 1 (CREB1), respectively. CONCLUSIONS: These results will help to understand the molecular mechanisms of intestinal response after LF and HF ingestions, and contribute to identify therapeutic targets for obesity and obesity-related diseases.

Impact of male fertility status on the transcriptome of the bovine epididymis.

STUDY QUESTION: Can region-specific transcriptional profiling of the epididymis from fertile and sub-fertile bulls predict the etiology of fertility/sub-fertility in males? SUMMARY ANSWER: The highly regulated gene expression along the bovine epididymis is affected by the fertility status of bulls used for artificial insemination. WHAT IS KNOWN ALREADY: In mammals, sperm maturation and storage occur in the epididymis. Each epididymal segment has his own transcriptomic signature that modulates the intraluminal composition and consequently governs sequential modifications of the maturing male gamete. STUDY DESIGN, SIZE, DURATION: Epididymides from six Holstein bulls with documented fertility were used. These bulls were divided into two groups: high fertility (n = 3), and medium-low fertility (n = 3) and their epididymal transcriptomic profiles were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Bovine cDNA microarray probing and bioinformatic tools were used to identify genes that are differentially expressed in caput, corpus and cauda epididymidal tissues of bulls with the documented fertility index. MAIN RESULTS AND THE ROLE OF CHANCE: Hierarchical clustering and principal component analysis revealed a clear separation between caput, corpus and cauda epididymides. Some transcripts characterize a particular anatomical segment, whereas others are expressed in two out of three epididymal segments. Gene ontology analysis allowed deduction of specific functions played by each epididymal segment. The transcriptional profiles between fertile versus sub-fertile conditions clustered most closely in the corpus and cauda segments, whereas the profiles in the caput segment were distinct between fertile and sub-fertile bulls. Of the differently expressed genes, 10 (AKAP4, SMCP, SPATA3, TCP11, ODF1, CTCFL, SPATA18, ADAM28, SORD and FAM161A) were found to exert functions related to reproductive systems and 5 genes (DEAD, CYST11, DEFB119, DEFB124 and MX1) were found to be associated with the defense response. LARGE SCALE DATA: The GEO number for public access of bovine epididymis microarray data is GSE96602. LIMITATIONS, REASONS FOR CAUTION: Further work is required to link these modulations of epididymal functions with sperm fertilizing ability in order to understand the etiology of certain cases of idiopathic infertility in livestock and men. WIDER IMPLICATIONS OF THE FINDINGS: As fertility can be quantified in bulls used for artificial insemination, this species is a unique model to aid in the understanding of male fertility/sub-fertility in man. Our data provide a molecular characterization that will facilitate advances in understanding the involvement of epididymal physiology in sub/infertility etiology. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant to R.S. from the Natural Sciences and Engineering Research Council (NSERC) of Canada. C.L., A.A., E.C. and R.S. have no conflict of interest to declare. P.B. is R&D director at Alliance Boviteq Inc., a bovine artificial insemination company.

Daylily protein constituents of the pollen and stigma a proteomics approach.

This study was aimed at the identification and quantification of the protein components of the pollen grains in parallel with the distal stigmatic tissue of tetraploid cultivars. Proteomes were analyzed using iTRAQ 4plex labeling, peptides separation by online RP-nano-LC and analysis by ESI-MS/MS. Protein identification and quantification were made using the Asparagales database as a reference. A total of 524,037 MS/MS spectra were produced from pollen and stigma samples. From these, a total of 8368 peptides wereidentified corresponding to 994 unique peptides and 432 protein groups. Among them, 128 differentially expressed proteins were retained for further analysis. In absence of the daylily genome availability, we exploited numerous databases and bioinformatics resources to exploring the putative biological functions of these proteins. The profile of differentially expressed proteins suggests an important representation of functions associated to the signalling and response against endogenous and environmental stresses, including several enzymes implicated in the biosynthesis of antibiotics. The abundance in stigma of several structural proteins of the ribosomal sub-units as well as of the core histones suggest that the translation processes and the regulation of gene expression in stigma is a more active mechanism than in pollen. In addition, pollen prioritizes the synthesis of fructose and glucose as opposed to sucrose in stigma as a source of energy. Finally, the modulated proteins in Hemerocallis point to several pathways that give potential clues concerning the molecular mechanisms underlying the functions of the pollen and the stigmatic fluid in daylily reproduction.

Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1.

17beta-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a steroidal enzyme which, in breast cancer cells, mainly synthesizes 17-beta-estradiol (E2), an estrogenic hormone that stimulates breast cancer cell growth. We previously showed that the enzyme increased breast cancer cell proliferation via a dual effect on E2 and 5α-dihydrotestosterone (DHT) levels and impacted gene expression and protein profile of breast cancer cells cultured in E2-contained medium. Here, we used RNA interference technique combined with microarray analyses to investigate the effect of 17ß-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition. Our data revealed that knockdown of 17ß-HSD1 gene, HSD17B1, modulates the transcript profile of the hormone-dependent breast cancer cell line T47D, with 105 genes regulated 1.5 fold or higher (p < 0.05) in estradiol-independent manner. Using Ingenuity Pathway Analysis (IPA), we additionally assessed functional enrichment analyses, including biological functions and canonical pathways, and found that, in concordance with the role of 17ß-HSD1 in cancer cell growth, most regulated genes are cancer-related genes. Genes that primarily involved in the cell cycle progression, such as the cyclin A2 gene, CCNA2, are generally down-regulated whereas genes involved in apoptosis and cell death, including the pro-apoptotic gene XAF1, IFIH1 and FGF12, are on the contrary up-regulated by 17ß-HSD1 knockdown, and 21% of the modulated genes belong to this latter functional category. This indicates that 17ß-HSD1 may be involved in oncogenesis by favoring anti-apoptosis pathway in breast cancer cells and correborates with its previously shown role in increasing breast cancer cell proliferation. The gene regulation occurring in steroid-deprived conditions showed that 17ß-HSD1 can modulate endogenous gene expression in steroid-independent manners. Besides, we tested the ability of estrogen to induce or repress endogenous genes of T47D by microarray analysis. Expression of a total of 130 genes were found to increase or decrease 1.5-fold or higher (p < 0.05) in response to E2 treatment (1 nM for 48 h), revealing a list of potential new estrogen-responsive genes and providing useful information for further studies of estrogen-dependent breast cancer mechanisms. In conclusion, in breast cancer cells, in addition to its implication in the E2-dependent gene transcription, the present study demonstrates that 17ß-HSD1 also modulates gene expression via mechanisms independent of steroid actions. Those mechanisms that may include the ligand-independent gene transcription of estrogen receptor alpha (ERα), whose expression is positively correlated with that of the enzyme, and that may implicate 17ß-HSD1 in anti-apoptosis pathways, have been discussed.

Genomic data on breast cancer transcript profile modulation by 17beta-hydroxysteroid dehydrogenase type 1 and 17-beta-estradiol.

The data presented here are related to the research article entitled "Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1" (J.A. Aka, E.L. Calvo, S.X. Lin, 2016) [1]. We evaluated the effect of the steroidal enzyme 17ß-HSD1 and its product, the estrogenic hormone 17-beta-estradiol (E2), on gene transcription profile of breast cancer cells. RNA interference technique was used to knock down the 17ß-HSD1 gene (HSD17B1) in the hormone-dependent breast cancer cell line T47D in steroid-deprived medium. Transfected cells were subsequently treated with E2, and microarray analyses (with three contrasts) were used to investigate (i) the effect of 17ß-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition, (ii) the effect of E2 on breast cancer gene expression and (iii) if E2 affects gene regulation by 17ß-HSD1. Functional enrichments of the differentially expressed genes were assessed using Ingenuity Pathway Analysis (IPA). Here, we showed data on 140 genes that are induced or repressed 1.5 time or higher (p < 0.05) in the HSD17B1-silenced and E2-treated T47D cells revealed by microarray analysis, and presented the 14 functional terms found in the cancer and in the cell death and survival categories revealed by the IPA biological function analysis. Data on IPA Canonical Pathway and network analyses is also presented. Further discussion on gene regulation by 17ß-HSD1 and E2 is provided in the accompanying publication [1].

Role of Dicer1-Dependent Factors in the Paracrine Regulation of Epididymal Gene Expression.

Dicer1 is an endoribonuclease involved in the biogenesis of functional molecules such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs). These small non-coding RNAs are important regulators of post-transcriptional gene expression and participate in the control of male fertility. With the knowledge that 1) Dicer1-dependent factors are required for proper sperm maturation in the epididymis, and that 2) miRNAs are potent mediators of intercellular communication in most biological systems, we investigated the role of Dicer1-dependent factors produced by the proximal epididymis (initial segment/caput)- including miRNAs- on the regulation of epididymal gene expression in the distal epididymis regions (i.e. corpus and cauda). To this end, we performed comparative microarray and ANOVA analyses on control vs. Defb41iCre/wt;Dicer1fl/fl mice in which functional Dicer1 is absent from the principal cells of the proximal epididymis. We identified 35 and 33 transcripts that displayed significant expression level changes in the corpus and cauda regions (Fold change > 2 or < -2; p < 0.002), respectively. Among these transcripts, Zn-alpha 2-glycoprotein (Azgp1) encodes for a sperm equatorial protein whose expression in the epididymis of Dicer1 cKO mice is significantly increased compared to controls. In addition, 154 miRNAs, including miR-210, miR-672, miR-191 and miR-204, showed significantly impaired biogenesis in the absence of Dicer1 from the principal cells of the proximal epididymis (Fold change > 2 or < -2; p < 0.01). These miRNAs are secreted via extracellular vesicles (EVs) derived from the DC2 epididymal principal cell line, and their expression correlates with target transcripts involved in distinct biological pathways, as evidenced by in silico analysis. Albeit correlative and based on in silico approach, our study proposes that Dicer1-dependent factors trigger- directly or not-significant genes expression changes in distinct regions of this organ. The paracrine control of functions important to post-testicular sperm maturation by Dicer1-dependent factors may open new avenues for the identification of molecular targets important to male fertility control.

Phenotypic Characterization of Mice Carrying Homozygous Deletion of KLF11, a Gene in Which Mutations Cause Human Neonatal and MODY VII Diabetes.

We have previously shown that amino acid changes in the human Kruppel-Like Factor (KLF) 11 protein is associated with the development of maturity onset diabetes of the young VII, whereas complete inactivation of this pathway by the -331 human insulin mutation causes neonatal diabetes mellitus. Here, we report that Klf11-/- mice have decreased circulating insulin levels, alterations in the control of blood glucose and body weight, as well as serum dyslipidemia, but do not develop diabetes. Functional assays using ex vivo liver tissue sections demonstrate that Klf11-/- mice display increased insulin sensitivity. Genome-wide experiments validated by pathway-specific quantitative PCR arrays reveal that the Klf11-/- phenotype associates to alterations in the regulation of gene networks involved in lipid metabolism, in particular those regulated by peroxisome proliferator-activated receptor-γ. Combined, these results demonstrate that the major phenotype given by the whole-body deletion of Klf11 in mouse is not diabetes but increased insulin sensitivity, likely due to altered transcriptional regulation in target tissues. The absence of diabetes in the Klf11-/- mouse either indicates an interspecies difference for the role of this transcription factor in metabolic homeostasis between mouse and humans, or potentially highlights the fact that other molecular factors can compensate for its absence. Nevertheless, the data of this study, gathered at the whole-organism level, further support a role for KLF11 in metabolic processes like insulin sensitivity, which regulation is critical in several forms of diabetes.

The Aurora A-HP1γ pathway regulates gene expression and mitosis in cells from the sperm lineage.

BACKGROUND: HP1γ, a well-known regulator of gene expression, has been recently identified to be a target of Aurora A, a mitotic kinase which is important for both gametogenesis and embryogenesis. The purpose of this study was to define whether the Aurora A-HP1γ pathway supports cell division of gametes and/or early embryos, using western blot, immunofluorescence, immunohistochemistry, electron microscopy, shRNA-based knockdown, site-directed mutagenesis, and Affymetrix-based genome-wide expression profiles. RESULTS: We find that the form of HP1γ phosphorylated by Aurora A, P-Ser83 HP1γ, is a passenger protein, which localizes to the spermatozoa centriole and axoneme. In addition, disruption in this pathway causes centrosomal abnormalities and aberrations in cell division. Expression profiling of male germ cell lines demonstrates that HP1γ phosphorylation is critical for the regulation of mitosis-associated gene expression networks. In female gametes, we observe that P-Ser83-HP1γ is not present in meiotic centrosomes of M2 oocytes, but after syngamy, it becomes detectable during cleavage divisions, coinciding with early embryonic genome activation. CONCLUSIONS: These results support the idea that phosphorylation of HP1γ by Aurora A plays a role in the regulation of gene expression and mitotic cell division in cells from the sperm lineage and in early embryos. Combined, this data is relevant to better understanding the function of HP1γ in reproductive biology.

Transcriptomic analysis predicts survival and sensitivity to anticancer drugs of patients with a pancreatic adenocarcinoma.

A major impediment to the effective treatment of patients with pancreatic ductal adenocarcinoma (PDAC) is the molecular heterogeneity of this disease, which is reflected in an equally diverse pattern of clinical outcome and in responses to therapies. We developed an efficient strategy in which PDAC samples from 17 consecutive patients were collected by endoscopic ultrasound-guided fine-needle aspiration or surgery and were preserved as breathing tumors by xenografting and as a primary culture of epithelial cells. Transcriptomic analysis was performed from breathing tumors by an Affymetrix approach. We observed significant heterogeneity in the RNA expression profile of tumors. However, the bioinformatic analysis of these data was able to discriminate between patients with long- and short-term survival corresponding to patients with moderately or poorly differentiated PDAC tumors, respectively. Primary culture of cells allowed us to analyze their relative sensitivity to anticancer drugs in vitro using a chemogram, similar to the antibiogram for microorganisms, establishing an individual profile of drug sensitivity. As expected, the response was patient dependent. We also found that transcriptomic analysis predicts the sensitivity of cells to the five anticancer drugs most frequently used to treat patients with PDAC. In conclusion, using this approach, we found that transcriptomic analysis could predict the sensitivity to anticancer drugs and the clinical outcome of patients with PDAC.

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

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.

A subgroup of pancreatic adenocarcinoma is sensitive to the 5-aza-dC DNA methyltransferase inhibitor.

Pancreatic Ductal Adenocarcinoma (PDAC) is a disease with a great heterogeneity in the response to treatments. To improve the responsiveness to treatments there are two different approaches, the first one consist to develop new and more efficient drugs that intent to cure all patients and the second one is to use already-approved drugs, alone or in combination, but selecting beforehand the most sensitive patients. In this work we explored the efficiency of the second possibility. We developed a collection of 17 PDAC samples collected by Endoscopic Ultrasound-Guided Fine-Needle Aspiration (EUS-FNA) or surgery and preserved as xenografts and as primary cultures. This collection was characterized at molecular level by a transcriptomic analysis using an Affymetrix approach. In this paper we present data demonstrating that a subgroup of PDAC responds to low doses of 5-aza-dC. These tumors show a specific RNA expression profile that could serve as a marker, but there is no correlation with Dnmt1, Dnmt3A or Dnmt3B expression. Responder tumors corresponded to well-differentiated samples and longer survival patients. In conclusion, we present data obtained with the well-known drug 5-aza-dC as a proof of concept that a drug that seems to be inefficient in solid tumors in general could be applicable to a particular subgroup of patients with PDAC.

Single and combinatorial chromatin coupling events underlies the function of transcript factor Krüppel-like factor 11 in the regulation of gene networks.

BACKGROUND: Krüppel-like factors (KLFs) are a group of master regulators of gene expression conserved from flies to human. However, scant information is available on either the mechanisms or functional impact of the coupling of KLF proteins to chromatin remodeling machines, a deterministic step in transcriptional regulation. RESULTS AND DISCUSSION: In the current study, we use genome-wide analyses of chromatin immunoprecipitation (ChIP-on-Chip) and Affymetrix-based expression profiling to gain insight into how KLF11, a human transcription factor involved in tumor suppression and metabolic diseases, works by coupling to three co-factor groups: the Sin3-histone deacetylase system, WD40-domain containing proteins, and the HP1-histone methyltransferase system. Our results reveal that KLF11 regulates distinct gene networks involved in metabolism and growth by using single or combinatorial coupling events. CONCLUSION: This study, the first of its type for any KLF protein, reveals that interactions with multiple chromatin systems are required for the full gene regulatory function of these proteins.