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As many other cancers, pancreatic ductal adenocarcinoma (PDAC) progression is associated with a series of hallmark changes for cancer cells to secure their own growth success. Yet, these very changes render cancer cells highly sensitive to viral infection. A promising strategy may rely on and exploit viral replication for tumor destruction, whereby infection of tumor cells by a replication-conditional virus may lead to cell destruction and simultaneous release of progeny particles that can spread and infect adjacent tumor cells, while sparing healthy tissues. In the present study, we used Myb34.5, a second-generation replication-conditional herpes simplex virus type 1 (HSV-1) mutant in which ICP6 gene expression is defective and expression of the HSV-1 γ134.5 gene is regulated by the cellular B-myb promoter. We found that B-myb is present in experimental PDAC and tumors, and is overexpressed in patients' tumors, as compared with normal adjacent pancreas. Myb34.5 replicates to high level in human PDAC cell lines and is associated with cell death by apoptosis. In experimental models of PDAC, mice receiving intratumoral Myb34.5 injections appeared healthy and tumor progression was inhibited, with evidence of tumor necrosis, hemorrhage, viral replication, and cancer cell death by apoptosis. Combining standard-of-care chemotherapy with Myb34.5 successfully led to a very impressive antitumoral effect that is rarely achieved in this experimental model, and resulted in a greater reduction in tumor growth than chemotherapy alone. These promising results warrant further evaluation in early phase clinical trial for patients diagnosed with PDAC for whom no effective treatment is available.

Despite tremendous efforts from scientists and clinicians worldwide, pancreatic adenocarcinoma (PDAC) remains a deadly disease due to the lack of early diagnostic tools and reliable therapeutic approaches. Consequently, a majority of patients (80%) display an advanced disease that results in a low resection rate leading to an overall median survival of less than 6 months. Accordingly, robust markers for the early diagnosis and prognosis of pancreatic cancer, or markers indicative of survival and/or metastatic disease are desperately needed to help alleviate the dismal prognosis of this cancer. In addition, the discovery of new therapeutic targets is mandatory to design effective treatments. In this review, we will highlight the translational studies demonstrating that microRNAs may soon translate into clinical applications as long-awaited screening tools and therapeutic targets for PDAC.

Goals and background Mutation of the KRAS oncogene is present in 75% to 95% of pancreatic cancer tissues. This study aimed to evaluate whether endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), combined with analysis of the KRAS mutation, improves the diagnosis of pancreatic cancer in cases of inconclusive or doubtful cytopathologic analysis. Patients and methods We prospectively included 186 patients with a pancreatic mass (103 men; mean age: 62 y). Cytopathology and KRAS mutations, using TaqMan MGB allelic discrimination, were performed on EUS-FNA material. A final diagnosis was obtained from EUS-FNA analysis and/or a subsequent biopsy if necessary, and/or surgery, and follow-up: these were pancreatic adenocarcinoma (n=104), other malignant pancreatic tumors (n=22), and benign lesions (n=60, including 35 cases of chronic pancreatitis). Results Inconclusive or doubtful (low-grade dysplasia or atypia) cytopathology was found in 68 cases. Of these, 29 patients who had adenocarcinoma were subsequently diagnosed, including 19 cases with a former KRAS mutation. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of cytopathology alone to diagnose adenocarcinoma were 73%, 100%, 100%, 75%, and 85%, respectively. When KRAS mutation analysis was combined with pathology, these values reached 88%, 99%, 99%, 89%, and 93%, respectively. The performance of EUS-FNA to diagnose malignancy was similarly improved after the KRAS-mutation assay (negative predictive value increased from 67% to 88%; accuracy increased from 85% to 94%). Conclusions EUS-FNA plus KRAS-mutation analysis, using allelic discrimination, is accurate and improves the diagnosis of pancreatic adenocarcinoma when pathology is inconclusive or doubtful.

The incidence of pancreatic cancer is increasing but the prognosis remains poor. The lack of clearly identified risk factors hinders screening and early diagnosis (when the disease is still curable). The sole curative treatment is surgical resection, which is feasible in only 15% of patients. Chemotherapy regimens are being developed in an attempt to prolong the survival of patients who do not quality for surgery. Recent advances include better knowledge and treatment of pre-neoplastic pancreatic lesions such as mucinous tumors. Basic and translational researchers are attempting to identify tumoral or circulating antigens that could serve as diagnostic markers and/or therapeutic targets.

SPECIFIC AIMSThe sst2 somatostatin receptor is an inhibitory G-protein-coupled receptor that inhibits normal and tumor cell growth by a mechanism involving activation of the tyrosine phosphatase SHP-1. We addressed whether nitric oxide synthase (NOS) is involved in sst2 signaling. We first investigated whether an NOS isoform interacts with SHP-1 and is activated by somatostatin. We further studied NOS role for sst2-mediated negative growth signal regulation and identified the associated molecular mechanism.PRINCIPAL FINDINGS1. SHP-1 associates with neuronal NOS (nNOS) and is involved in somatostatin-induced dephosphorylation of nNOSTo investigate whether SHP-1 interacts with NOS, we performed coimmunoprecipitation and immunoblotting studies in CHO cells expressing sst2 (CHO/sst2). nNOS, but not endothelial NOS, was detected within anti-SHP-1 immunoprecipitates in resting cells. SHP-1 was also coprecipitated within anti-nNOS immunoprecipitates. CHO/sst2 cell treatment with RC-160 (1 nM), a sst2 agonist, in...

Somatostatin acts as an inhibitory peptide of various secretory and proliferative responses. Its effects are mediated by a family of G-protein-coupled receptors (sst1-5) that can couple to diverse signal transduction pathways such as inhibition of adenylate cyclase and guanylate cyclase, modulation of ionic conductance channels, and protein dephosphorylation. The five receptors bind the natural peptide with high affinity but only sst2, sst5 and sst3 bind the short synthetic analogues. Somatostatin negatively regulates the growth of various normal and tumour cells. This effect is mediated indirectly through inhibition of secretion of growth-promoting factors, angiogenesis and modulation of the immune system. Somatostatin can also act directly through sst receptors present on target cells. The five receptors are expressed in various normal and tumour cells, the expression of each receptor being receptor subtype and cell type specific. According to the receptor subtypes, distinct signal transduction pathways are involved in the antiproliferative action of somatostatin. Sst1, 4 and 5 modulate the MAP kinase pathway and induce G1 cell cycle arrest. Sst3 and sst2 promote apoptosis by p53-dependent and -independent mechanisms, respectively.

In CHO cells we had found that CCK positively regulated cell proliferation via the activation of a soluble guanylate cyclase. Here we demonstrate that CCK stimulated a nitric oxide synthase (NOS) activity. The production of NO was involved in the proliferative response elicited by CCK regarding the inhibitory effect of NOS inhibitors L-NAME and alpha-guanidinoglutaric acid. We identified the NOS activated by the peptide as the neuronal isoform: the expression of the C415A neuronal NOS mutant inhibited both CCK-induced stimulation of NOS activity and cell proliferation. These two effects were also inhibited after expression of the C459S tyrosine phosphatase SHP-2 mutant and the betaARK1 (495-689) sequestrant peptide, indicating the requirement of activated SHP-2 and G-betagamma subunit. Kinetic analysis (Western blot after coimmunoprecipitation and specific SHP-2 activity) revealed that in response to CCK-treatment, SHP-2 associated to G-beta1 subunit, became activated, and then dephosphorylated the neuronal NOS through a direct association. These data demonstrate that the neuronal NOS is implicated in proliferative effect evoked by CCK. A novel growth signaling pathway is described, involving the activation of neuronal NOS by dephosphorylation of tyrosyl residues.

Pancreatic cancer is the fourth leading cause of death by cancer worldwide. There is currently no curative treatment excepting surgery for 15 % of patients. Consequently, it is necessary to identify new therapeutic targets such as microRNAs to help manage this disease. Interestingly, these short non-coding RNAs can negatively control the expression of hundreds of genes, and thus are key regulators of tumor progression and dissemination. In addition, they are implicated in cancer cell resistance to treatment. Taken together, microRNAs can represent a new class of molecular targets. MicroRNAs can be combined with different carriers (either non viral or viral) to increase their stability and specificity. Successful examples of microRNA targeting in vivo for the therapy of experimental models of pancreatic cancer have recently emerged. Nevertheless, clinical trials based on microRNA targeting for cancer are still lacking while their interest as biomarkers is emerging. Importantly, improved delivery and specificity to reduce off-target effects must be controlled to accelerate the use of microRNA as new therapeutic targets in oncology.

Point mutations of the Kras oncogene induce in cancerous cells an uncontrolled increase of cell proliferation and invasiveness. Mutation of Kras appears early during the process of the pancreatic carcinogenesis and is the most frequent genetic alteration in pancreatic adenocarcinoma (75 to 95 % of cases) as well as in precancerous lesions such as PanIN and IMPN. These latter lesions and tumour microenvironment are reproduced in transgenic models developed in mice. These models are induced on the basis of Kras mutation (Pdx1-Cre ; Kras(G12D) mice) associated or not to the inactivation of tumour suppressor genes (TP53, DPC4, INK4A). Kras mutation assay is easily performed in human biological samples, especially in the cellular material sampled in pancreatic masses under endoscopic ultrasound by fine needle aspiration biopsy. In the near future, searching for Kras mutation could be useful in clinical practice either for positive diagnosis of pancreatic adenocarcinoma in case of unconclusive/doubtful cytopathological analysis or for the differential diagnosis with chronic pancreatitis especially in its pseudotumoural form.

Pancreatic ductal adenocarcinoma remains one of the most deadly types of tumor. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is a safe, cost-effective, and accurate technique for evaluating and staging pancreatic tumors. However, EUS-FNA may be inconclusive or doubtful in up to 20% of cases. This review underlines the clinical interest of the molecular analysis of samples obtained by EUS-FNA in assessing diagnosis or prognosis of pancreatic cancer, especially in locally advanced tumors. On EUS-FNA materials DNA, mRNA and miRNA can be extracted, amplified, quantified and subjected to methylation assay. Kras mutation assay, improves diagnosis of pancreatic cancer. When facing to clinical and radiological presentations of pseudo-tumorous chronic pancreatitis, wild-type Kras is evocative of benignity. Conversely, in front of a pancreatic mass suspected of malignancy, a mutated Kras is highly evocative of pancreatic adenocarcinoma. This strategy can reduce false-negative diagnoses, avoids the delay of making decisions and reduces loss of surgical resectability. Similar approaches are conducted using analysis of miRNA expression as well as Mucin or markers of invasion (S100P, S100A6, PLAT or PLAU). Beyond the diagnosis approach, the prediction of response to treatment can be also investigated form biomarkers expression within EUS-FNA materials.

Pancreatic cancer (PDAC) remains a deadly disease with no cure. In this dismal context, we demonstrated that non-viral gene therapy to sensitize cancer cells to chemotherapy was safe and feasible, and offers therapeutic benefit in patients (Thergap trial 2010-2012). However, alternative molecular pathways must be targeted to relieve resistance to treatment for best therapeutic benefit. We identified cytidine deaminase (CDA) that catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, as overexpressed (i) in cohorts of patients resisting to gemcitabine, (ii) in PDAC as compared to normal parenchyma, and (iii) in patients receiving gene therapy. Targeting CDA using genetic tools sensitizes cancer cells to chemotherapy both in vitro and in vivo, with very high efficacy in preclinical models of cancer. On the other hand, loss of CDA per se unexpectedly alters cell proliferation, cell cycle progression and tumor growth in vivo, when more than half of mice engrafted with CDA-null human pancreatic cancer cells remained free of tumors nor developed metastasis. Using a multi-omics approach, we found that both pyrimidine and purine levels are decreased, inducing replicative stress hallmarks in CDA-null cells, and TCA cycle is altered, strongly suggesting mitochondrial dysfunction, as we further demonstrate decreased mitochondrial ATP and down expression of key proteins of the mitochondrial OXPHOS complexes and β-oxidation. Consequently, mitochondrial ROS are elevated with major changes in the redox balance in PDAC cells. Remarkably, tumor cells retaliate using compensatory mechanisms resembling Pasteur effect with elevated lactate levels. These compensatory mechanisms served as secondary targets to (i) further strengthen the inhibition of cell proliferation and migration seen in CDA-null cells, and (ii) annihilate potential mechanisms of resistance to treatment. Taken together, our aforementioned results demonstrate for the first time that targeting CDA, an enzyme primarily thought to be restricted to the pyrimidine salvage pathway, strongly alters PDAC proliferation and tumor progression, via massive modification of cancer cell metabolism and alteration of cell cycle, in the absence of chemotherapy. To the best of our knowledge, the crosstalk between replicative and energetic pathways has been thus far totally underexplored in cancer. As specific inhibitors of CDA are underway, we postulate that PDAC rewiring following CDA targeting can be considered exploitable Achille’s eel or vulnerabilities for novel, DNA and metabolic-based, synthetic lethality strategies and the promise of targeted-approaches with high efficacy for PDAC therapy. Note: This abstract was not presented at the conference. Citation Format: Marion Gayral, Audrey Frances, Estelle Saland, Hubert Lulka, Naima Hanoun, Anthony Lemarié, Laetitia Ligat, Nathalie Saint-Laurent, Frédéric Lopez, Louis Buscail, Jean-Emmanuel Sarry, Pierre Cordelier.{Authors}. Characterization of novel molecular vulnerabilities provoking replicative and energetic stresses in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A23.

International audience

Fil: Loncle, Celine. Aix-Marseille Universite; Francia. Centre National de la Recherche Scientifique; Francia

Despite tremendous efforts from clinician and cancer scientists worldwide, cancer still remains a deadly disease. Aside from conventional radiotherapy of chemotherapy, targeted therapies based on the molecular signature of cancer-derived cells (so called biotherapies) have recently emerged as promising new approaches to help fight this disease. In this context, microRNAs, a class of endogenous, noncoding albeit functional small RNAs, have been recently identified as key players in oncogenesis. They participate in well-characterized oncogenic networks, are induced by oncogenes, such as C-Myc or KRAS, to favor cancer cell proliferation, invasion, survival and escape from apoptosis. However, their potential as therapeutic targets for cancer in relevant preclinical models has been scarcely investigated to date. Consequently, we establish herein for the first time the proof-of-concept of targeting a pleiotropic oncogenic microRNA (miR-21) for the therapy of pancreatic cancer, a deadly disease for which no cure is available. We generated lentiviral particles to achieve highly efficient and long-lasting knockdown of miR-21 in pancreatic cancer-derived cells. Following miR-21 depletion, cancer cells stopped to grow both in 2-D and 3-D settings, and died by apoptosis. For in vivo studies, we generated Gaussia luciferase-secreting pancreatic cancer cells to create the first noninvasive model for orthotopic pancreatic cancer growth monitoring. In this model, targeting miR-21 in exponentially growing tumors using a single injection of lentiviral particles stopped tumor growth, inhibited tumor cell proliferation and induced tumor cells’ apoptosis. Interestingly depleting miR21 stimulated tumor angiogenesis but failed to enhance chemotherapy perfusion and efficacy in this model. Taken together, our study raises many important issues: 1) We demonstrate for the first time that in vivo targeting of oncogenic microRNA is feasible using lentiviral particles; 2) For tumors addicted to miR-21 such as pancreatic cancer, depletion of oncogenic microRNA leads to tumor progression inhibition in a very aggressive model of cancer. Because miR-21 is overexpressed ubiquitously in malignant diseases, our approach can be applied for the treatment of many cancers. In addition, targeting miR21 should not impact the function of the normal adjacent tissue that doesn’t express this therapeutic target, thus reinforcing the safety of our approach. Last, targeting miR-21 offers the unique opportunity to knockdown several oncogenic pathways involved in cancer cells proliferation, survival and escape from apoptosis “all at once.” This may prove essential to prevent tumor clonality and resistance to treatment as previously observed with other targeted biotherapies such as antiangiogenic agents. We are currently performing a unique gene therapy clinical trial for pancreatic cancer (http://clinicaltrials.gov/ct2/show/NCT01274 455?term=thergap&rank=1). Based on our experience and on the results presented herein, it is tempting to speculate that targeting oncogenic microRNA such as miR-21 will be applied in the near future in forthcoming gene therapy clinical trials for cancer, including pancreatic adenocarcinoma. Citation Format: Flavie Sicard, Hubert Lulka, Louis Buscail, Pierre Cordelier. In vivo targeting of oncogenic microRNA for the therapy of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A27.

International audience; Endothelial-mesenchymal transition (EndMT) has a significant role in embryonic heart formation and in various pathologies. However, the molecular mechanisms that regulate EndMT induction remain to be elucidated. We show that suppression of receptor tyrosine kinase Tie1 but not Tie2 induces human endothelial cells to undergo EndMT and that Slug deficiency reverts this process. We find that Erk1/2, Erk5 and Akt cascades control Slug promoter activity induced by Tie1 deficiency. Interestingly, EndMT is present in human pancreatic tumour. We propose that EndMT associated with Tie1 downregulation participates in the pathological development of stroma observed in tumours.

Aims The purpose of this study was to investigate the clinical feasibility and utility of low-density array analysis on samples obtained from endoscopic ultrasound-guided fine needle aspiration biopsy in locally advanced and/or metastatic pancreatic ductal adenocarcinoma and chronic pancreatitis. Patients and methods In this prospective multicenter study, we quantified candidate gene expression in biopsies sampled from 44 locally advanced and/or metastatic pancreatic carcinoma and from 17 pseudotumoural chronic pancreatitis using dedicated low-density array microfluidic plates. Results We first demonstrated that 18S gene expression is stable and comparable in normal pancreas and pancreatic cancer tissues. Next, we found that eight genes ( S100P , PLAT , PLAU , MSLN , MMP-11 , MMP-7 , KRT7 , KRT17 ) were significantly over expressed in pancreatic cancer samples when compared to pseudotumoural chronic pancreatitis ( p value ranging from 0.0007 to 0.0215): Linear discriminative analysis identified S100P , PLAT , MSLN , MMP-7 , KRT7 as highly explicative variables. The area under receiver operating curve establishes the clinical validity of the potential diagnostic markers identified in this study (values ranging from 0.69 to 0.76). In addition, combination of S100P and KRT7 gave better diagnosis performances (Area Under Receiver Operating Curve 0.81, sensitivity 81%, specificity 77%). Conclusion We demonstrate that molecular studies on EUS-guided FNA material are feasible for the identification and quantification of markers in PDAC patients diagnosed with non-resectable tumours. Using low-density array, we isolated a molecular signature of advanced pancreatic carcinoma including mostly cancer invasion-related genes. This work stems for the use of novel biomarkers for the molecular diagnosis of patient with solid pancreatic masses.

Background The multicellular tumor spheroid (MCTS) is an in vitro model associating malignant-cell microenvironment and 3D organization as currently observed in avascular tumors. Methods In order to evaluate the relevance of this model for pre-clinical studies of drug combinations, we analyzed the effect of gemcitabine alone and in combination with the CHIR-124 CHK1 inhibitor in a Capan-2 pancreatic cell MCTS model. Results Compared to monolayer cultures, Capan-2 MCTS exhibited resistance to gemcitabine cytotoxic effect. This resistance was amplified in EGF-deprived quiescent spheroid suggesting that quiescent cells are playing a role in gemcitabine multicellular resistance. After a prolonged incubation with gemcitabine, DNA damages and massive apoptosis were observed throughout the spheroid while cell cycle arrest was restricted to the outer cell layer, indicating that gemcitabine-induced apoptosis is directly correlated to DNA damages. The combination of gemcitabine and CHIR-124 in this MCTS model, enhanced the sensitivity to the gemcitabine antiproliferative effect in correlation with an increase in DNA damage and apoptosis. Conclusions These results demonstrate that our pancreatic MCTS model, suitable for both screening and imaging analysis, is a valuable advanced tool for evaluating the spatio-temporal effect of drugs and drug combinations in a chemoresistant and microenvironment-depending tumor model.

International audience; Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of β-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or β-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and β-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for β-arrestin recruitment. However, this region and β-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R*β-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth cause of death by cancer in Western countries. Its poor prognosis is primarily explained by a lack of early diagnostic markers and efficient therapeutic treatments. PDAC does not appear de novo but rather originates of an accumulation of genetic and epigenetic alterations that leads to an aberrant production of diverse molecules such as RNA and proteins. These altered expression profiles result in a multi-step progression of precursor lesions to invasive PDAC. Therefore, a better understanding of the early genetic and epigenetic alterations occurring in PDAC development is valuable for diagnostic and new therapeutic strategies. MicroRNAs (miRNAs) are small endogenous RNA molecules that function as translation inhibitors of messenger RNA by binding to their 3′ untranslated region. These molecules are tightly involved in the regulation of many physiological processes such as development, proliferation, invasion, and apoptosis among others. Their expressions are profoundly altered in PDAC and are strongly involved in PDAC carcinogenesis. In this chapter, we describe the miRNAs for which the expression is altered in PDAC and PDAC pre-neoplastic lesions. We outline the different molecular mechanisms that lead to altered miRNA expression in PDAC cells as well as the signaling pathways affected in response to altered miRNA expression. Lastly, we review the potential interests of miRNA as biological markers and therapeutic tools for PDAC.

Background: The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is accounted for by the absence of early diagnostic markers and effective treatments. MicroRNAs inhibit the translation of their target mRNAs. The production of microRNAs is strongly altered in cancers, but the causes of these alterations are only partially known. DNA hypermethylation is a major cause of gene inactivation in cancer. Our aims were to identify microRNAs whose gene expression is inactivated by hypermethylation in PDAC and to determine whether this hypermethylation-mediated repression is an early event during pancreatic carcinogenesis. We also sought to investigate whether these differentially methylated regions can serve as a diagnostic marker for PDAC.Methods: MicroRNA production was measured by microarray hybridization and reverse-transcription quantitative PCR. The level of DNA methylation was measured by bisulfite mapping and semiquantitative methylation-specific PCR.Results: We identified 29 microRNAs encoded by genes whose expression is potentially inactivated by DNA hypermethylation. We focused our study on microRNA 148a (miR-148a) and found its production to be repressed, not only in PDAC samples but also in preneoplastic pancreatic intraepithelial neoplasia (PanIN) lesions. More importantly, we found that hypermethylation of the DNA region encoding miR-148a is responsible for its repression, which occurs in PanIN preneoplastic lesions. Finally, we show that the hypermethylated DNA region encoding miR-148a can serve as an ancillary marker for the differential diagnosis of PDAC and chronic pancreatitis (CP).Conclusions: We show that the hypermethylation of the DNA region encoding miR-148a is responsible for its repression in PDAC precursor lesions and can be a useful tool for the differential diagnosis of PDAC and CP.

International audience; Pancreatic cancer (PC) remains a life-threatening disease. Efficient therapeutic gene delivery to PC-derived cells continues to present challenges. We used self-inactivated lentiviral vectors to transduce PC-derived cells in vitro and in vivo. We showed that lentiviral vectors transduce PC-derived cell lines with high efficiency (>90%), regardless of the differentiation state of the cell. Next, we transferred human interferon beta (hIFN-beta) gene. Expression of hIFN-beta in PC cells using lentiviral vectors resulted in the inhibition of cell proliferation and the induction of cell death by apoptosis. In vivo, lentiviral administration of hIFN-beta prevented PC tumor progression for up to 15 days following gene therapy, and induced tumor regression/stabilization in 50% of the mice treated. Again, hIFN-beta expression resulted in cancer cell proliferation inhibition and apoptosis induction. We provide evidence that human immunodeficiency virus (HIV)-1-based lentiviral vectors are very efficient for gene transfer in PC-derived cells in vitro and in vivo. As a consequence, delivery of hIFN-beta stopped PC tumor progression. Thus, our approach could be applied to the 85% of PC patients with a locally advanced disease.

International audience; Krüppel-like factor (KLF) family members share a three C2H2 zinc finger DNA binding domain, and are involved in cell proliferation and differentiation control in normal as in pathological situations. Studies over the past several years support a significant role for this family of transcription factors in carcinogenesis. KLFs can both activate and repress genes that participate in cell-cycle regulation. Among them, many up-regulated genes are inhibitors of proliferation, whereas genes that promote cell proliferation are repressed. However, several studies do present KLFs as positive regulator of cell proliferation. KLFs can be deregulated in multiple cancers either by loss of heterozygosity (LOH), somatic mutation or transcriptional silencing by promoter hypermethylation. Accordingly, KLF expression was shown to mediate growth inhibition when ectopically expressed in multiple cancer-derived cell lines through the inhibition of a number of key oncogenic signaling pathways, and to revert the tumorogenic phenotype in vivo. Taken together, these observations suggest that KLFs act as tumor suppressor. However, in some occasion, KLFs could act as tumor promoters, depending on "cellular context". Thus, this review will discuss the roles and the functions of KLF family members in carcinogenesis, with a special focus on cancers from epithelial origin.

International audience; Pancreatic ductal adenocarcinoma (PDAC) is still the fourth leading cause of cancer-related deaths in Western countries, with increasing incidence. Neither effective prognostic markers nor therapies exist for this cancer. MicroRNAs are potent inhibitors of protein translation, and aberrantly expressed in many cancers. Because let-7 microRNA targets the K-ras oncogene, we aimed to characterize let-7 expression and function in PDAC in vitro and in vivo. Let-7 expression was quantified by real-time RT-PCR from resected tumors and matching adjacent tissue, and in endoscopic ultrasound-guided fine needle aspiration material from patients with PDAC. Let-7 is detected by reverse transcription in situ PCR in a PDAC tissue microarray. PDAC-derived cells were transfected with plasmid-based synthetic microRNAs or by lentiviral transduction, in vitro and in vivo. Let-7 microRNA expression is strongly reduced in PDAC samples, as compared with adjacent tissue. Let-7 is present in normal acinar pancreatic cells, and lost in poorly differentiated cancer samples. In addition, let-7 expression was repressed in patients with PDAC not eligible for surgery. Restoring let-7 levels in cancer-derived cell lines strongly inhibits cell proliferation, K-ras expression, and mitogen-activated protein kinase activation, but fails to impede tumor growth progression after intratumoral gene transfer or after implantation of Capan-1 cells stably overexpressing let-7 microRNA. We describe here for the first time the extensive loss of expression of let-7 in PDAC. In addition, this study provides the initial steps for a microRNA replacement therapy for this cancer.

1932-6203 (Electronic) Journal Article Research Support, Non-U.S. Gov't; International audience; BACKGROUND: Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation. PRINCIPAL FINDINGS: Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth. CONCLUSION: These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.

Gene therapy is defined as the transduction of organisms or cells with replication-deficient recombinant viruses containing the desired gene or genes. Many replication-deficient viruses are currently used or have been proposed as gene transduction vectors. Examples include retroviruses, adenoviruses, adeno-associated viruses and herpesviruses. Although gene therapy seems to be feasible using recombinant viruses, all involve significant problems that limit or preclude their applicability to gene therapy in a clinically relevant setting. Many reasons for these difficulties lie in the nature of the viral agents used to introduce the foreign gene(s) in question. SV40 (Simian Virus-40) has been demonstrated to provide a unique vector for gene therapy which has several advantages over any of the currently available viral vectors. This review will discuss recent patents on making SV40 vectors from SV40 virus, on designing improved packaging cell lines for SV40 production, and ultimately, on producing SV40-like nanoparticles devoid of SV40 genome sequences. These patents and patent applications provide valuable information essential for fighting multiple diseases using gene therapy.

Macronodules (MN) occurring in cirrhosis are considered to be precursor lesions for hepatocellular carcinoma (HCC). However, early molecular events in hepatocellular carcinogenesis are poorly understood. The aim of this study was to compare gene expression profiling between cirrhotic tissues, MN, and HCC, to identify genes early involved in liver carcinogenesis.Tissues were obtained from explanted livers: nine cirrhosis, 10 MN, and seven HCC. Total RNAs were extracted by RNeasy and reverse transcribed with labelled [(33)P]-alpha ATP. Hybridations were performed on Atlas Human Cancer 1.2 membranes (1176 genes).A two-way hierarchical clustering algorithm successfully isolated specific gene expression profiles when comparing MN, cirrhosis, and HCC. A total of 16 and 14 genes were up- and down-expressed, respectively, in HCC as compared to cirrhotic tissues. The molecular signature of MN was characterized by the down-expression of 23 and 42 genes as compared to cirrhosis and HCC, respectively. Among them, Klf6 was down-expressed in all MN samples whereas it was over-expressed in cirrhosis and HCC. This result was confirmed at RNA level by quantitative real time-polymerase chain reaction and at protein level by Western blotting. However, no mutation in the exon 2 of Klf6 was detected.We identified a molecular signature of MN characterized by a down-expression of several genes. One of them, Klf6 was found to be down-expressed in all MN without evidence of somatic mutations in the exon 2. This gene could be involved at an early stage of hepatocarcinogenesis.

International audience; Hepatocellular carcinoma (HCC) is a major public health concern because of the absence of early diagnosis and effective treatments. Efficient diagnosis modalities and therapies to treat HCC are needed. Kruppel-like factor (KLF) family members, such as KLF6, are involved in cell proliferation and differentiation. KLF6 is inactivated in solid tumors, which may contribute to pathogenesis. However, KLF6 status in HCC is controversial. Thus, we undertook the characterization of KLF6 expression and function in HCC and HCC-derived cell lines. We found that HCC, HepG2 and HuH7 cells expressed KLF6 messenger ribonucleic acid and protein. Next, using RNA interference, we demonstrated that inhibiting KLF6 expression in vitro strongly impaired cell proliferation-induced G1-phase arrest, inhibited cyclin-dependent kinase 4 and cyclin D1 expression, and subsequent retinoblastoma phosphorylation. Finally, KLF6 silencing caused p53 upregulation and inhibited Bcl-xL expression, to induce cell death by apoptosis. Taken together, these data demonstrated that KLF6 is essential for HCC-derived cells to evade apoptosis.

To date, pancreatic adenocarcinoma (PDAC) can’t be diagnosed early. Consequently, a majority of patient (80%) display an advanced disease that results in a low resection rate leading to a dismal overall median survival of less than 6 months. We extensively demonstrated that delivering SSTR2, DCK and UMK genes using non-viral vectors strongly inhibit tumor progression and dissemination in relevant experimental models of PDAC. Consequently, we designed a GMP-grade gene therapy product, namely CYL-02, encoding for the above mentioned therapeutic proteins, delivered by a non-viral vector for the management of 24 patients diagnosed with advanced PDAC. In this phase I clinical trial, gene therapy was administered in dose-escalation in the tumors using endoscopic ultrasound. Patients received gemcitabine during this protocol. We demonstrate that the intratumoral injection of the gene therapy product is feasible, well tolerated and safe, and results in the presence and the expression of CYL-02 in the tumors. Consequently, tumor progression is inhibited during the course of the protocol. In patients with locally advanced PDAC at the time of diagnosis, tumor size and serum levels of CA 19-9 significantly decreased following gene therapy regardless of the previous lines of treatment and median progression free survival and median overall survival reaches 6.4 and 11.4 months respectively. In addition, we carried out several advanced proteomic and circulating miRNAs analyses to identify diagnostic biomarkers predictive and/or indicative of treatment response and follow-up, to select clinical trial participants and/or to tailor therapy for individual patient. We already identified several biomarkers of clinical interest using either CE-MS (capillary electrophoresis coupled with mass spectrometry) or high throughput q(RT)PCR for peptide and miRNA identification, respectively, to create a clear prescription path for gene therapy in forthcoming clinical trials. Based on these preliminary yet very encouraging results, we propose that patients may clinically benefit from this approach. Accordingly, we are now entering phase II clinical trial using CYL-02 gene therapy product in patients diagnosed with locally advanced PDAC. Citation Format: Louis Buscail, Barbara Bournet, Fabienne Vernejoul, Gilles Cambois, Hubert Lulka, Naima Hanoun, Aline Meulle, Alix Vignolle-Vidoni, Odile Barbey, Fabian Gross, Rosine Guimbaud, Philippe Ottal, Gérard Tiraby, Pierre Cordelier. Non-viral gene therapy for pancreatic cancer, from preclinical models to phase II clinical trial. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B96.

Pancreatic cancer is one of the most aggressive and devastating human malignancies. There is an urgent need for more effective therapy for patients with advanced disease. In this context, genetic therapy potentially represents a rational new approach to treating pancreatic cancer, which could provide an adjunct to conventional options. Because of the promise of recombinant SV40 vectors, we tested their ability to deliver a transgene, and to target a transcript, so as to inhibit pancreatic tumors growth in vivo. BxPC3 and Capan-1 cells were efficiently transduced using SV40 vectors without selection, as compared to synthetic vectors PEI. SV40 vectors were as efficient as adenoviral vectors, and provided long-term transgene expression. Next, we devised a SV40-derived, targeted gene therapy approach of pancreatic cancer, by combining hTR tumor-specific promoter with sst2 somatostatin receptor tumor-suppressor gene. In vitro cell proliferation was strongly impaired following administration of SV(hTR-sst2). SV40-derived sst2-mediated antiproliferative effect was dependent on the local production of somatostatin. In vivo, intratumoral gene transfer of sst2 using rSV40 vectors resulted in a marked inhibition of Capan-1 tumor progression, and proliferation. These results represent the initial steps toward a novel approach to the gene therapy of pancreatic cancer using SV40 as a vector.

Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.

Excepting surgical resection, there is no efficient treatment against pancreatic cancer. The chemotherapeutic agent gemcitabine improves the patient's clinical status but survival is not prolonged. The aim of this study was to design a new strategy to render gemcitabine more efficient in the treatment of pancreatic cancer using gene therapy. We have generated a fusion gene (DCK::UMK) combining deoxycytidine kinase (DCK) and uridine monophosphate kinase (UMK), which converts gemcitabine into its toxic phosphorylated metabolite. Antitumor effects of DCK::UMK gene expression were tested in vitro and in vivo in an orthotopic transplantable model of pancreatic cancer established in hamsters. DCK::UMK sensitizes pancreatic cancer cells to gemcitabine by reducing dramatically both in vitro cell viability and in vivo tumor volume. We found that in vivo expression of DCK::UMK resulted in an antitumor bystander effect due to apoptosis of untransduced cells. In vivo intratumoral gene transfer of DCK::UMK using the synthetic carrier PEI induced a potent tumor regression. Taken together, the results show that the fusion gene DCK::UMK sensitizes pancreatic cancer cells to gemcitabine treatment to induce cell death by apoptosis and tumor regression. Intratumoral delivery of the DCK::UMK gene in combination with gemcitabine might be of high interest for pancreatic cancer management.

Transducing macrophages and other phagocytic cells has been problematic because these cells are largely nondividing and can phagocytose and degrade viral gene delivery vectors. Because of their carriage of the CCR5 chemokine receptor that functions as a coreceptor for most clinical strains of HIV, these cells are also key targets in early HIV infection and dissemination. We describe here a strategy to transduce these phagocytes, reduce cell membrane CCR5, and protect from infection with R5-tropic HIV. Recombinant Tag-deleted SV40 vectors were used to transduce unselected CCR5-bearing cell lines and primary cells with98% efficiency. rSV40s were designed to express two different anti-CCR5 small interfering RNAs (siRNAs), driven by the adenoviral VA1 polymerase III (pol III) promoter, which localizes the transcripts in the cytoplasm. Transduction with both siRNAs substantially reduced CCR5 mRNA, which in turn decreased detectable cell membrane CCR5. Both CCR5+ cell lines and primary cells were used: SupT1/CCR5 cells, monocyte-derived macrophages (MDM), and primary human brain microglia. In addition, one siRNA, siRNA R5 #5, was designed to recognize conserved sequences in both murine and human CCR5 mRNA and effectively reduced CCR5 transcript in cells of both species. These siRNAs largely protected CCR5+ cell lines and primary human macrophages and brain microglia from challenge with R5-tropic HIV. Therefore, strategies to target CCR5 using rSV40-delivered, VA promoter-driven siRNAs may be useful therapeutic options for treating HIV infection.

Chronic infection with hepatitis B virus (HBV) has potentially devastating consequences and is very difficult to treat. Therapy with recombinant interferons (IFN), especially IFN-alpha, may be effective. The blood IFN-alpha levels that are needed to maintain therapeutic IFN-alpha levels in the liver, however, often cause severe side effects. Gene delivery to the liver may provide a solution. Using a long-term expression construct could provide the desired levels of IFN locally without the need to maintain potentially problematic blood levels. Recombinant, Tag-deleted SV40-derived vectors transduce hepatocytes efficiently and provide permanent transgene expression. We designed an expression construct that was effective against HBV and whose activity was limited to HBV-infected cells. To do this, we exploited the ability of HBV X protein to activate NF-kappaB and, via NF-kappaB, to activate promoter activity of HIV long terminal repeat (LTR) in hepatocytes. Using HIVLTR as a conditional promoter upstream of human and murine IFN-alpha and IFN-gamma cDNAs, rSV40 vectors were used to test the responsiveness of IFN to HBV and the ability of these IFNs to inhibit HBV transcripts and protein production and to activate IFN signaling in neighboring untransduced cells. We found that in hepatocyte cell lines and in primary hepatocytes, HBV activated the promoter activity of the HIVLTR via NF-kappaB. When whole HBV genome was delivered to cells by transfection to simulate HBV infection, IFN expression was activated, IFNs were produced and secreted, and they protected cells from HBV. Levels of IFN proteins that were secreted in this context were comparable to targeted blood levels needed to control chronic hepatitis viral infection. Further, IFNs that were elicited and secreted in this manner were able to activate IFN-induced signaling pathways in neighboring, untransduced cells and so were likely to provide protection even to cells that the rSV40 vector did not transduce. Gene delivery using such rSV40 vectors expressing IFNs conditionally in response to HBV may be an attractive therapeutic option for the treatment of chronic hepatitis B.

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.

Gene therapy to treat primary and secondary CNS diseases, including neuro-AIDS, has not yet been effective. New approaches to delivering therapeutic genes to the central nervous system are therefore required. Recombinant SV40 vectors (rSV40) transduce both dividing and quiescent cells efficiently, and so we tested them for their ability to deliver anti-HIV-1 transgenes to terminally differentiated human NT2-derived neurons (NT2-N). These vectors transduced >95% of immature as well as mature human neurons efficiently, without detectable toxicity and without requiring selection. rSV40 gene delivery was stable to retinoic acid-induced neuronal differentiation. The rSV40 vectors used in these studies, SV(RevM10) and SV(AT), respectively carried the cDNAs for RevM10, a trans-dominant mutant of HIV-1 Rev, and human alpha1-antitrypsin. As measured by HIV-1 p24 antigen assays and by immunostaining for gp120, NT2-N treated with these vectors strongly resisted challenge with different strains of HIV-1. Protection from HIV replication and HIV-induced cytotoxicity was conferred by SV(AT) and SV(RevM10) and remained constant throughout retinoic acid-induced neuronal differentiation and for the duration of these studies (> or =11 weeks). rSV40 transduction of human neurons might therefore be a practicable approach to gene delivery for the treatment of CNS diseases, including neuro-AIDS.

Recombinant gene delivery vectors derived from Tag -deleted SV40 viruses (rSV40s) are potentially useful tools for transduction of many cell types, both in culture and in animals. Characteristically, these vectors: (i) can be made to very high titers (>10 12 infectious units/ml); (ii) infect almost all nucleated mammalian cell types, whether resting or dividing, with high efficiency such that selection is not necessary; (iii) integrate rapidly into the cellular genome and provide for permanent transgene expression without detectable diminution over time; (iv) lack immunogenicity, neither imparting antigenicity to transduced cells nor eliciting neutralizing immune responses against themselves and thus; (v) can be administered multiple times in vivo to normal, immunocompetent animals; (vi) can carry up to ≈5 kb of foreign DNA; and (vii) are safe. Recombinant SV40-derived vectors have been used with pol II promoters to deliver transgenes that encode intracellular, cell membrane, and secreted proteins. They have also been used with pol III promoters to deliver untranslated RNAs, including ribozymes, antisense, RNA decoys, and small inhibitory RNAs. Experimental therapeutic applications of these vectors have included inhibiting HIV in culture and in vivo ; delivering transgenes encoding important missing enzymes, in animal models of human diseases; protecting cells from free radical-induced damage; immunizing against lentiviral antigens; and manipulating the balance of neurotransmitters in the brain. These vectors are limited by their cloning capacity of 5 kb and by the fact that they do not easily express commonly used marker genes such as fluorescent proteins and β-galactosidase. Currently, human trials of rSV40 vectors are being planned.

Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45-60 years were excluded to help define "young" and "old" groups. Thirty-nine cases of sporadic EOCRC (patients ≤ 45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways including the TNFR1 pathway have been identified as potential biomarkers for both the diagnosis and treatment of this disease.

To evaluate the in vivo efficacy of gene therapy for treating human immunodeficiency virus type 1 (HIV-1) infection, a novel simian virus (SV) 40-derived vector gene delivery system that efficiently transduces human leukocytes was combined with a model using severe combined immunodeficiency mice infected with HIV-1 and implanted with human fetal thymic and liver tissue (thy/liv-SCID-hu mice). The SV40-derived vector, SV(Aw), which encodes a variable fragment antibody recognizing HIV-1 integrase (IN#33),was injected into the human thymic grafts of thy/ liv-SCID-hu mice and induced IN#33 expression in most of the thymocytes in the graft. After in vivo challenge with HIV-1, IN#33 expression inhibited in vivo HIV-1 infection, as evidenced by the markedly lower number of HIV-1-infected thymocytes detected in human thymic grafts injected with the SV(Aw) vector, compared with those injected with a control SV40-derived vector. Thus, these findings demonstrate the utility of this new mouse model system for assessing the in vivo efficacy of HIV-1-specific gene therapy. In addition, these data indicate that SV40-derived vectors may provide a system capable of efficient in vivo gene delivery.

Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tag-deleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCK A receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCK A and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.

Background: the median survival of patients diagnosed with locally advanced or metastatic pancreatic adenocarcinoma (PDA) is 4 to 6 months. Chemotherapies (gemcitabine, FOLFIRINOX) and molecular targeted therapies only offer a marginal survival benefit. Consequently, we developed new strategies based on therapeutic gene transfer to help alleviate the dismal prognosis of PDA. We demonstrated that transferring SSTR2 gene in PDA experimental models strongly inhibited tumor progression. We generated DCK::UMK fusion gene to chemosensitize tumor cells to gemcitabine to provoke tumor shrinkage in immune competent animals. Based on these promising results, we asked whether combining SSTR2 and DCK::UMK genes delivered by a non-viral vector may impede PDA growth in patients. Methods: in this first-in-man clinical trial, 22 patients (12 first line, 10 second line) were included in the CHU of Toulouse from 12/2010 to 9/2012. Four different doses (125, 250, 500 and 1000μg) of the gene therapy product (GTP) were injected twice using endoscopic ultrasound (EUS) (day 1 and day 28). Patients concomitantly received gemcitabine for 2 months. Results: the feasibility of this approach is excellent, as all the patients were successfully injected despite differences in the site and the size of the primary tumor, and pre-existing treatments (biliary stent, surgical bypass). No serious adverse events related to the GTP were recorded. We found that less than 0.001% of GTP was detected in blood, while urine was negative in all cases. We found that CA 19.19 levels decreased in 68% (15/22) of patients following gene therapy. In first line patients, we demonstrated partial tumor shrinkage in 2 cases (16%), stable disease in 9 cases (75%), and tumor progression in 1 case (8%). In these latter patients, progression-free survival and median survival following gene therapy reached 7 and 11.3 months, respectively. Conclusions: all together, we demonstrated for the first time that delivering antitumoral genes using non-viral vectors by EUS is feasible and safe in patients with advanced PDA. As our preliminary results tend to demonstrate therapeutic efficacy, we are currently designing a phase II trial based on this approach. Citation Format: Louis Buscail, Barbara Bournet, Fabienne Vernejoul, Hubert Lulka, Gilles Cambois, Naima Hanoun, Fabian Gross, Jean Tiraby, Pierre Cordelier. Development of gene therapy for pancreatic carcinoma: from experimental models to phase I clinical trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3532A. doi:10.1158/1538-7445.AM2013-3532A

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.

s / Pancreatology 12 (2012) 502–597 514 Dept. of Medicine, Div. of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany 2 Institute for General Pathology, University Hospital, Tuebingen, Germany Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive pancreatic cancer with an overall 5-year survival rate of