The β-interferon scaffold attachment region confers high-level transgene expression and avoids extinction by epigenetic modifications of integrated provirus in adipose tissue-derived human mesenchymal stem cells
Moreno R.,Institute Dinvestigacio Biomedica Of Bellvitge Idibell |
Martinez I.,Institute Dinvestigacio Biomedica Of Bellvitge Idibell |
Petriz J.,Universitari Vall dHebron |
Nadal M.,IDIBELL Institute Catala dOncologia |
And 4 more authors.
Tissue Engineering - Part C: Methods | Year: 2011
Because of their abundance and ease of isolation, multilineage differentiation, and paracrine and immunoregulatory capabilities, genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) might combine cell- and gene therapy-based strategies for efficacious tissue repair/regeneration. In this report, we aimed to analyze and influence the long-term dynamics of transgene expression in ASCs transduced with different gammaretroviral vector configurations incorporating the human β-interferon scaffold attachment region (IFN-SAR) and/or chicken 5′HS4 β-globin insulator sequences. In our undifferentiated ASC culture model, naked retroviral vectors experienced EGFP transgene extinction correlating with increases in both H3 histone deacetylation and CpG dinucleotide methylation within the 5′ long terminal repeat-primer-binding site proviral region. Retroviral configurations incorporating the referred boundary elements alone or combined were able to prevent the development of the above epigenetic events and to reduce transgene extinction to different degrees. Particularly, the IFN-SAR sustained the highest levels of H3 histone acetylation and transgene expression throughout the study. Analogously, ASCs differentiating to adipocytes or osteocytes experienced a gradual decline of EGFP expression using naked retroviral vectors. In contrast, only retroviral configurations including the IFN-SAR alone were able to overcome the epigenetic pressure, yielding high-level, uniform transgene expression throughout both lineage differentiation processes. Thus, embedding the IFN-SAR in retroviral vectors should have positive implications in gene repair strategies using ASCs. © Mary Ann Liebert, Inc. 2011.
PubMed | University of California at San Diego, IDIBELL Institute Catala dOncologia, University of California at Los Angeles, Emory University and Sanford Burnham Institute for Medical Research
Type: Journal Article | Journal: Journal of virology | Year: 2015
Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo.Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.
Babel I.,CSIC - Biological Research Center |
Barderas R.,CSIC - Biological Research Center |
Diaz-Uriarte R.,CSIC - National Center for Metallurgical Research |
Moreno V.,IDIBELL Institute Catala dOncologia |
And 6 more authors.
Molecular and Cellular Proteomics | Year: 2011
The characterization of the humoral response in cancer patients is becoming a practical alternative to improve early detection. We prepared phage microarrays containing colorectal cancer cDNA libraries to identify phage-expressed peptides recognized by tumor-specific autoantibodies from patient sera. From a total of 1536 printed phages, 128 gave statistically significant values to discriminate cancer patients from control samples. From this, 43 peptide sequences were unique following DNA sequencing. Six phages containing homologous sequences to STK4/MST1, SULF1, NHSL1, SREBF2, GRN, and GTF2I were selected to build up a predictor panel. A previous study with high-density protein microarrays had identified STK4/MST1 as a candidate biomarker. An independent collection of 153 serum samples (50 colorectal cancer sera and 103 reference samples, including healthy donors and sera from other related pathologies) was used as a validation set to study prediction capability. A combination of four phages and two recombinant proteins, corresponding to MST1 and SULF1, achieved an area under the curve of 0.86 to correctly discriminate cancer from healthy sera. Inclusion of sera from other different neoplasias did not change significantly this value. For early stages (A+B), the corrected area under the curve was 0.786. Moreover, we have demonstrated that MST1 and SULF1 proteins, homologous to phage-peptide sequences, can replace the original phages in the predictor panel, improving their diagnostic accuracy. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Rodriguez-Paredes M.,Hospital Duran i Reynals |
Martinez De Paz A.,Hospital Duran i Reynals |
Simo-Riudalbas L.,Hospital Duran i Reynals |
Sayols S.,Hospital Duran i Reynals |
And 14 more authors.
Oncogene | Year: 2014
Disruption of the histone modification patterns is one of the most common features of human tumors. However, few genetic alterations in the histone modifier genes have been described in tumorigenesis. Herein we show that the histone methyltransferase SETDB1 undergoes gene amplification in non-small and small lung cancer cell lines and primary tumors. The existence of additional copies of the SETDB1 gene in these transformed cells is associated with higher levels of the corresponding mRNA and protein. From a functional standpoint, the depletion of SETDB1 expression in amplified cells reduces cancer growth in cell culture and nude mice models, whereas its overexpression increases the tumor invasiveness. The increased gene dosage of SETDB1 is also associated with enhanced sensitivity to the growth inhibitory effect mediated by the SETDB1-interfering drug mithramycin. Overall, the findings identify SETDB1 as a bona fide oncogene undergoing gene amplification-associated activation in lung cancer and suggest its potential for new therapeutic strategies.© 2014 Macmillan Publishers Limited All rights reserved.
PubMed | IDIBELL Institute Catala dOncologia, Barcelona Institute for Research in Biomedicine, National Cancer Center Research Institute, University of Salamanca and 3 more.
Type: Journal Article | Journal: Oncogene | Year: 2016
The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.
Perez-Perarnau A.,University of Barcelona |
Coll-Mulet L.,University of Barcelona |
Rubio-Patino C.,University of Barcelona |
Iglesias-Serret D.,University of Barcelona |
And 7 more authors.
Epigenetics | Year: 2011
Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells. © 2011 Landes Bioscience.
Davalos V.,Hospital Duran I Reynals |
Moutinho C.,Hospital Duran I Reynals |
Villanueva A.,IDIBELL Institute Catala dOncologia |
Boque R.,Hospital Duran I Reynals |
And 5 more authors.
Oncogene | Year: 2012
Epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transitions occur in the development of human tumorigenesis and are part of the natural history of the process to adapt to the changing microenvironment. In this setting, the miR-200 family is recognized as a master regulator of the epithelial phenotype by targeting ZEB1 and ZEB2, two important transcriptional repressors of the cell adherence (E-cadherin) and polarity (CRB3 and LGL2) genes. Recently, the putative DNA methylation associated inactivation of various miR-200 members has been described in cancer. Herein, we show that the miR-200ba429 and miR-200c141 transcripts undergo a dynamic epigenetic regulation linked to EMT or MET phenotypes in tumor progression. The 5′-CpG islands of both miR-200 loci were found unmethylated and coupled to the expression of the corresponding miRNAs in human cancer cell lines with epithelial features, such as low levels of ZEB1/ZEB2 and high expression of E-cadherin, CRB3 and LGL2, while CpG island hypermethylation-associated silencing was observed in transformed cells with mesenchymal characteristics. The recovery of miR-200ba429 and miR-200c141 expression by stable transfection in the hypermethylated cells restored the epithelial markers and inhibited migration in cell culture and tumoral growth and metastasis formation in nude mice. We also discovered, using both cell culture and animal models, that the miR-200 epigenetic silencing is not an static and fixed process but it can be shifted to hypermethylated or unmethylated 5′-CpG island status corresponding to the EMT and MET phenotypes, respectively. In fact, careful laser microdissection in human primary colorectal tumorigenesis unveiled that in normal colon mucosa crypts (epithelia) and stroma (mesenchyma) already are unmethylated and methylated at these loci, respectively; and that the colorectal tumors undergo selective miR-200 hypermethylation of their epithelial component. These findings indicate that the epigenetic silencing plasticity of the miR-200 family contributes to the evolving and adapting phenotypes of human tumors. © 2012 Macmillan Publishers Limited All rights reserved.
PubMed | IDIBELL Institute Catala dOncologia and Baylor College of Medicine
Type: Journal Article | Journal: Molecular therapy : the journal of the American Society of Gene Therapy | Year: 2015
Oncolytic adenoviruses (OAdV) represent a promising strategy for cancer therapy. Despite their activity in preclinical models, to date the clinical efficacy remains confined to minor responses after intratumor injection. To overcome these limitations, we developed an alternative approach using the combination of the OAdv ICOVIR15 with a replication incompetent adenoviral vector carrying the suicide gene of inducible Caspase 9 (Ad.iC9), both of which are delivered by mesenchymal stromal cells (MSCs). We hypothesized that coinfection with ICOVIR15 and Ad.iC9 would allow MSCs to replicate both vectors and deliver two distinct types of antitumor therapy to the tumor, amplifying the cytotoxic effects of the two viruses, in a non-small-cell lung cancer (NSCLC) model. We showed that MSCs can replicate and release both vectors, enabling significant transduction of the iC9 gene in tumor cells. In the in vivo model using human NSCLC xenografts, MSCs homed to lung tumors where they released both viruses. The activation of iC9 by the chemical inducer of dimerization (CID) significantly enhanced the antitumor activity of the ICOVIR15, increasing the tumor control and translating into improved overall survival of tumor-bearing mice. These data support the use of this innovative approach for the treatment of NSCLC.
PubMed | IDIBELL Institute Catala dOncologia and Autonomous University of Barcelona
Type: Journal Article | Journal: The pharmacogenomics journal | Year: 2015
Epidermal growth factor receptor (EGFR) activation by radiation leads to increased cell proliferation and acts as a radioresistance mechanism. Neoadjuvant chemoradiation is the standard of care for locally advanced rectal cancer, and to date, no biomarkers of response have been found. We analyzed polymorphisms in the EGFR and its ligands, DNA repair genes and the thymidylate synthase in 84 stages II and III rectal cancer patients treated with neoadjuvant capecitabine plus radiotherapy. The rs11942466 polymorphism in the amphiregulin (AREG) gene region was associated with a pathological complete response (ypCR) (odds ratio: 0.26; 95% confidence interval: 0.06-0.79; P=0.014). The rs11615 C>T polymorphism in the ERCC1 gene also correlated with the ypCR as no patients with a C/C genotype achieved ypCR; P=0.023. This is the first work to propose variants within the AREG and the ERCC1 genes as promising predictive biomarkers of ypCR in rectal cancer.
PubMed | Uppsala University and IDIBELL Institute Catala dOncologia
Type: | Journal: Gene therapy | Year: 2016
CD40 is an interesting target in cancer immunotherapy due to its ability to stimulate T-helper 1 immunity via maturation of dendritic cells and to drive M2 to M1 macrophage differentiation. Pancreatic cancer has a high M2 content that has shown responsive to anti-CD40 agonist therapy and CD40 may thus be a suitable target for immune activation in these patients. In this study, a novel oncolytic adenovirus armed with a trimerized membrane-bound extracellular CD40L (TMZ-CD40L) was evaluated as a treatment of pancreatic cancer. Further, the CD40L mechanisms of action were elucidated in cancer models. The results demonstrated that the virus transferring TMZ-CD40L had oncolytic capacity in pancreatic cancer cells and could control tumor progression. TMZ-CD40L was a potent stimulator of human myeloid cells and T-cell responses. Further, CD40L-mediated stimulation increased tumor-infiltrating T cells in vivo, which may be due to a direct activation of endothelial cells to upregulate receptors for lymphocyte attachment and transmigration. In conclusion, CD40L-mediated gene therapy is an interesting concept for the treatment of tumors with high levels of M2 macrophages, such as pancreatic cancer, and an oncolytic virus as carrier of CD40L may further boost tumor killing and immune activation.Gene Therapy advance online publication, 5 January 2017; doi:10.1038/gt.2016.80.