To improve the safety profile and specificity in cancer gene therapy applications, а set of singleplasmid vectors for expression of suicide thymidine kinase gene of the herpes simplex virus (HSVtk) and mouse OX40L gene (m0X40L) involved in regulation of immune response was constructed. Therapeutic transgene expression was directed by a strong constitutive promoter of human cytomegalovirus (CMV), while translation occurred on an RNA template due to an encephalomyocarditis virus internal ribosome entry site (construct CMV-HSVtk-IRES-mOX40L) or 2A-peptide of Porcine Teschovirus-1 (construct CMV-HSVtk-2AmOX40L) inserted between them. The constructs obtained were functionally validated by transient transfection of C26 cells (mouse colon carcinoma). Both bicystronic vectors demonstrated strong cytotoxic activity and produced cytotoxic protein HSVtk at the similar level. To analyze efficiency of mOX40L expression, C26 transfected cells were immunofluorescence-labelled with anti-OX40L-antibodies conjugated with phycoerythrin following flow cytometric analysis. Production of mOX40L was higher in the case of CMV-HSVtk- 2A-mOX40L. Better performance of this expression vector was correlated with our data. Previously, we have demonstrated that a higher level of transgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence. Thus, transfection with the vector based on the 2A peptide sequence is optimal for achieving high efficiency of expression of both oncotherapeutic genes.

One problem in the study of regulatory mechanisms in living systems is the difficulty in analysis of regulatory elements in their natural context. One promising way to solve this problem is direct in situ modification of regulatory element sequences. The new technology of gene modification based on the bacterial CRISPR/Cas system allows one to quickly and accurately modify any genomic fragment, in particular, in cells of an adult organism. This review considers principles of the CRISPR/Cas technology and its application to the study of regulatory elements, as well as the potential of using this technology in studies of regulatory systems in pancreas tumors.

In this study, we evaluated the antitumor activity of a gene therapy complex in which the tumor-specific control of the expression of the effector suicide gene FCU1 was performed using a two-vector system based on the site-specific Cre–LoxP recombinase system. The complex of interest showed a high therapeutic potential in a mouse colon adenocarcinoma model.

A set of vectors for Cre recombinase-dependent expression of the hybrid suicidal FCU1 transgene was constructed, including a two-plasmid system wherein the FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants in which a single plasmid bears both transgenes. To improve the safety profile and specificity in cancer gene therapy applications, as well as to ensure stable propagation of plasmids in bacterial cells, the Cre/LoxP system components were optimized. A bicistronic vector with the Cre expression cassette placed between the LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared with the double-plasmid system in an enzyme-prodrug suicide cancer gene therapy scheme. Therefore, the feasibility of a single-plasmid approach in the development of cancer gene therapy with hierarchical enhancement of therapeutic transgene expression has been demonstrated.

Despite substantial progress in understanding the mechanisms of carcinogenesis and fighting oncology diseases, cancer mortality remains rather high. Therefore, there is a striving to reduce this mortality to the level determined by endogenous biological factors. The review analyzes the mutations that lead to cell malignant transformation and describes the contribution that self-renewal of adult tissues makes to tumorigenesis. Cancer progression is considered as a development of a complicated system where cells mutate, evolve, and are subject to selection. Cancer paradoxes are described in conclusion.

Antitumor efficacy of the combined suicide gene therapy and radiotherapy was studied on the model of CT26 murine colon adenocarcinoma. CMV-FCU1-IRES-mGM-CSF-pGL3 construct with PEG-PEI-TAT (FCU1–mGM/5-FC) block copolymer as a vector was used for intratumoral administration. Tumors were irradiated with a single 5 Gy dose. The efficacy was evaluated according to the grade of tumor growth inhibition (T/C) and lifespan of the animals. Pronounced antitumor activity of the combined use of FCU1–mGM/5-FC system with radiotherapy on the background of prolonged lifespan and the synergism of the applied methods was revealed.

Pancreatic cancer is one of the most aggressive tumor types characterized by chemotherapy resistance and high metastatic activity. Recent studies revealed new genes, which are likely to be actively involved in the regulation of the processes occurring in the pancreas, as well as in the development of cancer in this organ. This review is devoted to the description of one of the recently revealed genes, KLF5, which seems to be a promising target for therapeutic intervention in the most widespread type of pancreatic cancer, ductal adenocarcinoma.

Despite substantial progress in understanding the mechanisms of carcinogenesis and fighting oncology diseases, cancer mortality remains rather high. Therefore, there is a striving to reduce this mortality to the level determined by endogenous biological factors. The review analyzes the mutations that lead to cell malignant transformation and describes the contribution that self-renewal of adult tissues makes to tumorigenesis. Cancer progression is considered as a development of a complicated system where cells mutate, evolve, and are subject to selection. Cancer paradoxes are described in conclusion.

Hybrid therapeutic gene FCU1 gene was cloned into a lentiviral expression vector and the therapeutic effect of its expression was studied in three pancreatic cancer cell lines. Expression of FCU1 gene sensitized cells of two of three studied pancreatic cancer cell lines to 5-fluorocytosine. In addition, uracil phosphoribosyl transferase activity of the hybrid FCU1 protein increased sensitivity of transfected cells of all three studied pancreatic cancer cell lines to 5-fluorouracil, a standard chemotherapeutic agent.

Abstract Background: Insulin-like growth factor 1 (IGF1) exerts a broad anti-apoptotic function, and IGF1/IGF1-receptor (IGF-1R) signaling pathway plays a crucial role in human cancer development and progression. Stromal cells associated with cancer cells are considered an important component of tumor microenvironment, including pancreatic cancers. Previously we have shown an increased expression of IGF1 in pancreatic ductal adenocarcinoma stroma. The goal of our study was to investigate the effect of IGF1 on expression of important for tumor progression genes that regulate epithelial-mesenchymal transition (EMT) in cancer and stromal cells of pancreatic tumors. Methods: The effect of IGF1 was tested on five pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-2, MiaPaCa-2 and Panc1), all cultivated on cell medium with reduced levels of serum. Also, we used primary cultures of pancreatic ductal adenocarcinoma stromal cells on early passages. Gene expression was analyzed via Western Blotting and qRT-PCR. Results: In reduced serum medium IGF1 has been shown to stimulate proliferation in all cell lines and primary cultures. In cancer cell lines MiaPaCa-2 and Panc1 incubation with IGF1 lead to an increase in levels of SNAIL and Zeb1 proteins after 120 hours of incubation. There was an elevation in expression of Twist protein in all cancer cell lines after 120 hours of incubation. However, we have not detected a decrease in levels of epithelial differentiation marker E-cadherin, nor a notable increase in levels of mesenchymal differentiation markers N-cadherin and Vimentin in IGF1-treated cancer cell lines. Only a slight decrease in E-cadherin expression was shown in IGF1-treated AsPC-1 and Panc1 cells. Treatment of primary stromal cells of pancreatic ductal adenocarcinoma with IGF1 results in a slight elevation of N-cadherin and Vimentin expression, as well as a significant increase in anti-apoptotic protein survivin levels. Incubation of stromal cells with specific low-molecular weight inhibitor of IGF-1R was shown to significantly increase levels of SNAI1 gene expression and increase SNAIL protein levels. Conclusions: Analysis of EMT markers and transcription factors in pancreatic cancer cell lines does not allow us to draw conclusions about the ability of IGF1 to cause EMT. Despite increased levels of SNAIL, Zeb1 and Twist proteins, epithelial cancer cells retained stable expression of E-cadherin and did not show an increase in mesenchymal marker expression. In pancreatic tumor stromal cultures IGF1 increases mesenchymal cell phenotype and, evidently, increases cell survival rate by stimulating survivin expression. The effect of IGF-1R inhibitor on the activation of SNAIL expression, as we assume, was caused by interactions between signaling pathways of IGF1 and SHH in stromal cell cultures. The work is supported by grant of Russian Foundation of Basic Research 13-04-40171-KOMFI Note: This abstract was not presented at the meeting. Citation Format: Marina R. Kopantseva, Eugenia Usova, Arsen Mikaelyan, Maria Kostina, Olga Melekhina, Vyacheslav Egorov, Eugene P. Kopantzev. Effect of IGF1 on cancer and stromal cells of human pancreatic tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2015-1540

Background: Genetic targeting of tumor-surrounding tissue is a promising direction of the development of new methods of anticancer therapy, especially for solid tumor types with a pronounced stromal component. Specific expression of suicide genes, such as HSVtk or CD, in the tumor microenvironment potentially can inhibit the growth of tumor cells within stroma and restrict their metastasizing potential. We studied the cytotoxic effect of expressing the therapeutic gene FCU1 on pancreatic ductal adenocarcinoma stromal cells, as well as on co-cultured tumor cells, in different culturing systems and in the presence of various low-molecular inhibitors of signal pathways. Methods: For experiments, we used primary cultures of pancreatic ductal adenocarcinoma stromal cells at early passages. The stromal cells were transfected with a lentivirus vector carrying the chimeric suicide gene FCU1 (the fusion of yeast FCY1 and FUR1 genes). In the experiments with co-culturing, we used tumor cells of Panc1 and AsPC-1 lines labeled with a reporter gene. We studied the effect of low-molecular inhibitors of IGF signal pathway (picropodophyllin and PQ4011) and inhibitors of Shh signal pathway (cyclopamine and GANT61). Results: The transfection of pancreatic ductal adenocarcinoma stromal cells with the lentivirus carrying FCU1 induced proliferation arrest and stromal cell death in the presence of 5-FC prodrug. The cytotoxic effect was stronger for tumor stromal cells as compared with stromal cells of the normal pancreas. It should be noted that 5-FC-containing conditioned medium from tumor stromal cells transfected with FCU1-containing construct, but not with the empty vector, also had a cytotoxic effect on tumor cells. In the co-culturing system, transfection of tumor stromal cells with the lentivirus vector with FCU1 in the presence of 5-FC induced a pronounced cytotoxic effect on the co-cultured pancreas tumor cells. Addition of low-molecular inhibitors of IGF signal pathway to the co-cultured cells enhanced the cytotoxic effect of the suicide gene expression. On the contrary, addition of cyclopamine (an inhibitor of Shh signal pathway) to the co-culturing system reduced the cytotoxic effect of the stromal suicide gene FCU1 expression on tumor cells. Conclusions: Expression of the suicide gene FCU1 in the tumor microenvironment can induce considerable depletion of the tumor stroma of pancreatic ductal adenocarcinoma that is supposed to essentially enhance the antitumor effect of standard chemotherapy. The use of low-molecular inhibitors of the signal pathways activated in tumors does not always increase the antitumor efficiency of gene therapeutic constructs with suicide genes. The work is supported by grant of Russian Foundation for Basic Research 13-04-40171-H. Citation Format: Marina Kopantseva, Eugenia Usova, Maria Kostina, Olga Melekhina, Vyacheslav Egorov, Eugene P. Kopantzev, Eugene D. Sverdlov. Stromal expression of suicide gene FCU1 affects proliferation of tumor cells and depletes stromal cells in co-culture models of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 188. doi:10.1158/1538-7445.AM2014-188

Abstract Introduction: Stromal cells associated with cancer cells are considered an important component of tumor microenvironment of pancreatic cancer. The intratumoral desmoplasia characteristic of pancreatic cancer is a result of growing carcinoma paracrine action on surrounding normal tissue cells. Selective targeting of tumor stroma cells can markedly increase the effectiveness of currently used chemotherapeutic agents against pancreatic cancer. Therefore, a search for new targets in stromal cells is an important part of developing new combined therapies of this disease. Our work was aimed at comparative studying of gene expression in stromal cells of normal pancreas, pancreatitis and pancreatic ductal adenocarcinoma tissues. Methods: Cultured stromal cells were obtained from samples of normal pancreatic (n=3), pancreatitis (n=3) and ductal adenocarcinoma tissues (n=4). The cell cultures obtained were preliminary characterized by immunofluorescence, RT-PCR and western blotting. A full genome gene expression analysis was performed using a modified SAGE technique. Expression of selected dysregulated genes was analyzed by quantitative PCR. Results: Morphological and immunofluorescent analyses of the obtained primary stromal cultures indicated that they could be assigned to pancreatic stellate cells (PSC). The SAGE analysis allowed to quantitatively estimate expression of 9860 genes in normal PSC, 8744 PSC genes in pancreatitis, and 9311 genes in tumor PSC. A comparison of normal and tumor PSC revealed statistically significant (P<0.001) differences in expression of 270 genes. Of them, 146 genes were upregulated and 124 genes downregulated in tumor PSC as compared to normal PSC. An expression analysis of selected dysregulated genes by RT-PCR detected changes in the activity of some genes involved in tumor progression of pancreatic cancer. In particular, the genes of two chemokines (CXCL12 and CX3CL1) and the JAG1 Notch ligand were upregulated in tumor PSC. A similar comparison of tumor PSC and PSC from pancreatitis tissue detected 68 differentially expressed genes (P<0.001), 40 of which were upregulated and 28 downregulated in tumor PSC as compared to pancreatitis PSC. Expression of 23 genes was upregulated in tumor PSC as compared with PSC both from pancreas and pancreatitis. On the whole, the expression profile of tumor PSC was markedly more similar to that of PSC from pancreatitis tissues than from normal PSC. Conclusions: The results obtained in our work revealed statistically significant changes in the expression of some PSC genes in stroma of ductal pancreatic adenocarcinoma as compared to normal PSC. The similarity of tumor and pancreatitis PSC expression profiles may suggest mutual mechanisms of forming the fibrous tissue of pancreatitis and tumor stroma of pancreatic carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4277. doi:1538-7445.AM2012-4277

Members of the 14-3-3 protein family are known to be important regulators of plant primary metabolism, hormonal signal transduction, and ion homeostasis. We identified nine isoforms of 14-3-3 genes of Thellungiella salsuginea, an extremophile relative of Arabidopsis thaliana. All the identified isoforms were designated according to their Arabidopsis orthologs: Chi, Omega, Psi, Phi, Upsilon, Lambda, Mu, Epsilon, and Omicron. Comparison of the deduced amino acid sequences reveals high degree of identity between the members of this protein family. Isoforms, designated as Ts14-3-3 Chi, Omicron, and Mu, display noticeable differences in their C-terminal domain as compared to their Arabidopsis homologs. Phylogenetic analysis demonstrated that the identified isoforms split into two groups, epsilon and non-epsilon, according to the common classification of the 14-3-3 family genes. The Thellungiella 14-3-3 isoforms are differentially expressed in various plant tissues, and real-time RT-PCR revealed that most of the isoforms are highly expressed even under normal growth conditions. In response to abiotic stress, low temperatures and high concentrations of salts, 14-3-3 genes exhibited different expression patterns. Our data suggest that, due to the high expression levels of the 14-3-3 genes, Thellungiella plants are likely pre-adapted to the stress conditions. Differences between the C-terminal domains of some Thellungiella 14-3-3 proteins and their Arabidopsis homologs may result in differences in target protein specificity.