Activation of IGF/IGF-IR signaling pathway fails to induce epithelial-mesenchymal transition in pancreatic cancer cells.

BACKGROUND: Pancreatic cancer stromal cells produce various protein factors, which presumably provide cancer cells with drug resistance and may influence their ability to form metastasis via induction of epithelial-mesenchymal transition (ЕМТ). The goal of our project was to study the effects of IGF-I on expression of protein markers of epithelial and mesenchymal differentiation, and on expression of transcriptional regulators of EMT in pancreatic cancer cell lines. METHODS: We used Western blot analysis to study the expression patterns of epithelial and mesenchymal protein markers in pancreatic cancer cell lines, which have been stimulated with IGF-I for various periods of time. The ELISA technique was employed to determine the concentration of IGF-I in conditioned media. Additionally, the effect of IGF-I on proliferation of pancreatic cancer cells was measured via MTS technique. RESULTS: We investigated the effect of IGF/IGF-IR signaling pathway activation on expression levels of cell differentiation markers in five pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-2, MiaPaCa-2 and Panc1). The IGF-I stimulation led to phosphorylation of IGF-IR and activation of PI-3K/Akt signaling cascade. At the same time our results reveal that the activation of IGF/IGF-IR signaling pathway in pancreatic cancer cells does not induce a significant shift in cell phenotype towards mesenchymal differentiation and does not induce a decrease in expression levels of epithelial protein markers. CONCLUSIONS: Our results demonstrate that IGF-I does not function as an effective inductor of EMT in pancreatic cancer cell lines and that stimulation of IGF-I/IGF-IR signaling pathway does not lead to EMT associated changes in cell differentiation.

Heterogeneous Expression of Embryonal Development Master Regulator SOX9 in Patients with Pancreatic Cancer.

The expression levels of the SOX9 gene in fetal, postnatal, and neoplastic pancreatic tissues were compared. In the fetal pancreatic samples, the mean relative level of the SOX9 gene expression was 8 times greater than the normal level. The tumor samples were divided into three groups depending on the SOX9 expression level. The first group showed a 6.5-fold increased expression level of SOX9 with respect to the normal one. The second and normal groups had approximately equal levels expression. The third group showed a 25-fold decreased expression level of SOX9. The discrepancy in the SOX9 expression, associated with the predominance of different functions of this master gene, depends on the poorly predictable individual factors and indicates that SOX9 should be excluded from the potential diagnostic biomarkers of pancreatic cancer.

Variable Effects of Growth Factors on Developmental Gene Expression in Pancreatic Cancer Cells.

Stimulation of BxPC-3, Panc-1, and MIA PaCA-2 pancreatic cancer cells with EGF, HGF, FGF-1, FGF-2, FGF-7, and FGF-10 growth factors caused changes in the expression of master genes regulating pancreatic development (SOX9, HNF3b, GATA-4, GATA-6, and HES1). This, in turn, caused changes in the expression profile of important transcription factors, embryonic development regulators. It was also found that the master genes belonging to the same family may cause opposite effects (suppression or enhancement of expression of a particular transcriptional regulator) in the same cell line.

Dependence of expression of regulatory master genes of embryonic development in pancreatic cancer cells on the intracellular concentration of the master regulator PDX1.

Exogenous expression of the gene encoding the pancreatic master regulator PDX1 in cell lines with different degrees of differentiation of pancreatic cancer cells is accompanied by changes in the expression of known master genes involved in cancer progression. In BxPC3PDX+ cells, as compared to BxPC3PDX-, we detected an increased expression of the following genes: NKX6.1 (2 times), NR5A2 (2.5 times), KLF5 (1.8 times), ZEB1 (3 times), and ONECUT1 (1.3 times), as well as a decreased expression of MUC1 and SLUG genes (3 and 2 times, respectively). In PANC1PDX+ cells, as compared to the control PANC1PDX- cells, we detected a decreased expression of ISL1 (2 times) and an increased expressed of KRT8 (2 times) and MUC1 (by 30%). In the high-grade cell lines (including the BxPC3 line studied), the total content of sites containing the marks of active enhancers was higher than that in the low-grade cell lines (PANC1).

Expression of master regulatory genes of embryonic development in pancreatic tumors.

The expression level of some important master regulators of embryonic development of the pancreas in the tumor samples of this human organ was determined. We found that the transcription of SOX9, GATA4, PDX1, PTF1a, and HNF1b genes in the tumor samples was reduced as compared to the samples of normal pancreatic tissues, and the KLF5 gene expression in the tumor cells was elevated. We assume that all the studied genes, except KLF5, form a single regulatory module that supports the identity of tumor progenitor cells. A simultaneous suppression of expression of these master factors may be critical for the neoplastic transformation of pancreatic cells.

Expression of transcription factor genes in cell lines corresponding to different stages of pancreatic cancer progression.

The expression level of six transcription factor genes and the content of their protein products in five pancreatic cancer cell lines with parallel control of expression of three marker genes reflecting epithelial or mesenchymal state of cells was investigated. Cell lines MIA PaCa-2 and Capan-2 represented the best models of quasi-mesenchymal and epithelial, respectively, types of progression of the pancreatic ductal adenocarcinoma, according to the content of E-cadherin and vimentin and the expression of KLF5 and ZEB1 transcription factors.

[Fibroblast growth factors and their effects in pancreas organogenesis]. / Vliianie faktorov rosta fibroblastov na organogenez podzheludochnoi zhelezy.

Fibroblast growth factors (FGF) - growth factors that regulate many important biological processes, including proliferation and differentiation of embryonic cells during organogenesis. In this review, we will summarize current information about the involvement of FGFs in the pancreas organogenesis. Pancreas organogenesis is a complex process, which involves constant signaling from mesenchymal tissue. This orchestrates the activation of various regulator genes at specific stages, determining the specification of progenitor cells. Alterations in FGF/FGFR signaling pathway during this process lead to incorrect activation of the master genes, which leads to different pathologies during pancreas development. Understanding the full picture about role of FGF factors in pancreas development will make it possible to more accurately understand their role in other pathologies of this organ, including carcinogenesis.

Downregulation of expression of mater genes SOX9, FOXA2, and GATA4 in pancreatic cancer cells stimulated with TGFß1 epithelial-mesenchymal transition.

We show characteristic morphological changes corresponding to epithelial-mesenchymal transition (EMT) program fulfillment in PANC1 cell line stimulated with TGFß1. Our results support downregulation of E-cadherin protein. We show 5- and 28-fold increase in SNAI1 and SNAI2 expression levels and 25- and 15-fold decrease in CDH1 and KRT8 expression levels, respectively, which confirms the EMT-program fulfillment. We demonstrate downregulation of expression of pancreatic master genes SOX9, FOXA2, and GATA4 (2-, 5-, and 4-fold, respectively) and absence of significant changes in HES1, NR5A2, and GATA6 expression levels in the cells stimulated with TGFß1. Our results indicate the absence of induction of expression of PTF1A, PDX1, HNF1b, NEUROG3, RPBJL, NKX6.1, and ONECUT1 genes, which are inactive in PANC1 cell line after the EMT stimulated by TGFß1.

[ADULT STEM CELLS AND CELLS OF MALIGNANT ORIGIN. PART I].

Recent data on adult stem cells are reviewed. According to the present dominant paradigm, it is most probable that cancer predisposition arises or cancer is initiated in these cells.

[SNAI1 and SNAI2 - transcriptional master-regulators of epithelial-mesenchimal transition].

Epithelial-mesenchymal transition is a result of cellular epigenetic reprogramming. During this process differentiated epithelial cells lose specific markers of epithelial phenotype and gradually start displaying qualities of poorly differentiated mesenchymal cells, resistant to apoptosis and capable of local invasion. Despite their obvious importance for biology and medicine, many aspects of epithelial-mesenchymal transition, especially those related to its genetic regulation, remain poorly characterized. In this review we analyze molecular structure and mechanisms of regulation of two closely-related transcription factors SNAI1 and SNAI2, which play an important role in induction and progression of epithelial-mesenchymal transition during both normal development and carcinogenesis. Special attention is paid to the role of SNAI1 and SNAI2 and their active co-reeressors in initiation of epigenetic repression of epithelial differentiation marker genes.

[ADULT STEM CELLS AND CELLS OF MALIGNANT ORIGIN. PART II].

Recent data on adult stem cells are reviewed. According to the present dominant paradigm, it is most probable that cancer predisposition arises or cancer is initiated in these cells.

Cancer specificity of promoters of the genes involved in cell proliferation control.

Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a "head-to-head" position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.

Cellular and molecular phenotypes of proliferating stromal cells from human carcinomas.

BACKGROUND: Stromal cells are a functionally important component of human carcinomas. The aim of this study was to obtain and characterise primary cultures of stromal cells from human carcinomas and the corresponding surrounding normal tissue. METHODS: Primary stromal cell cultures from tumours of lung, oesophagus and pancreas were obtained using a mild tissue dissociation method and a medium for culturing mesenchymal cells. Immunofluorescence staining and western blotting were used to analyse the expression of differentiation markers and selected known oncoproteins in the cell cultures obtained. RESULTS: A panel of stromal primary cultures was prepared from different human tumours and from matched normal cancer-free tissues. The in vitro proliferative potential of tumour-associated fibroblasts was shown to be higher than that of matched normal stromal cells. A mutational analysis of the TP53 and KRAS2 genes in a number of stromal cultures did not reveal known mutations in most cells of the cultures studied. Western blot analysis showed that stromal cells of lung tumours were characterised by a statistically significantly lower expression level of the p16 protein as compared with that in normal lung stromal cells. An important finding of our study was that, according to immunofluorescence assay, a fraction of fibroblast-like vimentin-positive cells in some tumour and normal stromal cell cultures expressed an epithelial marker - cytokeratins. CONCLUSIONS: Proliferating stromal cells from the carcinomas studied proved to be genetically normal cells with altered expression profiles of some genes involved in carcinogenesis, as compared with normal stromal cells. Epithelial-mesenchymal transition may lead to the emergence of transdifferentiated fibroblast-like cells in tumour stroma and in the tumour-surrounding tissue.

Enhancer element potentially involved in human survivin gene promoter regulation in lung cancer cell lines.

We have revealed evolutionarily conserved regions in a 4500-bp DNA sequence 5'-adjacent to the survivin (BIRC5) gene. In the transcribed region of the BIRC5 gene we have detected and characterized in detail a 3'-fragment of the CpG island that stimulated in enhancer-like way the gene promoter activity in normal cells and in a number of cancer, in particular lung cancer, cell lines. When added to the initial 1498-bp survivin promoter region, a transcribed DNA fragment of a CpG island approximately twofold enhanced the promoter activity in cancer cells and in normal lung fibroblasts. The observed effect did not depend upon the orientation of the fragment and distances between the fragment and the transcription initiation site. In the case of a heterologous SV40 virus promoter, the effect was less pronounced. In addition to earlier reports, the results provide new information on the BIRC5 gene expression regulation and also demonstrate that this gene exon sequences can play a significant role in BIRC5 gene expression regulation. The data provide another possibility to increase survivin promoter activity without changing its cancer specificity for application in cancer (in particular, lung cancer) gene therapy.

Identification of some human genes oppositely regulated during esophageal squamous cell carcinoma formation and human embryonic esophagus development.

Here we directly compared gene expression profiles in human esophageal squamous cell carcinomas and in human fetal esophagus development. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from tumor and normal human esophageal samples. cDNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analysis of RNAs from human tumor and the normal esophagus revealed 10 differentially transcribed genes: CSTA, CRNN, CEACAM1, MAL, EMP1, ECRG2, and SPRR downregulated, and PLAUR, SFRP4, and secreted protein that is acidic and rich in cysteine upregulated in tumor tissue as compared with surrounding normal tissue. In turn, genes up- and downregulated in tumor tissue were down- and upregulated, respectively, during development from the fetal to adult esophagus. Thus, we demonstrated that, as reported for other tumors, gene transcriptional activation and/or suppression events in esophageal tumor progression were opposite to those observed during development from the fetal to adult esophagus. This tumor 'embryonization' supports the idea that stem or progenitor cells are implicated in esophageal cancer emergence.