Cancer Stem Complex, Not a Cancer Stem Cell, Is the Driver of Cancer Evolution.

Here, we put forward the hypothesis on the mechanism of functioning of cancer stem cells, provided that they exist. The hypothesis is based on the following postulates. 1) Paracrine exchange between cancer and stromal cells is efficient only if they are in a close contact and form a synapse-like cleft between them for the cell-cell crosstalk. The concentration of paracrine signaling molecules in the cleft is high because of the cleft small volume. 2) Cancer stem cells per se do not exist. Instead, there are cancer stem complexes formed by cancer cells tightly bound to stromal cells (portable niches) that exchange paracrine signals. 3) Cancer stem complex is a complex system with newly emerged properties, such as a stemness and resistance to external impacts, including therapeutic interventions. 4) The stemness manifests itself as the ability of cancer cells within the complex to divide asymmetrically: one daughter cell remains in the complex forming a renewed stem complex, whereas the other daughter cell detaches from the complex and transforms in a non-stem cell capable of differentiation. 5) An increased resistance of a cancer stem complex is due to the integration of its intrinsic defense systems through the exchange of paracrine signals, i.e., represents a microresistance at the cell level. 6) Cancer stem complexes can stochastically dissociate with the formation of non-stem cancer cells. Partially differentiated non-stem cancer cells are able to stochastically bind to the stromal component, dedifferentiate under the action of paracrine signals, and form new cancer stem complexes. Therefore, a tumor is a flexible system existing in the pseudo-equilibrium state. Such systems comply with the Le Chatelier's principle stating that an equilibrium system under the action of external factors activates the processes antagonistic to the changes (homeostasis). This promotes tumor resistance at the level of cell populations, i.e., the macroresistance. 7) The portable niche travels with the cancer cell during metastasis. We propose a general therapeutic strategy targeting the contacts between cancer and stromal cells. The disruption of these contacts should lead to the destruction of cancer stem complexes and elimination of tumors.

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.

Correlation between Expression of KLF5 and ZEB1 Transcription Factor Genes in Pancreatic Cancer.

The mRNA content of the transcription factors KLF5 and ZEB1 was studied in pancreatic tumor tissues and in fetal and normal pancreas. Transcription of these factors was not high and similar in normal and fetal pancreatic tissues but greatly increased in the pancreatic ductal adenocarcinoma tissues. A significant positive correlation between the KLF5 and ZEB1 transcription levels in the pancreatic tumor tissues was observed.

A Universal Tumor-Specific Promoter for Cancer Gene Therapy.

An artificial double tandem tumor-specific promoter based on survivin and human telomerase reverse transcriptase gene promoters was constructed. Studies in in vitro and ex vivo therapeutic systems showed that the designed promoter exhibits a high activity in tumor cells, which is comparable to the activity of the CMV constitutive promoter.

Unsolvable Problems of Biology: It Is Impossible to Create Two Identical Organisms, to Defeat Cancer, or to Map Organisms onto Their Genomes.

The review is devoted to unsolvable problems of biology. 1) Problems unsolvable due to stochastic mutations occurring during DNA replication that make it impossible to create two identical organisms or even two identical complex cells (Sverdlov, E. D. (2009) Biochemistry (Moscow), 74, 939-944) and to "defeat" cancer. 2) Problems unsolvable due to multiple interactions in complex systems leading to the appearance of unpredictable emergent properties that prevent establishment of unambiguous relationships between the genetic architecture and phenotypic manifestation of the genome and make impossible to predict with certainty responses of the organism, its parts, or pathological processes to external factors. 3) Problems unsolvable because of the uncertainty principle and observer effect in biology, due to which it is impossible to obtain adequate information about cells in their tissue microenvironment by isolating and analyzing individual cells. In particular, we cannot draw conclusions on the properties of stem cells in their niches based on the properties of stem cell cultures. A strategy is proposed for constructing the pattern most closely approximated to the relationship of genotypes with their phenotypes by designing networks of intermediate phenotypes (endophenotypes).

The Antitumor Efficacy of a Complex Based on Two-Vector System for Coexpression of the Suicide Gene Fcu1 and Cre Recombinase.

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.

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.

PDX1: A Unique Pancreatic Master Regulator Constantly Changes Its Functions during Embryonic Development and Progression of Pancreatic Cancer.

Multifunctional activity of the PDX1 gene product is reviewed. The PDX1 protein is unique in that being expressed exclusively in the pancreas it exhibits various functional activities in this organ both during embryonic development and during induction and progression of pancreatic cancer. Hence, PDX1 belongs to the family of master regulators with multiple and often antagonistic functions.

[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.

Enhanced Vector Design for Cancer Gene Therapy with Hierarchical Enhancement of Therapeutic Transgene Expression.

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.

[Role of fibroblast growth factors in pancreatic cancer]. / Rol' signal'nogo puti FGF/FGFR v kantserogeneze podzheludochnoi zhelezy.

Fibroblast growth factors belong to a family of growth factors that are involved in various processes in organism and have a wide range of biological functions. Specifically for pancreas, FGFs are important during both organogenesis and carcinogenesis. One of the main characteristic of pancreatic cancer, is it close interaction between cancer and stromal cells via different factors, including FGF. Pathological changes in FGF/FGFR signaling pathway is a complex process. The remodeling effects and stimulation of tumor growth are mostly depend not only on types of receptors, but also from their isoforms. FGF/FGFR signaling pathway is a perspective specific marker for cancer progression, and a potential drug target, which can be used for treatment of pancreatic cancer.

Antitumor efficacy of combined gene and radiotherapy in animals.

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.

Expression of the FAP gene in non-fibroblast human cell lines. Development of cancer-associated fibroblast models.

The fibroblast activation protein (FAP) is selectively expressed in cancer-associated fibroblasts (CAFs) and facilitates tumor progression, which makes this protein an attractive therapeutic target. There are difficulties in obtaining CAFs for studying the function and suppression of FAP. In this work, the expression level of FAP was determined by PCR assay in 25 human cell lines and 8 surgical samples of tumor stroma. The expression of FAP was observed in all tumor stroma samples and in four cell lines: NGP-127, SJCRH30, SJSA-1, and A375. The level of FAP expression in NGP-127, SJCRH30, and SJSA-1 lines as well as in CAFs of patients was comparable, which makes these cell lines a possible model for studying FAP.

Expression of Therapeutic Gene FCU1 Sensitizes Pancreatic Cancer Cells to 5-Fluorocytosine and Enhances the Cytotoxic Effect of 5-Fluorouracil.

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.

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.

Multidimensional Complexity of Cancer. Simple Solutions Are Needed.

Cancer is a complex system. Tumor complexity is determined not only by genetic and epigenetic heterogeneity, but also by a huge number of interactions between cancer and normal cells. The heterogeneity and complexity of a tumor causes failure of molecular targeting therapy as a tool for fighting cancer. This review considers the concepts of malignant tumors as organisms that have common characteristics despite all heterogeneity. This leads to the idea that one of the most promising strategies for fighting cancer is the use of the patient's immune system.