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Abstract Recent advances in tumor biology have revealed that a detailed analysis of the complex interactions of tumor cells with their adjacent microenvironment (tumor stroma) is mandatory in order to understand the various mechanisms involved in tumor growth and the development of metastasis. The mutual interactions between tumor cells and cellular and non-cellular components (extracellular matrix = ECM) of the tumor microenvironment will eventually lead to a loss of tissue homeostasis and promote tumor development and progression. Thus, interactions of genetically altered tumor cells and the ECM on the one hand and reactive non-neoplastic cells on the other hand essentially control most aspects of tumorigenesis such as epithelial-mesenchymal-transition (EMT), migration, invasion (i.e. migration through connective tissue), metastasis formation, neovascularisation, apoptosis and chemotherapeutic drug resistance. In this mini-review we will focus on these issues that were recently raised by two review articles in CCS.

Abstract Background Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-β)/Smad signalling pathway, eventually resulting in loss of TGF-β-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-β are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-β type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-β1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-β1-mediated growth inhibition and cell migration (chemokinesis). Results SiRNA-mediated silencing of Smad3 in the TGF-β responsive PDAC cell line PANC-1 reduced TGF-β1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-β1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-β1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-β signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-β growth inhibition. Conclusions In malignant cells with a functional TGF-β signalling pathway Rac1 antagonizes the TGF-β1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 and Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-β signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression.

Abstract Background Because specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues. Results We introduced the mesenchymal microsphere method as a feasible time- and cell saving screening method to analyse multilineage differentiation properties of adult progenitor cells in a three-dimensional system. For this purpose we isolated, characterized and analyzed new sources of adult murine mesenchymal progenitor cells from perirenal adipose tissue and mediastinal stromal tissue in comparison to bone marrow progenitor cells. The proliferation capacity of the cells was demonstrated by determination of the daily doubling index. Although the flow cytometry analysis of undifferentiated cells revealed differences in the expression of CD marker molecules, all isolates have the capacity for multilineage differentiation following the mesenchymal microsphere protocol as well as the classical "micro mass body" protocol for chondrogenic and the monolayer cultivation protocol for osteogenic and adipogenic differentiation. Differentiation was characterized using histochemical and immunhistochemical staining as well as RT-PCR. Conclusions We were able to show that the mesenchymal microsphere method is an efficient test system for chondro-, osteo- and adipogenic differentiation of adult progenitor cells. The advantage of this system in comparison to classical protocols is that approximately 7 times lower cell numbers are necessary. Since classical culture procedures are time intensive because high cell numbers have to be obtained, the new differentiation method may also save cells and time in future clinical applications using human mesenchymal stromal cells.

Abstract Background miRNAs play important roles in the regulation of gene functions. Maternal dietary modifications during pregnancy and gestation have long-term effects on the offspring, but it is not known whether a maternal high fat (HF) diet during pregnancy and lactation alters expression of key miRNAs in the offspring. Results We studied the effects of maternal HF diet on the adult offspring by feeding mice with either a HF or a chow diet prior to conception, during pregnancy and lactation, and all offspring were weaned onto the same chow diet until adulthood. Maternal HF fed offspring had markedly increased hepatic mRNA levels of peroxisome proliferator activated receptor-alpha (ppar-alpha) and carnitine palmitoyl transferase-1a (cpt-1a) as well as insulin like growth factor-2 (Igf2). A HF diet induced up-regulation of ppar-alpha and cpt-1a expression in the wild type but not in Igf2 knock out mice. Furthermore, hepatic expression of let-7c was also reduced in maternal HF fed offspring. Among 579 miRNAs measured with microarray, ~23 miRNA levels were reduced by ~1.5-4.9-fold. Reduced expression of miR-709 (a highly expressed miRNA), miR-122, miR-192, miR-194, miR-26a, let-7a, let7b and let-7c, miR-494 and miR-483* (reduced by ~4.9 fold) was validated by qPCR. We found that methyl-CpG binding protein 2 was the common predicted target for miR-709, miR-let7s, miR-122, miR-194 and miR-26a using our own purpose-built computer program. Conclusion Maternal HF feeding during pregnancy and lactation induced co-ordinated and long-lasting changes in expression of Igf2, fat metabolic genes and several important miRNAs in the offspring.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, due in part to early invasion and metastasis, which in turn involves epithelial–mesenchymal transition (EMT) of the cancer cells. Prompted by the discovery that two PDAC cell lines of the quasi-mesenchymal subtype (PANC-1, MIA PaCa-2) exhibit neuroendocrine differentiation (NED), we asked whether NED is associated with EMT. Using real-time PCR and immunoblotting, we initially verified endogenous expressions of various NED markers, i.e., chromogranin A (CHGA), synaptophysin (SYP), somatostatin receptor 2 (SSTR2), and SSTR5 in PANC-1 and MIA PaCa-2 cells. By means of immunohistochemistry, the expressions of CHGA, SYP, SSTR2, and the EMT markers cytokeratin 7 (CK7) and vimentin could be allocated to the neoplastic ductal epithelial cells of pancreatic ducts in surgically resected tissues from patients with PDAC. In HPDE6c7 normal pancreatic duct epithelial cells and in epithelial subtype BxPC-3 PDAC cells, the expression of CHGA, SYP, and neuron-specific enolase 2 (NSE) was either undetectable or much lower than in PANC-1 and MIA PaCa-2 cells. Parental cultures of PANC-1 cells exhibit EM plasticity (EMP) and harbor clonal subpopulations with both M- and E-phenotypes. Of note, M-type clones were found to display more pronounced NED than E-type clones. Inducing EMT in parental cultures of PANC-1 cells by treatment with transforming growth factor-β1 (TGF-β1) repressed epithelial genes and co-induced mesenchymal and NED genes, except for SSTR5. Surprisingly, treatment with bone morphogenetic protein (BMP)-7 differentially affected gene expressions in PANC-1, MIA PaCa-2, BxPC-3, and HPDE cells. It synergized with TGF-β1 in the induction of vimentin, SNAIL, SSTR2, and NSE but antagonized it in the regulation of CHGA and SSTR5. Phospho-immunoblotting in M- and E-type PANC-1 clones revealed that both TGF-β1 and, surprisingly, also BMP-7 activated SMAD2 and SMAD3 and that in M- but not E-type clones BMP-7 was able to dramatically enhance the activation of SMAD3. From these data, we conclude that in EMT of PDAC cells mesenchymal and NED markers are co-regulated, and that mesenchymal–epithelial transition (MET) is associated with a loss of both the mesenchymal and NED phenotypes. Analyzing NED in another tumor type, small cell carcinoma of the ovary hypercalcemic type (SCCOHT), revealed that two model cell lines of this disease (SCCOHT-1, BIN-67) do express CDH1, SNAI1, VIM, CHGA, SYP, ENO2, and SSTR2, but that in contrast to BMP-7, none of these genes was transcriptionally regulated by TGF-β1. Likewise, in BIN-67 cells, BMP-7 was able to reduce proliferation, while in SCCOHT-1 cells this occurred only upon combined treatment with TGF-β and BMP-7. We conclude that in PDAC-derived tumor cells, NED is closely linked to EMT and TGF-β signaling, which may have implications for the therapeutic use of TGF-β inhibitors in PDAC management. [ABSTRACT FROM AUTHOR]