Bram De Craene

Regulatory networks defining EMT during cancer initiation and progression

Epithelial to mesenchymal transition (EMT) is essential for driving plasticity during development, but is an unintentional behaviour of cells during cancer progression. The EMT-associated reprogramming of cells not only suggests that fundamental changes may occur to several regulatory networks but also that an intimate interplay exists between them. Disturbance of a controlled epithelial balance is triggered by altering several layers of regulation, including the transcriptional and translational machinery, expression of non-coding RNAs, alternative splicing and protein stability.

SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions

SIP1/ZEB2 is a member of the δEF-1 family of two-handed zinc finger nuclear factors. The expression of these transcription factors is associated with epithelial mesenchymal transitions (EMT) during development. SIP1 is also expressed in some breast cancer cell lines and was detected in intestinal gastric carcinomas, where its expression is inversely correlated with that of E-cadherin. Here, we show that expression of SIP1 in human epithelial cells results in a clear morphological change from an epithelial to a mesenchymal phenotype. Induction of this epithelial dedifferentiation was accompanied by repression of several cell junctional proteins, with concomitant repression of their mRNA levels. Besides E-cadherin, other genes coding for crucial proteins of tight junctions, desmosomes and gap junctions were found to be transcriptionally regulated by the transcriptional repressor SIP1. Moreover, study of the promoter regions of selected genes by luciferase reporter assays and chromatin immunoprecipitation shows that repression is directly mediated by SIP1. These data indicate that, during epithelial dedifferentiation, SIP1 represses in a coordinated manner the transcription of genes coding for junctional proteins contributing to the dedifferentiated state; this repression occurs by a general mechanism mediated by Smad Interacting Protein 1 (SIP1)-binding sites.

The Transcription Factor Snail Induces Tumor Cell Invasion through Modulation of the Epithelial Cell Differentiation Program

Abberant activation of the process of epithelial-mesenchymal transition in cancer cells is a late event in tumor progression. A key inducer of this transition is the transcription factor Snail, which represses E-cadherin. We report that conditional expression of the human transcriptional repressor Snail in colorectal cancer cells induces an epithelial dedifferentiation program that coincides with a drastic change in cell morphology. Snail target genes control the establishment of several junctional complexes, intermediate filament networks, and the actin cytoskeleton. Modulation of the expression of these genes is associated with loss of cell aggregation and induction of invasion. Chromatin immunoprecipitation experiments showed that repression of selected target genes is associated with increased binding of Snail to their promoters, which contain consensus Snail-binding sites. Thus, Snail constitutes a master switch that directly represses the epithelial phenotype, resulting in malignant carcinoma cells.

Molecular and pathological signatures of epithelial–mesenchymal transitions at the cancer invasion front

Reduction of epithelial cell–cell adhesion via the transcriptional repression of cadherins in combination with the acquisition of mesenchymal properties are key determinants of epithelial–mesenchymal transition (EMT). EMT is associated with early stages of carcinogenesis, cancer invasion and recurrence. Furthermore, the tumor stroma dictates EMT through intensive bidirectional communication. The pathological analysis of EMT signatures is critically, especially to determine the presence of cancer cells at the resection margins of a tumor. When diffusion barriers disappear, EMT markers may be detected in sera from cancer patients. The detection of EMT signatures is not only important for diagnosis but can also be exploited to enhance classical chemotherapy treatments. In conclusion, further detailed understanding of the contextual cues and molecular mediators that control EMT will be required in order to develop diagnostic tools and small molecule inhibitors with potential clinical implications.

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.

Evolutionary functional analysis and molecular regulation of the ZEB transcription factors

ZEB1 and ZEB2, which are members of the ZEB family of transcription factors, play a pivotal role in the development of the vertebrate embryo. However, recent evidence shows that both proteins can also drive the process of epithelial-mesenchymal transition during malignant cancer progression. The understanding of how both ZEBs act as transcription factors opens up new possibilities for future treatment of advanced carcinomas. This review gives insight into the molecular mechanisms that form the basis of the multitude of cellular processes controlled by both ZEB factors. By using an evolutionary approach, we analyzed how the specific organization of the different domains and regulatory sites in ZEB1 and ZEB2 came into existence. On the basis of this analysis, a detailed overview is provided of the different cofactors and post-translational mechanisms that are associated with ZEB protein functionality.

Key signalling nodes in mammary gland development and cancer. The Snail1-Twist1 conspiracy in malignant breast cancer progression.

Breast cancer is the most common cancer among women, and despite significant advances in diagnosing and treating it, metastatic spread of cancer cells results in a high mortality rate. Epithelial-to-mesenchymal transition (EMT) is an embryonic program in which epithelial cells lose their characteristics and gain mesenchymal features. Therefore, EMT might play a very important role during malignant tumour progression. In this review we summarise recent advances in breast cancer research with a particular focus on the transcription factors Snail1 and Twist1. Besides discussing the role of EMT in normal mammary gland development, we describe regulatory mechanisms involving newly discovered upstream regulators and microRNAs, the association of EMT with breast cancer stem cells, and the involvement of the tumour microenvironment in breast cancer progression.

Strategies for immortalization of primary hepatocytes

The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications.

Snail in the frame of malignant tumor recurrence.

Snail plays an important role in the epithelial-to-mesenchymal transition during development and in tumor progression. Induction of Snail expression coincides with drastic morphological changes in cultured epithelial cells. Recently, a new role for Snail in tumor recurrence has been inferred from a reversible HER-2/neu-induced breast cancer mouse model. Comparative transcriptome analysis of human primary breast cancers suggests that elevated Snail expression is correlated with decreased relapse-free survival. Further characterization of Snail as master regulator in this process might enhance our understanding of the molecular and cellular changes during and after breast tumor recurrence.

ZEB2‑transgene expression in the epidermis compromises the integrity of the epidermal barrier through the repression of different tight junction proteins

Abstract Epithelial homeostasis within the epidermis is maintained by means of multiple cell–cell adhesion complexes such as adherens junctions, tight junctions, gap junctions, and desmosomes. These complexes co-operate in the formation and the regulation of the epidermal barrier. Disruption of the epidermal barrier through the deregulation of the above complexes is the cause behind a number of skin disorders such as psoriasis, dermatitis, keratosis, and others. During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their adhesive capacities and gain mesenchymal properties. ZEB transcription factors are key inducers of EMT. In order to gain a better understanding of the functional role of ZEB2 in epidermal homeostasis, we generated a mouse model with conditional overexpression of Zeb2 in the epidermis. Our analysis revealed that Zeb2 expression in the epidermis leads to hyperproliferation due to the combined downregulation of different tight junction proteins compromising the epidermal barrier. Using two epidermis-specific in vivo models and in vitro promoter assays, we identified occludin as a new Zeb2 target gene. Immunohistological analysis performed on human skin biopsies covering various pathogeneses revealed ZEB2 expression in the epidermis of pemphigus vulgaris. Collectively, our data support the notion for a potential role of ZEB2 in intracellular signaling of this disease.