Tissue-selective expression of a conditionally-active ROCK2-estrogen receptor fusion protein

The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models

The Role of the Extracellular Matrix and Its Molecular and Cellular Regulators in Cancer Cell Plasticity

The microenvironment encompasses all components of a tumor other than the cancer cells themselves. It is highly heterogenous, comprising a cellular component that includes immune cells, fibroblasts, adipocytes, and endothelial cells, and a non-cellular component, which is a meshwork of polymeric proteins and accessory molecules, termed the extracellular matrix (ECM). The ECM provides both a biochemical and biomechanical context within which cancer cells exist. Cancer progression is dependent on the ability of cancer cells to traverse the ECM barrier, access the circulation and establish distal metastases. Communication between cancer cells and the microenvironment is therefore an important aspect of tumor progression. Significant progress has been made in identifying the molecular mechanisms that enable cancer cells to subvert the immune component of the microenvironment to facilitate tumor growth and spread. While much less is known about how the tumor cells adapt to changes in the ECM nor indeed how they influence ECM structure and composition, the importance of the ECM to cancer progression is now well established. Plasticity refers to the ability of cancer cells to modify their physiological characteristics, permitting them to survive hostile microenvironments and resist therapy. Examples include the acquisition of stemness characteristics and the epithelial-mesenchymal and mesenchymal-epithelial transitions. There is emerging evidence that the biochemical and biomechanical properties of the ECM influence cancer cell plasticity and vice versa. Outstanding challenges for the field remain the identification of the cellular mechanisms by which cancer cells establish tumor-promoting ECM characteristics and delineating the key molecular mechanisms underlying ECM-induced cancer cell plasticity. Here we summarize the current state of understanding about the relationships between cancer cells and the main stromal cell types of the microenvironment that determine ECM characteristics, and the key molecular pathways that govern this three-way interaction to regulate cancer cell plasticity. We postulate that a comprehensive understanding of this dynamic system will be required to fully exploit opportunities for targeting the ECM regulators of cancer cell plasticity

CXCR4/CXCL12 Participate in Extravasation of Metastasizing Breast Cancer Cells within the Liver in a Rat Model

INTRODUCTION: Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer. METHODS: Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4. RESULTS: In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p,0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p < 0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. CONCLUSION: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.Claudia Wendel, André Hemping-Bovenkerk, Julia Krasnyanska, Sören Torge Mees, Marina Kochetkova, Sandra Stoeppeler and Jörg Haie

BLOCKCHAIN IN THE ART MARKET: Opportunities and Challenges

Blockchain is usually associated with cryptocurrencies. However, as a distributed ledger technology, it can have many other applications. For example, blockchain can bring changes to how the art market operates. It can be utilised for many types of digital transactions, including collection, authentication, tracking of provenance, and sharing ownership of artworks. This main purpose of this thesis is to provide perspectives on how and in what areas blockchain could be used to change the art market. It also examines how this technology may shift the balance of powers in the art market. The thesis further explores opportunities and challenges when using blockchain technology in the art market. The thesis utilises a narrative thematic literature research methodology and includes a qualitative analysis of blockchain technology. Due to the nature and novelty of this technology, the reviewed literature covers a different range of disciplines, in which blockchain can be utilised. The findings were extrapolated to the use of blockchain technology in the art market. The results demonstrate that blockchain can increase the speed, transparency, and volume of art sales worldwide and democratise the sector so that artists, collectors, and spectators can benefit from this technology. A blockchain platform can coexist with other traditional applications. However, before implementing this technology, we may need to overcome technological, governance, organisational, and societal barriers

Unusual Suspects: Bone and Cartilage ECM Proteins as Carcinoma Facilitators

The extracellular matrix (ECM) is the complex three-dimensional network of fibrous proteins and proteoglycans that constitutes an essential part of every tissue to provide support for normal tissue homeostasis. Tissue specificity of the ECM in its topology and structure supports unique biochemical and mechanical properties of each organ. Cancers, like normal tissues, require the ECM to maintain multiple processes governing tumor development, progression and spread. A large body of experimental and clinical evidence has now accumulated to demonstrate essential roles of numerous ECM components in all cancer types. Latest findings also suggest that multiple tumor types express, and use to their advantage, atypical ECM components that are not found in the cancer tissue of origin. However, the understanding of cancer-specific expression patterns of these ECM proteins and their exact roles in selected tumor types is still sketchy. In this review, we summarize the latest data on the aberrant expression of bone and cartilage ECM proteins in epithelial cancers and their specific functions in the pathogenesis of carcinomas and discuss future directions in exploring the utility of this selective group of ECM components as future drug targets

Additional file 1: of Interplay between CCR7 and Notch1 axes promotes stemness in MMTV-PyMT mammary cancer cells

Supplementary Figures. (PDF 17190 kb

In vivo migration of breast cancer cells within the liver sinusoids.

<p>Single cell suspensions of fluorescence-labelled tumour cells were injected into Spague-Dawley rats. (a) Example of an adherent cells (white arrow) and a cell starting to migrate (black arrow) into the liver parenchyma. Magnifications show location of the cells in relation to marked sinusoid-parenchyma borders. Intravital microscopy was done to analyse adhesion and migration properties of MDA-MB-231 (b) and MDA-MB-468 cells (c). Thirty microscopic fields were analysed in 5min observation periods for semiquantitative analysis of adherent (x) and migrated cells (□). The total numbers of arrested cells (▴) were calculated. <b>CXCR4 inhibition decreased tumour cell migration in vivo.</b> The MDA-MB-231 cells were transduced with shRNA to inhibit CXCR4 expression. Two clones 19 (◊) and 27 (x) were tested for in vivo adhesion (d) and migration (e) in the rat liver and compared with untreated cells (▪). Transfected cells showed significantly (*p<0.001) decreased relative migration rates into the liver parenchyma but cell adhesion within the hepatic microcirculation was only slightly influenced by CXCR4 reduction. Relative migration rates were based on the number of arrested cells and were calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030046#pone.0030046-Sahai1" target="_blank">[26]</a>.</p