A molecular toolbox to study progesterone receptor signaling.

Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells.

Fourteenth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The fourteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) on Methods in Mammary Gland Biology and Breast Cancer was held on April 26th - 29th in Weggis, Switzerland. For the first time, early career researchers organised and took part in an additional ECR workshop on the 26th of April, which was received with great enthusiasm. The topics of the main workshop included mammary branching and morphogenesis, novel experimental systems (model organisms), systemic influences on tumour progression and the tumour microenvironment. Novel and recent findings were shared across excellent oral and poster presentations.

Visualizing WNT signaling in mammalian systems.

WNT/CTNNB1 signaling plays a critical role in the development of all multicellular animals. Here, we include both the embryonic stages, during which tissue morphogenesis takes place, and the postnatal stages of development, during which tissue homeostasis occurs. Thus, embryonic development concerns lineage development and cell fate specification, while postnatal development involves tissue maintenance and regeneration. Multiple tools are available to researchers who want to investigate, and ideally visualize, the dynamic and pleiotropic involvement of WNT/CTNNB1 signaling in these processes. Here, we discuss and evaluate the decisions that researchers need to make in identifying the experimental system and appropriate tools for the specific question they want to address, covering different types of WNT/CTNNB1 reporters in cells and mice. At a molecular level, advanced quantitative imaging techniques can provide spatio-temporal information that cannot be provided by traditional biochemical assays. We therefore also highlight some recent studies to show their potential in deciphering the complex and dynamic mechanisms that drive WNT/CTNNB1 signaling.

Quantitative live-cell imaging and computational modeling shed new light on endogenous WNT/CTNNB1 signaling dynamics.

WNT/CTNNB1 signaling regulates tissue development and homeostasis in all multicellular animals, but the underlying molecular mechanism remains incompletely understood. Specifically, quantitative insight into endogenous protein behavior is missing. Here, we combine CRISPR/Cas9-mediated genome editing and quantitative live-cell microscopy to measure the dynamics, diffusion characteristics and absolute concentrations of fluorescently tagged, endogenous CTNNB1 in human cells under both physiological and oncogenic conditions. State-of-the-art imaging reveals that a substantial fraction of CTNNB1 resides in slow-diffusing cytoplasmic complexes, irrespective of the activation status of the pathway. This cytoplasmic CTNNB1 complex undergoes a major reduction in size when WNT/CTNNB1 is (hyper)activated. Based on our biophysical measurements, we build a computational model of WNT/CTNNB1 signaling. Our integrated experimental and computational approach reveals that WNT pathway activation regulates the dynamic distribution of free and complexed CTNNB1 across different subcellular compartments through three regulatory nodes: the destruction complex, nucleocytoplasmic shuttling, and nuclear retention.

Walking the tight wire between cell adhesion and WNT signalling: a balancing act for ß-catenin.

CTNNB1 (catenin ß-1, also known as ß-catenin) plays a dual role in the cell. It is the key effector of WNT/CTNNB1 signalling, acting as a transcriptional co-activator of TCF/LEF target genes. It is also crucial for cell adhesion and a critical component of cadherin-based adherens junctions. Two functional pools of CTNNB1, a transcriptionally active and an adhesive pool, can therefore be distinguished. Whether cells merely balance the distribution of available CTNNB1 between these functional pools or whether interplay occurs between them has long been studied and debated. While interplay has been indicated upon artificial modulation of cadherin expression levels and during epithelial-mesenchymal transition, it is unclear to what extent CTNNB1 exchange occurs under physiological conditions and in response to WNT stimulation. Here, we review the available evidence for both of these models, discuss how CTNNB1 binding to its many interaction partners is controlled and propose avenues for future studies.

Cell-cell junctions as sensors and transducers of mechanical forces.

Epithelial and endothelial monolayers are multicellular sheets that form barriers between the 'outside' and 'inside' of tissues. Cell-cell junctions, made by adherens junctions, tight junctions and desmosomes, hold together these monolayers. They form intercellular contacts by binding their receptor counterparts on neighboring cells and anchoring these structures intracellularly to the cytoskeleton. During tissue development, maintenance and pathogenesis, monolayers encounter a range of mechanical forces from the cells themselves and from external systemic forces, such as blood pressure or tissue stiffness. The molecular landscape of cell-cell junctions is diverse, containing transmembrane proteins that form intercellular bonds and a variety of cytoplasmic proteins that remodel the junctional connection to the cytoskeleton. Many junction-associated proteins participate in mechanotransduction cascades to confer mechanical cues into cellular responses that allow monolayers to maintain their structural integrity. We will discuss force-dependent junctional molecular events and their role in cell-cell contact organization and remodeling.

TMEM98 is a negative regulator of FRAT mediated Wnt/ß-catenin signalling.

Wnt/ß-catenin signalling is crucial for maintaining the balance between cell proliferation and differentiation, both during tissue morphogenesis and in tissue maintenance throughout postnatal life. Whereas the signalling activities of the core Wnt/ß-catenin pathway components are understood in great detail, far less is known about the precise role and regulation of the many different modulators of Wnt/ß-catenin signalling that have been identified to date. Here we describe TMEM98, a putative transmembrane protein of unknown function, as an interaction partner and regulator of the GSK3-binding protein FRAT2. We show that TMEM98 reduces FRAT2 protein levels and, accordingly, inhibits the FRAT2-mediated induction of ß-catenin/TCF signalling. We also characterize the intracellular trafficking of TMEM98 in more detail and show that it is recycled between the plasma membrane and the Golgi. Together, our findings not only reveal a new layer of regulation for Wnt/ß-catenin signalling, but also a new biological activity for TMEM98.