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Wnt Enhancer LandscapE CharacTerization

Periodic Reporting for period 1 - WntELECT (Wnt Enhancer LandscapE CharacTerization)

Reporting period: 2016-05-01 to 2018-04-30

Cancer remains one of the major health challenges in Europe. In recent years, a lot of clinical efforts have focused on targeting developmental signalling pathways for cancer therapy. One of the signalling cascades that has emerged as key regulator of both adult stem cells and cancer (stem) cells is the Wnt pathway.
In human breast tumours, deregulation of different Wnt signalling components is frequently observed, ranging from overexpression of individual Wnt genes to elevated levels of the downstream effector beta-catenin. However, in the absence of apparent dominant genetic mutations, the mechanism behind those alterations is unclear. In fact, very little is known about the precise spatio-temporal regulation of Wnt ligands during tissue homeostasis and disease to begin with.
The long-term goal of our research is to uncover how environmental and intrinsic signals are integrated to control dynamic Wnt expression patterns during both normal development and tumour formation. In this project, we aimed to combine bioinformatics, state-of-the-art genome engineering and an innovative screening approach to identify Wnt-associated enhancers in the mammary gland.
The major limitation in studying functional gene-enhancer interactions is the difficulty to establish a link between the enhancer activity of a particular sequence and the regulation of its endogenous target gene. To solve this problem, we established an efficient experimental pipeline, starting with the initial computational identification of a putative enhancer element and testing its functional activity in reporter assays, to finally linking that activity to regulation of an endogenous Wnt gene. Using this strategy, we discovered several enhancers that are capable of controlling Wnt gene expression in mammary epithelial cells. More detailed mechanistic analyses and in vivo validation experiments to probe activity of the identified Wnt enhancers in a complex tissue context are planned.

We also made important steps towards establishing a high-throughput screening system that will allow the identification of a larger number of active Wnt enhancers. Using a generic CRISPR/Cas9-mediated gene tagging approach, we created clonal cell lines that carry the bright red fluorescent protein mScarlet-I in the last exon of 2 different Wnt genes. Pilot experiments revealed that these cell lines allow us to faithfully measure changes in endogenous Wnt gene expression and will serve as useful tools for studying Wnt signalling biology in development, tissue homeostasis and cancer. Our efforts to optimize the precise screening conditions are still ongoing.
Conserved developmental signalling cascades governing three-dimensional tissue growth and organisation are often exploited by cancer cells to drive tumour progression. For example, Wnt ligands play crucial roles during both normal mammary gland development and breast cancer formation. Partially due to the lack of appropriate experimental tools, however, there is a significant knowledge gap concerning the upstream signals that are responsible for Wnt gene expression. By identifying novel Wnt-regulatory sequences, the work performed in this project will bring us one step closer towards understanding how complex spatio-temporal expression patterns of different Wnt ligands in the mammary gland are established. Appreciation of the fundamental processes controlling Wnt signalling during tissue homeostasis will fuel our continued efforts to effectively target Wnt pathway alterations in human tumours.