ABSTRACT
Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the ß-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP-TEAD and ß-catenin-TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/ß-catenin-TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP-p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional "switch" between pluripotency and initiation of differentiation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Embryonic Stem Cells/cytology , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Protein p73/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Cycle Proteins , Cell Differentiation , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/physiology , Female , Gene Expression Regulation, Developmental , Hippo Signaling Pathway , Humans , Male , Mice, Inbred C57BL , Mice, Inbred CBA , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phosphoproteins/genetics , Protein Serine-Threonine Kinases/genetics , Signal Transduction , TEA Domain Transcription Factors , Transcription Factors/metabolism , Tumor Protein p73/genetics , Tumor Suppressor Proteins/genetics , Wnt Proteins/metabolism , YAP-Signaling Proteins , beta Catenin/metabolismABSTRACT
Pharmaceutical research requires pre-clinical testing of new therapeutics using both in-vitro and in-vivo models. However, the species specificity of non-human in-vivo models and the inadequate recapitulation of physiological conditions in-vitro are intrinsic weaknesses. Here we show that perfusion is a vital factor for engineered human tissues to recapitulate key aspects of the tumour microenvironment. Organotypic culture and human tumour explants were allowed to grow long-term (14-35 days) and phenotypic features of perfused microtumours compared with those in the static culture. Differentiation status and therapeutic responses were significantly different under perfusion, indicating a distinct biological response of cultures grown under static conditions. Furthermore, heterogeneous co-culture of tumour and endothelial cells demonstrated selective cell-killing under therapeutic perfusion versus episodic delivery. We present a perfused 3D microtumour culture platform that sustains a more physiological tissue state and increased viability for long-term analyses. This system has the potential to tackle the disadvantages inherit of conventional pharmaceutical models and is suitable for precision medicine screening of tumour explants, particularly in hard-to-treat cancer types such as brain cancer which suffer from a lack of clinical samples.
