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The co-transcription factors YAP/TAZ are potent inducers of tissue and organ growth during development and tissue regeneration after damage. In this work we have expanded on the repertoire of YAP/TAZ regulations and on their biological properties. We found that these are regulated by the mechanical properties of the microenvironement and by the Wnt signaling cascade. The latter is induced by mutations in the tumor suppressor APC, a hallmark of intestinal cancer, and here we show that this is accompanied by YAP/TAZ stabilization and is functional for transformation of intestinal cells. Both the mechanical and Wnt regulations have been published (Azzolin et al., 2014; Aragona et al., 2013). Aberrant YAP/TAZ activation is known to be associated to tissue overgrowth, tumorigenesis and acquisition of cancer stem cell traits. Here we explore the role of YAP/TAZ as key factors in somatic stem cell self-renewal and somatic cell plasticity (unpublished).

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

In spite of tremendous advances made in the comprehension of mechanotransduction, implementation of mechanobiology assays remains challenging for the broad community of cell biologists. Hydrogel substrates with tunable stiffness are essential tool in mechanobiology, allowing to investigate the effects of mechanical signals on cell behavior. A bottleneck that slows down the popularization of hydrogel formulations for mechanobiology is the assessment of their stiffness, typically requiring expensive and sophisticated methodologies in the domain of material science. Here we overcome such barriers offering the reader protocols to set-up and interpret two straightforward, low cost and high-throughput tools to measure hydrogel stiffness: static macroindentation and micropipette aspiration. We advanced on how to build up these tools and on the underlying theoretical modeling. Specifically, we validated our tools by comparing them with leading techniques used for measuring hydrogel stiffness (atomic force microscopy, uniaxial compression and rheometric analysis) with consistent results on PAA hydrogels or their modification. In so doing, we also took advantage of YAP/TAZ nuclear localization as biologically validated and sensitive readers of mechanosensing, all in all presenting a suite of biologically and theoretically proven protocols to be implemented in most biological laboratories to approach mechanobiology.

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)–Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK–Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell’s environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies. Receptor tyrosine kinase (RTK)–Ras oncogenes have now been shown to reprogram normal primary human and mouse cells into tumour precursors by empowering cellular mechanotransduction, in a process requiring permissive extracellular-matrix rigidity and intracellular YAP/TAZ/Rac mechanical signalling sustained by activated oncogenes.