YAP/TAZ transcriptional activity in triple negative breast cancer cells

YAP and TAZ are two closely related transcriptional regulators involved in tissue growth, stem cell maintenance and cancer. YAP/TAZ are aberrantly activated in different tumors where they have causative roles in initiation, progression and metastasis. However, the transcriptional program they activate in cancer cells remains incompletely understood. Therefore, we tried to dissect YAP/TAZ direct target genes in a breast cancer cell line (MDAMB-231 cells) by genome-wide analysis. In so doing, we discovered that YAP/TAZ mainly bind distal enhancers that contact target promoters through chromatin looping to activate a broad transcriptional program activating cell proliferation. We assessed that YAP/TAZ exploit TEAD proteins as DNA binding partners in breast cancer cells. We then focused on the interaction of YAP/TAZ and TEAD with general transcriptional regulators, aiming at identifying indispensible co-factors for YAP/TAZ/TEAD transcriptional activity at enhancers. Our findings provide new details on YAP/TAZ behaviour, and open a new therapeutic perspective to achieve pharmacological inhibition of YAP/TAZ by impairing their nuclear function. Part of this work has been published in Nature Cell Biology (Zanconato et al., 2015). Part is unpublished

YAP/TAZ as therapeutic targets in cancer

The biology and regulation of YAP and TAZ, two closely related transcriptional regulators, are receiving increasing attention owing to their fundamental roles in organ growth, tissue repair and cancer. In particular, the widespread activation of YAP/TAZ in carcinomas, and the crucial role of YAP/TAZ activation for many 'hallmarks' of cancer are indicating YAP/TAZ as prime targets for designing anti-cancer drugs. Here, we start from the known modalities to regulate YAP/TAZ to highlight possible routes of therapeutic intervention

Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth

YAP/TAZ are nuclear effectors of the Hippo pathway regulating organ growth and tumorigenesis. Yet, their function as transcriptional regulators remains underinvestigated. By ChIP-seq analyses in breast cancer cells, we discovered that the YAP/TAZ transcriptional response is pervasively mediated by a dual element: TEAD factors, through which YAP/TAZ bind to DNA, co-occupying chromatin with activator protein-1 (AP-1, dimer of JUN and FOS proteins) at composite cis-regulatory elements harbouring both TEAD and AP-1 motifs. YAP/TAZ/TEAD and AP-1 form a complex that synergistically activates target genes directly involved in the control of S-phase entry and mitosis. This control occurs almost exclusively from distal enhancers that contact target promoters through chromatin looping. YAP/TAZ-induced oncogenic growth is strongly enhanced by gain of AP-1 and severely blunted by its loss. Conversely, AP-1-promoted skin tumorigenesis is prevented in YAP/TAZ conditional knockout mice. This work highlights a new layer of signalling integration, feeding on YAP/TAZ function at the chromatin level

Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4

Cancer cells rely on dysregulated gene expression. This establishes specific transcriptional addictions that may be therapeutically exploited. Yet, the mechanisms that are ultimately responsible for these addictions are poorly understood. Here, we investigated the transcriptional dependencies of transformed cells to the transcription factors YAP and TAZ. YAP/TAZ physically engage the general coactivator bromodomain-containing protein 4 (BRD4), dictating the genome-wide association of BRD4 to chromatin. YAP/TAZ flag a large set of enhancers with super-enhancer-like functional properties. YAP/TAZ-bound enhancers mediate the recruitment of BRD4 and RNA polymerase II at YAP/TAZ-regulated promoters, boosting the expression of a host of growth-regulating genes. Treatment with small-molecule inhibitors of BRD4 blunts YAP/TAZ pro-tumorigenic activity in several cell or tissue contexts, causes the regression of pre-established, YAP/TAZ-addicted neoplastic lesions and reverts drug resistance. This work sheds light on essential mediators, mechanisms and genome-wide regulatory elements that are responsible for transcriptional addiction in cancer and lays the groundwork for a rational use of BET inhibitors according to YAP/TAZ biology

Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties

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

YAP/TAZ transcriptional activity in triple negative breast cancer cells

YAP and TAZ are two closely related transcriptional regulators involved in tissue growth, stem cell maintenance and cancer. YAP/TAZ are aberrantly activated in different tumors where they have causative roles in initiation, progression and metastasis. However, the transcriptional program they activate in cancer cells remains incompletely understood. Therefore, we tried to dissect YAP/TAZ direct target genes in a breast cancer cell line (MDAMB-231 cells) by genome-wide analysis. In so doing, we discovered that YAP/TAZ mainly bind distal enhancers that contact target promoters through chromatin looping to activate a broad transcriptional program activating cell proliferation. We assessed that YAP/TAZ exploit TEAD proteins as DNA binding partners in breast cancer cells. We then focused on the interaction of YAP/TAZ and TEAD with general transcriptional regulators, aiming at identifying indispensible co-factors for YAP/TAZ/TEAD transcriptional activity at enhancers. Our findings provide new details on YAP/TAZ behaviour, and open a new therapeutic perspective to achieve pharmacological inhibition of YAP/TAZ by impairing their nuclear function. Part of this work has been published in Nature Cell Biology (Zanconato et al., 2015). Part is unpublished.YAP e TAZ sono due regolatori trascrizionali strettamente correlati, coinvolti nella crescita dei tessuti, nella biologia delle cellule staminali e nel cancro. Un’espressione anomala di YAP e TAZ è riscontrata in diversi tipi di tumori; YAP/TAZ sono infatti coinvolti nella formazione, nella progressione e nella crescita metastatica di molti tumori umani. Tuttavia, il programma trascrizionale attivato da YAP/TAZ nelle cellule tumorali non è ancora ben definito. Pertanto, noi abbiamo ricercato, attraverso un’analisi ad ampio spettro, i geni trascrizionalmente regolati da YAP/TAZ utilizzando la tecnologia della ChIP-Seq in una linea cellulare di tumore alla mammella (cellule MDA-MB-231). In questo modo, abbiamo scoperto che YAP/TAZ sono fattori che regolano la trascrizione genica prevalentemente legando siti enhancer che contattano i promotori dei geni regolati tramite il ripiegamento della cromatina. In particolare, YAP/TAZ attivano un programma di crescita cellulare, modulando l'espressione di centinaia di geni, come ad esempio MYC, nelle cellule MDA-MB-231. YAP/TAZ non possono legare direttamente il DNA, ma solo tramite l’interazione con fattori di trascrizione; in particolare, nelle cellule di tumore alla mammella, sono risultati interagire con i fattori di trascrizione appartenenti alla famiglia TEAD. In seguito, abbiamo ricercato possibili interazioni di YAP e TAZ con regolatori generali della trascrizione allo scopo di identificare co-fattori indispensabili per l’attività trascrizionale di YAP/TAZ/TEAD mediata da siti enhancer. I nostri risultati hanno meglio elucidato l’attività trascrizionale di YAP/TAZ, aprendo una nuova prospettiva terapeutica; inibire farmacologicamente YAP/TAZ, agendo sulla loro funzione nucleare, potrebbe infatti essere una possibile strategia di cura per il cancro

Phenotypic and functional characterization of Glioblastoma cancer stem cells identified through 5-aminolevulinic acid-assisted surgery [corrected]

5-aminolevulinic acid (5-ALA) introduction in the surgical management of Glioblastoma (GBM) enables the intra-operatively identification of cancer cells in the mass by means of fluorescence. Here, we analyzed the phenotype of GBM cells isolated from distinct tumour areas determined by 5-ALA (tumour core, 5-ALA intense and vague layers) and the potency of 5-ALA labelling in identifying GBM cells and cancer stem cells (CSCs) in the mass. 5-ALA identified distinct layers in the mass, with less differentiated cells residing in the core of the tumour. 5-ALA was able to stain up to 68.5 % of CD133(+) cells in the 5-ALA intense layer and, although 5-ALA(+) cells retrieved from different tumour areas contained a similar proportion of CD133(+) cells (range 27.5-35.6 %), those from the vague layer displayed the lowest ability to self-renew. In conclusion, our data demonstrate that a substantial amount of GBM cells and CSCs in the mass are able to avoid 5-ALA labelling and support the presence of heterogenic CSC populations in the GBM mass

Cell phenotypic plasticity requires autophagic flux driven by YAP/TAZ mechanotransduction

Autophagy, besides ensuring energy metabolism and organelle renewal, is crucial for the biology of adult normal and cancer stem cells. However, it remains incompletely understood how autophagy connects to stemness factors and the nature of the microenvironmental signals that pattern autophagy in different cell types. Here we advance in these directions by reporting that YAP/TAZ transcriptionally control autophagy, being critical for autophagosomal degradation into autolysosomes. YAP/TAZ are downstream effectors of cellular mechanotransduction and indeed we found that cell mechanics, dictated by the physical property of the ECM and cytoskeletal tension, profoundly impact on autophagic flux in a YAP/TAZ-mediated manner. Functionally, by using pancreatic and mammary organoid cultures, we found that YAP/TAZ-regulated autophagy is essential in normal cells for YAP/TAZ-mediated dedifferentiation and acquisition of self-renewing properties. In tumor cells, the YAP/TAZ-autophagy connection is key to sustain transformed traits and for acquisition of a cancer stem cell state by otherwise more benign cells. Mechanistically, YAP/TAZ promote autophagic flux by directly promoting the expression of Armus, a RAB7-GAP required for autophagosome turnover and whose add-back rescues autophagy in YAP/TAZ-depleted cells. These findings expand the influence of YAP/TAZ mechanotransduction to the control of autophagy and, vice versa, the role of autophagy in YAP/TAZ biology, and suggest a mechanism to coordinate transcriptional rewiring with cytoplasmic restructuring during cell reprogramming

Simple yet effective methods to probe hydrogel stiffness for mechanobiology

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