TAZ activation drives fibroblast spheroid growth, expression of profibrotic paracrine signals, and context-dependent ECM gene expression.

Recent studies have implicated the Hippo pathway and its transcriptional effectors YAP and TAZ as necessary for fibroblast activation and tissue fibrosis. To test the specific and sufficient roles for TAZ in driving autonomous fibroblast activation, we cultured NIH3T3 fibroblasts expressing a doxycycline-inducible nuclear-localized mutant of TAZ (TAZ4SA) in scaffold-free 3D hanging drop spheroids, or on matrices of specified mechanical rigidity. Control NIH3T3 fibroblasts formed spheroids in hanging drop culture that remained stable and neither increased nor decreased in size significantly over 15 days. In contrast, TAZ4SA-transduced fibroblasts grew robustly in spheroid culture, and expressed enhanced levels of genes encoding profibrotic soluble factors connective tissue growth factor (CTGF), endothelin-1 (Et-1), and plasminogen activator inhibitor 1 (PAI-1). However, TAZ4SA expression was unable to enhance expression of extracellular matrix (ECM)-encoding genes Col1a1, Col1a2, Col3a1, or Fn1 in spheroid culture. Micromechanical testing indicated that spheroids composed of either control or TAZ4SA-expressing cells were highly compliant and indistinguishable in mechanical properties. In fibroblasts cultured on 2D matrices of compliance similar to spheroids, TAZ4SA expression was able to enhance contractile force generation, but was unable to enhance ECM gene expression. In contrast, culture on stiff hydrogels potentiated TAZ4SA enhancement of ECM expression. TAZ4SA enhancement of Col1a1 expression on soft matrices was potentiated by TGF-ß1, while on stiff matrices it was abrogated by inhibition of myocardin-related transcription factor, demonstrating context-dependent crosstalk of TAZ with these pathways. These findings demonstrate sufficiency of TAZ activation for driving fibroblast proliferation, contraction, and soluble profibrotic factor expression, and mechanical context-dependent crosstalk of TAZ with other pathways in regulating Col1a1 expression.

The transcriptional regulators TAZ and YAP direct transforming growth factor ß-induced tumorigenic phenotypes in breast cancer cells.

Uncontrolled transforming growth factor-ß (TGFß) signaling promotes aggressive metastatic properties in late-stage breast cancers. However, how TGFß-mediated cues are directed to induce tumorigenic events is poorly understood, particularly given that TGFß has clear tumor suppressing activity in other contexts. Here, we demonstrate that the transcriptional regulators TAZ and YAP (TAZ/YAP), key effectors of the Hippo pathway, are necessary to promote and maintain TGFß-induced tumorigenic phenotypes in breast cancer cells. Interactions between TAZ/YAP, TGFß-activated SMAD2/3, and TEAD transcription factors reveal convergent roles for these factors in the nucleus. Genome-wide expression analyses indicate that TAZ/YAP, TEADs, and TGFß-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by TAZ/YAP, TEAD, and TGFß, such as the novel targets NEGR1 and UCA1, are necessary for maintaining tumorigenic activity in metastatic breast cancer cells. Nuclear TAZ/YAP also cooperate with TGFß signaling to promote phenotypic and transcriptional changes in nontumorigenic cells to overcome TGFß-repressive effects. Our work thus identifies cross-talk between nuclear TAZ/YAP and TGFß signaling in breast cancer cells, revealing novel insight into late-stage disease-driving mechanisms.

The Hippo pathway effectors TAZ/YAP regulate dicer expression and microRNA biogenesis through Let-7.

MicroRNAs (miRNAs) are genome-encoded small double-stranded RNAs that have emerged as key regulators of gene expression and are implicated in most aspects of human development and disease. Canonical miRNA biogenesis involves processing of ∼70-nucleotide pre-miRNA hairpins by Dicer to generate mature ∼22-nucleotide miRNAs, which target complementary RNA sequences. Despite the importance of miRNA biogenesis, signaling mechanisms controlling this process are poorly defined. Here we demonstrate that the post-transcriptional regulation of Dicer is controlled by the cell density-mediated localization of the Hippo pathway effectors TAZ (transcriptional co-activator with PDZ-binding motif) and YAP (Yes-associated protein) (TAZ/YAP). We show that nuclear TAZ/YAP, which are abundant at low cell density, are required for efficient pre-miRNA processing. Knockdown of TAZ/YAP in low density cells, or density-mediated sequestration of TAZ/YAP into the cytoplasm, results in the defective processing of pre-miRNAs. Strikingly, one exception is Let-7, which accumulates upon loss of nuclear TAZ/YAP, leading to Let-7-dependent reduction in Dicer levels. Accordingly, inhibition of Let-7 rescues the miRNA biogenesis defects observed following TAZ/YAP knockdown. Thus, density-regulated TAZ/YAP localization defines a critical and previously unrecognized mechanism by which cells relay cell contact-induced cues to control miRNA biogenesis.

Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis.

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-ß signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

Stem cell regulation by the Hippo pathway.

BACKGROUND: The Hippo pathway coordinates cell proliferation, apoptosis, and differentiation, and has emerged as a major regulator of organ development and regeneration. Central to the mammalian Hippo pathway is the action of the transcriptional regulators TAZ (also known as WWTR1) and YAP, which are controlled by a kinase cascade that is sensitive to mechanosensory and cell polarity cues. SCOPE OF REVIEW: We review recent studies focused on the Hippo pathway in embryonic and somatic stem cell renewal and differentiation. MAJOR CONCLUSIONS: Accurate control of TAZ and YAP is crucial for the self-renewal of stem cells and in guiding distinct cell fate decisions. In vivo studies have implicated YAP as a key regulator of tissue-specific progenitor cell proliferation and tissue regeneration. Misappropriate activation of nuclear TAZ and YAP transcriptional activity drives tissue overgrowth and is implicated in cancer stem cell-like properties that promote tumor initiation. GENERAL SIGNIFICANCE: Understanding the activity and regulation of Hippo pathway effectors will offer insight into human pathologies that evolve from the deregulation of stem cell populations. Given the roles of the Hippo pathway in directing cell fate and tissue regeneration, the discernment of Hippo pathway regulatory cues will be essential for the advancement of regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma.

UNLABELLED: Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here, we show that the transcriptional regulators YAP (YAP1) and TAZ (WWTR1), which are key effectors of the Hippo pathway, drive protumorigenic signals in OSCC. Regions of premalignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro, and is required for OSCC tumor growth and metastasis in vivo. Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in protumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by The Cancer Genome Atlas (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology. IMPLICATIONS: This study defines a YAP/TAZ-regulated transcriptional program in OSCC and reveals novel roles for nuclear YAP/TAZ activity in the onset and progression of this cancer.