Stefano Piccolo

Role of YAP/TAZ in mechanotransduction

Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

MicroRNA control of signal transduction

MicroRNAs (miRNAs) are integral elements in the post-transcriptional control of gene expression. After the identification of hundreds of miRNAs, the challenge is now to understand their specific biological function. Signalling pathways are ideal candidates for miRNA-mediated regulation owing to the sharp dose-sensitive nature of their effects. Indeed, emerging evidence suggests that miRNAs affect the responsiveness of cells to signalling molecules such as transforming growth factor-β, WNT, Notch and epidermal growth factor. As such, miRNAs serve as nodes of signalling networks that ensure homeostasis and regulate cancer, metastasis, fibrosis and stem cell biology.

The head inducer cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk–tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.

Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors.

Wnt/β-catenin signaling plays key roles in several developmental and pathological processes. Domains of Wnt expression have been extensively investigated in the mouse, but the tissues receiving the signal remain largely unidentified. To define which cells respond to activated β-catenin during mammalian development, we generated the β-catenin-activated transgene driving expression of nuclear β-galactosidase reporter (BAT-gal) transgenic mice, expressing the lacZ gene under the control of β-catenin/T cell factor responsive elements. Reporter gene activity is found in known organizing centers, such as the midhindbrain border and the limb apical ectodermal ridge. Moreover, BAT-gal expression identifies novel sites of Wnt signaling, like notochord, endothelia, and areas of the adult brain, revealing an unsuspected dynamic pattern of β-catenin transcriptional activity. Expression of the transgene was analyzed in mutant backgrounds. In lipoprotein receptor-related protein 6-null homozygous mice, which lack a Wnt coreceptor, BAT-gal staining is absent in mutant tissues, indicating that BAT-gal mice are bona fide in vivo indicators of Wnt/β-catenin signaling. Analyses of BAT-gal expression in the adenomatous polyposis coli (multiple intestinal neoplasia/+) background revealed βcatenin transcriptional activity in intestinal adenomas but surprisingly not in normal crypt cells. In summary, BAT-gal mice unveil the entire complexity of Wnt/β-catenin signaling in mammals and have broad application potentials for the identification of Wnt-responsive cell populations in development and disease.

The Hippo Transducer TAZ Confers Cancer Stem Cell-Related Traits on Breast Cancer Cells

Cancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble--or induction of the epithelial-mesenchymal transition (EMT)--disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.

A Mutant-p53/Smad Complex Opposes p63 to Empower TGFβ-Induced Metastasis

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

A MicroRNA Targeting Dicer for Metastasis Control

Although specific microRNAs (miRNAs) can be upregulated in cancer, global miRNA downregulation is a common trait of human malignancies. The mechanisms of this phenomenon and the advantages it affords remain poorly understood. Here we identify a microRNA family, miR-103/107, that attenuates miRNA biosynthesis by targeting Dicer, a key component of the miRNA processing machinery. In human breast cancer, high levels of miR-103/107 are associated with metastasis and poor outcome. Functionally, miR-103/107 confer migratory capacities in vitro and empower metastatic dissemination of otherwise nonaggressive cells in vivo. Inhibition of miR-103/107 opposes migration and metastasis of malignant cells. At the cellular level, a key event fostered by miR-103/107 is induction of epithelial-to-mesenchymal transition (EMT), attained by downregulating miR-200 levels. These findings suggest a new pathway by which Dicer inhibition drifts epithelial cancer toward a less-differentiated, mesenchymal fate to foster metastasis.

Transduction of mechanical and cytoskeletal cues by YAP and TAZ

The physical and mechanical properties of the cellular microenvironment regulate cell shape and can strongly influence cell fate. How mechanical cues are sensed and transduced to regulate gene expression has long remained elusive. Recently, cues from the extracellular matrix, cell adhesion sites, cell shape and the actomyosin cytoskeleton were found to converge on the regulation of the downstream effectors of the Hippo pathway YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) in vertebrates and Yorkie in flies. This convergence may explain how mechanical signals can direct normal and pathological cell behaviour.

Glycogen synthase kinase-3beta haploinsufficiency mimics the behavioral and molecular effects of lithium.

Lithium is widely used to treat bipolar disorder, but its mechanism of action in this disorder is unknown. Several molecular targets of lithium have been identified, but these putative targets have not been shown to be responsible for the behavioral effects of lithium in vivo. A robust model for the effects of chronic lithium on behavior in mice would greatly facilitate the characterization of lithium action. We describe behaviors in mice that are robustly affected by chronic lithium. Remarkably, these lithium-sensitive behaviors are also observed in mice lacking one copy of the gene encoding glycogen synthase kinase-3β (Gsk-3β), a well established direct target of lithium. In addition, chronic lithium induces molecular changes consistent with inhibition of GSK-3 within regions of the brain that are paralleled in Gsk-3β+/- heterozygous mice. We also show that lithium therapy activates Wnt signaling in vivo, as measured by increased Wnt-dependent gene expression in the amygdala, hippocampus, and hypothalamus. These observations support a central role for GSK-3β in mediating behavioral responses to lithium.

YAP/TAZ Incorporation in the β-Catenin Destruction Complex Orchestrates the Wnt Response

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the β-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for β-TrCP recruitment to the complex and β-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/β-catenin signaling. In line, the β-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.