Nicolas Tapon

The Salvador–Warts–Hippo pathway — an emerging tumour-suppressor network

Intense research over the past four years has led to the discovery and characterization of a novel signalling network, known as the Salvador–Warts–Hippo (SWH) pathway, involved in tissue growth control in Drosophila melanogaster. At present, eleven proteins have been implicated as members of this pathway, and several downstream effector genes have been characterized. The importance of this pathway is emphasized by its evolutionary conservation, and by increasing evidence that its deregulation occurs in human tumours. Here, we review the main findings from Drosophila and the implications that these have for tumorigenesis in mammals.

The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila

Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador (sav) gene, which encodes a scaffold protein, has been shown to restrict cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Here we identify Hippo (Hpo), the Drosophila orthologue of the mammalian MST1 and MST2 serine/threonine kinases, as a partner of Sav. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1, overexpression of Hpo destabilizes DIAP1 in cell culture. We show that DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, we show that Sav is an unstable protein that is stabilized by Hpo. We propose that Hpo and Sav function together to restrict tissue growth in vivo.

Kibra Is a Regulator of the Salvador/Warts/Hippo Signaling Network

Summary The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core of the pathway consists of a MST and LATS family kinase cascade that ultimately phosphorylates and inactivates the YAP/Yorkie (Yki) transcription coactivator. The FERM domain proteins Merlin (Mer) and Expanded (Ex) represent one mode of upstream regulation controlling pathway activity. Here, we identify Kibra as a member of the SWH network. Kibra, which colocalizes and associates with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kibra/Mer association is conserved in human cells. Finally, Kibra complexes with Wts and kibra depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. We suggest that Kibra is part of an apical scaffold that promotes SWH pathway activity.

The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration

Intestinal stem cells (ISCs) in the adult Drosophila midgut proliferate to self-renew and to produce differentiating daughter cells that replace those lost as part of normal gut function. Intestinal stress induces the activation of Upd/Jak/Stat signalling, which promotes intestinal regeneration by inducing rapid stem cell proliferation. We have investigated the role of the Hippo (Hpo) pathway in the Drosophila intestine (midgut). Hpo pathway inactivation in either the ISCs or the differentiated enterocytes induces a phenotype similar to that observed under stress situations, including increased stem cell proliferation and expression of Jak/Stat pathway ligands. Hpo pathway targets are induced by stresses such as bacterial infection, suggesting that the Hpo pathway functions as a sensor of cellular stress in the differentiated cells of the midgut. In addition, Yki, the pro-growth transcription factor target of the Hpo pathway, is required in ISCs to drive the proliferative response to stress. Our results suggest that the Hpo pathway is a mediator of the regenerative response in the Drosophila midgut.

A new rac target POSH is an SH3-containing scaffold protein involved in the JNK and NF-kappaB signalling pathways.

The Rho, Rac and Cdc42 GTPases coordinately regulate the organization of the actin cytoskeleton and the JNK MAP kinase pathway. Mutational analysis of Rac has previously shown that these two activities are mediated by distinct cellular targets, though their identity is not known. Two Rac targets, p65PAK and MLK, are ser/thr kinases that have been reported to be capable of activating the JNK pathway. We present evidence that neither is the Rac target mediating JNK activation in Cos‐1 cells. We have used yeast two‐hybrid selection and identified a new target of Rac, POSH. This protein consists of four SH3 domains and ectopic expression leads to the activation of the JNK pathway and to nuclear translocation of NF‐κB. When overexpressed in fibroblasts, POSH is a strong inducer of apoptosis. We propose that POSH acts as a scaffold protein and contributes to Rac‐induced signal transduction pathways leading to diverse gene transcriptional changes.

A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.

Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.

Planar polarization of the atypical myosin Dachs orients cell divisions in Drosophila

Tissues can grow in a particular direction by controlling the orientation of cell divisions. This phenomenon is evident in the developing Drosophila wing epithelium, where the tissue becomes elongated along the proximal-distal axis. We show that orientation of cell divisions in the wing requires planar polarization of an atypical myosin, Dachs. Our evidence suggests that Dachs constricts cell-cell junctions to alter the geometry of cell shapes at the apical surface, and that cell shape then determines the orientation of the mitotic spindle. Using a computational model of a growing epithelium, we show that polarized cell tension is sufficient to orient cell shapes, cell divisions, and tissue growth. Planar polarization of Dachs is ultimately oriented by long-range gradients emanating from compartment boundaries, and is therefore a mechanism linking these gradients with the control of tissue shape.

Combined Functional Genomic and Proteomic Approaches Identify a PP2A Complex as a Negative Regulator of Hippo Signaling

The Hippo (Hpo) pathway is a central determinant of tissue size in both Drosophila and higher organisms. The core of the pathway is a kinase cascade composed of an upstream kinase Hpo (MST1/2 in mammals) and a downstream kinase Warts (Wts, Lats1/2 in mammals), as well as several scaffold proteins, Sav, dRASSF, and Mats. Activation of the core kinase cassette results in phosphorylation and inactivation of the progrowth transcriptional coactivator Yki, leading to increased apoptosis and reduced tissue growth. The mechanisms that prevent inappropriate Hpo activation remain unclear, and in particular, the identity of the phosphatase that antagonizes Hpo is unknown. Using combined proteomic and RNAi screening approaches, we identify the dSTRIPAK PP2A complex as a major regulator of Hpo signaling. dSTRIPAK depletion leads to increased Hpo activatory phosphorylation and repression of Yki target genes in vivo, suggesting this phosphatase complex prevents Hpo activation during development.

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.

The Hippo pathway and apico-basal cell polarity.

The establishment and maintenance of apico-basal cell polarity is a pre-requisite for the formation of a functioning epithelial tissue. Many lines of evidence suggest that cell polarity perturbations favour cancer formation, even though the mechanistic basis for this link remains unclear. Studies in Drosophila have uncovered complex interactions between the conserved Hpo (Hippo) tumour suppressor pathway and apico-basal polarity determinants. The Hpo pathway is a crucial growth regulatory network whose inactivation in Drosophila epithelial tissues induces massive overproliferation. Its core consists of a phosphorylation cascade (comprising the kinases Hpo and Warts) that mediates the inactivation of the pro-growth transcriptional co-activator Yki [Yorkie; YAP (Yes-associated protein) in mammals]. Several apically located proteins, such as Merlin, Expanded or Kibra, have been identified as upstream regulators of the Hpo pathway, leading to the notion that an apical multi-molecular complex modulates core kinase activity and promotes Yki/YAP inactivation. In the present review, we explore the links between apico-basal polarity and Hpo signalling. We focus on the regulation of Yki/YAP by apical proteins, but also on how the Hpo pathway might in turn influence apical domain size as part of a regulatory feedback loop.