Kieran F. Harvey

The Hippo pathway and human cancer

The Hippo pathway controls organ size in diverse species, whereas pathway deregulation can induce tumours in model organisms and occurs in a broad range of human carcinomas, including lung, colorectal, ovarian and liver cancer. Despite this, somatic or germline mutations in Hippo pathway genes are uncommon, with only the upstream pathway gene neurofibromin 2 (NF2) recognized as a bona fide tumour suppressor gene. In this Review, we appraise the evidence for the Hippo pathway as a cancer signalling network, and discuss cancer-relevant biological functions, potential mechanisms by which Hippo pathway activity is altered in cancer and emerging therapeutic strategies.

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.

Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms

BACKGROUND The Drosophila neoplastic tumor suppressor Lethal (2) giant larvae (Lgl) controls apicobasal cell polarity and proliferation. We have previously shown that lgl(-) clones in the developing eye exhibit ectopic proliferation and suppress apoptosis without affecting apicobasal cell polarity. Ectopic expression of the apical polarity regulators atypical protein kinase C (aPKC) and Crumbs also leads to increased cell proliferation and/or survival. Here we investigate how these cell polarity regulators control proliferation and survival. RESULTS We report that depletion of lgl in eye epithelial tissue, where polarity is maintained, results in upregulation of targets of the Salvador/Warts/Hippo (SWH) tumor suppressor pathway. Consistent with this, the SWH pathway transcriptional coactivator Yorkie is hyperactivated in Lgl-deficient tissue and is rate limiting for lgl(-) phenotypes. Overexpression of the apical polarity regulators Crumbs or aPKC also leads to ectopic expression of SWH pathway targets without affecting polarity. We show that Lgl depletion or aPKC overexpression results in comislocalization of Hippo and Ras-associated domain family protein (RASSF), consistent with RASSFs ability to block Hippo activation by Salvador. In contrast, Crumbs overexpression leads to mislocalization of Expanded away from the apical cortex, which is predicted to deregulate the pathway. CONCLUSIONS Collectively, our data reveal that the cell polarity regulators Lgl, aPKC, and Crumbs regulate the SWH pathway by two distinct pathways: Lgl acts antagonistically to aPKC to regulate Hippo and RASSF localization, whereas Crumbs regulates Expanded localization. Thus, our study implicates Lgl, aPKC, and Crumbs as regulators of tissue growth via the SWH pathway.

Fat Cadherin Modulates Organ Size in Drosophila via the Salvador/Warts/Hippo Signaling Pathway

BACKGROUND The atypical Fat cadherin has long been known to control cell proliferation and organ size in Drosophila, but the mechanism by which Fat controls these processes has remained elusive. A newly emerging signaling pathway that controls organ size during development is the Salvador/Warts/Hippo pathway. RESULTS Here we demonstrate that Fat limits organ size by modulating activity of the Salvador/Warts/Hippo pathway. ft interacts genetically with positive and negative regulators of this pathway, and tissue lacking fat closely phenocopies tissue deficient for genes that normally promote Salvador/Warts/Hippo pathway activity. Cells lacking fat grow and proliferate more quickly than their wild-type counterparts and exhibit delayed cell-cycle exit as a result of elevated expression of Cyclin E. fat mutant cells display partial insensitivity to normal developmental apoptosis cues and express increased levels of the anti-apoptotic DIAP1 protein. Collectively, these defects lead to increased organ size and organism lethality in fat mutant animals. Fat modulates Salvador/Warts/Hippo pathway activity by promoting abundance and localization of Expanded protein at the apical membrane of epithelial tissues. CONCLUSIONS Fat restricts organ size during Drosophila development via the Salvador/Warts/Hippo pathway. These studies aid our understanding of developmental organ size control and have implications for human hyperproliferative disorders, such as cancers.

Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions

The members of an emerging family of proteins similar to Nedd4 have a unique modular structure consisting of a Ca2+/lipid-binding domain, multiple protein-protein interaction modules and a ubiquitin-protein ligase domain. Although little is known about the physiological roles of these proteins, studies in both mammals and yeast are providing evidence that members of this family might be involved in diverse cellular functions, such as regulation of membrane channels and permeases, the cell cycle and transcription. This article attempts to bring together what is currently known about these evolutionarily conserved ubiquitin-protein ligases.

Upstream Regulation of the Hippo Size Control Pathway

The newly discovered Salvador-Warts-Hippo (SWH) pathway is a key regulator of tissue growth during both development and disease. The first identified components of this pathway represent core downstream effector proteins: the kinases Warts and Hippo; the adaptor proteins Salvador and Mats; and the transcriptional co-activator Yorkie. More recently, a surprising number of proteins have been implicated as upstream regulators of the SWH pathway, including: the planar cell polarity cadherins Fat and Dachsous; the FERM-domain proteins Expanded and Merlin; the WW-domain protein Kibra; the Ras-association family protein dRASSF; and the apicobasal polarity proteins lethal giant larvae, atypical protein kinase C and Crumbs. The identification of a large cohort of upstream regulatory proteins suggests that core SWH pathway proteins are poised to respond to diverse stimuli that must be integrated in a coordinated fashion. Here, we review the existing knowledge of upstream SWH pathway proteins and discuss possible mechanisms of action and signal integration.

The Hippo pathway transcriptional co-activator, YAP, is an ovarian cancer oncogene.

The Salvador-Warts-Hippo (SWH) pathway was first discovered in Drosophila melanogaster as a potent inhibitor of tissue growth. The SWH pathway is highly conserved between D. melanogaster and mammals, both in function and in the mechanism of signal transduction. The mammalian SWH pathway limits tissue growth by inhibiting the nuclear access and expression of the transcriptional co-activator, Yes-associated protein (YAP). Mutation and altered expression of SWH pathway proteins has been observed in several types of human cancer, but the contribution of these events to tumorigenesis has been unclear. Here we show that YAP can enhance the transformed phenotype of ovarian cancer cell lines and that YAP confers resistance to chemotherapeutic agents that are commonly used to treat ovarian cancer. We find that high nuclear YAP expression correlates with poor patient prognosis in a cohort of 268 invasive epithelial ovarian cancer samples. Segregation by histotype shows that the correlation between nuclear YAP and poor survival is predominantly associated with clear cell tumors, independent of stage. Collectively our findings suggest that YAP derepression contributes to the genesis of ovarian clear cell carcinoma and that the SWH pathway is an attractive therapeutic target.

The Nedd4-like Protein KIAA0439 Is a Potential Regulator of the Epithelial Sodium Channel

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of α, β, and γ subunits. The carboxyl terminus of each ENaC subunit contains a PPxY, motif which is believed to be important for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddles syndrome, where mutations delete or alter the PPxY motif of either the β or γ subunits, has been proposed to result in increased ENaC activity. Here we present evidence that KIAA0439 protein, a close relative of Nedd4, is also a potential regulator of ENaC. We demonstrate that KIAA0439 WW domains bind all three ENaC subunits. We show that a recombinant KIAA0439 WW domain protein acts as a dominant negative mutant that can interfere with the Na+-dependent feedback inhibition of ENaC in whole-cell patch clamp experiments. We propose that KIAA0439 and Nedd4 proteins either play a redundant role in ENaC regulation or function in a tissue- and/or signal-specific manner to down-regulate ENaC.

Modularity in the Hippo signaling pathway

Metazoans have evolved several pathways to regulate the size of organs and ultimately that of organisms. One such pathway is known as Salvador-Warts-Hippo, or simply Hippo. Research on the Hippo pathway has grown exponentially during the past 8 years, revealing a complex signaling network. Intriguingly, within this complexity, there are levels of modularity. One level of modularity is represented by the unusually wide occurrence of the WW module in the Hippo core kinase cassette, the upstream regulatory components and the downstream nuclear proteins. We suggest that the prevalence of WW domain-mediated complexes in the Hippo pathway should facilitate its molecular analysis and aid prediction of new pathway components.

All Three WW Domains of Murine Nedd4 Are Involved in the Regulation of Epithelial Sodium Channels by Intracellular Na

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of α, β, and γ subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle’s syndrome where mutations delete or alter the PY motif of either the β or γ subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind α-, β-, and γ-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+channel and of WW domain 1 to an unknown associated protein.