Zhipeng Meng

Mechanisms of Hippo pathway regulation

The Hippo pathway was initially identified in Drosophila melanogaster screens for tissue growth two decades ago and has been a subject extensively studied in both Drosophila and mammals in the last several years. The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. Recent studies have expanded the Hippo pathway as a complex signaling network with >30 components. This pathway is regulated by intrinsic cell machineries, such as cell-cell contact, cell polarity, and actin cytoskeleton, as well as a wide range of signals, including cellular energy status, mechanical cues, and hormonal signals that act through G-protein-coupled receptors. The major functions of the Hippo pathway have been defined to restrict tissue growth in adults and modulate cell proliferation, differentiation, and migration in developing organs. Furthermore, dysregulation of the Hippo pathway leads to aberrant cell growth and neoplasia. In this review, we focus on recent developments in our understanding of the molecular actions of the core Hippo kinase cascade and discuss key open questions in the regulation and function of the Hippo pathway.

Mutant Gq/11 Promote Uveal Melanoma Tumorigenesis by Activating YAP

Uveal melanoma (UM) is the most common cancer in adult eyes. Approximately 80% of UMs harbor somatic activating mutations in GNAQ or GNA11 (encoding Gq or G11, respectively). Herein, we show in both cell culture and human tumors that cancer-associated Gq/11 mutants activate YAP, a major effector of the Hippo tumor suppressor pathway that is also regulated by G protein-coupled receptor signaling. YAP mediates the oncogenic activity of mutant Gq/11 in UM development, and the YAP inhibitor verteporfin blocks tumor growth of UM cells containing Gq/11 mutations. This study reveals an essential role of the Hippo-YAP pathway in Gq/11-induced tumorigenesis and suggests YAP as a potential drug target for UM patients carrying mutations in GNAQ or GNA11.

MAP4K family kinases act in parallel to MST1 2 to activate LATS1 2 in the Hippo pathway

The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members—Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7—as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway.

A YAP/TAZ-induced feedback mechanism regulates Hippo pathway homeostasis

YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are major downstream effectors of the Hippo pathway that influences tissue homeostasis, organ size, and cancer development. Aberrant hyperactivation of YAP/TAZ causes tissue overgrowth and tumorigenesis, whereas their inactivation impairs tissue development and regeneration. Dynamic and precise control of YAP/TAZ activity is thus important to ensure proper physiological regulation and homeostasis of the cells. Here, we show that YAP/TAZ activation results in activation of their negative regulators, LATS1/2 (large tumor suppressor 1/2) kinases, to constitute a negative feedback loop of the Hippo pathway in both cultured cells and mouse tissues. YAP/TAZ in complex with the transcription factor TEAD (TEA domain family member) directly induce LATS2 expression. Furthermore, YAP/TAZ also stimulate the kinase activity of LATS1/2 through inducing NF2 (neurofibromin 2). This feedback regulation is responsible for the transient activation of YAP upon lysophosphatidic acid (LPA) stimulation and the inhibition of YAP-induced cell migration. Thus, this LATS-mediated feedback loop provides an efficient mechanism to establish the robustness and homeostasis of YAP/TAZ regulation.

A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening

With <2% of the human genome coding for proteins, a major challenge is to interpret the function of the noncoding DNA. Millions of regulatory sequences have been predicted in the human genome through analysis of DNA methylation, chromatin modification, hypersensitivity to nucleases, and transcription factor binding, but few have been shown to regulate transcription in their native contexts. We have developed a high-throughput CRISPR/Cas9-based genome-editing strategy and used it to interrogate 174 candidate regulatory sequences within the 1-Mbp POU5F1 locus in human embryonic stem cells (hESCs). We identified two classical regulatory elements, including a promoter and a proximal enhancer, that are essential for POU5F1 transcription in hESCs. Unexpectedly, we also discovered a new class of enhancers that contribute to POU5F1 transcription in an unusual way: Disruption of such sequences led to a temporary loss of POU5F1 transcription that is fully restored after a few rounds of cell division. These results demonstrate the utility of high-throughput screening for functional characterization of noncoding DNA and reveal a previously unrecognized layer of gene regulation in human cells.

Hippo Pathway Regulation of Gastrointestinal Tissues

The Hippo pathway plays a crucial role in regulating tissue homeostasis and organ size, and its deregulation is frequently observed in human cancer. Yap is the major effector of and is inhibited by the Hippo pathway. In mouse model studies, inducible Yap expression in multiple tissues results in organ overgrowth. In the liver, knockout of upstream Hippo pathway components or transgenic expression of Yap leads to liver enlargement and hepatocellular carcinoma. In the small intestine or colon, deletion of upstream Hippo pathway components also results in expansion of intestinal progenitor cells and eventual development of adenomas. Genetic deletion of Yap in the intestine does not change the intestinal structure, but Yap is essential for intestinal repair upon certain types of tissue injury. The function of the Hippo pathway has also been studied in other gastrointestinal tissues, including the pancreas and stomach. Here we provide a brief overview of the Hippo pathway and discuss the physiological and pathological functions of this tumor suppressor pathway in gastrointestinal tissues.

The Hippo pathway in intestinal regeneration and disease

The Hippo pathway is a signalling cascade conserved from Drosophila melanogaster to mammals. The mammalian core kinase components comprise MST1 and MST2, SAV1, LATS1 and LATS2 and MOB1A and MOB1B. The transcriptional co-activators YAP1 and TAZ are the downstream effectors of the Hippo pathway and regulate target gene expression. Hippo signalling has crucial roles in the control of organ size, tissue homeostasis and regeneration, and dysregulation of the Hippo pathway can lead to uncontrolled cell growth and malignant transformation. Mammalian intestine consists of a stem cell compartment as well as differentiated cells, and its ability to regenerate rapidly after injury makes it an excellent model system to study tissue homeostasis, regeneration and tumorigenesis. Several studies have established the important role of the Hippo pathway in these processes. In addition, crosstalk between Hippo and other signalling pathways provides tight, yet versatile, regulation of tissue homeostasis. In this Review, we summarize studies on the role of the Hippo pathway in the intestine on these physiological processes and the underlying mechanisms responsible, and discuss future research directions and potential therapeutic strategies targeting Hippo signalling in intestinal disease.

A tiling-deletion-based genetic screen for cis -regulatory element identification in mammalian cells

Millions of cis-regulatory elements are predicted to be present in the human genome, but direct evidence for their biological function is scarce. Here we report a high-throughput method, cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq), for the unbiased discovery and functional assessment of cis-regulatory sequences in the genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells, and identified 45 cis-regulatory elements. A majority of these elements have active chromatin marks, DNase hypersensitivity, and occupancy by multiple transcription factors, which confirms the utility of chromatin signatures in cis-element mapping. Notably, 17 of them are previously annotated promoters of functionally unrelated genes, and like typical enhancers, they form extensive spatial contacts with the POU5F1 promoter. These results point to the commonality of enhancer-like promoters in the human genome.

Characterization of Hippo Pathway Components by Gene Inactivation

The Hippo pathway is important for regulating tissue homeostasis, and its dysregulation has been implicated in human cancer. However, it is not well understood how the Hippo pathway becomes dysregulated because few mutations in core Hippo pathway components have been identified. Therefore, much work in the Hippo field has focused on identifying upstream regulators, and a complex Hippo interactome has been identified. Nevertheless, it is not always clear which components are the most physiologically relevant in regulating YAP/TAZ. To provide an overview of important Hippo pathway components, we created knockout cell lines for many of these components and compared their relative contributions to YAP/TAZ regulation in response to a wide range of physiological signals. By this approach, we provide an overview of the functional importance of many Hippo pathway components and demonstrate NF2 and RHOA as important regulators of YAP/TAZ and TAOK1/3 as direct kinases for LATS1/2.

Kaposi sarcoma-associated herpesvirus promotes tumorigenesis by modulating the Hippo pathway

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the culprit behind the human disease Kaposi sarcoma (KS), an AIDS-defining malignancy. KSHV encodes a viral G-protein-coupled receptor (vGPCR) critical for the initiation and progression of KS. In this study, we identified that YAP/TAZ, two homologous oncoproteins inhibited by the Hippo tumor suppressor pathway, are activated in KSHV-infected cells in vitro, KS-like mouse tumors and clinical human KS specimens. The KSHV-encoded vGPCR acts through Gq/11 and G12/13 to inhibit the Hippo pathway kinases Lats1/2, promoting the activation of YAP/TAZ. Furthermore, depletion of YAP/TAZ blocks vGPCR-induced cell proliferation and tumorigenesis in a xenograft mouse model. The vGPCR-transformed cells are sensitive to pharmacologic inhibition of YAP. Our study establishes a pivotal role of the Hippo pathway in mediating the oncogenic activity of KSHV and development of KS, and also suggests a potential of using YAP inhibitors for KS intervention.