Ras drives malignancy through stem cell crosstalk with the microenvironment.

Squamous cell carcinomas are triggered by marked elevation of RAS-MAPK signalling and progression from benign papilloma to invasive malignancy1-4. At tumour-stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6. The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7. Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFß signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)-AKT-mTOR signalling during the benign-to-malignant transition. By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy. These insights suggest broad implications for cancer therapeutics.

YAP mediates crosstalk between the Hippo and PI(3)K­TOR pathways by suppressing PTEN via miR-29.

Organ development is a complex process governed by the interplay of several signalling pathways that have critical functions in the regulation of cell growth and proliferation. Over the past years, the Hippo pathway has emerged as a key regulator of organ size. Perturbation of this pathway has been shown to play important roles in tumorigenesis. YAP, the main downstream target of the mammalian Hippo pathway, promotes organ growth, yet the underlying molecular mechanism of this regulation remains unclear. Here we provide evidence that YAP activates the mammalian target of rapamycin (mTOR), a major regulator of cell growth. We have identified the tumour suppressor PTEN, an upstream negative regulator of mTOR, as a critical mediator of YAP in mTOR regulation. We demonstrate that YAP downregulates PTEN by inducing miR-29 to inhibit PTEN translation. Last, we show that PI(3)K­mTOR is a pathway modulated by YAP to regulate cell size, tissue growth and hyperplasia. Our studies reveal a functional link between Hippo and PI(3)K­mTOR, providing a molecular basis for the coordination of these two pathways in organ size regulation.

Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.

The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.

Organ size control by Hippo and TOR pathways.

The determination of final organ size is a highly coordinated and complex process that relies on the precise regulation of cell number and/or cell size. Perturbation of organ size control contributes to many human diseases, including hypertrophy, degenerative diseases, and cancer. Hippo and TOR are among the key signaling pathways involved in the regulation of organ size through their respective functions in the regulation of cell number and cell size. Here, we review the general mechanisms that regulate organ growth, describe how Hippo and TOR control key aspects of growth, and discuss recent findings that highlight a possible coordination between Hippo and TOR in organ size regulation.

The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal.

Precise control of organ size is crucial during animal development and regeneration. In Drosophila and mammals, studies over the past decade have uncovered a critical role for the Hippo tumour-suppressor pathway in the regulation of organ size. Dysregulation of this pathway leads to massive overgrowth of tissue. The Hippo signalling pathway is highly conserved and limits organ size by phosphorylating and inhibiting the transcription co-activators YAP and TAZ in mammals and Yki in Drosophila, key regulators of proliferation and apoptosis. The Hippo pathway also has a critical role in the self-renewal and expansion of stem cells and tissue-specific progenitor cells, and has important functions in tissue regeneration. Emerging evidence shows that the Hippo pathway is regulated by cell polarity, cell adhesion and cell junction proteins. In this review we summarize current understanding of the composition and regulation of the Hippo pathway, and discuss how cell polarity and cell adhesion proteins inform the role of this pathway in organ size control and regeneration.

A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP).

The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).