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A YAP/TAZ-TEAD signaling module links endothelial nutrient acquisition to angiogenic growth

ABSTRACT: Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue's metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are dictated by YAP/TAZ-TEAD – a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2, and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fueling nutrient mTORC1 signaling. By orchestrating the transcription of a repertoire of cell-surface transporters, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 pathway activation. Dissociating mTORC1 from these nutrient inputs – elicited by the loss of Rag GTPases – inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. These findings define a pivotal role for YAP/TAZ-TEAD in steering endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell Of Umbilical Vein, Huvec Cell

SUBMITTER: Scientific Service Group Biomolecular Mass Spectrometry

LAB HEAD: Michael Potente

PROVIDER: PXD026872 | Pride | 2022-05-08

REPOSITORIES: Pride

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Project description:Uncontrolled Transforming growth factor-beta (TGFβ) signaling promotes aggressive metastatic properties in late-stage breast cancers. However, how TGFβ-mediated cues are directed to induce late-stage tumorigenic events is poorly understood, particularly given that TGFβ has clear tumor suppressing activity in other contexts. Here we demonstrate that the transcriptional regulators TAZ and YAP (TAZ/YAP), key effectors of the Hippo pathway, are necessary to promote and maintain TGFβ-induced tumorigenic phenotypes in breast cancer cells. Interactions between TAZ/YAP, TGFβ-activated SMAD2/3, and TEAD transcription factors reveal convergent roles for these factors in the nucleus. Genome-wide expression analyses indicate that TAZ/YAP, TEADs and TGFβ-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by TAZ/YAP, TEAD, and TGFβ, such as the novel targets NEGR1 and UCA1, are necessary for maintaining tumorigenic activity in metastatic breast cancer cells. Nuclear TAZ/YAP also cooperate with TGFβ signaling to promote phenotypic and transcriptional changes in non-tumorigenic cells to overcome TGFβ repressive effects. Our work thus identifies crosstalk between nuclear TAZ/YAP and TGFβ signaling in breast cancer cells, revealing novel insight into late-stage disease-driving mechanisms. Expression profiling was conducted following the repression of the transcriptional regulators TAZ and YAP (TAZ/YAP), the TEAD family of transcription factors (TEAD1/2/3/4), or the TGFb signaling pathway (with SB-431542, an inhibitor of the TBRI recpeptor) in human MDA-MB-231-LM2 breast cancer cells treated with TGFβ1. Human MDA-MB-231-LM2-4 breast cancer cells were transfected with control siRNA, or siRNAs targeting TAZ/YAP or all four TEADs and were treated 24 hours later with 500pM TGFβ1 or 5mM SB-431542 for an additional 24 hours. Total RNA was isolated and twelve microarrays in total were performed, with each condition carried out three times on separate days. The Boston University Microarray Core generated the data using the Affymetrix Human Gene 1.0 St Array.

Project description:Activation of endothelial YAP/TAZ signaling is crucial for physiological and pathological angiogenesis. The mechanisms of endothelial YAP/TAZ regulation are, however, incompletely understood. Here we report that the protocadherin FAT1 acts as a critical upstream regulator of endothelial YAP/TAZ which limits the activity of these transcriptional cofactors during developmental and tumor angiogenesis by promoting their degradation. We show that loss of endothelial FAT1 results in increased endothelial cell proliferation in vitro and in various angiogenesis models in vivo. This effect is due to perturbed YAP/TAZ protein degradation, leading to increased YAP/TAZ protein levels and expression of canonical YAP/TAZ target genes. We identified the E3 ubiquitin ligase Mind Bomb-2 (MIB2) as a novel FAT1-interacting protein mediating FAT1-induced YAP/TAZ ubiquitination and degradation. Loss of MIB2 expression in endothelial cells in vitro and in vivo recapitulated the effects of FAT1 depletion and caused decreased YAP/TAZ degradation and increased YAP/TAZ signaling. Our data identify a pivotal mechanism of YAP/TAZ regulation involving FAT1 and its associated E3 ligase MIB2, which is essential for YAP/TAZ-dependent angiogenesis. Samples for proteomics were obtained from HUVECs transduced with the FAT1 intracellular part fused with the extracellular part and transmembrane domain of the IL-2 receptor and carrying a Flag tag on the C terminus (see above) for 36 h followed by lysis in IP buffer. Lysates were incubated with magnetic anti-FLAG beads (M8823, Sigma/Aldrich) overnight at 4Deg. Affinity purified samples were subjected to in-gel (4-12% NuPAGE Bis-Tris gels, ThermoFisher Scientific) digestion as described (DOI: 10.1038/nprot.2006.468). In brief, gel lanes were cut into nine blocks/fractions and finely diced. Embedded proteins were reduced (10 mM dithiothreitol) and alkylated (55 mM iodoacetamide), followed by overnight digestion using trypsin (Serva). Peptides were extracted by increasing concentrations of acetonitrile and lyophilized. Final desalting, concentration and storage utilized "stop and go extraction" (STAGE) tips (DOI: 10.1021/ac026117i). Eluted peptides where subjected to electrospray ionization (ESI)-mediated liquid chromatography/tandem mass spectrometry (LC/MS2) using in house-packed column emitters (15 cm length, 70 um ID, 1.9 um ReprsoSil-Pur 120 C18-AQ, Dr. Maisch) and a buffer system comprising solvent A (0.1% formic acid) and solvent B (80% acetonitrile, 0.1% formic acid). Instrumentation details and parameters were extracted and summarized using MARMoSET (DOI: 10.1074/mcp.TIR119.001505) and are repository deposited. Peptide/spectrum matching and label free quantitation were performed using the MaxQuant suite of algorithms (DOIs: 10.1038/nbt.1511, 10.1021/pr101065j, 10.1074/mcp.M113.031591) against the human UniProt database (canonical & isoforms; downloaded on 2019/01/23). Parameters are included here. Downstream statistical analysis used the limma-based (DOI: 10.1093/nar/gkv007) in-house package autonomics (https://doi.org/doi:10.18129/B9.bioc.autonomics).

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A YAP/TAZ-TEAD signaling module links endothelial nutrient acquisition to angiogenic growth

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