YAP Accelerates Notch-Driven Cholangiocarcinogenesis via mTORC1 in Mice
2021; Elsevier BV; Volume: 191; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2021.05.017
ISSN1525-2191
AutoresXinjun Lu, Baogang Peng, Ge Chen, Giovanni Mario Pes, Silvia Ribback, Cindy Ament, Hongwei Xu, Rajesh Pal, Pedro M. Rodrigues, Jesús M. Bañales, Matthias Evert, Diego F. Calvisi, Xin Chen, Biao Fan, Jingxiao Wang,
Tópico(s)Cancer-related gene regulation
ResumoIntrahepatic cholangiocarcinoma (iCCA) is a lethal malignant neoplasm with limited therapeutic options. Previous studies have found that Notch1 overexpression alone suffices to induce iCCA in the mouse, albeit after long latency. The current study found that activation of the Yes-associated protein (Yap) proto-oncogene occurs during Notch1-driven iCCA progression. After co-expressing activated Notch1 intracellular domain (Nicd) and Yap (YapS127A) in the mouse liver, rapid iCCA formation and progression occurred in Nicd/Yap mice. Mechanistically, an increased expression of amino acid transporters and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway was detected in Nicd/Yap mouse liver tumors. Significantly, the genetic deletion of Raptor, the major mTORC1 component, completely suppressed iCCA development in Nicd/Yap mice. Elevated expression of Notch1, YAP, amino acid transporters, and members of the mTORC1 pathway was also detected ubiquitously in a collection of human iCCA specimens. Their levels were associated with a poor patient outcome. This study demonstrates that Notch and YAP concomitant activation is frequent in human cholangiocarcinogenesis. Notch and YAP synergize to promote iCCA formation by activating the mTORC1 pathway. Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignant neoplasm with limited therapeutic options. Previous studies have found that Notch1 overexpression alone suffices to induce iCCA in the mouse, albeit after long latency. The current study found that activation of the Yes-associated protein (Yap) proto-oncogene occurs during Notch1-driven iCCA progression. After co-expressing activated Notch1 intracellular domain (Nicd) and Yap (YapS127A) in the mouse liver, rapid iCCA formation and progression occurred in Nicd/Yap mice. Mechanistically, an increased expression of amino acid transporters and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway was detected in Nicd/Yap mouse liver tumors. Significantly, the genetic deletion of Raptor, the major mTORC1 component, completely suppressed iCCA development in Nicd/Yap mice. Elevated expression of Notch1, YAP, amino acid transporters, and members of the mTORC1 pathway was also detected ubiquitously in a collection of human iCCA specimens. Their levels were associated with a poor patient outcome. This study demonstrates that Notch and YAP concomitant activation is frequent in human cholangiocarcinogenesis. Notch and YAP synergize to promote iCCA formation by activating the mTORC1 pathway. Intrahepatic cholangiocarcinoma (iCCA) is a deadly tumor and the second most common primary liver cancer type.1Razumilava N. Gores G.J. 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In human iCCA specimens, the YAP, Notch1, and mTOR pathways were coordinately activated, especially in the most biologically aggressive tumors. Thus, the Nicd/Yap model might represent a valuable tool for the study of human iCCA. Wild-type FVB/N mice and Raptorfl/fl mice were from the Jackson Laboratory (Sacramento, CA). Mice were housed and monitored following protocols approved by the Committee for Animal Research at the University of California, San Francisco. The plasmids, including pT3EF1α-NICD (MYC tagged), pT3EF1α-YAPS127A, pCMV, pCMV-Cre (Cre), and pCMV–sleeping beauty transposase (SB), have been described previously in detail.26Wang J. Dong M. Xu Z. Song X. Zhang S. Qiao Y. Che L. Gordan J. Hu K. Liu Y. Calvisi D.F. Chen X. Notch2 controls hepatocyte-derived cholangiocarcinoma formation in mice.Oncogene. 2018; 37: 3229-3242Crossref PubMed Scopus (43) Google Scholar,37Zhang S. Song X. Cao D. Xu Z. Fan B. Che L. Hu J. Chen B. Dong M. Pilo M.G. Cigliano A. Evert K. Ribback S. 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Inhibition of metalloprotease hyperactivity in cystic cholangiocytes halts the development of polycystic liver diseases.Gut. 2014; 63: 1658-1667Crossref PubMed Scopus (35) Google Scholar Briefly, 20 μg of pT3EF1α-NICD and pT3EF1α-YAPS127A constructs and the SB plasmid at the ratio of 25:1 to all oncogenes were diluted in 2 mL of 0.9% sodium chloride solution and injected into the tail vein of 20-g FVB/N mice in approximately 5 seconds. Concerning Raptor ablation experiments, 20 μg of pT3EF1α-NICD and pT3EF1α-YAPS127A, with 60 μg of pCMV or Cre, and SB plasmids were injected into Raptorfl/fl mice. All plasmids were extracted using the Endotoxin-free Maxi prep kit (Sigma-Aldrich, St. Louis, MO) before injection. Mice were sacrificed at indicated time points or when mice became moribund or developed large abdominal masses. Mouse liver tissues and human cell lines were homogenized in mammalian protein extraction reagent (catalog number 78503; Thermo Fisher Scientific, Waltham, MA) plus protease inhibitor cocktail (catalog number 78440; Thermo Fisher Scientific). Proteins were quantified with the BCA assay (catalog number 23225; Thermo Fisher Scientific). The antibodies used in this study are listed in Table 1.Table 1Antibody ListAntibodySourceCatalog no.SpeciesApplicationDilutionRetrieval bufferp-AKTS473Cell Signaling Technology (Danvers, MA)9271MouseWestern blot1:1000NATotal AKTCell Signaling Technology9272MouseWestern blot1:1000NAmTORCell Signaling Technology2983MouseWestern blot1:1000NAp-4EBP1Cell Signaling Technology9451MouseWestern blot1:1000NA4EBP1Cell Signaling Technology9644MouseWestern blot1:1000NAp-RPS6Cell Signaling Technology4858MouseWestern blot1:1000NARPS6Cell Signaling Technology2217MouseWestern blot1:1000NAp-ERK1/2Cell Signaling Technology9101MouseWestern blot1:1000NATotal ERK1/2Cell Signaling Technology9102MouseWestern blot1:1000NAYAPCell Signaling Technology14074MouseWestern blot1:1000NANotch1Cell Signaling Technology4380MouseWestern blot1:1000NAMYC taggedCell Signaling Technology2278MouseWestern blot1:1000NARICTORCell Signaling Technology2114MouseWestern blot1:500NARAPTORCell Signaling Technology2280MouseWestern blot1:1000NAGAPDHCell Signaling Technology5174MouseWestern blot1:3000NACK19Abcam (Cambridge, UK)ab181604MouseImmunohistochemistry1:800Sodium citrateHNF4αAbcamab181604MouseImmunohistochemistry1:2000Tris-EDTASOX9Abcamab185230MouseImmunohistochemistry1:2000Sodium citrateKi-67Cell Signaling Technology12202MouseImmunohistochemistry1:150Sodium citratep-ERK1/2Cell Signaling Technology4370MouseImmunohistochemistry1:200Sodium citratep-mTOR Ser2448Cell Signaling Technology2976MouseImmunohistochemistry1:150Sodium citrateMYC-taggedCell Signaling Technology2278MouseImmunohistochemistry1:100Sodium citrateCK19Cell Signaling Technology12434HumanImmunohistochemistry1:500Sodium citrateRAPTORAbcamab40768HumanImmunohistochemistry1:200Sodium citratep-4EBP1Cell Signaling Technology2855HumanImmunohistochemistry1:300Sodium citrateNotch1Lifespan Biosciences (Seattle, WA)LS-C114369HumanImmunohistochemistry1:100Sodium citrateYAPCell Signaling Technology14074HumanImmunohistochemistry1:200Sodium citratep-YAPCell Signaling Technology13008HumanImmunohistochemistry1:300Sodium citrateCTGFAtlas Antibodies (Stockholm, Sweden)AMAb91366HumanImmunohistochemistry1:100Sodium citrateCK, cytokeratin; CTGF, connective tissue growth factor; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNF4α, hepatocyte nuclear factor 4α; mTOR, mammalian target of rapamycin; NA, not applicable; p-, phosphorylated; MYC tagged, pT3EF1α-NICD; YAP, Yes-associated protein. Open table in a new tab CK, cytokeratin; CTGF, connective tissue growth factor; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNF4α, hepatocyte nuclear factor 4α; mTOR, mammalian target of rapamycin; NA, not applicable; p-, phosphorylated; MYC tagged, pT3EF1α-NICD; YAP, Yes-associated protein. Two experienced pathologists and liver experts (M.E. and S.R.) analyzed hematoxylin and eosin–stained liver sections based on the criteria established by Frith and Ward.47Frith C.H. Ward J.M. A morphologic classification of proliferative and neoplastic hepatic lesions in mice.J Environ Pathol Toxicol. 1979; 3: 329-351PubMed Google Scholar Cholangiomas, alias biliary adenomas, are benign cholangiocellular lesions that compress the surrounding parenchyma but lack cytologic atypia, substantial mitotic/apoptotic activity, necrosis, infiltration/invasion, and fibrosis. When cystic, cholangiomas are referred to as cystic cholangiomas or cystadenomas. Cholangiocellular lesions characterized by cytologic atypia and extensive proliferation are defined as intracystic papillary neoplasms with intraepithelial neoplasia. If these neoplasms result from malignant transformation of benign cystadenomas, they are referred to as cystadenocarcinomas. Cystadenocarcinomas that inva
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