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L-type amino acid transporter 1 promotes proliferation and invasion of human chorionic trophoblast and choriocarcinoma cells through mTORC1.

ABSTRACT: L-type amino acid transporter 1 (LAT1) is a neutral amino acid transporter expressed in trophoblast giant cells onembryonic day 8 in mice. LAT1 is responsible for metabolism in blastocysts and cancer cells. Despite research concerning the aberrant high expression and indispensable function of LAT1 in various cancers, little is known about the role of LAT1 in regulating the behaviors of human trophoblast cells under different physiological and pathological conditions. The HTR8-SVneo human trophoblast cell line and JEG-3 and JAR choriocarcinoma cell lines are used as models for trophoblast cell biological research. The proliferation and apoptosis of these cells were assayed using the CCK-8 assay and flow cytometry, respectively. Transwell-chambers were used to observed migration and invasion of the cells. Immunofluorescent staining, western blot, and RT-PCR assays were used to determine the possible mechanism of LAT1 on human trophoblast cell behaviors with small interfering RNA or signal agonists and antagonist treatments. LAT1 was expressed in the trophoblast and choriocarcinoma cells. LAT1 was involved in regulating behaviors of these cells, such as cell proliferation, apoptosis, migration, and invasion. Detailed results suggested that LAT1 modulated trophoblast cell functions by mediation of mTORC1 signaling pathways. Our results implicate LAT1 as a very important regulator in human trophoblast cell behaviors at the maternal-fetal interface.

SUBMITTER: Luo W

PROVIDER: S-EPMC7653574 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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L-type amino acid transporter 1 promotes proliferation and invasion of human chorionic trophoblast and choriocarcinoma cells through mTORC1.

Luo Wenping W Zhang Hongmei H Zhang Yan Y Liang Panpan P Wang Xiaojie X Ma Jing J Tan Dongmei D Tan Yi Y Song Jinlin J Ji Ping P Zhao Tianyu T

American journal of translational research 20201015 10

L-type amino acid transporter 1 (LAT1) is a neutral amino acid transporter expressed in trophoblast giant cells onembryonic day 8 in mice. LAT1 is responsible for metabolism in blastocysts and cancer cells. Despite research concerning the aberrant high expression and indispensable function of LAT1 in various cancers, little is known about the role of LAT1 in regulating the behaviors of human trophoblast cells under different physiological and pathological conditions. The HTR8-SVneo human trophob ...[more]

PMID: 33194063

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Project description:BACKGROUND:Tumor angiogenesis is regarded as a rational anti-cancer target. The efficacy and indications of anti-angiogenic therapies in clinical practice, however, are relatively limited. Therefore, there still exists a demand for revealing the distinct characteristics of tumor endothelium that is crucial for the pathological angiogenesis. L-type amino acid transporter 1 (LAT1) is well known to be highly and broadly upregulated in tumor cells to support their growth and proliferation. In this study, we aimed to establish the upregulation of LAT1 as a novel general characteristic of tumor-associated endothelial cells as well, and to explore the functional relevance in tumor angiogenesis. METHODS:Expression of LAT1 in tumor-associated endothelial cells was immunohistologically investigated in human pancreatic ductal adenocarcinoma (PDA) and xenograft- and syngeneic mouse tumor models. The effects of pharmacological and genetic ablation of endothelial LAT1 were examined in aortic ring assay, Matrigel plug assay, and mouse tumor models. The effects of LAT1 inhibitors and gene knockdown on cell proliferation, regulation of translation, as well as on the VEGF-A-dependent angiogenic processes and intracellular signaling were investigated in in vitro by using human umbilical vein endothelial cells. RESULTS:LAT1 was highly expressed in vascular endothelial cells of human PDA but not in normal pancreas. Similarly, high endothelial LAT1 expression was observed in mouse tumor models. The angiogenesis in ex/in vivo assays was suppressed by abrogating the function or expression of LAT1. Tumor growth in mice was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. LAT1-mediated amino acid transport was fundamental to support endothelial cell proliferation and translation initiation in vitro. Furthermore, LAT1 was required for the VEGF-A-dependent migration, invasion, tube formation, and activation of mTORC1, suggesting a novel cross-talk between pro-angiogenic signaling and nutrient-sensing in endothelial cells. CONCLUSIONS:These results demonstrate that the endothelial LAT1 is a novel key player in tumor angiogenesis, which regulates proliferation, translation, and pro-angiogenic VEGF-A signaling. This study furthermore indicates a new insight into the dual functioning of LAT1 in tumor progression both in tumor cells and stromal endothelium. Therapeutic inhibition of LAT1 may offer an ideal option to potentiate anti-angiogenic therapies.

Project description:Choriocarcinoma (CC) is characterized by earlier blood metastasis compared with other female genital tumors and a high incidence of massive hemorrhage. Vasculogenic mimicry (VM) is highly associated with metastasis, and syncytiotrophoblast is involved in the formation of VM in CC. Forskolin is a typical activator of the cAMP pathway, which is involved in the syncytiolization of trophoblastic cells. In the present study, to determine the effects and mechanism of forskolin on cell invasion and VM during syncytiolization in vitro and in vivo, JEG?3 and JAR cell lines were treated with 100 µM forskolin for 48 h, and wound healing and invasion assays were used to verify cell migratory and invasive capacities. A 3D culture and tube formation assays were established to detect VM. Variation of morphology and markers of the epithelial?to?mesenchymal transition (EMT) were assessed, and the role of the Notch signaling pathway was investigated in CC cells treated with forskolin. The results of the present study demonstrated that 100 µM forskolin induced syncytiolization of trophoblastic cells and enhanced the migratory and invasive abilities of JEG?3 and JAR cell lines. In addition, the capacity of VM was significantly increased, whereas tube formation ability was decreased by forskolin in vitro and in vivo compared with the respective control groups. The cellular morphology exhibited EMT during the syncytiolization process, which was further supported by the changes in EMT marker expression, including downregulation of E?cadherin and cytokeratin and upregulation of N?cadherin, vimentin and zinc finger E?box?binding homeobox 1. The Notch?1 signaling pathway was activated to induce EMT in forskolin?induced VM process in CC cells, and VM and EMT could be reversed by using the ??secretase inhibitor DAPT to block the Notch?1 pathway. Overall, the results of the present study demonstrated that forskolin enhanced the capacity of VM formation and metastasis through Notch?1?activated EMT in the syncytiolization of trophoblastic cells.

Dataset Information

L-type amino acid transporter 1 promotes proliferation and invasion of human chorionic trophoblast and choriocarcinoma cells through mTORC1.

Publications

L-type amino acid transporter 1 promotes proliferation and invasion of human chorionic trophoblast and choriocarcinoma cells through mTORC1.

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