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AKT hyper-phosphorylation associated with PI3K mutations in lymphatic endothelial cells from a patient with lymphatic malformation

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Abstract

Lymphatic malformations (LM) are characterized by abnormal formation of lymphatic vessels and tissue overgrowth. The lymphatic vessels present in LM lesions may become blocked and enlarged as lymphatic fluid collects, forming a mass or cyst. Lesions are typically diagnosed during childhood and are often disfiguring and life threatening. Available treatments consist of sclerotherapy, surgical removal and therapies to diminish complications. We isolated lymphatic endothelial cells (LM-LEC) from a surgically removed microcystic LM lesion. LM-LEC and normal human dermal-LEC (HD-LEC) expressed endothelial (CD31, VE-Cadherin) as well as lymphatic endothelial (Podoplanin, PROX1, LYVE1)-specific markers. Targeted gene sequencing analysis in patient-derived LM-LEC revealed the presence of two mutations in class I phosphoinositide 3-kinases (PI3K) genes. One is an inherited, premature stop codon in the PI3K regulatory subunit PIK3R3. The second is a somatic missense mutation in the PI3K catalytic subunit PIK3CA; this mutation has been found in association with overgrowth syndromes and cancer growth. LM-LEC exhibited angiogenic properties: both cellular proliferation and sprouting in collagen were significantly increased compared with HD-LEC. AKT-Thr308 was constitutively hyper-phosphorylated in LM-LEC. Treatment of LM-LEC with PI3-Kinase inhibitors Wortmannin and LY294 decreased cellular proliferation and prevented the phosphorylation of AKT-Thr308 in both HD-LEC and LM-LEC. Treatment with the mTOR inhibitor rapamycin also diminished cellular proliferation, sprouting and AKT phosphorylation, but only in LM-LEC. Our results implicate disrupted PI3K-AKT signaling in LEC isolated from a human lymphatic malformation lesion.

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Abbreviations

LM:

Lymphatic malformation

LEC:

Lymphatic endothelial cells

PI3K:

Phosphoinositide 3-kinase

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Acknowledgments

Research reported in this manuscript was supported by a Translational Research Program Pilot Study Grant from Boston Children’s Hospital (J.B.), the Charles Hood Foundation (E.B.), the Manton Center for Orphan Disease Research (E.B.) and the National Heart, Lung, and Blood Institute, part of the National Institutes of Health, under Award Number R01 HL117952 (E.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Dr. Steven Fishman, and Lan Huang for helpful discussions, Drs. Camille L. Stewart and Annie Kulungowski for initial characterization of the LM-LEC, Dr. Tanya Mayadas for providing HUVECs, the Cytogenetics Core of Dana Farber Harvard Cancer Center (P30 CA006516), Jill Wylie-Sears for technical assistance and Kristin Johnson for the preparation of figures.

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The authors declare that they have no conflict of interests.

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The experiments in this manuscript comply with the current laws of the United States of America.

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  1. Elisa Boscolo

    Present address: Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

Authors and Affiliations

  1. Vascular Biology Program and Department of Surgery, Boston Children’s Hospital, Harvard Medical School, Boston, MA, 02115, USA

    Elisa Boscolo, Silvia Coma, Michael Klagsbrun & Joyce Bischoff

  2. Departments of Orthopedic Surgery and Genetics and the Vascular Anomalies Center, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Valerie L. Luks & Matthew L. Warman

  3. Department of Plastic and Oral Surgery and Vascular Anomalies Center, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Arin K. Greene

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  1. Elisa Boscolo
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Boscolo, E., Coma, S., Luks, V.L. et al. AKT hyper-phosphorylation associated with PI3K mutations in lymphatic endothelial cells from a patient with lymphatic malformation. Angiogenesis 18, 151–162 (2015). https://doi.org/10.1007/s10456-014-9453-2

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