PIK3CA mutations are specifically localized to lymphatic endothelial cells of lymphatic malformations

PLoS One. 2018 Jul 9;13(7):e0200343. doi: 10.1371/journal.pone.0200343. eCollection 2018.

Abstract

Lymphatic malformations (LM) are characterized by the overgrowth of lymphatic vessels during pre- and postnatal development. Macrocystic, microcystic and combined forms of LM are known. The cysts are lined by lymphatic endothelial cells (LECs). Resection and sclerotherapy are the most common treatment methods. Recent studies performed on LM specimens in the United States of America have identified activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene in LM. However, whole tissue but not isolated cell types were studied. Here, we studied LM tissues resected at the University Hospitals Freiburg and Regensburg, Germany. We isolated LECs and fibroblasts separately, and sequenced the commonly affected exons 8, 10, and 21 of the PIK3CA gene. We confirm typical monoallelic mutations in 4 out of 6 LM-derived LEC lines, and describe two new mutations i.) in exon 10 (c.1636C>A; p.Gln546Lys), and ii.) a 3bp in-frame deletion of GAA (Glu109del). LM-derived fibroblasts did not possess such mutations, showing cell-type specificity of the gene defect. High activity of the PIK3CA-AKT- mTOR pathway was demonstrated by hyperphosphorylation of AKT-Ser473 in all LM-derived LECs (including the ones with newly identified mutations), as compared to normal LECs. Additionally, hyperphosphorylation of ERK was seen in all LM-derived LECs, except for the one with Glu109del. In vitro, the small molecule kinase inhibitors Buparlisib/BKM-120, Wortmannin, and Ly294002, (all inhibitors of PIK3CA), CAL-101 (inhibitor of PIK3CD), MK-2206 (AKT inhibitor), Sorafenib (multiple kinases inhibitor), and rapamycin (mTOR inhibitor) significantly blocked proliferation of LM-derived LECs in a concentration-dependent manner, but also blocked proliferation of normal LECs. However, MK-2206 appeared to be more specific for mutated LECs, except in case of Glu109 deletion. In sum, children that are, or will be, treated with kinase inhibitors must be monitored closely.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Proliferation / drug effects
  • Cell Proliferation / physiology
  • Class I Phosphatidylinositol 3-Kinases / antagonists & inhibitors
  • Class I Phosphatidylinositol 3-Kinases / genetics*
  • Class I Phosphatidylinositol 3-Kinases / metabolism*
  • Endothelial Cells / drug effects
  • Endothelial Cells / enzymology*
  • Endothelial Cells / pathology
  • Enzyme Inhibitors / pharmacology
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Female
  • Fibroblasts / enzymology
  • Humans
  • Lymphatic Vessels / abnormalities*
  • Lymphatic Vessels / drug effects
  • Lymphatic Vessels / enzymology*
  • Lymphatic Vessels / pathology
  • Male
  • Mutation*
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors
  • Proto-Oncogene Proteins c-akt / metabolism
  • TOR Serine-Threonine Kinases / antagonists & inhibitors
  • TOR Serine-Threonine Kinases / metabolism

Substances

  • Enzyme Inhibitors
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human
  • Proto-Oncogene Proteins c-akt
  • Extracellular Signal-Regulated MAP Kinases

Grant support

The studies were funded by a grant from the Deutsche Gesellschaft für Lymphologie (DGL) to JW. The DGL did not have any influence on the performance of the study and the interpretation of the results.