13 research outputs found
Investigation of specific inhibitors of the PI3K signal cascade for the therapy of lymphangioma
PIK3CA mutations are specifically localized to lymphatic endothelial cells of lymphatic malformations.
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
Phospho-ERK Western blot analysis of lymphatic endothelial cells.
<p>Note that phospho-ERK (pERK) is highly expressed in lymphatic malformation-derived Ly-LECs (Ly-LEC1, 10, and 12), but not in normal HD-LECc3 and c4. Ly-LEC2, which contain a Glu109del in <i>PIK3CA</i>, are similar to normal LECs, which contain clear levels of total ERK (tERK). Antibodies against α-tubulin were used as loading control.</p
Proliferation studies with PIK3CA inhibitor Buparlisib (BKM120) on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
<p>0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.</p
Primers used for mutation analyses of <i>PIK3CA</i>.
<p>Primers used for mutation analyses of <i>PIK3CA</i>.</p
Patient-derived fibroblasts (Ly-F-12).
<p><b>A</b>) Phase-contrast image showing typical morphology. <b>B</b>) Anti-αSMA staining. <b>C</b>) Anti-vimentin staining. <b>D</b>) Negative control without primary antibodies. Note that all fibroblasts express αSMA and vimentin. Bar = 50μm.</p
Culture of lymphatic endothelial cells and fibroblasts.
<p>Immunocytology showing CD31 (green) and PROX1 (magenta). Nuclei are counter-stained with DAPI (blue). <b>A</b>) Foreskin-derived HD-LECc4; <b>B</b>) Foreskin-derived HD-LECc2; <b>C</b>) Patient-derived Ly-LEC-12, <b>D</b>) Patient-derived Ly-LEC-2; <b>E</b>) Patient-derived Ly-LEC-10; <b>F</b>) Patient-derived fibroblasts (Ly-F-12) do not express CD31 or PROX1. Bar = 45 μm in A-D,F, and 90 μm in E.</p
Proliferation studies with PIK3CD inhibitor CAL-101 (Idealisib) on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
<p>0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.</p
Proliferation studies with PIK3CA inhibitor Wortmannin on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
<p>0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.</p
Proliferation studies with PIK3CA inhibitor Ly294002 on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
<p>0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.</p