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

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 to 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.

Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors

“…It remains a possibility that signaling through AKT may be utilized downstream of Flt4 at later stages of lymphatic morphogenesis or maintenance. Indeed, signaling through the phosphoinositide 3-kinase (PI3K) pathway, an essential upstream activator of AKT, has been implicated in both mice and humans at later stages of lymphatic morphogenesis and maintenance, and in an organ-specific manner (Boscolo et al, 2015;Mouta-Bellum et al, 2009). Thus, although these studies suggest that it is likely that AKT plays an important role in lymphatic development, our findings rule out this effector during the earliest steps of trunk lymphatic development.…”

Rapamycin reversal of VEGF-C–driven lymphatic anomalies in the respiratory tract

“…To build on evidence that rapamycin can block sprouting of lymphatic endothelial cells in vitro (20) and in zebrafish (36), we determined whether rapamycin can prevent lymphatic sprouting in CCSP/VEGF-C mice on doxycycline for 2 days (P21-P23). Lymphatic sprouts were abundant at 2 days but were reduced by 72% when doxycycline was given together with rapamycin ( Figure 6, A and B).…”

“…Our rationale for comparing the efficacy of six therapeutic strategies for reversing established lymphangiectasia was based on published reports of their use in prevention or treatment of lymphatic malformations, hemangiomas, and related conditions (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Inhibition of VEGFR-2 and VEGFR-3 signaling by function-blocking antibodies prevents lymphatic growth in inflammation and in CCSP/VEGF-C mice (6,13).…”

“…Inhibition of VEGFR-2 and VEGFR-3 by function-blocking antibodies (6,13) was compared with inhibition of Notch signaling by the γ-secretase inhibitor DAPT (14), suppression of β-adrenergic receptor signaling by propranolol (15,16), inhibition of autophagy by chloroquine (17,18), suppression of inflammation by dexamethasone (13,19), or inhibition of mTOR by rapamycin (18,20). Surprisingly, only rapamycin led to significant reversal of lymphangiectasia in this model, and the efficacy of rapamycin was not improved by coadministration of anti-VEGFR-2/VEGFR-3 antibodies, propranolol, or chloroquine.…”

Natural History of Lymphatic Malformation Progression in Children: A Retrospective Study

“…Recent literature has demonstrated a significant percentage of LMs have documented somatic PIK3CA mutations [4], resulting in uncontrolled local proliferation of dysfunctional lymphatic tissue [5,6].…”