Altered miRNA Expression Drives Proliferation of Lymphatic Malformation by Activating Pro-Growth Signaling Cascades

PROJECT SUMMARY/ABSTRACT Lymphatic malformations (LMs) are complex congenital lesions composed of dilated, abnormal lymphatic channels that can result in life-threatening morbidity due to their propensity to enlarge, encroach on nearby anatomical structures, become infected, and cause significant pain and disfigurement. Although somatic, gain- of-function mutations in the PIK3CA gene have been identified in LM endothelial cells (LM-ECs) and are thought to drive aberrant lymphangiogenesis through overactivation of the PI3K/Akt pathway, the mechanisms underlying many phenotypic abnormalities apparent in LMs, such as abnormal vessel formation and permeability, are not fully understood. Considering the significant complications and recurrence rates of traditional treatments for LMs, greater insight into the molecular mechanisms underlying LM pathogenesis is needed to identify novel therapeutic targets and develop improved molecular therapies. Angiopoietin-2 (Ang-2) is a vascular growth factor that plays a critical role in lymphatic development and homeostasis; however, its functions in LMs are unknown. Utilizing a multi-omics approach (miRNA-seq, mRNA-seq, proteomics) to generate a comprehensive network of miRNA-mRNA-protein expression in LM-ECs with gain-of-function PIK3CA mutations compared to normal human dermal lymphatic endothelial cells, we have identified significant downregulation of Ang-2 mRNA and protein in LM-ECs in parallel with significant upregulation of miRNAs in LM-ECs that are predicted post- transcriptional suppressors of Ang-2, yet their function in LM-ECs is unknown. Ang-2 is also downregulated in PIK3CA-mutant blood endothelial cells through Akt-mediated inactivation of its transcription factor, Forkhead box O1. Ang-2 expression can be rescued with PI3K pathway inhibitors; however, this mechanism has not been demonstrated in LMs. Considering the critical role of Ang-2 in lymphatic endothelial cell function, we hypothesize that alternations in Ang-2 expression in LM-ECs drive their abnormal lymphangiogenic phenotype and may be a viable therapeutic target. This hypothesis will be tested with the following specific aims: (1) define the regulatory mechanisms driving differential expression of Ang-2 in LM-ECs and (2) define the impact of aberrant Ang-2 expression on LM-EC proliferation, migration, tube formation, and permeability. This proposal will be the first investigation into the regulation and function of Ang-2 in LMs, potentially uncovering novel mechanisms underlying the pathogenesis of LMs and lymphatic and vascular endothelial cell dysfunction which may improve clinical practice and thus has significant relevance to the field of vascular biology. The overarching goal of this proposal is to identify suitable targets for the development of deliverable, molecular therapeutics. The ACRI Vascular Anomalies Laboratory provides an exceptional training environment, and we have assembled a mentoring team of leaders in vascular anomalies, systems biology, and translational medicine that will facilitate this unique and rigorous training, with the principal goal of preparing for a successful career as an independent physician scientist.