ABSTRACT
BACKGROUND: Patients with lymph node (LN) metastatic bladder cancer (BCa) present with extremely poor prognosis. BCa-derived exosomes function as crucial bioactive cargo carriers to mediate the signal transduction in tumor microenvironment triggering tumor metastasis. However, the mechanisms underlying exosome-mediated LN metastasis in BCa are unclear. METHODS: We conducted the high-throughput sequencing to explore the expression profile of long noncoding RNA (lncRNA) in urinary exosomes (urinary-EXO) from patients with BCa and further evaluated the clinical relevance of exosomal lncRNA BCYRN1 in a larger 210-case cohort. The functional role of exosomal BCYRN1 was evaluated through the migration and tube formation assays in vitro and the footpad-popliteal LN metastasis model in vivo. RNA pull-down assays, luciferase assays, and actinomycin assays were conducted to detect the regulatory mechanism of exosomal BCYRN1. RESULTS: LncRNA BCYRN1 was substantially upregulated in urinary-EXO from patients with BCa, and associated with the LN metastasis of BCa. We demonstrated that exosomal BCYRN1 markedly promoted tube formation and migration of human lymphatic endothelial cells (HLECs) in vitro and lymphangiogenesis and LN metastasis of BCa in vivo. Mechanistically, BCYRN1 epigenetically upregulated WNT5A expression by inducing hnRNPA1-associated H3K4 trimethylation in WNT5A promoter, which activated Wnt/ß-catenin signaling to facilitate the secretion of VEGF-C in BCa. Moreover, exosomal BCYRN1 was transmitted to HLECs to stabilize the VEGFR3 mRNA and thus formed an hnRNPA1/WNT5A/VEGFR3 feedforward regulatory loop, ultimately promoting the lymphatic metastasis of BCa. Importantly, blocking VEGFR3 with specific inhibitor, SAR131675 significantly impaired exosomal BCYRN1-induced the LN metastasis in vivo. Clinically, exosomal BCYRN1 was positively associated with the shorter survival of BCa patients and identified as a poor prognostic factor of patients. CONCLUSION: Our results uncover a novel mechanism by which exosomal BCYRN1 synergistically enhances VEGF-C/VEGFR3 signaling-induced lymphatic metastasis of BCa, indicating that BCYRN1 may serve as an encouraging therapeutic target for patients with BCa.
Subject(s)
Exosomes/metabolism , RNA, Long Noncoding/metabolism , Urinary Bladder Neoplasms/pathology , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolism , Wnt-5a Protein/metabolism , Animals , Cell Line, Tumor , Endothelial Cells/cytology , Endothelial Cells/metabolism , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Histones/metabolism , Humans , Lymphangiogenesis , Lymphatic Metastasis , Mice , Mice, Nude , Promoter Regions, Genetic , Protein Kinase Inhibitors/pharmacology , RNA, Long Noncoding/genetics , Signal Transduction/drug effects , Survival Rate , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/mortality , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-3/genetics , Wnt-5a Protein/geneticsABSTRACT
While both blood and lymphatic vessels transport fluids and thus share many similarities, they also show functional and structural differences, which can be used to differentiate them. Specific visualization of lymphatic vessels has historically been and still is a pivot point in lymphatic research. Many of the proteins that are investigated by molecular biologists in lymphatic research have been defined as marker molecules, i.e. to visualize and distinguish lymphatic endothelial cells (LECs) from other cell types, most notably from blood vascular endothelial cells (BECs) and cells of the hematopoietic lineage. Among the factors that drive the developmental differentiation of lymphatic structures from venous endothelium, Prospero homeobox protein 1 (PROX1) is the master transcriptional regulator. PROX1 maintains lymphatic identity also in the adult organism and thus is a universal LEC marker. Vascular endothelial growth factor receptor-3 (VEGFR-3) is the major tyrosine kinase receptor that drives LEC proliferation and migration. The major activator for VEGFR-3 is vascular endothelial growth factor-C (VEGF-C). However, before VEGF-C can signal, it needs to be proteolytically activated by an extracellular protein complex comprised of Collagen and calcium binding EGF domains 1 (CCBE1) protein and the protease A disintegrin and metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). This minireview attempts to give an overview of these and a few other central proteins that scientific inquiry has linked specifically to the lymphatic vasculature. It is limited in scope to a brief description of their main functions, properties and developmental roles.
