Identification, functional characterization and clinical relevance of neuropilin-2 (NRP2) in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is the major histological subtype of esophagus cancer. The disease is particularly prevalent in Asia, and ranks the 8th most common cause of cancer mortality in Hong Kong. The overall prognosis for ESCC is poor in part due to the high incidences of metastasis. Therefore, the identification of valid targets and the development of new therapies are necessary for more effective clinical management of this highly aggressive disease. Here, we performed an integrative transcriptome sequencing (RNA-Seq) analysis on three pairs of patient-derived non-tumor and ESCC clinical samples and successfully identified a number of significantly differentially expressed genes, including neuropilin-2 (NRP2) overexpression. Although NRP2 has been reported to be overexpressed in a number of cancer types, its clinical significance and functional role in ESCC has not been explored to date. Subsequent validation in a larger cohort of clinical samples collected from different cities in China known to be troubled with high prevalence of ESCC (Guangzhou p < 0.001, Hong Kong p = 0.025 and Henan p = 0.002) further confirmed the frequent upregulation of NRP2. By immunohistochemistry analysis, we also likewise found NRP2 to be frequently overexpressed in ESCC at the protein level and that its overexpression was correlated with advanced tumor stage (p = 0.002), lymph node metastasis (p = 0.020) and, in the age group ≥ 59, poor survival (p = 0.049). Further, secretory NRP2 was also found to be present in ESCC patients' sera, but not in the sera collected from healthy normal individuals (p < 0.001). Elevated serum NRP2 was associated with less favorable R category (p = 0.035) and worst overall survival (p = 0.035). The functional role of NRP2 was then subsequently evaluated by cell-based in vitro and in vivo assays using lentiviral-based NRP2 modulation approach. Foci formation, chemotaxis migration, Matrigel invasion and capillary tube formation assays were performed and results suggested that NRP2 can promote ESCC proliferation, migration, invasion and tube formation in endothelial cells in vitro. These observations were further validated in vivo with an experimental tail vein injection model where KYSE150 ESCC cells with NRP2 stably expressed displayed an enhanced ability to promote lung metastasis than as compared to empty vector control cells. Treatment of NRP2 expressing ESCC cells with an anti-human NRP2 antibody resulted in attenuated ability of the cells to migrate and induce angiogenesis in vitro. To delineate the molecular mechanism underlying NRP2-driven ESCC, real-time quantitative PCR array profiling was performed. Two metastasis-related arrays (cell motility and tumor metastasis) were utilized and the transcript factor Ets variant 4 (ETV4) was found to be significantly downregulated in ESCC cells with NRP2 repressed, with the reverse observed in NRP2 overexpressing cells. E-cadherin, a hallmark of epithelial-mesenchymal transition, was also identified to be altered following NRP2 deregulation in ESCC. Collectively, our findings provide evidence that NRP2 functions as an important oncogene in ESCC development and metastasis through modulating ETV4 and E-cadherin expression. Clinically, NRP2 represents a potential diagnostic and prognostic biomarker and therapeutic target in ESCC.