부산대학교 도서관

Byline: , , Man Tong, Jin-Na Chen, Stella Chai, Kin-Tak Chan, Simon Law, Nikki P Lee, Mei Yuk Choi, Bin Li, Annie L Cheung, Sai Wah Tsao, Yan-Ru Qin, Xin-Yuan Guan, Kwok Wah Chan, Stephanie Ma Keywords: NRP2; ETV4; ESCC; RNA-Seq; metastasis Abstract Oesophageal squamous cell carcinoma (ESCC) is the most common histological subtype of oesophageal cancer. The disease is particularly prevalent in southern China. The incidence of the disease is on the rise and its overall survival rate remains dismal. Identification and characterization of better molecular markers for early detection and therapeutic targeting are urgently needed. Here, we report levels of transmembrane and soluble neuropilin-2 (NRP2) to be significantly up-regulated in ESCC, and to correlate positively with advanced tumour stage, lymph node metastasis, less favourable R category and worse overall patient survival. NRP2 up-regulation in ESCC was in part a result of gene amplification at chromosome 2q. NRP2 overexpression promoted clonogenicity, angiogenesis and metastasis in ESCC in vitro, while NRP2 silencing by lentiviral knockdown or neutralizing antibody resulted in a contrary effect. This observation was extended in vivo in animal models of subcutaneous tumourigenicity and tail vein metastasis. Mechanistically, overexpression of NRP2 induced expression of ERK MAP kinase and the transcription factor ETV4, leading to enhanced MMP-2 and MMP-9 activity and, as a consequence, suppression of E-cadherin. In summary, NRP2 promotes tumourigenesis and metastasis in ESCC through deregulation of ERK-MAPK-ETV4-MMP-E-cadherin signalling. NRP2 represents a potential diagnostic or prognostic biomarker and therapeutic target for ESCC. Copyright [c] 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. CAPTION(S): Table S1. Clinico-pathological correlation of transmembrane NRP2 expression in 208 ESCC tissue samples Table S2. Clinico-pathological correlation of soluble NRP2 expression in 105 ESCC serum samples Table S3. Qiagen SABiosciences array profiling (cell motility and metastasis) of KYSE510 cells with or without NRP2 stably repressed (NTC versus NRP2 KD) (A) Expression tiling of the NRP2 (NM_003872) gene in three pairs of non-tumour (N) and ESCC (T) clinical samples examined by RNA-Seq (patients 16, 18 and 19). (B) RNA-Seq RPKM values of NRP1 (NM_003873) in three pairs of non-tumour (N) and ESCC (T) clinical samples (patients 16, 18 and 19). (C) Validation of NRP1 expression by RT-qPCR in the original three ESCC (T) and their adjacent non-tumour (N) samples; error bars represent standard error (SE) of the mean. (D) Relative NRP1 expression in matched non-tumour (N) and primary ESCC obtained from three independent sample cohorts, as detected by RT-qPCR, Guangzhou (n=38; p=0.032), Hong Kong (n=28; p NRP1 expression by RT-qPCR in ESCC cell lines with NRP2 overexpressed [KYSE150 and EC109; empty vector (EV) or overexpressing clones 1 and 2 (OE 1 and OE 2)] or repressed [KYSE510 and KYSE410; non-target control (NTC) or knockdown (KD)] Quantification of capillary tubes formed by HUVECs following treatment with plain medium supplemented with PBS control, VEGF165, goat IgG control (IgG) or anti-NRP2 neutralizing antibody ([alpha]NRP2); *p (A) Molecular size (kDa) of transmembrane and soluble NRP2, harvested from KYSE150 cells transfected with empty vector (EV) or NRP2 overexpressing clones 1 and 2 (OE 1 and OE 2) cell lysate and conditioned medium, respectively. (B) (Top left) Confirmation of enhanced soluble NRP2 levels in KYSE150 and EC109 NRP2 overexpressing cells by western blot; (bottom) proteomic and genomic endogenous NRP2 expression levels following treatment of ESCC parental cells with conditioned medium collected from KYSE150 and EC109, with or without NRP2 stably overexpressed; *p