NETO2 PROMOTES ESOPHAGEAL CANCER PROGRESSION BY INDUCING PROLIFERATION AND METASTASIS VIA PI3K/AKT AND ERK PATHWAYS
Jia-Cheng Xu*1, Tao Chen2, Yi-Qun Zhang1, Ping-Hong Zhou1
1Zhongshan Hospital, Shanghai, China; 2Shanghai East Hospital affiliated to Tongji University, Shanghai, China
Esophageal squamous cell carcinoma (ESCC) causes aggressive and lethal malignancies with extremely poor prognoses, and accounts for about 90% of cases of esophageal cancer. Neuropilin and tolloid-like 2 (NETO2) encodes transmembrane proteins that belong to the CUB domain and LDLa-containing unique subfamily, which was originally thought to be a neuron-specific protein. Increasing evidence indicates NETO2 expression is also detectable in several types of cancers. In this study, we performed gene expression profiling and the most significantly enriched pathway was the pathways of cancer. Nevertheless, little information is reported about the phenotypic expression and its clinical significance in ESCC progression. Here, our research revealed that NETO2 expression was markedly elevated in ESCC as compared with adjacent control tissues. High levels of NETO2 were also a marker of poor survival in ESCC patients.(FigA) Gain-of-function and loss-of-function analyses showed that NETO2 stimulated ESCC cell proliferation while suppressing apoptosis in vitro and enhanced tumor growth in vivo. We then measured cell apoptosis-related signaling molecules and the data demonstrated that NETO2 antagonized apoptosis by regulating the intrinsic apoptotic pathway.(FigB.C) Moreover, knockdown of NETO2 significantly inhibited migration and invasion in combination with regulation of epithelial-mesenchymal transition (EMT) related markers such as E-cadherin, vimetin, N-cadherin, snail, slug and MMP2.(FigD) Mechanistically, overexpression of NETO2 increased the phosphorylation of ERK, PI3k/AKT and expression of Nuclear factor erythroid-2-related factor 2(Nrf2), whereas silencing NETO2 decreased these targets. What’s more, treatment with the inhibitor of PI3K and ERK abolished the upregulated P-AKT and P-ERK level induced by NETO2 and partially abrogated NETO2 overexpression-induced proliferation and metastasis. Therefore, NETO2 promotes ESCC cell growth and metastasis, at least partially, through activation of MAPK/ERK and PI3K/AKT signaling pathways(FigE). Nrf2, a member of the NF-E2 family, is a cap ‘n’ collar basic leucine-zipper transcription factor. Our results indicated that Nrf2 and P-Nrf2 was dramatically downregulated by NETO2 knock down. ESCC cell proliferation was also prominently inhibited by Nrf2-siRNA treatment. Interestingly, in the hypoxic microenvironment, knockdown of Nrf2 led to obvious effects on cell migration and invasion compared with basal conditions. (FigF) A recent study have suggested that ERK and AKT activated Nrf2 expression. Therefore, our data suggest that Nrf2 was a critical downstream event responsible for triggering the PI3K/AKT and ERK signaling pathways and plays a crucial role in NETO2-mediated tumorigenesis. Taken together, NETO2 acts as an oncogene and might serve as a novel therapeutic target or prognostic biomarker in ESCC patients.
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