NETO2 promotes esophageal cancer progression by inducing proliferation and metastasis via PI3K/AKT and ERK pathway

Abstract

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) protein coding genes have been associated with various human cancers. Nevertheless, little information is reported about the phenotypic expression and its clinical significance in ESCC progression. Here, our study found that NETO2 expression in ESCC patients was associated with tumor clinical stage and lymph node metastasis status. 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. Moreover, knockdown of NETO2 significantly inhibited migration and invasion in combination with regulation of epithelial-mesenchymal transition (EMT) related markers. Mechanistically, overexpression of NETO2 increased the phosphorylation of ERK, PI3k/AKT, and Nuclear factor erythroid-2-related factor 2(Nrf2), whereas silencing NETO2 decreased the phosphorylation of these targets. 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.

Keywords: Esophageal squamous cell carcinoma; Metastasis; NETO2; PI3K/AKT and ERK; Proliferation.

Figure 1

NETO2 is highly expressed and associated with progression and poor prognosis of ESCC patients. A, NETO2 mRNA levels were significantly higher in ESCC compared with paired non-tumor tissues. B, Protein expression levels of NETO2 in eight pairs of ESCC tissues. C, mRNA level of NETO2 in all tumor samples and paired normal tissues (TCGA). D, mRNA level of NETO2 in ESCC and normal tissues (TCGA). E, Representative images of weak, moderate, strong and nagative NETO2 IHC staining. F, Scatterplots of the average staining scores of NETO2 expression in ESCC and normal tissues. G, Kaplan-Meier overall survival curve of ESCC patients correlated with NETO2 expression. *P < 0.05, **P < 0.01.

Figure 2

NETO2 promotes ESCC cell proliferation and viability. A and B, NETO2 expression in 293T and ESCC cell lines. C, NETO2 expression in ECA109 and KYSE150 cell lines transfected with NC and siNETO. D, Knockdown on NETO2 protein levels in ESCC cells infected with shNETO2 lentivirus. E, Effects of NETO2 overexpression in ECA109 and KYSE150 cells. F and G, CCK-8 assays and plate colony formation assays were conducted to determine the function of NETO2 on proliferation capabilities. All data are presented as mean ± SD and all the experiments were repeated 3 times. Significant differences are indicated by *P < 0.05, **P < 0.01.

Figure 3

NETO2 silencing retards ECA109 cells tumorigenesis in vivo. A-D, Images of 5 representative mice from Lv-shNC/Lv-shNETO2 group are shown. Tumor sizes and tumor weights were measured. E and F, The expression of c Ki67, p-AKT and p-ERK were evaluated by IHC analysis in xenograft tumor; scale bar = 100 µm. All data are presented as mean ± SD and all the experiments were repeated 3 times. Significant differences are indicated by *P < 0.05, **P < 0.01.

Figure 4

Apoptosis of ESCC cells after NETO2-knockdown. A, Apoptosis was assessed by flow cytometry with annexin V-FITC/propidium iodide (PI) staining. The x-axis represents annexinV-FITC, and the y-axis represents propidium iodide (PI) staining. B, There was a significant difference of apoptosis rate between Lv-shNC and Lv-shNETO2 groups. C and D, Nuclear morphological changes associated with apoptosis were evaluated by Hoechst 33342 staining. E. NETO2-knockdown increased C-PARP, C-caspase3 but suppressed Bcl-2 expression and the indicated factors was examined by western blot. GAPDH was used as the loading control. All data are presented as mean ± SD and all the experiments were repeated 3 times. Significant differences are indicated by *P < 0.05, **P < 0.01.

Figure 5

NETO2 promotes ESCC cell migration and invasion by inducing EMT. A and B, Wound healing assay was used to examine cell migration in ECA109 and KYSE150 cells transfected with Lv-shNETO2 or the NETO2 overexpression plasmid. C, Transwell assays were used to analyze ESCC cells motility. D, Morphologies of ECA109 and KYSE150 cells were examined under a microscope. E and F, Western blot analysis of E-cadherin, N-cadherin and snail slug and MMP2 levels in ESCC cells transfected with siNETO2 or the NETO2 overexpression plasmid. G, Effects of NETO2 knockdown and overexpression on ESCC cells motility. All data are presented as mean ± SD and all the experiments were repeated 3 times. Significant differences are indicated by *P < 0.05, **P < 0.01.

Figure 6

Inhibition of PI3K and ERK antagonizes NETO2-mediated proliferation, migration and invasion of ESCC cells. Nrf2 is associated with NETO2 to influence ESCC cell tumorigenesis ability. A, Western blot analysis of PI3K, p-AKT and p-ERK expression in indicated cells. GAPDH was used as the loading control. B, CCK8 assay for NETO2 overexpressing and vector cells with or without inhibitor treatment. C, Western blot analysis of EMT-related proteins for NETO2- overexpressed cells with or without inhibitor treatment. D and E, Transwell assays were used to analyze ESCC cell migration and invasion ability. F, Western blot analysis of expression of Nrf2 and p-Nrf2 with NETO2 knockdown and overexpression in ESCC cells. G, Cell proliferation assay were conducted to analyze the effects of Nrf2 on ESCC cell proliferation ability. H, In the hypoxic microenvironment, knockdown of Nrf2 led to an obvious effect on cell migration and invasion compared with basal conditions. I, E-cadherin, N-cadherin, MMP2 and snail expression levels were analyzed by Western blot analysis in cells transfected with siNrf2-mix. GAPDH was used as a loading control. J, Number of migrated cells according to Transwell data. All data are presented as mean ± SD and all the experiments were repeated 3 times. Significant differences are indicated by *P < 0.05, **P < 0.01.

Figure 7

Schematic diagram summarizing the NETO2-induced ERK/PI3K pathway.