Elevated EGFL6 modulates cell metastasis and growth via AKT pathway in nasopharyngeal carcinoma

Abstract

Epidermal growth factor-like domain multiple 6 (EGFL6) is a secreted protein, regulates maintenance and metastasis of cancer cells. Nevertheless, how EGFL6 participates in the progression and tumorigenesis of nasopharyngeal carcinoma (NPC) remains unclear. In our study, EGFL6 was detected highly expressed in 20 NPC tissues compared with normal tissues by IHC assay. Then, the level of EGFL6 in NPC serum and NPC cells was explored through enzyme-linked immunosorbent assay and western blot, the results consistent with IHC. More interestingly, EGFL6 accelerated the migration and growth of NPC in vitro assays. Considering the mechanism of migration, NPC cells were cultured with AKT activator, revealing EGFL6 facilitated the progression of NPC via AKT. Moreover, the same effect of EGFL6 in promoting NPC growth was proved in nude mice. Furthermore, heat-shock zebrafish model was established with EGFL6 overexpression. Then, CNE2 cells were injected into the model and cells mass was observed, showing that EGFL6 enhanced the migration and metastasis of NPC. Currently, as the prognosis of NPC is severely affected by distant metastasis, it might be a new therapeutic target toward EGFL6. Taken together, our results suggested that EGFL6 acts as a potential positive regulator in the migration and proliferation of NPC.

Keywords: AKT; EGFL6; growth; metastasis; nasopharyngeal carcinoma.

Figure 1

The expression of EGFL6 in nasopharyngeal carcinoma (NPC). A, High expression of EGFL6 was observed in NPC tissues compared with non‐cancerous nasopharyngeal (N) tissues by immunohistochemistry analysis. B, High level of EGFL6 in NPC serum compared with N serum. **P < 0.01. C, The standard curve analysis of EGFL6 in serum

Figure 2

EGFL6 promoted the migration and proliferation of nasopharyngeal carcinoma (NPC) cells. A, Western blot analysis of the expression of EGFL6 in NPC cell lines and normal nasopharyngeal epithelial (NP69) cells. Actin was used as a loading control. Data are the means ± SD. **P < 0.01, ***P < 0.001. B, CNE2 and 5‐8F cells were transfected with shEGFL6 (1, 2, 3) and shNC. 48 hours later, total RNA was extracted from those cells and the levels of EGFL6 were measured by qRT‐PCR. *P < 0.05, **P < 0.01. C, The fluorescent transfection efficiency of NPC cells was captured by inverted microscope. D, The effect of EGFL6 on migration of NPC cells was observed by transwell. **P < 0.01. E, Flow cytometry was performed to quantify the progress of cell cycle, *P < 0.05, **P < 0.01, ***P < 0.001. F, Immunofluorescence analysis of KI67 expression in NPC cells of shEGFL6 and shNC group. The nucleus was counterstained with Hoechst in blue. KI67 staining was in red

Figure 3

EGFL6 mediated the axis on migration of AKT signaling. A, P‐AKT and AKT in NPC cells transfected with shEGFL6 and shNC was measured by Western blot analysis. β‐actin was used as a loading control. *P < 0.05, **P < 0.01. B, After treated with SC79 or DMSO, the level of p‐AKT and AKT in shEGFL6 NPC cells was detected by Western blot analysis. *P < 0.05. C, ShEGFL6 NPC cells treated with SC79 or DMSO were subjected to transwell assay. **P < 0.01, ***P < 0.001

Figure 4

The silence of EGFL6 reduced the growth of NPC in nude mice. A, Representative examples of nude mice subcutaneous injection of CNE2 cells. B, Tumor volumes were evaluated every two days. C, The final tumor volume of sacrificed mice at day 12 was obtained. Mean ± SEM, *P < 0.05

Figure 5

EGFL6 gain of function increased the migration of NPC cells in zebrafish. We selected embryos at 24 h for green hearts, and observed the red fluorescence in embryos after heat‐shocked for 1 h. A, hsp70l:EGFL6‐mcherry embryos at 48 hpf (hours post fertilization), after heat‐shocked for 1 h at 24 hpf. B, hsp70l:EGFL6‐mcherry :: fli1ep: CAAX‐EGFP transgenic line embryos at 48 hours, after heat‐shocked for 1 h at 24 hpf. C and D, CNE2 cells labeled with dil were injected into hsp70l:EGFL6‐mcherry :: fli1ep: CAAX‐EGFP transgenic line embryos at 48 hpf. Then, the migration of CNE2 cells was detected at 2 d post injection. Dpf (days post fertilization). Arrows indicate tumor foci. **P < 0.01