Epidermal growth factor-like domain-containing protein 7 (EGFL7) enhances EGF receptor-AKT signaling, epithelial-mesenchymal transition, and metastasis of gastric cancer cells

Abstract

Epidermal growth factor-like domain-containing protein 7 (EGFL7) is upregulated in human epithelial tumors and so is a potential biomarker for malignancy. Indeed, previous studies have shown that high EGFL7 expression promotes infiltration and metastasis of gastric carcinoma. The epithelial-mesenchymal transition (EMT) initiates the metastatic cascade and endows cancer cells with invasive and migratory capacity; however, it is not known if EGFL7 promotes metastasis by triggering EMT. We found that EGFL7 was overexpressed in multiple human gastric cancer (GC) cell lines and that overexpression promoted cell invasion and migration as revealed by scratch wound and transwell migration assays. Conversely, shRNA-mediated EGFL7 knockdown reduced invasion and migration. Furthermore, EGFL7-overexpressing cells grew into larger tumors and were more likely to metastasize to the liver compared to underexpressing CG cells following subcutaneous injection in mice. EGFL7 overexpression protected GC cell lines against anoikis, providing a plausible mechanism for this enhanced metastatic capacity. In excised human gastric tumors, expression of EGFL7 was positively correlated with expression levels of the mesenchymal marker vimentin and the EMT-associated transcription repressor Snail, and negatively correlated with expression of the epithelial cell marker E-cadherin. In GC cell lines, EGFL7 knockdown reversed morphological signs of EMT and decreased both vimentin and Snail expression. In addition, EGFL7 overexpression promoted EGF receptor (EGFR) and protein kinase B (AKT) phospho-activation, effects markedly suppressed by the EGFR tyrosine kinase inhibitor AG1478. Moreover, AG1478 also reduced the elevated invasive and migratory capacity of GC cell lines overexpressing EGFL7. Collectively, these results strongly suggest that EGFL7 promotes metastasis by activating EMT through an EGFR-AKT-Snail signaling pathway. Disruption of EGFL7-EGFR-AKT-Snail signaling may a promising therapeutic strategy for gastric cancer.

Figure 1. EGFL7 expression in native gastric cancer (GC) cell lines, normal gastric cells, and GC cell lines stably expressing an EGFL7 expression vector or targeted shRNA.

(A) Expression levels of EGFL7 protein in the GC cell lines SGC7901, BGC823, MKN45, and MKN28, and the normal gastric cell line GES-1 were evaluated by Western blot. The GC cell lines showed higher EGFL7 protein expression levels than GES-1 cells, with BGC823 cells having the highest EGFL7 protein expression levels. (B) qRT-PCR revealed that BGC823 had the highest EGFL7 mRNA expression. Western blot and PCR experiments were performed in triplicate, and GAPDH was used as the internal control. (C) qRT-PCR showed that the shRNA1 sequence resulted in 75% inhibition of EGFL7 mRNA expression, whereas the shRNA2 sequence resulted in only 30% inhibition. (D) BGC823 cell lines stably transfected with pGPU6/GFP/Neo-EGFL7-shRNA1 or pGPU6/GFP/Neo-nonspecific-shRNA are designated BGC2-13 and BGC-NC, respectively. MKN28 cell lines transfected with pEX-2-EGFL7 or pEX-2-nonspecific are designated MKN28-EGFL7 and MKN28-NC, respectively. Expression of EGFL7 protein was markedly lower in BGC2-13 cells compared to BGC823 and BGC-NC cells, and markedly higher in MKN28-EGFL7 cells compared to MKN28 and MKN28-NC cells. (E) EGFL7 expression levels were also analyzed by qRT-PCR, and results confirmed Western blot data. GAPDH served as the internal control for both qRT-PCR and Western blot. Error bars represent the SD of triplicate experiments (*P<0.05).

Figure 2. EGFL7 expression regulates GC cell migration and invasiveness.

(A) Scratch wound healing was used to determine migration behavior in cell lines underexpressing or overexpressing EGFL7. A wound was generated and imaged at 0 and 48 h. Wound closure was significantly slower in (underexpressing) BGC2-13 cultures compared to BGC823 and BGC-NC cultures (32% vs. 100% and 98%, *P<0.05). (B) Wound closure was significantly faster in (overexpressing) MKN28-EGFL7 cultures compared to MKN28 and MKN28-NC cultures (99% vs. 49% and 50%, *P<0.05). (C) Invasive potential was assessed by the Matrigel invasion assay. Significantly fewer BGC2-13 cells passed through the Matrigel compared to BGC-NC and BGC823 cells (25±3.1 vs. 76±2.9 and 79±5.7, *P<0.05). For the transwell migration assay, cell lines were seeded in the upper chamber of the transwell. Significantly fewer BGC2-13 cells traveled through the transwell filter compared to BGC823 and BGC-NC cells (19±1.1 vs. 53±2.9 and 54±2.6, **P<0.05). (D) Significantly more MKN28-EGFL7 cells passed through the Matrigel to the lower side of the filter compared to MKN28-NC and MKN28 cells (79±6.19 vs. 40±2.00 and 41±4.22, *P<0.05). Similarly, significantly more MKN28-EGFL7 cells that traveled through the transwell filter compared to MKN28 and MKN28-NC cells (67±4.16 vs. 33±5.92 and 35±3.7, **P<0.05).

Figure 3. EGFL7 expression does not alter GC cell proliferation.

(A) Effect of EGFL7 underexpression on cell proliferation as determined by the adherent plate colony formation assay. BGC823, BGC-NC, and BGC2-13 cells were plated at low density and colonies counted after 10 days. There was no significant difference in the number of colonies formed (52±1.1 vs. 58±2.2 and 55±0.8, *P>0.05) (mean ± SD from three independent experiments). (B) Effect of EGFL7 overexpression on cell proliferation. MKN28-EGFL7, MKN28-NC, and MKN28 cells were treated as above. There was no significant difference in the number of colonies formed (42±1.91 vs. 39±1.04 and 45±3.00, *P>0.05) (mean ± SD from three independent experiments). (C) Effect of EGFL7 underexpression on cell proliferation as measured by MTT assay. There was no significant difference in proliferation rate among BGC823, BGC-NC, and BGC2-13 cells. (D) Effect of EGFL7 overexpression on cell proliferation. There were also no significant differences in proliferation rates among the MKN28-EGFL7, MKN28-NC, and MKN28 cells. All data were expressed as mean ± SD and were obtained from three independent experiments.

Figure 4. EGFL7 induces anoikis resistance of GC cells in suspension culture.

(A) Effect of EGFL7 underexpression on anoikis resistance. Representative cytograms from flow cytometry analysis of apoptotic cells revealed by Annexin V-PE/7-AAD staining of BGC823, BGC-NC, and BGC2-13 cells after 24 h in suspension culture. A greater percentage of BGC2-13 cells (22.95±1.72%) were apoptotic compared to BGC823 (11.83% ±0.99%) and BGC-NC cells (9.36% ±1.65%). (B) Effect of EGFL7 overexpression on anoikis resistance. Representative cytograms from flow cytometry analysis of apoptotic cells revealed by Annexin V-PE/7-AAD staining of MKN28-EGFL7, MKN28-NC, and MKN28 cells after 24 h of suspension culture. The number of apoptotic MKN28-EGFL7 cells (5.13% ±0.65%) was lower than the number of apoptotic MKN28 (29.53% ±0.68%) and MKN28-NC cells (35.98% ±1.77%). (C) and (D) Flow cytometry results plotted as the mean ± SD of triplicate experiments. *P<0.05 considered significant. All experiments were performed in triplicate and repeated at least three times.

Figure 5. EGFL7 modulates the growth and metastasis of GC xenograft tumors in nude mice.

(A) Subcutaneous xenograft tumors were significantly smaller in BGC2-13 cell-injected mice compared to BGC823 cell- and BGC-NC cell-injected mice, while MKN28-EGFL7 cell-injected mice exhibited significantly larger tumors than MKN28 cell- and MKN28-NC cell-injected mice. Tumor volume was calculated according to tumor volume (mm³) = 0.5×length×width² (*P<0.05, **P<0.01). (B) Subcutaneous xenograft tumors were analyzed by H&E staining. Liver metastasis was observed in mice injected with high EGFL7-expressing cells (BGC823, BGC-NC, and MKN28-EGFL7). Black arrow, metastatic cancer cells in the liver tissues of nude mice (H&E staining, original magnification×200). (C) Average MVD of tumors was lower in the BGC2-13 group compared to the BGC823 and BGC-NC groups (4±1.2 vs. 15±2 and 13±1, *P<0.05), while the average MVD of tumors was higher in the MKN28-EGFL7 group than the MKN28 and MKN28-NC groups (28.7±6.02 vs. 4.3±1.53 and 5.0±2.0, **P<0.05).

Figure 6. EGFL7 promotes Epithelial−Mesenchymal transition (EMT) of GC cells through the EGFR–AKT signaling pathway.

(A) Immunohistochemistry showing EGFL7, E-cadherin, vimentin, and Snail expression in gastric carcinoma tissues. (B) Cellular morphology of EGFL7-underexpressing cells (BGC2-13, MKN28, and MKN28-NC) was distinct from that of EGFL7-overexpressed cells (BGC823, BGCNC, and MKN28-EGFL7). BGC823, BGCNC, and MKN28-EGFL7 cells exhibited loss of intercellular contacts and typical spindle-shaped mesenchymal cell morphology, whereas BGC2-13, MKN28, and MKN28-NC cells exhibited an epithelial cell-like morphology with small cell size and cobblestone-like shape with tightly arranged intercellular contacts. (C) and (D) Expression levels of EMT-related molecules in cell lines analyzed by qRT-PCR (*P<0.05, **P<0.05, ***P<0.05). (E) Western blot was used to confirm changes in expression of EMT-related molecules. qRT-PCR and Western blot results showed higher E-cadherin mRNA and protein expression levels in BGC2-13 cells and lower mRNA and protein expression levels of the mesenchymal markers vimentin and Snail. GAPDH served as an internal control for qRT-PCR reactions and Western blot. Error bars represent SD of triplicate experiments (*P<0.05, **P<0.05, ***P<0.05 compared to BGC823 and BGC-NC cells). Conversely, qRT-PCR and Western blot results showed lower E-cadherin mRNA and protein expression levels in MKN28-EGFL7 cells and higher mRNA and protein expression levels of mesenchymal markers vimentin and Snail. Error bars represent SD of triplicate experiments (*P<0.05, **P<0.05, ***P<0.05, compared to MKN28 and MKN28-NC cells). (F) Western blots showing both total and phosphorylated levels of EGFR, ERK1/2, and AKT in BGC823, BGC-NC, BGC2-13, MKN28-EGFL7, MKN28-NC, and MKN28 cells. Phosphorylated EGFR and AKT levels (pEGFR and pAKT) were significantly lower in BGC2-13 cells than in BGC823 and BGC-NC cells, while pEGFR and pAKT expression levels were significantly higher in MKN28-EGFL7 cells compared to MKN28 and MKN28-NC cells. Total EGFR and AKT levels did not differ significantly among cell lines. Neither total ERK1/2 nor pERK1/2 differed significantly. Western blots were performed in triplicate.

Figure 7. An EGFR tyrosine kinase inhibitor (Tyrphostin AG1478) blocks the effect of EGFL7 on EGFR phosphorylation, AKT phosphorylation, and cell motility.

(A) Western blots showing that expression of EMT-related proteins changed significantly in BGC823 and MKN28-EGFL7 cells after treatment with the EGFR inhibitor Tyrphostin AG1478 (20 µM) for 1 h compared to vehicle (0.25% DMSO). E-cadherin protein expression levels increased in BGC823 and MKN28-EGFL7 cells after Tyrphostin AG1478 treatment, while expression levels of the mesenchymal marker proteins vimentin and Snail decreased. pEGFR and pAKT expression levels were significantly lower in BGC823 and MKN28-EGFL7 cells treated with Tyrphostin AG1478 than in BGC823 and MKN28-EGFL7 cells treated with 0.25% DMSO. No changes in EGFL7 protein expression levels were observed in BGC823 and MKN28-EGFL7 cells after treatment with Tyrphostin AG1478. (B) Migration of BGC823 and MKN28-EGFL7 cells was inhibited by Tyrphostin AG1478 treatment in the scratch wound assay (79% vs. 49%, *P<0.05; 97% vs. 26%, **P<0.05). (C) Tyrphostin AG1478 also reduced migration in the transwell assay (37±3.9 vs. 18±1.5, *P<0.05; 58±3.7 vs. 15±4.0, **P<0.05; vehicle-treated vs. Tyrphostin AG1478-treated).