Exosomal epidermal growth factor receptor is involved in HPV-16 E7-induced epithelial-mesenchymal transition of non-small cell lung cancer cells: A driver of signaling in vivo

Abstract

Our previous studies have demonstrated that human papillomavirus (HPV)-16 E7 oncoprotein promoted epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells. Moreover, recent studies have found that exosomes can mediate EMT of NSCLC cells and epidermal growth factor receptor (EGFR) is related to the progression of NSCLC. Here, we further investigated the role of exosomal EGFR in HPV-16 E7-induced EMT of NSCLC cells. Our results showed that the exosomes derived from the stable HPV-16 E7-overexpressing A549 and NCI-H460 NSCLC cells (E7 Exo) significantly increased migration, invasion, and proliferation abilities of NSCLC cells as compared with the exosomes derived from empty vector-infected NSCLC cells (ev Exo). Moreover, both in vitro and in vivo results demonstrated that E7 Exo dramatically enhanced EMT of NSCLC cells and promoted the growth of subcutaneous NSCLC xenografts. Additionally, HPV-16 E7 enhanced the expression of EGFR and p-EGFR in both NSCLC cells and exosomes. Furthermore, the inhibition of EGFR activation or exosome secretion suppressed E7 Exo-induced migration, invasion, and EMT of NSCLC. Moreover, 12 kinds of differentially expressed miRNAs between E7 Exo and ev Exo (fold change≥2, P ≤ .05) were screened out, of which 7 miRNAs were up-regulated while 5 miRNAs were down-regulated in A549 E7 Exo. Taken together, our findings suggest that exosomal EGFR is involved in HPV-16 E7-induced EMT of NSCLC cells, which may play a key role in the progression of HPV-related NSCLC.

Keywords: EGFR; EMT; Exosomes; HPV-16 E7; NSCLC.

Figure 1.

Identification and uptake of exosomes. (a) The images of transmission electron microscope of exosomes derived from the stable HPV-16 E7-overexpressing A549 and NCI-H460 cells (E7 Exo) and empty vector A549 and NCI-H460 cells (ev Exo), scale bar = 200 nm. (b) The expression of exosomal markers including CD9, CD81, and TSG101 and intracellular protein Grp94 (absence in exosomes) was analyzed by Western blot. (c) The fluorescent results of exosome uptake analysis, scale bar = 100 μm.

Figure 2.

Exosomes derived from the stable HPV-16 E7-overexpressing A549 and NCI-H460 cells enhanced migration, invasion, proliferation, and EMT of NSCLC cells. (a) Wound healing results for A549 cells, scale bar = 500 μm. (b) Transwell results for the migration and invasion abilities of NCI-H460 cells, scale bar = 100 μm. (c) Colony formation results for the proliferation abilities of A549 cells. (d) Western blot analysis of expression of EMT related markers in A549 cells. E7 Exo: the cells treated with the exosomes derived from the stable HPV-16 E7-overexpressing cells; ev Exo: the cells treated with the exosomes derived from the empty vector-infected cells; PBS: the cells treated with PBS buffer. Left: one representative result of three independent experiments; right: the results for three independent experiments. All data are expressed as mean ± SD of three independent experiments. **P < .01.

Figure 3.

Exosomes derived from stable HPV-16 E7-overexpressing NCI-H460 cells promoted tumor growth and EMT of NSCLC in vivo. (a) The representative results of subcutaneous tumors. (b) Tumor volume (mm³). (c) Tumor weight (g). (d) Nude mouse weight (g). (e) The representative results of HE and immunohistochemical staining, scale bar = 100 μm.

Figure 4.

EGFR induced by HPV-16 E7 can be delivered to NSCLC cells through exosomes. (a) The expression of EGFR and p-EGFR in HPV-16 E7-overexpressing cells (E7 cells) and empty vector cells (ev cells). (b) The expression of EGFR in the exosomes derived from the stable HPV-16 E7-overexpressing cells (E7 Exo) and the exosomes derived from empty vector-infected cells (ev Exo). (c,d) The analysis of the protein levels of EGFR and p-EGFR in A549 (c) and NCI-H460 cells (d) co-cultured with ev cells and E7 cells treated with DMSO, PD168393, and GW4869, respectively. Left: one representative result of three independent experiments; right: the results of density for three independent experiments. All data are expressed as mean ± SD of three independent experiments. *P < .05, **P < .01.

Figure 5.

Exosomal EGFR was involved in migration, invasion, and EMT of NSCLC cells induced by HPV-16 E7. (a) Transwell assays were performed to analyze the migration and invasion abilities of A549 and NCI-H460 cells after co-cultured with HPV-16 E7-overexpressing cells (E7 cells) and empty vector cells (ev cells) treated with DMSO, PD168393, and GW4869, respectively; Up: one representative result of three independent experiments, scale bar = 100 μm; down: the number of migratory cells (left) and invasive cells (right) for three independent experiments. (b-d) EMT-related mRNA (b) and protein (c,d) levels were determined by RT-qPCR and Western blot after co-cultured with ev cells and E7 cells treated with DMSO, PD168393, and GW4869, respectively. The left of c (A549) and d (NCI-H460): one representative result of three independent experiments; the right of c (A549) and d (NCI-H460): the results of density for three independent experiments. All data are expressed as mean ± SD of three independent experiments. *P < .05, **P < .01.

Figure 6.

Screening of the differentially expressed miRNAs. The analysis of difference of miRNA expression between exosomes derived from the stable HPV-16 E7-overexpressing A549 cells (E7 Exo) and empty vector-infected A549 cells (ev Exo). (a) Scatter plots. (b) Volcano plots. (c) Clustering was performed on differentially expressed miRNAs between E7 Exo and ev Exo, fold change≥2 and P ≤ .05. (d) Pathway analysis of differentially expressed miRNAs in E7 Exo and ev Exo. All data are expressed as mean ± SD of three independent experiments.