Androgen receptor promotes oral squamous cell carcinoma cell migration by increasing EGFR phosphorylation

Abstract

Objectives: This study is aimed to investigate the role of androgen receptor (AR) in regulating oral squamous cell carcinoma (OSCC) cells migration. Materials and methods: Tumors from 23 patients with OSCC and five OSCC cell lines were used for analyzing AR expression. The effects of AR agonist and antagonist were used to examine the role of AR in regulating the migration of OSCC cells. Results: Ten of 23 tumors from patients with OSCC were AR positive. There was no significant difference in total EGFR (tEGFR) expression between AR-positive tumors and AR-negative tumors. However, the expression of phosphorylated EGFR (pEGFR) in AR-positive tumors was significantly higher than that in AR-negative tumors (p<0.01). Stimulation of AR by dihydrotestosterone in SCC9 (AR-positive OSCC cell) caused an increase in pEGFR and pAKT expression and promoted cell migration without changed tEGFR expression, whereas treatment with bicalutamide led to a decrement in pEGFR expression and pAKT and inhibited cell migration. No effects were found in SCC25 cell line (AR-negative) either treated by dihydrotestosterone or bicalutamide. Furthermore, SCC9 cell line treated by EGF or cetuximab (EGFR inhibitor) significantly promoted or inhibited cell migration. Conclusion: Our data indicate that OSSC tumors and OSCC cell lines express AR which is critical for promoting cell migration by increasing EGFR phosphorylation.

Keywords: EGFR; androgen receptor; migration; oral squamous cell carcinoma; phosphorylation.

Figure 1

Expression of AR, EGFR, and pEGFR in OSCC tumors. (A) AR-positive staining in OSCC tumor (red arrow: cancer cell). (B) AR-positive staining in OSCC tumor. (C) AR staining in prostate cancer. (D and E) tEGFR staining in AR-positive OSCC tumor and AR-negative OSCC tumor. (F) No difference in tEGFR expression between AR-positive tumors and AR-positive tumors (*p>0.05). (G and H) pEGFR staining in AR-positive OSCC tumor and AR-negative OSCC tumor. (I) AR-positive tumors express significantly higher pEGFR (^#p<0.01). Scale bar in A–E, G, and H, 50 µm. Abbreviations: AR, androgen receptor; OSCC, oral squamous cell carcinoma; EGFR, epidermal growth factor receptor; pEGFR, phosphorylated EGFR; tEGFR, total EGFR.

Figure 2

Expression of AR in different OSCC cell lines and LNCaP cells. (A) AR mRNA in LNCaP was used as a positive control, AR mRNA was detected in four OSCC cell lines, not SCC25. (B) Different levels of AR proteins expressed in these cell lines. Abbreviations: AR, androgen receptor; OSCC, oral squamous cell carcinoma.

Figure 3

AR ligand (DHT) promoted the migration of SCC9 cells via increasing EGFR phosphorylation. (A, B, E, F, and I) DHT increased the migration of SCC9 cells following treatment with DHT for 20 h. (C, D, and G–I) DHT did not change the migration of SCC25 cells. (J and K) In SCC9 cells, DHT increased expression of pEGFR and pAKT proteins with no changing of tEGFR mRNA and protein. DHT did not cause any change of tEGFR, pEGFR and pAKT in SCC25 (scale bar, 500 µm).Abbreviations: AR, androgen; EGFR, epidermal growth factor receptor; pEGFR, phosphorylated EGFR; tEGFR, total EGFR.

Figure 4

AR inhibitor (bicalutamide) inhibited the migration of SCC9 cells via decreasing EGFR phosphorylation. (A, B, E, F, and I) Bicalutamide decreased the migration of SCC9 cells following treatment for 20 h. (C, D, and G–I) Bicalutamide did not change the migration of SCC25 cells. (J and K) In SCC9 cells, bicalutamide decreased expression of pEGFR and pAKT proteins with no changing of tEGFR mRNA and protein. Bicalutamide did not cause any change of tEGFR, pEGFR and pAKT in SCC25 (scale bar, 500 µm). Abbreviations: AR, androgen receptor; EGFR, epidermal growth factor receptor; pEGFR, phosphorylated EGFR; tEGFR, total EGFR.

Figure 5

EGF promoted and cetuximab inhibited SCC9 migration. (A, B, E, F, and I) EGF increased the migration of SCC9 cells following treatment for 20 h. (C, D, and G–I) Cetuximab decreased the migration of SCC9 cells following treatment for 20 h (scale bar, 500 µm).