SOD2 deregulation enhances migration, invasion and has poor prognosis in salivary adenoid cystic carcinoma

Abstract

This study aimed to investigate the role of SOD2 in the progression and metastasis of salivary adenoid cystic carcinoma (SACC). We analyzed the expression of SOD2 in 50 SACC patients. Then, the effects and mechanism of SOD2 on cell metastasis in a pair of different metastatic potential cell lines was investigated. SOD2 was deregulated in patients with SACC. Up-regulation of SOD2 was associated with distant metastasis and reduced overall survival and disease free - survival. Compared to SACC-83 cells (lower metastasis ability), SACC-LM cells (higher metastasis ability) had higher SOD2 activity and intracellular H2O2 concentrations, and protein levels of pERK1/2 and Slug, but had similar catalase protein level and activity. In SACC-LM, reducing the expression of SOD2 by SiRNA inhibited the metastasis ability and reduced the SOD2 activities, intracellular H2O2 concentrations, and protein levels of pERK1/2 and Slug. These effects were revised in SACC-83 after SOD2 overexpression. Moreover, in SACC-83, treated with H2O2, the metastasis was enhanced accompanied by increased protein levels of pERK1/2 and Slug. We confirmed that SOD2 play an important role in the development and prognosis of SACC and SOD2-dependent production of H2O2 contributes to metastasis of SACC through the ERK-Slug signaling pathway.

Figure 1. The deregulation of SOD2 in the development of SACC tissue.

Immunohistochemical analyses for SOD2 expression were performed in normal salivary gland (A) and SACC samples (B). (Scale bar: 50 μm) Box plots compare the expression of SOD2 between 20 normal salivary glands and 50 SACC samples (C), P = 0.000), and SACC with (n = 8) or without (n = 42) distant metastasis (D), P = 0.000). Boxes represent the 25^th and 75^th percentiles. The lines in the middle of the box represent medians. *P < 0.001. Patients were divided into SOD2 low expression group (n = 28) and SOD2 high expression group (n = 22). The 5-year overall survival rate (E), P = 0.017) and 5-year disease-free survival rate (F), P = 0.038) were significantly higher in SOD2 low expression group. *P < 0.05. Student’s T test was used to compare the difference between groups (C,D). Survival curves were plotted using the Kaplan-Meier method and compared with the log-rank test (E,F).

Figure 2. The expression of SOD2 in pair SACC cell lines with different migration and invasion ability.

Migration and invasion of SACC cells were assessed using a transwell migration and invasion assay. Migration (A) and invasion (B) were significantly higher in SACC-LM cells than in SACC-83 cells. SOD2 protein expression and activity were assessed with Western blot (C) and SOD activity assays (D), respectively. SOD2 expression and activity were significantly higher in SACC-LM cells than in SACC-83 cells. H₂O₂ concentrations were measured as described in the text. SACC-LM cells produced significantly more H₂O₂ than SACC-83 cells (E). Concentrations of CAT (catalase) and activity, assessed with Western blot (C) and a CAT activity assay (F), respectively, did not differ between the two cell lines (C,F). Vimentin, Slug, Snail, ERK, p-ERK, and MMP-2 protein expression were higher in SACC-LM cells, and E-cadherin protein expressions were lower in SACC-83 cells (C). *P < 0.05.

Figure 3. SOD2 knockdown inhibits the migration and invasion of SACC cells.

To characterize the function of SOD2 in aiding metastasis, SOD2 siRNA was transfected into SACC-LM cells using Lipofectamine^TM RNAiMAX. Cells were collected and tested 24 h after transfection. SOD2 protein concentrations and activities were significantly lower in the SOD2 siRNA-transfected SACC-LM cells than in the control transfected cells (A,B). SOD2 knockdown inhibited the migration and invasion of SACC-LM cells (C,D). H₂O₂ production was significantly lower in SACC-LM cells after transfection with the SOD2 siRNA (E). CAT protein concentrations and activity in SACC-LM cells did not differ after transfection with SOD2 siRNA (B,F). Vimentin, Slug, Snail, ERK, p-ERK, and MMP-2 protein concentrations were lower in SACC-LM cells but E-cadherin protein concentrations were higher with SOD2 knockdown (B). *P < 0.05.

Figure 4. SOD2 overexpression promotes the migration and invasion SACC.

To characterize the function of SOD2 in aiding metastasis, lentivirus containing SOD2 overexpression was transfected into SACC-83 cells. SOD2 protein concentrations and activities were significantly higher in the SOD2 overexpression-transfected SACC-83 cells than in the vector control transfected cells (A,B). SOD2 overexpression promoted the migration and invasion of SACC-83 cells (C,D). SACC-83 cells significant increased production of H₂O₂ after SOD2 overexpression (E). CAT protein concentrations and activity in SACC-83 cells did not differ after SOD2 overexpression (B,F). Vimentin, Slug, Snail, ERK, p-ERK, and MMP-2 protein concentrations were higher in SACC-83 cells, and E-cadherin protein concentrations were lower with SOD2 overexpression (B). *P < 0.05.

Figure 5. SOD2-dependent production of H₂O₂ increases migration and invasion of SACC.

To confirm that H₂O₂ production induces migration and invasion of SACC, SACC-83 cells were treated with H₂O₂ for 24 h. The migration and invasion of SACC-83 cells were significantly higher after treatment with 25 μM H₂O₂ once every 6 h (A,B). Adding H₂O₂ increased the protein concentrations of Vimentin, Slug, Snail, ERK, p-ERK, and MMP-2 and decreased E-cadherin protein concentrations and had no affection on expression of SOD2 (C).