Thioredoxin 1 mediates TGF-β-induced epithelial-mesenchymal transition in salivary adenoid cystic carcinoma

Abstract

Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) which is characterized by wide local infiltration, perineural spread, a propensity to local recurrence and late distant metastasis. Our recent studies have disclosed that TGF-β is a crucial factor for EMT in metastatic SACC. In this study, we further uncovered small redox protein thioredoxin 1 (TXN) as a critical mediator of TGF-β induced EMT. Immunohistochemistry analysis revealed significantly higher expressions of TXN, thioredoxin reductase 1 (TXNRD1) and N-cadherin, and lower expression of E-cadherin in human metastatic SACC compared to non-metastatic SACC tissues. Consistently, cultured SACC cells with stable TXN overexpression had decreased E-cadherin and increased N-cadherin as well as Snail and Slug expressions. The enhanced migration and invasion potential of these cells was abrogated by Akt or TXNRD1 inhibitors. Expression of N-cadherin and Akt p-Akt decreased, whereas E-cadherin expression increased in a BBSKE (TXNRD1 inhibitor)-dose-dependent manner. In a xenograft mouse model, TXN overexpression facilitated the metastatic potential of SACC-83 cells to the lung. Our results indicate that TXN plays a key role in SACC invasion and metastasis through the modulation of TGF-β-Akt/GSK-3β on EMT. TXN could be a potential therapeutic target for SACC.

Keywords: TGF-β; epithelial-mesenchymal transition; metastasis; salivary adenoid cystic carcinoma; thioredoxin 1.

Figure 1. Immunohistochemical staining reveals differential expressions of thioredoxin 1 (TXN), thioredoxin reductase 1 (TXNRD1) and epithelial-mesenchymal transition signs in salivary adenoid cystic carcinoma (SACC) samples from patients with/without lung metastasis

Higher expression of TXN (AA’), TXNRD1 (CC’) and N-cadherin (GG’) in SACC tissues with metastasis compared to those without metastasis (BB’, DD’, HH’), respectively. Lower expression of E-cadherin in SACC tissues with metastasis (EE’) compared to those without metastasis (FF’). Scale bar = 20 μm, A’-H’ show enlarged fields of the inset squares in panels A–H.

Figure 2. TXN and TXNRD1 expression are correlated with survival rate of patients with SACC

Kaplan-Meier survival curves for cumulative survival rate of 47 patients with SACC. Expressions of TXN A. and TXNRD1 B. were positively correlated with poor survival rate.

Figure 3. TXN modulates epithelial and mesenchymal proteins and promotes migration and invasion in SACC cell lines

A. Immunoblotting analysis of TXN, TXNRD1, E-cadherin, and N-cadherin expression in SACC-83 and SACC-LM tumor cell lines. B. Phase contrast microscopy revealed fibroblastic morphological changes of epithelial-mesenchymal transition (EMT) in TXN-overexpressed SACC-83 cells (SACC-TXN) compared to control cells (Mock). Knocking down TXN in SACC-LM (siTXN) induced no apparent morphological changes compared with control cells (siCon). Scale bar = 100 μm. C. Wound closure assays in Mock, SACC-TXN, siCon, and siTXN cells. Images were captured at 0 and 9 hours after wounding in serum-free media. N = 3, scale bar = 50 μm. D. Invasion assays (24 hours) for Mock, SACC-TXN, siCon, and siTXN cells. Numbers of invaded cells were compared. N = 3, scale bar = 200 μm. E. Immunoblotting analysis of TXN, TXNRD1, E-cadherin, and N-cadherin expressions in Mock, SACC-TXN, siCon, and siTXNs (three different TXN targeting siRNAs – siTXN1, siTXN2, and siTXN3) cells. Data represent means ± standard deviation (SD) of three independent experiments (*P < 0.05).

Figure 4. TXN is crucial for EMT induced by TGF-β in SACC cell lines

A. Decreased TGF-β, TXN, TXNRD1, and N-cadherin expression and increased E-cadherin expression on immunoblots in TGF-β knockdown SACC-LM cells (si1, si2, si3) compared with control cells (siCon). B. Immunoblot analysis of expression of TXN, TXNRD1, E-cadherin, and N-cadherin in SACC-83 cells treated with or without TGF-β (2, 5 10 ng/ml) for 24 hours. C. After pre-transfection of TXN or Con siRNAs for 24 hours, siTXN or siCon cells were treated with or without TGF-β (10 ng/ml) for another 24 hours. Then expressions of TXN, TXNRD1, E-cadherin, and N-cadherin were analyzed. β-actin was used as loading control. Data represent means ± SD of three independent experiments (*P < 0.05).

Figure 5. TXN regulates EMT in SACC cells through the Akt/GSK-3β/Snail signaling pathway

Immunoblotting analysis of Snail, Slug, p-Akt/Akt, and p-GSK3β/GSK3β expression in Mock, SACC-TXN A. siCon, and siTXN B. cells. Wound closure assays (C. scale bar = 100 μm) and invasion assays (D. scale bar = 200 μm) in SACC-TXN treated with or without PI3K inhibitor LY294002 (25 μM, 50 μM) for 12 hours. Effects of LY294002 on expressions of Snail, Slug, p-Akt/Akt, and p-GSK3β/GSK3β E. or TXN, TXNRD1, E-cadherin, and N-cadherin F. in SACC-TXN cells. β-actin was used as loading control. Data represent means ± SD of three independent experiments (*P < 0.05).

Figure 6. TXNRD1 inhibitor BBSKE inhibits EMT in SACC cells through the Akt/GSK-3β signaling pathway

A. Effect of BBSKE on TXNRD1 activity in SACC-LM cells. TXNRD1 activity was measured of treatment with 1 μM, 5 μM, and 10 μM BBSKE after 48 h by DTNB assay. B. Immunoblotting analysis of E-cadherin, N-cadherin, p-Akt/Akt, and p-GSK3β/GSK-3β expressions in SACC-LM cells treated with BBSKE (1 μM, 5 μM, or 10 μM) for 24 hours. β-actin was used as loading control. Data represent means ± SD of three independent experiments (*P < 0.05).

Figure 7. A hypothetical model demonstrating the essential roles of TXN in mediation of TGF-β induced EMT and SACC metastasis

TXN in the cytoplasm mediated TGF-β induced EMT through Akt/GSK-3β signaling pathway, which could be blocked by TXNRD1 inhibitor (BBSKE) and siTXN. Overexpression of TXN leads to activation of Akt induced inactivation (phosphorylation) of GSK-3β, and repression of E-cadherin expression and increase of N-cadherin expression by transcription factor Snail and Slug. LY294002 could suppress Akt/GSK-3β, Snail and Slug which are the downstream signaling pathways of TXN in TXN-overexpressing cells.

Figure 8. Overexpression of TXN enhances lung metastasis of SACC-83 cells in vivo

A. Mice were inoculated intravenously with 3 × 10⁶ Mock or SACC-TXN cells. Eight weeks later, lung surface metastatic nodules (arrows) were observed and quantified. B. Representative hematoxylin and eosin staining showed that metastatic tumor cells in lung tissues section of mice injected intravenously with Mock or SACC-TXN cells. Scale bar = 100 μm.