High mobility group box 1 regulates tumor metastasis in cutaneous squamous cell carcinoma via the PI3K/AKT and MAPK signaling pathways

Abstract

The present study examined the mechanisms of high mobility group box 1 (HMGB1)-induced cell migration in human cutaneous squamous cell carcinoma (CSCC) SCC13 cells. Western blotting, a chemotaxis assay and ELISA were performed to analyze HMGB1 level in SCC13 cells and its ability to regulate tumor metastasis. The results demonstrated a significantly higher level of HMGB1 in the SCC13 cell supernatant compared with the human epidermoid carcinoma A431 cell supernatant. Administration of HMGB1 to the SCC13 cells caused cell migration, which occurred in a time- and dose-dependent manner. Moreover, HMGB1 significantly activated the phosphosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) signaling pathways by an increased level of phosphorylation in p85PI3K, AKT, p38 and p42/44 MAPK. Taken together, these data suggest that HMGB1 regulates tumor metastasis in CSCC via the PI3K/AKT and MAPK signaling pathways.

Keywords: HMGB1; MAPK pathway; PI3K/AKT pathway; cutaneous squamous cell carcinoma; migration.

Figure 1.

CSCC SCC13 cells secrete more HMGB1 than human epidermoid carcinoma A431 cells. (A) Human CSCC SCC13 and epidermoid carcinoma A431 cells were cultured with regular medium for 24 h. Following this, concentrated media was prepared, resolved by SDS-PAGE and subjected to western blot analysis of HMGB1. Results shown are representative of three independent experiments and were qualified by densitometry. *P<0.01. (B) Enzyme-linked immunosorbent assay data of the supernatant showing the HMGB1 concentration in the two different cell lines. Data from three independent experiments were pooled together. CSCC, cutaneous squamous cell carcinoma; HMGB1, high-mobility group box 1.

Figure 2.

HMGB1 induces the cell migration of human CSCC cells. (A) CSCC SCC13 cells were treated for the indicated times (3, 6 and 12 h). The number of migrated cells were counted and the results expressed as the mean number of migrated cells ± SEM/microscopic field (n=3). A significantly higher level of cell migration was found at the 12-h time-point (*P<0.05, ****P<0.0001). (B) The SCC13 cells were treated with the indicated concentration of HMGB1 for 12 h. The number of migrated cells was counted and the results expressed as the mean number of migrated cells ± SEM/microscopic field (n=3). A significantly higher level of cell migration was found at a HMGB1 concentration of 100 ng/ml (****P<0.0001). CSCC, cutaneous squamous cell carcinoma; HMGB1, high-mobility group box 1.

Figure 3.

PI3K/AKT and MAPK signaling pathways are involved in HMGB1-induced migration. CSCC SCC13 cells were treated with/without HMGB1 (100 ng/ml) or GR (100 µM) for 24 h. At the end of treatment, the activation [phosphorylation (P)] of AKT, p85 PI3K, p38 and p42/44 MAPK was analyzed by western blotting. The whole cell lysates were prepared, resolved by SDS-PAGE and subjected to western blot analysis. The total (T)-AKT, −p85 PI3K, −p38 and −p42/44 MAPK were used as a loading control. Results shown are representative of three independent experiments and were qualified by densitometry. (A) AKT and p85 PI3K; (B) p38 MAPK and p42/44 MAPK. CSCC, cutaneous squamous cell carcinoma; HMGB1, high-mobility group box 1; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; GR, glycyrrhizin.