Arsenic activates STAT3 signaling during the transformation of the human bronchial epithelial cells

Abstract

Arsenic (As³⁺), a metalloid abundant in environment, is classified as a group I carcinogen associated with several common human cancers, including cancers in lung, skin, bladder, liver, and prostate (Wei et al., 2019). The mechanisms of As³⁺-induced carcinogenesis had been extensively studied, and different mechanisms might be involved in different types of cancer (Wei et al., 2019). Recent studies showed that exposure to a high dose of arsenic is able to induce lung cancer. Meanwhile, prolonged exposure to a low concentration of arsenic can increase the risk of lung cancer also (Liao et al., 2009; Fernández et al., 2012). Emerging evidence indicated that prolonged exposure to arsenic promotes malignant transformation and some of the transformed cells have cancer-stem-like properties (Ngalame et al., 2014). In the present report, we revealed that exposure to As³⁺ for short time period inhibited tyrosine-705 phosphorylation of signal transducer and activator of transcription 3 (pSTAT3^Y705) and induced Src homology region 2 domain-containing phosphatase-1 (SHP-1) in bronchial epithelial cell line, BEAS-2B. In addition, we found that long term exposure of the cells to As³⁺ activates phosphorylation of STAT3 at serine 727 (pSTAT3^S727) as well as pSTAT3^Y705. Moreover, As³⁺ is able to induce the expression of miRNA-21 (miR-21) and decrease the expression of PDCD4. Taken together, our data suggest that activation of STAT3 and induction of miR-21 are important contributing factors to the reduced expression of PDCD4, which may play significant role in As³⁺-induced transformation of BEAS-2B cells.

Keywords: Arsenic; PDCD4; STAT3; Transformation; miR-21.

Fig. 1.

Protein expression of pSTAT3^S727, STAT3, pJNK^Th183/Th185, JNK, and pSTAT3^Y705 in BEAS-2B cells. BEAS-2B cells were treated with various concentrations of As³⁺ for the indicated times followed by Western blotting using the indicated antibodies. The cell lysates were prepared following treatment of the cells with As³⁺ and immunoblotted on PVDF membranes. Membranes were probed for A: pSTAT3^S727, B: STAT3, C: pJNK^Th183/Th185, D: JNK, and E: pSTAT3^Y705 proteins. Experiments were repeated three times, and the mean percent of protein/GAPDH ratio ± SEM was presented. One way ANOVA was used for statistics analysis *: p < 0.05; **: p < 0.01; ***: p < 0.001, ****: p < 0.0001.

Fig. 2.

Dose and time-dependent effects of As³⁺ on STAT3, and JNK activation. A, B, and C: BEAS-2B cells were starved overnight then treated with various concentrations of As³⁺ (0, 5, 10, 20, 40, 80 μM) for 6 h. D: BEAS-2B cells were treated with various concentrations of As³⁺ for 6 h without starvation. A: pSTAT3^Y705, B: pSTAT3^S727, C: pJNK^Th183/Th185, and D: pSTAT3^Y705. E: BEAS-2B cells were starved overnight then treated with 10 μM As³⁺ for the indicated times. The levels of pSTAT3^S727 and pSTAT3^Y705 proteins expression were confirmed by Western blotting. Experiments were repeated three times, and the mean percent of protein/GAPDH ratio ± SEM was presented. One way ANOVA was used for statistics analysis *: p < 0.05; **: p < 0.01; ***: p < 0.001, ****: p < 0.0001.

Fig. 3.

As³⁺ activates JAK1 and JAK2: BEAS-2B cells were starved overnight then treated with the indicated concentrations of As³⁺ for 6 h. BEAS-2B cell lysates were prepared following As³⁺ treatment and immunoblotted on PVDF membranes. Membranes were probed with the indicated antibodies. The levels of protein expression were confirmed by Western blotting.

Fig. 4.

The effect of As³⁺ on SOCS1, SOCS3, SHP-1, SHP-2, and pSTAT3^Y705: A & B: BEAS-2B cells were treated with various concentrations of As³⁺ (0, 5, 10, 20, 40, 80 μM) for 6 h. The levels of SOCS1, SOCS3, SHP-2 (A), and SHP-1 (B) protein expression were confirmed by Western blotting. C: Transformed Cells were treated with various concentrations of As³⁺ (0, 5, 10, 20, 40, 80 μM) for 6 h. The levels of pSTAT3^Y705, and SHP-1 were determined by western blotting.

Fig. 5.

Regulatory role of As³⁺ on mRNAs of SHP-1, STAT3, and miR-21. A: BEAS-2B cells were starved overnight then treated with the indicated concentrations of As³⁺ for 6 h. The mRNA level of SHP-1 were determined by real-time PCR. B: Transformed cells starved overnight then treated with As³⁺ as in A. The mRNA level of SHP-1 were determined. C: BEAS-2B cells were starved overnight then treated with As³⁺ as in A. The mRNA level of STAT3 were determined. D: BEAS-2B cells were starved overnight then treated with the indicated concentrations of As³⁺ for 12 h. The level miR-21 were determined by QPCR. All experiments were repeated three times, and the mean percent of mRNA/GAPDH mRNA ratio ± SEM was presented. One way ANOVA was used for statistics analysis. *: p 0.05; **: p < 0.01; ***: p < 0.001, ****: p < 0.0001.

Fig. 6.

Increased protein levels of pSTAT3^Y705 and pSTAT3^S727 in transformed cells relative to the BEAS-2B cells. Cell lysates were prepared from both BEAS-2B cells and the As³⁺-transformed cells for Western blotting using antibodies against pSTAT3^Y705 (A), pSTAT3^S727 (B), SHP-1 (C), and PDCD4 (D). Experiments were repeated three times, and the mean percent of protein/GAPDH ratio ± SEM was presented. One way ANOVA was used for statistics analysis. *:p < 0.05; **: p < 0.01; ***: p < 0.001, ****: p < 0.0001.

Fig. 7.

Increased mRNAs of STAT3 and miR-21, but decreased mRNAs of SHP-1 and PDCD4 in As³⁺-transformed cells relative to the BEAS-2B cells. Total RNAs were prepared from the BEAS-2B cells and the transformed cells and subjected to QPCR for STAT3 (A), miR-21 (B), SHP-1 (C), and PDCD4 (D). Experiments were repeated three times, and the mean percent of mRNA/GAPDH mRNA ratio ± SEM was presented. One way ANOVA was used for statistics analysis. *:p < 0.05; **: p < 0.01; ***: p < 0.001, ****: p < 0.0001.

Fig. 8.

Diagram shows differences in the activation of STAT3 signaling between short-term and long-term As³⁺ treatment. In short-term treatment, As³⁺ activates JNK that linked to pSTAT3^S727 and stabilizes SHP-1 protein that down-regulates pSTAT3^Y705. In long-term treatment, due to the inhibition of SHP-1 at both the mRNA level and protein level, As³⁺ induces both pSTAT3^S727 and pSTAT3^Y705. One of the oncogenic activities of the activated STAT3, either pSTAT3^S727 or pSTAT3^Y705, or both, is achieved through the expression of an oncogenic non-coding RNA, miR-21 that negatively regulates several tumor suppressors, such as PTEN, PDCD4 and SPRY2, leading to an enhanced oncogenesis and the generation of the CSCs.