Carcinogenic metalloid arsenic induces expression of mdig oncogene through JNK and STAT3 activation

Abstract

Environmental or occupational exposure to arsenic, a chemical element classified as metalloid, has been associated with cancer of the lung, skin, bladder, liver, etc. Mdig (mineral dust-induced gene) is a newly identified oncogene linked to occupational lung diseases and lung cancer. It is unclear whether mdig is also involved in arsenic-induced malignant transformation of the lung cells. By using human bronchial epithelial cells and human lung cancer cell lines, we showed that arsenic was able to induce expression of mdig. We further demonstrated that this mdig induction by arsenic was partially dependent on the JNK and STAT3 signaling pathways. Disruption of the JNK or STAT3 by either chemical inhibitors or siRNAs diminished arsenic-induced accumulation of mdig mRNA and protein. Furthermore, we also showed that microRNA-21 (miR-21) and Akt were down-stream effectors of the JNK and STAT3 signaling pathways in arsenic-induced mdig expression. Transfection of the cells with anti-miR-21 or pre-treatment of the cells with Akt inhibitor blunted mdig induction by arsenic. Clinically, the levels of mdig can be applied to predict the disease progression, the first progression (FP), in non-small cell lung cancer (NSCLC) patients. Taken together, our data suggest that mdig may play important roles on the pathogenesis of arsenic-induced lung cancer and that JNK and STAT3 signaling pathways are essential in mediating arsenic-induced mdig expression.

Keywords: Arsenic; JNK; Mdig; Metalloid; STAT3.

Fig. 1

As³⁺ induces mdig expression in A549 cells and BEAS-2B cells. A. RT-PCR analyses showed dose- and time-dependent induction of mdig mRNA by As³⁺. B. Dose- and time-dependent induction of mdig protein by As³⁺ as revealed by Westernblotting assay. C. As³⁺ induced mdig mRNA in BEAS-2B cells as revealed by RT-PCR. D. Westernblotting shows dose- and time-dependent induction of mdig protein by As³⁺ in BEAS-2B cells.

Fig. 2

JNK and STAT3 inhibitors decrease As³⁺-induced mdig mRNA expression. A549 cells were cultured in the presence or absence of 20 μM JNK inhibitor SP600125 (SP, A) or STAT3 inhibitor V (InV, B) for 4 h followed by the indicated concentrations of As³⁺ for an additional 2 h.

Fig. 3

JNK, STAT3 and Akt contribute to As³⁺-induced mdig expression. A. BEAS-2B cells were pre-treated with 20 μM JNK inhibitor, SP600125, STAT3 inhibitor V (InV) or PI3K/Akt inhibitor, Wortmannin (Wort) for 1 h, followed by culturing the cells in the presence of 20 μM As³⁺ for an additional 2 h. The levels of mdig protein, activation of STAT3, JNK and Akt were determined by Westernblotting. B. Inhibition of STAT3 by STAT3 inhibitor V (InV) diminished dose-dependent activation of JNK2 and Akt, and reduced mdig expression induced by As³⁺ in the BEAS-2B cells.

Fig. 4

Silencing STAT3 and JNK repressed mdig expression. A. BEAS-2B cells were transfected with 50 nM control siRNA (siCtrl), or STAT3 specific siRNA (siSTAT3) and cultured for 24 h. Cells were cultured in the presence of 20 μM As³⁺ for an additional 2 h. The levels of STAT3 activation, mdig protein, JNK and Akt activation were determined by Westernblotting. B. Silencing JNK2, but not JNK1, reduced the levels of basal and As³⁺-induced mdig. Cell transfection with siRNA and treatment are the same as in A.

Fig. 5

As³⁺-induced mdig requires miR-21. A. Dose-dependent induction of miR-21 by As³⁺. Relative levels (fold changes) of miR-21 were determined by quantitative real-time PCR. B. As³⁺ induces miR-21 through JNK and STAT3 activation. BEAS-2B cells were pretreated with inhibitors for JNK or STAT3 for 2 h and then treated with 20 μM As³⁺ for an additional 2 h. The levels of miR-21 were determined as in A. C. Repressing miR-21 by anti-miR-21 prevented mdig mRNA induction by As³⁺. BEAS-2B cells were pre-transfected with 100 nM anti-miR-21 for 24 h and then treated with As³⁺ for an additional 2 h. The relative levels of mdig mRNA and GAPDH mRNA were determined by regular RT-PCR.

Fig. 6

Kaplan-Meier probability analyses of the first progression (FP) for the patients with NSCLC and higher or lower mdig expression (mdig^high or mdig^low). (A) Higher mdig expression shortens the times to FP of the patients with Adenocarcinoma (Ad, left panel) but not the patients with squamous cell carcinoma (Sq, right panel). (B) Higher mdig expression predicts earlier FP of the male patients with Ad (left panel) but later FP of the female patients with Ad (right panel).

Fig. 7

Schematic diagram showing As³⁺-induced JNK-STAT3-Akt signaling on the expression of mdig that contributes to initiation and progression of the non-small cell lung cancers (NSCLC), esp., for male adenocarcinoma.