USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

Abstract

Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22) with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

Keywords: DNA damage repair; apoptosis; cisplatin resistance; lung adenocarcinoma; ubiquitin-specific peptidase 22.

FIGURE 1

Overexpression of USP22 was correlated with acquired resistance to cisplatin in A549 cells. (A) Western blot analysis of USP22 expression in A549-WT, A549-vector, and A549-USP22 cells (the cell clones which were stably transfected with vector or USP22 overexpression plasmids). (B) The sensitivity to cisplatin of A549-WT, A549-vector, and A549-USP22 cells was detected by CCK-8. Cells were exposed to various concentrations of cisplatin for 24 and 48 h. (C) The response of A549-vector and A549-USP22 cells to cisplatin was tested by colony-forming assay. Cell lines were treated continuously with either 0 or 0.33 μM of cisplatin for 14 days. (D) A549-vector and A549-USP22 cells were treated with 0 or 0.33 μM of cisplatin for 48 h and subjected to flow cytometry analysis of cell cycle distribution. Percentage change in G1 phase is shown. (E) Cisplatin-induced apoptosis in A549-vector and A549-USP22 cells was demonstrated by flow cytometric analysis. Cell lines were treated continuously with either 0 or 0.33 μM cisplatin for 48 h. Every experiment was conducted at least three times, and the average is shown (mean ± SD). ^∗P < 0.05, ^∗∗P < 0.01, significant.

FIGURE 2

USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin-resistant A549/CDDP cell line. (A) The sensitivity to cisplatin of A549-WT and A549/CDDP cells was detected by CCK-8. Cells were exposed to various concentrations of cisplatin for 24 and 48 h. (B) Western blot analysis of USP22, γH2AX and Sirt1 in A549-WT, A549/CDDP, A549-vector, and A549-USP22 cells. (C) Western blot analysis of the proteins of USP22, γH2AX and Sirt1 in A549-WT and A549/CDDP cells. Cell lines were treated continuously with either 0 or 0.33 μM cisplatin for 48 h. Every experiment was conducted at least three times, and the average is shown (mean ± SD). ^∗P < 0.05, ^∗∗P < 0.01, significant.

FIGURE 3

USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. (A) Comet assay was performed to assess DNA damage after 0.33 μM cisplatin treatment for 6 h in A549-WT, A549-USP22, and A549/CDDP cells (scale bar 20 μM). (B,C) H2A interacts with USP22 in A549-WT cells. A549-WT cells lysates were subjected to immunoprecipitation (IP) with control IgG, anti-USP22 (B), and anti-H2A (C) antibodies. The immunoprecipitates were then blotted with the indicated antibodies. (D) H2A binding to USP22, γH2AX, and the ubiquitination of H2A (Lys119) was detected by IP and western blot after 0.33 μM cisplatin treatment for 48 h in A549-WT, A549/CDDP and A549-USP22 cells. (E) Western blot analysis of the proteins of USP22, γH2AX and Ubi-H2A after various concentrations of cisplatin treatment for 48 h. Every experiment was conducted at least three times, and the average is shown (mean ± SD). ^∗P < 0.05, ^∗∗P < 0.01, significant.

FIGURE 4

USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. (A) Acetylated Ku70 and the Bax binding to Ku70 were detected by IP and cytochrome C was detected by western blot analysis in A549-WT, A549/CDDP and A549-USP22 cells, after 0.33 μM cisplatin treatment for 48 h. (B) Acetylated Ku70 and the Bax binding to Ku70 were detected by IP and cytochrome C was detected by western blot analysis in A549 cells which were transfected by vector, USP22, USP22+Sirt1-siRNA, after 0.33 μM cisplatin treatment for 48 h. (C) Apoptosis in A549 cells which were transfected by vector, USP22, USP22+Sirt1-siRNA was demonstrated by flow cytometric analysis, after 0.33 μM cisplatin treatment for 48 h. (D) Apoptosis in A549 cells which were transfected by si-control, sh-USP22, Sirt1-si and sh-USP22+Sirt1-si, after 0.33 μM cisplatin treatment for 48 h. Every experiment was conducted at least three times, and the average is shown (mean ± SD). ^∗P < 0.05, ^∗∗P < 0.01, significant.

FIGURE 5

The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition. (A) The sensitivity to cisplatin of A549/CDDP and A549/CDDP-sh-USP22 (the cell clones which were stably transfected with sh-USP22) cells was detected by CCK-8. Cells were exposed to various concentrations of cisplatin for 48 h. (B) The response of A549/CDDP and A549/CDDP-sh-USP22 cells to cisplatin was tested by colony-forming assay. Cell lines were treated continuously with 0 or 0.33 μM cisplatin for 14 days. (C) A549/CDDP and A549/CDDP-sh-USP22 cells were treated with 0.33 μM of cisplatin for 48 h and subjected to flow cytometry analysis of cell cycle distribution. Percentage change in G1 phase is shown. (D) Cisplatin-induced apoptosis in A549/CDDP and A549/CDDP-sh-USP22 cells was demonstrated by flow cytometric analysis. Cell lines were treated continuously with 0.33 μM of cisplatin for 48 h. (E) Every experiment was conducted at least three times, and the average is shown (mean ± SD). ^∗P < 0.05, ^∗∗P < 0.01, significant.

FIGURE 6

Inhibiting USP22 expression enhanced cisplatin sensitivity in cisplatin-resistant A549/CDDP cell nude mouse xenografts. (A) Representative figures and growth curve of tumor by subcutaneous injection of A549/CDDP and A549/CDDP-sh-USP22 cells. (B) IHC staining of Sirt1, γH2AX and Ki67 in tumor nodules from A549/CDDP and A549/CDDP-sh-USP22 cells derived xenografts. Bar: 200 μm. (C) Western blot analysis of USP22, Sirt1, γH2AX, Ubi-H2A, and Ki67 in A549/CDDP and A549/CDDP-sh-USP22 cells derived xenografts. Every experiment was conducted at least three times, and the average is shown (mean ± SD). ^∗P < 0.05, ^∗∗P < 0.01, significant.