ABCF2, an Nrf2 target gene, contributes to cisplatin resistance in ovarian cancer cells

Abstract

Previously, we have demonstrated that NRF2 plays a key role in mediating cisplatin resistance in ovarian cancer. To further explore the mechanism underlying NRF2-dependent cisplatin resistance, we stably overexpressed or knocked down NRF2 in parental and cisplatin-resistant human ovarian cancer cells, respectively. These two pairs of stable cell lines were then subjected to microarray analysis, where we identified 18 putative NRF2 target genes. Among these genes, ABCF2, a cytosolic member of the ABC superfamily of transporters, has previously been reported to contribute to chemoresistance in clear cell ovarian cancer. A detailed analysis on ABCF2 revealed a functional antioxidant response element (ARE) in its promoter region, establishing ABCF2 as an NRF2 target gene. Next, we investigated the contribution of ABCF2 in NRF2-mediated cisplatin resistance using our stable ovarian cancer cell lines. The NRF2-overexpressing cell line, containing high levels of ABCF2, was more resistant to cisplatin-induced apoptosis compared to its control cell line; whereas the NRF2 knockdown cell line with low levels of ABCF2, was more sensitive to cisplatin treatment than its control cell line. Furthermore, transient overexpression of ABCF2 in the parental cells decreased apoptosis and increased cell viability following cisplatin treatment. Conversely, knockdown of ABCF2 using specific siRNA notably increased apoptosis and decreased cell viability in cisplatin-resistant cells treated with cisplatin. This data indicate that the novel NRF2 target gene, ABCF2, plays a critical role in cisplatin resistance in ovarian cancer, and that targeting ABCF2 may be a new strategy to improve chemotherapeutic efficiency.

Keywords: ABCF2; NRF2; chemoresistance; cisplatin; ovarian cancer.

Figure 1

NRF2 expression in high-grade serous ovarian cancer tissue and A2780 ovarian cancer cells. (A) Representative IHC images of NRF2 levels in 9 pairs of HGSOC and corresponding normal ovarian tissue samples (magnification, 400x). The scale bars represent 50 μm. Negative control (NC): rabbit immunoglobulin G. NOT: normal ovarian tissues. (B) NRF2 IHC score in HGSOC and corresponding NOT samples. **P<0.01. (C) IF staining of NRF2 was performed in A2780 and A2780cp cell lines. The red signal represents NRF2 (magnification, 400x) (left panel). Total cell lysates were collected for immunoblot analysis of NRF2 (right panel).

Figure 2

Establishment of NRF2 overexpression and knockdown stable cell lines. (A and B) A2780 cells were transfected with NRF2 plasmid or NRF2 shRNA by lentiviral packaging system to generate the stable cell lines. NRF2 and NQO1 protein levels were detected in A2780, A2780-NRF2, A2780cp, and A2780cp-NRF2-shRNA by immunoblot analysis. (C and D) Cell viability assay. A2780-NRF2 and A2780 cells (C) and A2780cp-NRF2-shRNA and A2780cp cells (D) were treated with the indicated doses of cisplatin for 48 h to determine cell viability. (E and F) Analysis of apoptosis. A2780-NRF2 and A2780 cells were treated with 1 μg/ml cisplatin (E) and A2780cp-NRF2-shRNA and A2780cp cells were treated with 10 μg/ml cisplatin (F) for 24 h to determine apoptosis by TUNEL assay. All data are presented as mean ± SD from three independent experiments, n=3. *P<0.05. **P<0.01.

Figure 3

Microarray analysis of NRF2 stable cell lines. (A) Venn diagrams of upregulated and downregulated genes in A2780-NRF2 vs. A2780 and A2780cp-NRF2-shRNA vs. A2780cp cells. (B) Heat map of the 18 genes that significantly changed in both sets of stable cell lines. (C) Gene Ontology enrichment analysis of the biological process of the downregulated genes from the knockdown stable cell lines. Each cell sample was done in triplicate.

Figure 4

ARE identification in the promoter of ABCF2. (A) Schematic representation of the luciferase constructs generated and their relative luciferase activities. (1) Wild-type ARE-luciferase plasmid: DNA fragment containing a putative ARE (−1292 bp to +1069 bp). (2) Mutated ARE-luciferase plasmid: two nucleotides in the wild-type ARE-luciferase plasmid were mutated (GTGACTTTGCA to GgGcCTTTGCA). (3) No ARE-luciferase plasmid: DNA fragment without the putative ARE (−893 bp to +1069 bp). These constructs were cotransfected with either a NRF2 plasmid or a control vector into HEK293T cells for 24 h, and dual luciferase assay was performed. Samples were prepared in triplicate. Data are represented as mean ± SD. **P<0.01. (B) CHIP assay was performed in A2780cp cells. Rabbit immunoglobulin G as negative control. RNA polymerase II as positive control. (C) Schematic representation of the NRF2 binding site which starts from −1213 bp to −1017 bp in ABCF2 promoter region.

Figure 5

NRF2 regulates ABCF2 expression. (A) ABCF2 mRNA and protein levels in A2780-NRF2 and A2780 cells, A2780cp-NRF2-shRNA and A2780cp cells. (B) The protein level of ABCF2 in A2780 and A2780cp cells. (C) A2780 cells were transfected with 5 nM KEAP1 siRNA or nonspecific control siRNA for 48 h. (D) A2780cp cells were transfected with 5 nM NRF2 siRNA, or nonspecific control siRNA for 48 h. Cell lysates were subjected to immunoblot analysis. Band intensity was analyzed by densitometry and values were normalized to β-actin. (E) Cell viability assay. A2780, A2780-NRF2 and A2780-NRF2 cells transfected with ABCF2 siRNA for 48 h, A2780cp, A2780cp-NRF2-shRNA and A2780cp-NRF2-shRNA transfected with HA-ABCF2 for 24 h were treated with doses of cisplatin for 48 h to determine cell viability. Data are represented as mean ± SD, from two independent experiments, n = 3. *P<0.05. **P<0.01.

Figure 6

Role of ABCF2 in cisplatin resistance of ovarian cancer cells. (A and B) Cell viability assay. A2780 cells were transfected with a HA-ABCF2 plasmid or an empty vector for 24 h (A), and A2780cp cells were transfected with ABCF2 siRNA or nonspecific siRNA for 48 h (B), then treated with the indicated doses of cisplatin for 48 h to determine cell viability. Data are represented as mean ± SD from three independent experiments, n = 3. *P<0.05. (C and D) Analysis of apoptosis. A2780 cells were transfected with HA-ABCF2 or control vector, and either left untreated or treated with cisplatin (1 μg/ml) for 24 h (C) and A2780cp cells were transfected with #3 ABCF2 siRNA or nonspecific control siRNA, and either left untreated or treated with (30 μg/ml) for 24 h (D). Apoptosis was determined with Annexin V-PI and flow cytometry. **P<0.01. (E) Representative IHC images of ABCF2 levels in HGSOC and corresponding normal ovarian tissue samples (magnification, 400x). The scale bars represent 50 μm. (F) ABCF2 IHC score in HGSOC and corresponding normal ovarian tissue samples. **P<0.01.