Short-hairpin RNA-induced suppression of adenine nucleotide translocase-2 in breast cancer cells restores their susceptibility to TRAIL-induced apoptosis by activating JNK and modulating TRAIL receptor expression

Abstract

Background: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL; apo2 ligand) induces apoptosis in cancer cells but has little effect on normal cells. However, many cancer cell types are resistant to TRAIL-induced apoptosis, limiting the clinical utility of TRAIL as an anti-cancer agent. We previously reported that the suppression of adenine nucleotide translocase-2 (ANT2) by short-hairpin RNA (shRNA) induces apoptosis of breast cancer cells, which frequently express high levels of ANT2. In the present study, we examined the effect of RNA shRNA-induced suppression of ANT2 on the resistance of breast cancer cells to TRAIL-induced apoptosis in vitro and in vivo.

Results: ANT2 shRNA treatment sensitized MCF7, T47 D, and BT474 cells to TRAIL-induced apoptosis by up-regulating the expression of TRAIL death receptors 4 and 5 (DR4 and DR5) and down-regulating the TRAIL decoy receptor 2 (DcR2). In MCF7 cells, ANT2 knockdown activated the stress kinase c-Jun N-terminal kinase (JNK), subsequently stabilizing and increasing the transcriptional activity of p53 by phosphorylating it at Thr81; it also enhanced the expression and activity of DNA methyltransferase 1 (DNMT1). ANT2 shRNA-induced overexpression of DR4/DR5 and TRAIL sensitization were blocked by a p53 inhibitor, suggesting that p53 activation plays an important role in the transcriptional up-regulation of DR4/DR5. However, ANT2 knockdown also up-regulated DR4/DR5 in the p53-mutant cell lines BT474 and T47 D. In MCF7 cells, ANT2 shRNA treatment led to DcR2 promoter methylation and concomitant down-regulation of DcR2 expression, consistent with the observed activation of DNMT1. Treatment of the cells with a demethylating agent or JNK inhibitor prevented the ANT2 shRNA-induced down-regulation of DcR2 and activation of both p53 and DNMT1. In in vivo experiments using nude mice, ANT2 shRNA caused TRAIL-resistant MCF7 xenografts to undergo TRAIL-induced cell death, up-regulated DR4/DR5, and down-regulated DcR2. Co-treatment with ANT2 shRNA and TRAIL efficiently suppressed tumor growth in these mice.

Conclusions: ANT2 suppression by shRNA might be exploited to overcome TRAIL-resistance in cancer.

Figure 1

TRAIL cytotoxicity and expression of TRAIL receptors in the TRAIL-resistant breast cancer cell line MCF7 and the TRAIL-sensitive breast cancer cell line MDA-MB-231. (A) Cell viability assays were performed 12 h after treatment of MCF7 and MDA-MB-231 cells with recombinant human TRAIL. (B) MCF7 and MDA-MB-231 cells were transfected with 1 μg of scrambled or ANT2 shRNA. After 24 h, they were treated with recombinant human TRAIL (100 ng/ml). Cell viability assays were performed 12 h after TRAIL treatment. (C) Morphologic analysis of TRAIL-resistant MCF7 cells transfected with scrambled or ANT2 shRNA. After 24 h, the cells were treated with recombinant human TRAIL (100 ng/ml) for 12 h and then examined under a phase contrast microscope. (D) The TRAIL-resistant cell lines T47 D and BT474 were treated with shRNA and TRAIL and examined for cell viability as in (A). (E) MCF7 cells were transfected with scrambled or ANT2 shRNA. After 24 h, they were transfected with pcDNA3.1 or pcDNA-ANT2. After another 24 h, they were treated with recombinant human TRAIL (100 ng/ml) for 12 h and assayed for cell viability as in (B). (F) Immunoblot analysis of effect of ANT2 shRNA on TRAIL sensitivity. MCF7 cells were transfected with scrambled or ANT2 shRNA, treated with TRAIL for 12 h as in (A). Cell extracts were prepared, subjected to SDS-PAGE, and immunoblotted with antibodies as indicated.

Figure 2

Effect of ANT2 shRNA on expression of TRAIL receptors in breast cancer cell lines. TRAIL receptor expression in MCF7 and MDA-MB-231 cells (A), MCF7 cells transfected with scrambled or ANT2 shRNA for 24 h (B), and T47 D and BT474 cells transfected with scrambled or ANT2 shRNA for 24 h (C) was analyzed by SDS-PAGE followed by immunoblotting of cell extracts with antibodies against DR4, DR5, DcR1, DcR2, and β-actin (control) as indicated.

Figure 3

Up-regulation of DR4 and DR5 expression by ANT2 shRNA-induced activation of p53 in MCF7 cells. (A) Immunoblot analysis of p53 expression and phosphorylation. MCF7 cells were transfected with scrambled shRNA or ANT2 shRNA. After 24 h, cells extracts were prepared, subjected to SDS-PAGE, and immunoblotted with antibodies against phospho-p53, p53, α-tubulin, ANT2, and β-actin (control). Band intensities were calculated using Image Gauge v4.0 (Fujifilm) (B) Reporter gene analysis of p53 transcriptional activity. MCF7 cells were transfected with a p53-luciferase-reporter construct and scrambled or ANT2 shRNA. After 18 h, cell lysates were prepared and analyzed for luciferase activities using a luminometer. (C) Immunoblot analysis of p53-mediated up-regulation of DR4/DR5. Cells were pre-treated with the p53 inhibitor pifithrin-α for 2 h and then transfected with scrambled or ANT2 shRNA. After 24 h, cell lysates were prepared, subjected to SDS-PAGE, and immunoblotted with antibodies against DR4, DR5, and α-tubulin. (D) Cells were treated with pifithrin-α and scrambled or ANT2 shRNA as in (C) and then analyzed for cell viability using a CCK8 assay kit.

Figure 4

Transfection of ANT2 shRNA leads to JNK activation and subsequent up-regulation and activation of p53 in MCF7 cells. (A) Analysis of ATP levels in lysates of MCF7 cells pre-treated with the JNK inhibitor SP600125 for 2 h and then transfected with scrambled or ANT2 shRNA for 24 h. The data are shown in relative luminescence units (RLU) calculated by normalizing total intracellular ATP to total protein. (B) MCF7 cells treated as in (A) were analyzed for JNK phosphorylation by immunoblotting using anti-phospho JNK and anti-JNK antibodies. (C) Effect of JNK inhibition on ANT2 shRNA-mediated upregulation of DR4 and DR5. MCF7 cells were treated with SP600125 and scrambled or ANT2 shRNA as in (A). Cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against phospho-p53, p53, DR4, DR5, and β-actin (control).

Figure 5

ANT2 knockdown-induced JNK signaling up-regulates DNMT1, leading to methylation of the DcR2 promoter in MCF7 cells. (A) Methylation-specific PCR analysis of effect of ANT2 shRNA on DcR2 genomic DNA of MCF7 cells. After cells were transfected with scrambled or ANT2 shRNA for 24 h, they were lysed, and the genomic DNA was purified. Unmethylated cytosines were converted to uracils using an Invitrogen MethylCode Bisulfite Conversion Kit, and the converted genomic DNA was used for PCR with methylation-specific primers. The cell extracts were also analyzed for ANT2 and β-actin expression by immunoblotting. (B) RT-PCR analysis of the effect of ANT2 shRNA-induced methylation on DcR2 expression. Cells were pre-treated with the methylation inhibitor 5-aza-dC for 2 h and then transfected with scrambled or ANT2 shRNA. (C) RT-PCR analysis of effect of ANT2 shRNA-induced JNK signaling on DNMT1 expression. MCF7 cells were pre-treated with SP600125 for 2 h and then transfected with scrambled or ANT2 shRNA. Cell extracts were also analyzed for DNMT1 and β-actin expression by immunoblotting. (D) Effect of ANT2 shRNA-induced JNK signaling on DNMT1 activity. MCF7 cells were treated as in (C), and nuclear extracts were evaluated for DNMT1 activity. (E) Effect of JNK inhibition on ANT2 shRNA-induced sensitization to TRAIL. MCF7 cells were treated with SP600125 and scrambled or ANT2 shRNA as in (C), and 24 h after transfection, they were treated with recombinant human TRAIL (100 ng/ml) for 12 h. Cell viability was measured using a CCK8 assay kit.

Figure 6

Anti-tumor effects of ANT2 shRNA and TRAIL in a breast cancer xenograft model in vivo. (A) Experimental protocol for tumor challenge and shRNA and TRAIL treatment. BALB/c nude mice were challenged with 5 × 10⁶ MCF7 cells by subcutaneous injection into the right flanks. After 21 days, they began 3 days of treatment with thrice daily intraperitoneal injections of PBS or human recombinant TRAIL with or without thrice daily intratumoral injections of Lipofectamine 2000-supplemented scrambled or ANT2 shRNA vector. Calipers were used to make weekly measurements of tumor dimensions, and tumor volumes were calculated as described in the Methods. Measurements continued until day 45 after tumor challenge. Data were analyzed using the Student t test. Differences are considered statistically significant at P < 0.05. (B) Expression of TRAIL receptors in tumors after shRNA and TRAIL treatment. Tumors were isolated on day 45 after tumor challenge and subjected to RT-PCR using primers specific for human DR4, DR5, DcR2, and GAPDH (internal control).