Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner

Abstract

ID1 (inhibitor of differentiation/DNA binding 1) acts an important role in metastasis, tumorigenesis, and maintenance of cell viability. It has been shown that the up-regulation of ID1 is correlated with poor prognosis and the resistance to chemotherapy of human cancers. However, the underlying molecular mechanism remains elusive. Here, we determined for the first time that up-regulating ID1 upon etoposide activation was mediated through AP-1 binding sites within theID1promoter and confirmed that ID1 enhanced cell resistance to DNA damage-induced apoptosis in esophageal squamous cell carcinoma cells. Ablation of c-Jun/c-Fos or ID1 expression enhanced etoposide-mediated apoptosis through increasing activity of caspase 3 and PARP cleavage. Moreover, c-Jun/c-Fos and ID1 were positively correlated in human cancers. More importantly, simultaneous high expression of ID1 and c-Jun or c-Fos was correlated with poor survival in cancer patients. Collectively, we demonstrate the importance of c-Jun/c-Fos-ID1 signaling pathway in chemoresistance of esophageal cancer cells and provide considerable insight into understanding the underlying molecular mechanisms in esophageal squamous cell carcinoma cell biology.

Keywords: AP-1 transcription factor (AP-1); ID1; apoptosis; c-Fos; c-Jun; chemoresistance; etoposide; promoter.

FIGURE 1.

ID1 expression was induced by etoposide in ESCC cell lines. A, up-regulated ID1 mRNA level was detected in 34 tumors compared with normal adjacent epithelia by qRT-PCR (paired t test). B, an example case showed the expression of ID1 in ESCC tumors and normal counterparts by immunohistochemistry staining on the tissue microarray (upper panels). Quantitative analysis of the ID1 staining between ESCC tissues and the matched normal esophageal epithelia is shown in the lower panel (paired t test). C, mRNA and protein level of endogenous ID1 was detected in ESCC cell lines by qRT-PCR (left panel) and Western blot (right panel). D, KYSE140, KYSE150, and KYSE450 cells were treated with 10 μm etoposide for the indicated time and harvested. ID1 expression was determined by qRT-PCR (upper panels) and Western blot (lower panels). β-Actin was used as a loading control. The data are shown as means ± S.E. from multiple independent experiments, one-way analysis of variance test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

Overexpression of ID1 enhances cellular resistance to etoposide. A, KYSE150, KYSE140, KYSE450, and KYSE180 cells were treated with increasing concentrations of etoposide for 48 h, and then cell viability was examined by MTS assay. B, KYSE150 and KYSE450 cells stably transfected with empty vector (pLVX) or ID1 (pLVX-ID1) were incubated with DMSO (control) or etoposide (10 μm) for the indicated time, and cell growth was detected using MTS assay. The values are the means ± S.D. of absorbance at 490 nm for three independent experiments. C and D, ID1 transfectants and empty vector controls were treated with 10 μm etoposide for 48 h and then subjected to annexin V-FITC and propidium iodide (PI) staining. The values are expressed as percentages of annexin V-positive versus total cells (C). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (D). β-Actin was used as a loading control. E and F, KYSE450 cells were transiently transfected with negative control (NC) or ID1 siRNA, followed by 10 μm etoposide treatment. The cells were labeled with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The values are expressed as percentages of annexin V-positive versus total cells (E). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (F). β-Actin was used as a loading control. The data are expressed as means ± S.D. *, p < 0.05; **, p < 0.01, one-way analysis of variance test.

FIGURE 3.

Up-regulating ID1 upon etoposide activation is mediated through AP-1 binding sites. A, KYSE450 cells were transiently transfected with the promoter construct of ID1 for 24 h and treated with 10 or 20 μm etoposide. After 24 h, the luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. B, comparison of nucleotide sequences among seven different species. The AP-1 DNA binding site is represented with a shaded box. * indicates the same nucleotide sequence. C, schematic representation depicts the location of the mutant variant in the 2-kb ID1 promoter. TSS stands for transcription start site (upper panel). KYSE450 cells were cotransfected with ID1 wild-type or mutant luciferase reporters, together with c-Jun/c-Fos or control vector for 24 h. Then luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. The data are shown as means ± S.E. from multiple independent experiments (lower panel). D, KYSE450 cells were transfected with ID1 promoter reporter construct containing either wild-type or mutant putative AP-1 binding site and treated with or without 10 μm etoposide, and then the luciferase activity was determined. E, KYSE450 cells were treated with 10 μm etoposide for 4 h, and then ChIP assays were carried out with antibody against c-Jun, c-Fos, or IgG. The percentages of input of coprecipitating DNAs were calculated by qRT-PCR. The data represent the means ± S.D. of triplicate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

FIGURE 4.

The activation of ID1 required c-Jun/c-Fos in response to etoposide. A and B, KYSE450 cells were treated with 10 μm etoposide for the indicated time, and the expression of c-Jun/c-Fos and ID1 was determined by qRT-PCR (A) and Western blot (B). C, KYSE450 cells were transiently transfected with c-Jun, c-Fos, c-Jun/c-Fos, and TAM67 as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by qRT-PCR and Western blot. D, KYSE450 and KYSE150 cells were transiently transfected with c-Jun/c-Fos siRNA as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by Western blot. The data represent the means ± S.D. of triplicate experiments. NC, negative control. **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

FIGURE 5.

ID1 inhibits etoposide-induced cell apoptosis in a c-Jun/c-Fos-dependent manner. A, KYSE450 cells were transiently transfected with either negative control (NC) or c-Jun/c-Fos siRNA as indicated. After 24 h, cells were incubated with DMSO or 10 μm etoposide. The expression of c-Jun, c-Fos, ID1, p53, cleaved caspase 3, and PARP was determined by Western blot. β-Actin was used as a loading control. B, KYSE450 cells were transiently transfected with c-Jun/c-Fos siRNA and rescued ID1 with pLVX-ID1 compared with pLVX for 24 h. After that, cells were treated with DMSO or 10 μm etoposide and then subjected to Annexin V-FITC and propidium iodide (PI) staining. The values are expressed as a percentage of annexin V-positive versus total cells. The data are expressed as means ± S.D. *, p < 0.05, one-way analysis of variance test.

FIGURE 6.

Positive correlation between c-Jun/c-Fos and ID1 in human cancers and prognostic value of high c-Jun/c-Fos and ID1 expression for cancer patients survive. A, a statistically significant positive correlation between c-Jun/c-Fos and ID1 mRNA was observed by Pearson's method in ESCC and patients in three independent published data sets including acute myeloid leukemia (GSE12417), ovarian cancer (GSE49997), and colorectal cancer (GSE24551), Pearson correlation analysis. B, clinical outcome data were analyzed by using PROGgeneV2 from published studies for correlations between c-Jun/c-Fos-ID1 expression levels and survival of cancer patients.