Interleukin enhancer-binding factor 3/NF110 is a target of YM155, a suppressant of survivin

Abstract

Survivin is responsible for cancer progression and drug resistance in many types of cancer. YM155 selectively suppresses the expression of survivin and induces apoptosis in cancer cells in vitro and in vivo. However, the mechanism underlying these effects of YM155 is unknown. Here, we show that a transcription factor, interleukin enhancer-binding factor 3 (ILF3)/NF110, is a direct binding target of YM155. The enhanced survivin promoter activity by overexpression of ILF3/NF110 was attenuated by YM155 in a concentration-dependent manner, suggesting that ILF3/NF110 is the physiological target through which YM155 mediates survivin suppression. The results also show that the unique C-terminal region of ILF3/NF110 is important for promoting survivin expression and for high affinity binding to YM155.

Fig. 1.

YM155 probe used for affinity purification. The figure shows the chemical structure and activity of YM155 and YM155 probe. In vitro activities are shown as growth inhibitory concentrations (GI₅₀) of PC-3 cells treated with YM155 or YM155 probe for 24 h and inhibitory concentrations (IC₅₀) determined in endogenous survivin mRNA expression and survivin promoter reporter assays using HEK-293 cells treated with YM155 or YM155 probe for 24 h.

Fig. 2.

Identification of proteins binding to the YM155 probe. a, elution profile of proteins labeled with the YM155 probe. Cell lysates of human hormone-refractory prostate cancer (PC-3), non-small cell lung cancer (Calu-6), and uterine cervix (HeLa) cell lines were used for affinity purification. The SDS-PAGE profile was visualized by silver staining. The proteins (∼30 kDa) purified using beads with or without the YM155 probe were identified as fragments of keratin 1 (NP_006112) and keratin 10 (NP_000412). b, Western blotting analysis of the proteins purified from the subcellular extracts of PC-3 cells. ILF3 was detected as two bands in the input lane of the nuclear extract. The upper and lower bands correspond to ILF3/NF110 and ILF3/NF90, respectively. The validity of the subcellular extracts is demonstrated in supplemental Fig. 2.

Fig. 3.

Pulldown assay using ILF3 isoforms and deletion mutants. a, the structure of ILF3 showing the predicted binding site of YM155. The ΔC fragment contains an ILF2 homology domain and two double-stranded RNA binding motifs (dsRBM). The ΔN fragment has an RGG motif and a GQSY-rich domain. b, pulldown assay using ILF3 isoforms. The amino acid sequences of the isoforms are shown in supplemental Fig. 3. The isolated proteins from YM155 beads were detected by Western blotting using an antibody against the FLAG tag. c, pulldown assay using ILF3 deletion mutants. Wild-type (WT), ΔC, and ΔN consist of Met¹–Arg⁸⁹⁴, Met¹–Val⁶⁰⁸, and Arg⁶⁰⁹–Arg⁸⁹⁴ residues of ILF3/NF110 (NP_036350), respectively.

Fig. 4.

Enhancement of survivin expression by ILF3/NF110 overexpression. a, promoter activities were examined using various lengths of the survivin gene promoter region in HEK-293 cells. Luciferase activity was measured in the presence of ILF3–1a (white) or control plasmid (black). The firefly luciferase enzyme activity was normalized to Renilla luciferase enzyme activity. The values are the means ± S.D. from three independent experiments. b, promoter activities of D3 containing −109 to +16 bp relative to the transcription start site were examined at different concentrations of YM155 in the presence of ILF3–1a or control plasmid in HEK-293 cells. c, the promoter activities of D3 (−109 to +16 bp) were examined in the presence of ILF3–1a, -2a, or -3a or control plasmid in HEK-293 cells. Comparable protein expression levels following overexpression were confirmed by Western blotting (supplemental Fig. 4).

Fig. 5.

Inhibition of survivin expression by ILF3/NF110 knockdown. a, PC-3 cells were treated with the indicated siRNAs. The mRNA levels of survivin were measured by real time RT-PCR (n = 3). The values are the means ± S.D. The expression levels of survivin mRNA are normalized to those of GAPDH and expressed relative to those cells receiving negative siRNA treatment. b, protein expression levels following siRNA treatment were confirmed by Western blotting.