KLF4 and SOX9 transcription factors antagonize β-catenin and inhibit TCF-activity in cancer cells

Abstract

The transcriptional activator β-catenin is a key mediator of the canonical Wnt signaling pathway. β-catenin itself does not bind DNA but functions via interaction with T-cell factor (TCF)/lymphoid-enhancing factor (LEF) transcription factors. Thus, in the case of active Wnt signaling, β-catenin, in cooperation with TCF/LEF proteins family, activates the expression of a wide variety of genes. To date, the list of established β-catenin interacting targets is far from complete. In this study, we aimed to establish the interaction between β-catenin and transcription factors that might affect TCF activity. We took advantage of EMSA, using TCF as a probe, to screen oligonucleotides known to bind specific transcription factors that might dislodge or antagonize β-catenin/TCF binding. We found that Sox9 and KLF4 antagonize β-catenin/TCF binding in HEK293, A549, SW480, and T47D cells. This inhibition of TCF binding was concentration-dependent and correlated to the in vitro TCF-luciferase functional assays. Overexpression of Sox9 and KLF4 transcription factors in cancer cells shows a concentration-dependent reduction of TCF-luciferase as well as the TCF-binding activities. In addition, we demonstrated that both Sox9 and KLF4 interact with β-catenin in an immunoprecipitation assay and reduce its binding to TCF4. Together, these results demonstrate that Sox9 and KLF4 transcription factors antagonize β-catenin/TCF in cancer cells.

Figure 1

Panel A. Competition of TCF-binding with different consensus oligonucleotide sequences. Nuclear extract proteins were prepared from SW480 colon cancer cells and EMSA were performed as described in Methods. Excess of unlabeled oligonucleotides were preincubated with nuclear protein extracts (10 μg) 30 min before adding TCF-radiolabeled probe. After 30 min incubation in the presence of TCF-radiolabeled probe, proteins were resolved on native polyacrylamide gel. 0: free probe, 1: Control, 2: TCF, 3: CREB, 4: Sp1, 5: Sox9, 6: NF-κB, 7: NFAT, 8: TFIID, 9: AP1, 10: KLF4, 11: SRF, 12: OCT1, 13: AP2, 14: SREB; 15, GATA. Panel B. The reduction of TCF binding by Sox9 and KLF4 oligonucleotides is Concentration dependent. Nuclear extracts prepared from SW480 colon cancer cells were incubated with increasing amounts of TCF, Sox9, and KLF4 cold oligonucleorides for 30 minutes. TCF probe was added and incubation was carried for another 30 minutes before resolving proteins on native polyacrylamide gel. C: control, 1: 10-, 2: 25-, 3: 50-, 4: 100-fold excess in cold probes. Figures are representative of three independent experiments.

Figure 2

TCF does not abolish Sox9 and KLF4 binding. Nuclear protein extracts were prepared from SW480 colon cancer cells and EMSA were performed as described in Methods. Oligonucleotides at increasing concentration (10-, 25-, 50-, or 100-fold excess) were incubated with nuc lear extract proteins (10 μg) for 30 min before adding either radiolabeled KLF4 (Panel A) or Sox9 (Panel B) probes and resolving proteins on native polyacrylamide gel. Figures are representative of three independent experiments.

Figure 3

Sox9 and KLF4 oligonucleotides abolish TCF-binding activity in cancer cells. Nuclear protein extracts (10 μg) were prepared from the indicated cancer cells and gel shift assays were performed as described in Methods. Excess of TCF (lane 2), Sox9 (lane 3), or KLF4 (lane 4) oligonucleotides were preincubated with nuclear extract proteins (10 μg) for 30 min. After adding TCF-radiolabeled probe and additional 30 minutes incubation, proteins were resolved on native polyacylamide gel. The square under the name of each cell represent a western blot for β-catenin expression. The figure is representative of three independent experiments.

Figure 4

Overexpression of Sox9 or KLF4 inhibits both TCF-binding and activity. Panel A, SW480, A549, and T47D cells were co-transfected with either Sox9/TCF-luciferase or KLF4/TCF-luciferase expression vectors. Forty-eight hours later, cells were lysed and luciferase assays were performed as described in Methods. Figures are a representative of three independent experiments performed in triplicates. **p<0.01. Panel B, in a parallel experiment, cells were transfected with either Sox9 or KLF4 expression vectors and 48 hours later nuclear extract proteins (10 μg) were prepared and incubated with TCF-radiolabeled probe for 30 min before resolving proteins on native polyacrylamide gel. 1: control; 2: Sox9; 3: KLF4 expression vectors. The figure is representative of three independent experiments.

Figure 5

Overexpression of Sox9 or KLF4 concentration dependently inhibits TCF-luciferase activity. HEK293, SW480, A549, and T47D cells were co-transfected with either Sox9/TCF-luciferase or KLF4/lTCF-luciferase constructs at concentrations ranging from 0 to 200 ng. Forty eight hours later, luciferase assays were performed as described in methods. The figure is representative of four independent experiments performed in triplicates. *p<0.05, **p<0.01.

Figure 6

Sox9 and KLF4 oligonucleotides decreased TCF-binding activity in HEK293 cells overexpressing β-catenin. Nuclear extract proteins were prepared from non-transfected HEK293 (Panel A) or HEK293 cells transfected with 1 μg of β-catenin expression vector (Panel B) and incubated with excess of TCF, Sox9, or KLF4 oligonucleotides for 30 min. TCF-radiolabeled probe was added and incubated for another 30 min before resolving proteins on native polyacrylamide gel. The Figure is representative of at least three independent experiments.

Figure 7

Panel A and B. Overexpression of KLF4 and Sox9 decreased β-catenin binding to TCF. HEK293 (Panel A) or SW480 (Panel B) cells were transfected with Sox9 (lane 2), KLF4 (lane 3), β-catenin (lane 4), β-catenin/Sox9 (lane 5), β-catenin/KLF4 (lane 6), or β-catenin/Sox9/KLF4 (lane 7) expression vectors (1 μg each). Nuclear extract proteins were prepared 48 hours post transfection and gel shift assays were performed as described in Methods using TCF as a radiolabeled probe. Lane 1 corresponds to control. Panel C. Western Blot showing the overexpression (2) of Sox9, KLF4, and β-catenin in HEK283 and SW480 cells compared to non-transfected cells (1). Panel DE. Sox9 and KLF4 interact with β-catenin. HEK293 cells were transiently cotransfected (2) for 48 hours with Sox9, β-catenin, or KLF4 expression vectors (1 μg each). Immunoprecipitation (IP) was performed 48 hours later with either Sox9 (Panel D) or KLF4 (Panel E) antibodies. After running the precipitate on 10% SDS-PAGE gel and transfer, nitrocellulose membranes were analyzed with anti-β-catenin antibody. Panel F. Sox9 and KLF4 reduce the interaction of β-catenin with TCF4. SW480 cells were transfected with Sox9 or KLF4 expression vectors at different amounts (1: 0 μg, 2: 1 μg, 3: 2 μg) for 48 hours. Nuclear extracts were prepared as mentioned in Material and Methods, Immucoprecipitation was performed using anti-TCF4 antibody (Santa-Cruz) followed with immublot using anti-β-catenin antibody. Results in figure 7F show that overexpression of Sox9 or KLF4 in SW480 results in a decrease of the amount of β-catenin interacting with TCF. Histone H1 was used as a loading control for nuclear extract proteins using anti-Histone H1 antibody (Santa Cruz). The figure is a representative of three independent Experiments.

Figure 7

Panel A and B. Overexpression of KLF4 and Sox9 decreased β-catenin binding to TCF. HEK293 (Panel A) or SW480 (Panel B) cells were transfected with Sox9 (lane 2), KLF4 (lane 3), β-catenin (lane 4), β-catenin/Sox9 (lane 5), β-catenin/KLF4 (lane 6), or β-catenin/Sox9/KLF4 (lane 7) expression vectors (1 μg each). Nuclear extract proteins were prepared 48 hours post transfection and gel shift assays were performed as described in Methods using TCF as a radiolabeled probe. Lane 1 corresponds to control. Panel C. Western Blot showing the overexpression (2) of Sox9, KLF4, and β-catenin in HEK283 and SW480 cells compared to non-transfected cells (1). Panel DE. Sox9 and KLF4 interact with β-catenin. HEK293 cells were transiently cotransfected (2) for 48 hours with Sox9, β-catenin, or KLF4 expression vectors (1 μg each). Immunoprecipitation (IP) was performed 48 hours later with either Sox9 (Panel D) or KLF4 (Panel E) antibodies. After running the precipitate on 10% SDS-PAGE gel and transfer, nitrocellulose membranes were analyzed with anti-β-catenin antibody. Panel F. Sox9 and KLF4 reduce the interaction of β-catenin with TCF4. SW480 cells were transfected with Sox9 or KLF4 expression vectors at different amounts (1: 0 μg, 2: 1 μg, 3: 2 μg) for 48 hours. Nuclear extracts were prepared as mentioned in Material and Methods, Immucoprecipitation was performed using anti-TCF4 antibody (Santa-Cruz) followed with immublot using anti-β-catenin antibody. Results in figure 7F show that overexpression of Sox9 or KLF4 in SW480 results in a decrease of the amount of β-catenin interacting with TCF. Histone H1 was used as a loading control for nuclear extract proteins using anti-Histone H1 antibody (Santa Cruz). The figure is a representative of three independent Experiments.