Interplay between transforming growth factor-β and Nur77 in dual regulations of inhibitor of differentiation 1 for colonic tumorigenesis

Abstract

The paradoxical roles of transforming growth factor-β (TGFβ) signaling and nuclear receptor Nur77 in colon cancer development are known but the underlying mechanisms remain obscure. Inhibitor of differentiation 1 (ID1) is a target gene of TGFβ and a key promoter for colon cancer progression. Here, we show that Nur77 enhances TGFβ/Smad3-induced ID1 mRNA expression through hindering Smurf2-mediated Smad3 mono-ubiquitylation, resulting in ID1 upregulation. In the absence of TGFβ, however, Nur77 destabilizes ID1 protein by promoting Smurf2-mediated ID1 poly-ubiquitylation, resulting in ID1 downregulation. Interestingly, TGFβ stabilizes ID1 protein by switching Nur77 interaction partners to inhibit ID1 ubiquitylation. This also endows TGFβ with an active pro-tumorigenic action in Smad4-deficient colon cancers. Thus, TGFβ converts Nur77's role from destabilizing ID1 protein and cancer inhibition to inducing ID1 mRNA expression and cancer promotion, which is highly relevant to colon cancer stemness, metastasis and oxaliplatin resistance. Our data therefore define the integrated duality of Nur77 and TGFβ signaling in regulating ID1 expression and provide mechanistic insights into the paradoxical roles of TGFβ and Nur77 in colon cancer progression.

Fig. 1. Nur77 transcriptionally upregulates TGFβ-induced ID1 expression through inhibiting Smurf2-mediated mono-ubiquitylation of Smad3.

a, b LS174T cells transfected with the indicated siRNAs for 48 h were treated with TGFβ (10 ng/mL) for the indicated time. ID1 mRNA and protein expressions were examined by qRT-PCR (a) and immunoblotting (IB) (b), respectively. si-ctr control siRNA, si-Nur77 Nur77 siRNA, hr hour. Two-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis, and data are presented as means ± SD (n = 3 biologically independent samples). c–e HCT116 cells were transfected with the indicated expression plasmids for 24 h or siRNAs for 48 h before SB431542 (10 μM) treatment for 1 h. Cells were then treated with TGFβ (10 ng/mL) for 1 h. Protein interactions were examined by co-immunoprecipitation (co-IP) using specific antibodies. IgG control IgG. f–l LS174T cells were transfected with the indicated expression plasmids, siRNAs, or shRNAs before TGFβ (10 ng/mL) treatment for 1 h. Chromatin immunoprecipitations were performed using control IgG or anti-Smad3 antibody followed by PCR (f). Smad3 ubiquitylation was examined by IP and IB with specific antibodies (g, i, k). Protein interactions were examined by co-IP (h, j, l). sh-ctr control shRNA, sh-Nur77 Nur77 shRNA, Ub ubiquitin. Two-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis, and data are presented as means ± SD (n = 4 biologically independent samples). Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 2. Nur77 post-translationally downregulates ID1 through mediating its association with and ubiquitylation by Smurf2.

a, c, f, g SW620 cells were transfected with the indicated expression plasmids, shRNAs, or siRNAs followed by cycloheximide (CHX) (10 μM) treatment for the indicated time. Protein expressions were examined by IB. The anti-ID1 antibody signal normalized relative to the anti-actin signal is expressed as a percentage of that present at the start of the chase. si-ctr control siRNA, si-Nur77 Nur77 siRNA, si-Smurf2 Smurf2 siRNA, sh-ctr control shRNA, sh-Nur77 Nur77 shRNA, hr hour, min minute. b, d, e SW620 cells transfected with the indicated expression plasmids, shRNAs, or siRNAs were treated with MG132 (20 μM) for 2 h. ID1 ubiquitylation was examined by IP with anti-ID1 antibody and IB with anti-ubiquitin (Ub) antibody. LC light chain, HC heavy chain. Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 3. Molecular mechanisms underlying Nur77’s effects on the interactions of Smurf2 with Smad3 and ID1.

a, g Re-co-immunoprecipitation assay to detect the trimeric interaction of Nur77, ID1, and Smurf2 in SW620 cells. The detailed procedure was presented in Supplementary Figure 2b and described in “Methods” section. LC light chain, HC heavy chain. b, c SW620 cells transfected with the indicated expression plasmids or siRNAs were treated with MG132 (20 μM) for 2 h. Protein interactions were examined by co-IP. si-ctr control siRNA, si-Nur77 Nur77 siRNA. d–f Protein interactions were examined in mouse colon tissues (d) and in SW620 cells (e, f). Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 4. TGFβ stabilizes ID1 protein through preventing its Nur77-mediated interaction with and ubiquitylation by Smurf2.

a SW620 cells were treated with TGFβ (10 ng/mL) for the indicated time. IB and qRT-PCR were applied to examine protein and ID1 mRNA expressions, respectively. hr hour. Two-tailed unpaired Student’s t test were used for statistical analysis, and data are presented as means ± SD (n = 3 biologically independent samples). b Cycloheximide (CHX) chase assay to determine ID1 turnover in the presence of TGFβ (10 ng/mL) in SW620 cells. min minute. c–e, g–i SW620 cells were transfected with the indicated expression plasmids, siRNAs, or shRNAs followed by MG132 treatment for 2 h and then TGFβ (10 ng/mL) treatment for 1 h. Protein ubiquitylation was examined by IP using the indicated antibodies and IB using anti-Ubiquitin (Ub) antibody. LC light chain, HC heavy chain, sh-ctr control shRNA, sh-Nur77 Nur77 shRNA. f SW620 cells were transfected with siRNAs before treatment with TGFβ (10 ng/mL) for the indicated times. Protein expressions were examined by IB. si-ctr control siRNA, si-Nur77 Nur77 siRNA. j SW620 cells transfected with the indicated shRNAs were treated with MG132 for 2 h and then TGFβ (10 ng/mL) for 1 h. Co-IP was applied to detect protein interactions. Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 5. TGFβ converts Nur77 role in regulating ID1 expression.

a, b, d, f, h HCT116 cells transfected with the indicated expression plasmids or siRNA were treated with TGFβ (10 ng/mL) for 1 h. Protein interactions were examined by co-IP. si-ctr control siRNA, si-Smad3 Smad3 siRNA. c HCT116 cells transfected with the indicated siRNAs were treated with MG132 for 2 h prior to TGFβ (10 ng/mL) treatment for 1 h. ID1 ubiquitylation was examined. si-Nur77 Nur77 siRNA. e, g LS174T cells were pretreated with flavopiridol (0.25 μM) for 1 h (g), and stimulated by TGFβ (10 ng/mL) for 1 h. Protein interactions were examined by co-IP. Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 6. Pathophysiological relevance of the TGFβ/Nur77/ID1 axis in colon cancer.

a Cells pretreated with MG132 (20 μM) for 2 h were then treated with TGFβ (10 ng/mL) for 1 h. ID1 ubiquitylation and mRNA expression were examined by co-immunoprecipitation and qRT-PCR, respectively. Ub ubiquitin. Two-tailed unpaired Student’s t test was used for statistical analysis, and data are presented as means ± SD (n = 3 biologically independent samples). b Cells were treated with TGFβ (10 ng/mL) for 1 h and protein expression was examined by immunoblotting using the indicated antibodies. c SW620 cells were treated with MG132 for 2 h and then with TGFβ at the indicated doses for 1 h. ID1 ubiquitylation and protein interactions were examined. d, e SW620 (d) and HCT116 (e) cells were treated with TGFβ at the indicated doses for 1 h. IB and qRT-PCR were applied to examine protein and ID1 mRNA expressions, respectively. The phosphorylation of Smad2 and Smad3 were detected using anti-p-Smad2(S465/467) and anti-p-Smad3(S423/425) antibodies, respectively. Two-tailed unpaired Student’s t test was used for statistical analysis, and data are presented as means ± SD (n = 3 biologically independent samples). f, g Immunohistochemistry analysis of clinical colon cancer samples showing correlations of ID1 expression with Nur77 expression and TGFβ signal activity. The green (n = 22) and red (n = 33) dots represented tissue samples with low and high TGFβ signal activity, respectively (f). The green (n = 5) and red (n = 11) dots represented tissue samples with low and high Nur77 expression, respectively (g). Two-tailed correlation analysis was used to indicate correlation (assume data were sampled from Gaussian population). Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 7. Involvement of the TGFβ/Nur77/ID1 axis in colon cancer stemness and metastasis.

a, b LS174T cells transfected with the indicated siRNAs were cultured for 7 days. Cell spheres were observed by microscope. Representative images were shown, and sphere numbers and diameters were counted and measured (a). Scale bars, 100 μm. The expression of stemness markers was determined by qRT-PCR (b). si-ctr control siRNA, si-ID1 ID1 siRNA, si-Nur77 Nur77 siRNA, si-Smurf2 Smurf2 siRNA. Two-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis, and data are presented as means ± SD. (a, top graph, n = 3 biologically independent samples; a, bottom graph, n = 37, 60, 9, 11, 35, 33, 7, 9, 32, 25, 6, 14, respectively; b, n = 5 biologically independent samples). c–f SW620/sh-ctr and SW620/sh-Nur77 cells were injected into spleens of nude mice. Mice were reared for 28 days and then sacrificed for analysis of tumor formation (c), tumor-bearing spleen and liver weight and coefficient (c), hematoxylin and eosin staining of tumor tissues (d), protein interactions and expressions in spleen tumors (e), and Smad3 phosphorylation status in spleen tumors comparing to in vitro SW620 cells treated with TGFβ (f). The white areas indicate tumors formed in spleens and livers (c). Tumor tissues are separated from normal tissues by dotted lines (d). S spleen, L liver, T tumor. Scale bars, 100 μm. Two-tailed unpaired Student’s t test was used for statistical analysis, and data are presented as means ± SD (n = 4 mice per group). Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 8. Involvement of the TGFβ/Nur77/ID1 axis in colon cancer resistance to oxaliplatin.

a–c LS174T cells transfected with siRNAs were treated with the indicated doses of oxaliplatin in the presence or absence of TGFβ (10 ng/mL) for 12 h. Protein expressions were examined by IB. si-ctr control siRNA, si-ID1 ID1 siRNA, si-Nur77 Nur77 siRNA. d–g The indicated LS174T cell lines were inoculated subcutaneously into flanks of nude mice. After 10 days, mice were intraperitoneally injected with oxaliplatin (5 mg/kg) daily. After 12 days, mice were sacrificed for analysis of dissected tumors (d), tumor weights (e), tumor inhibition ratios (f), and protein expressions in tumors (g). Two-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis, and data are presented as means ± SD (n = 5 mice per group). Data represent at least two independent experiments. Source data are provided as Source Data file.

Fig. 9. A working model.

The crosstalk of Nur77 and TGFβ on dual regulations of ID1 expression and the implications in colon cancer progression and oxaliplatin resistance. U ubiquitin, P phosphate.