ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor response to standard therapies and therefore unfavorable clinical outcomes. Better understanding of TNBC and new therapeutic strategies are urgently needed. ROR nuclear receptors are multifunctional transcription factors with important roles in circadian pathways and other processes including immunity and tumorigenesis. Nobiletin (NOB) is a natural compound known to display anticancer effects, and our previous studies showed that NOB activates RORs to enhance circadian rhythms and promote physiological fitness in mice. Here, we identified several TNBC cell lines being sensitive to NOB, by itself or in combination. Cell and xenograft experiments showed that NOB significantly inhibited TNBC cell proliferation and motility in vitro and in vivo. ROR loss- and gain-of-function studies showed concordant effects of the NOB-ROR axis on MDA-MB-231 cell growth. Mechanistically, we found that NOB activates ROR binding to the ROR response elements (RRE) of the IκBα promoter, and NOB strongly inhibited p65 nuclear translocation. Consistent with transcriptomic analysis indicating cancer and NF-κB signaling as major pathways altered by NOB, p65-inducible expression abolished NOB effects, illustrating a requisite role of NF-κB suppression mediating the anti-TNBC effect of NOB. Finally, in vivo mouse xenograft studies showed that NOB enhanced the antitumor efficacy in mammary fat pad implanted TNBC, as a single agent or in combination with the chemotherapy agent Docetaxel. Together, our study highlights an anti-TNBC mechanism of ROR-NOB via suppression of NF-κB signaling, suggesting novel preventive and chemotherapeutic strategies against this devastating disease.

Fig. 1. NOB suppress cell survival and motility of TNBC.

A TNBC cell lines (MDA-MB-231, BT549, and MDA-MB-468) and MCF10A were treated for 24, 48, and 72 h with 10 µM NOB or 0.1% DMSO as control. Cell proliferation was determined by WST-1 assays. Data represent mean ± SEM. Two-tailed Student’s t-test shows significant statistical difference between DMSO and NOB. *p < 0.05, **p < 0.01, and ***p < 0.001. B MBA-MB-231 cells were treated with various concentrations of NOB (0–30 µM) as indicated for 48 h and subjected to WST-1 assays. Data represent mean ± SEM. Two-tailed Student’s t-test, *p < 0.05 and **p < 0.01 vs control. C NOB suppressed MDA-MB-231 colony formation. Right panel: quantification. Data represent mean ± SEM. Two-tailed Student’s t-test, **p < 0.01. D NOB suppressed MDA-MB-468 colony formation. Right panel: quantification. Data represent mean ± SEM. Student t-test, **p < 0.01. E Wound-healing assay. The results are expressed as the percentage of motility compared with control cells at 0 h (100%). Data are shown as representative images or mean ± SEM from three independent experiments. Two-tailed Student’s t-test, **p < 0.01. Scale bar = 100 µm. F Wound-healing assays as above. Data represent mean ± SEM. Student's t-test, **p < 0.01, and ***p < 0.001. Scale bar = 277.3 μm. G Athymic nude mice were implanted with MDA-MB-231 by mammary fat pad injection. Tumor volume was measured regularly after treatment and the data shown are the mean tumor volumes ± SEM (n = 5/group). Two-tailed Student’s t-test, *p < 0.05; Ctrl vs NOB.

Fig. 2. ROR-dependent cell growth is inhibited by NOB.

A WT and derivative MDA-MB-231 cells with RORA or RORC knockdown were treated with 10 μM NOB for 24, 48, and 72 h. The data represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparison, *p < 0.05; **p < 0.01. B IκBα protein expression in the above ROR knockdown cells treated with DMSO or 10 µM NOB for 24 h. Right panel: quantification from four experiments. Data represent mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test, *p < 0.05, and two-tailed Student’s t-test, ^#p < 0.05 WT.DMSO vs sgRORC.DMSO. C MDA-MB-231 cells were treated with NOB or Doxorubicin (DOX) for 24 h and then stained with TUNEL and DAPI. Two-tailed Student’s t-test, *p < 0.05. D MDA-MB-231 cells were treated with NOB (10 or 20 µM) or DOX, and apoptosis was examined by detecting caspase-3/7 activity. Data represent the mean ± SEM of three experiments. Two-tailed Student’s t-test, *p < 0.05. E MDA-MB-231 cells were transfected with control siRNA, siRORA, or siRORC and treated with 10 μM NOB for 24 h. Right panel: quantification data are presented as the percentage of the corresponding phase of the cells and the mean ± SEM. Two-tailed Student’s t-test, ***p < 0.001.

Fig. 3. Combination of NOB and ROR ectopic expression in MDA-MB-231 cells suppresses cell growth and motility.

A WST-1 assays. Mock indicates P3Xflag-CMV10-Neo^r (Empty vector). Data represent mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test showed significant difference. ***p < 0.001. B Wound healing assays showed retarded motility in RORA- or RORC-expressing MDA-MB-231 cells. The results were expressed as the percentage of the motility of the control cells (100%, upper panel) (×100 magnification, scale bar = 277.3 μm). Data represent mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons, *p < 0.05 and ***p < 0.001. C NOB and RORA/C expression in MDA-MB-231 showed a combination effect in colony formation assays. Right panel: quantification. Data represent mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test, **p < 0.01 and ***p < 0.001. Interaction between RORA/C expression and NOB, p = 0.0026 via two-way ANOVA.

Fig. 4. The NF-κB pathway is a direct target of the NOB–RORs axis.

A MDA-MB-231 cells were treated with NOB. IkBα mRNA expression was measured by qRT-PCR. Data represent mean ± SEM. Two-tailed Student’s t-test, *p < 0.05. B MDA-MB-231 cells were treated with NOB for 24 h. IkBα protein expression was measured by western blotting. In all, 4.4-fold change, p < 0.01 compared to DMSO. C Schematic representation of putative RRE sites found in the IκBα promoter (TRANSFAC). The distance in bp from the transcription start site of IkBα gene is shown. MDA-MB-231 cells were treated with 20 µM NOB for 12 h. Samples were normalized to input chromatin and expressed as % input. Error bars represent mean ± SD; n = 2; two-tailed Student’s t-test. *p < 0.05; **p < 0.01. D MDA-MB-231 cells were treated with 10 µM NOB for 12 h prior to TNF-α treatment (10 ng/ml). Whole-cell lysates were subjected to western blot analysis using anti-IκBα, phospho-p65 (Ser536), total p65, and GAPDH antibodies. E MDA-MB-231 cells were transfected with pGL4.32 [luc2P/NF-κB-RE/Hygro] vector and treated with NOB (+, 10 µM; ++, 20 µM; +++, 30 µM) for 12 h prior to TNF-α treatment. Data represent mean ± SEM. Two-tailed Student’s t-test, ***p < 0.001. Compared with the TNF-α control, ^##p < 0.01, ^###p < 0.001. F p65 nuclear localization by TNFα was inhibited by NOB in MDA-MB-231. Representative immunofluorescence images of endogenous p65 in MDA-MB-231 with NOB pretreatment 12 h prior to TNF-α treatment (×400 magnification, scale bar = 10 µm). Two-tailed Student’s t-test, ***p < 0.001. G MDA-MB-231 cells were transfected with control siRNA, siRORA or siRORC and treated 10 µM NOB for 12 h prior to TNF-α treatment. Representative confocal images are shown (×400 magnification, scale bar = 10 µm). Cell extracts were subjected to dual luciferase assays. N nucleus; C cytoplasm.

Fig. 5. p65 is targeted by NOB to inhibit TNBC.

A Diagram indicating the p65-inducible system. B MDA-MB-231 cells were grown to confluence and treated NOB with (20 µM) and tetracycline (100 nM) for 12 h. WST-1 assays were performed at 72 h after NOB treatment. Mock indicates pCW 57.1 vector (Empty template vector). Data represent mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test, **p < 0.01, and ***p < 0.001. C Wound-healing assay. The closure of wounds in MDA-MB-231 cells after 16 h of p65 induction by tetracycline (100 nM). The results are presented as relative motility to the width at 0 h (100%). Data represent mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test, *p < 0.05 and ***p < 0.001. D Colony formation assays were performed with 20 µM NOB. Data represent mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test. **p < 0.01 and ***p < 0.001. E–I RNA-seq analysis. E Volcano plot of 4937 differential expressed genes (DEGs) between DMSO and NOB treatment MDA-MB-231 cells (p < 0.05 and fold change >2). Plots highlighted with red and green represent up- and downregulated genes. Multiple NF-κB pathway genes are marked. F Pie chart of protein-coding genes, LncRNA, and other genes in the DEG set. G Gene ontology analysis of DEGs with DAVID. Top-ranked pathways are presented, including NF-κB signaling indicated with red. H KEGG analysis of DEGs with ClusterProfiler. Top-ranked pathways are presented, including “Pathway in cancer ”. I Gene set enrichment analysis demonstrated the enrichment of gene sets related to cancer.

Fig. 6. Combination studies show the efficacy of NOB with DTX or CAR in TNBC cells.

A MDA-MB-231 cells were treated with the NOB and/or DTX for 24, 48, and 72 h and cell proliferation was determined by WST-1 assays. Data represent mean ± SEM. One-way ANOVA with Tukey’s post hoc test showed significant difference compared to DMSO, ***p < 0.001, compared to DTX, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001. Interaction, p < 0.01 via two-way ANOVA. B MDA-MB-231 cells were treated with NOB and/or CAR for 24, 48, and 72 h and cell proliferation was determined by WST-1 assay. Data represent mean ± SEM. Two-tailed Student’s t-test showed significant difference compared to DMSO, ***p < 0.001, compared to CAR 10 µM, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001. C MDA-MB-468 cells were treated with NOB (100 µM) and/or DTX (5 nM) for 48 h and cell death was determined by FACS analysis. Left panel; Representative images of flow cytometric analysis; right panel; quantification. Data represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test showed significant difference compared to DMSO, ***p < 0.001, NOB, ^###p < 0.001, and compared to DTX, ^†††p < 0.001 (interaction, p < 0.001 via two-way ANOVA). Student t-test compared to DMSO, ^§p < 0.05, ^§§p < 0.01. D DB7 cells were treated with NOB (20, 40, and 80 µM) and/or DTX (2.5 nM) for 24 and 72 h and cell proliferation was determined by WST-1 assays. Data represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test showed significant difference compared to DMSO, **p < 0.01; ****p < 0.0001.

Fig. 7. NOB suppresses tumorigenesis in MDA-MB-231 xenograft mice.

A DTX and NOB showed synergistic inhibitory effects in MDA-MB-231 xenograft tumor growth (n = 6 for Ctrl and NOB, n = 7 for DTX.Ctrl, and n = 8 for DTX.NOB). The black arrows show DTX injection time point (18, 25, 32, 39 days). Data represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test showed significant difference compared to Ctrl, *p < 0.05, **p < 0.01, and ***p < 0.001, compared with NOB, ^#p < 0.05 and ^##p < 0.01, and compared with DTX.Ctrl, ^†p < 0.05, CI = 1.22. B TNF-α levels in plasma (left panel) and tumor (right panel) of MDA-MB-231 xenograft mice model. Data represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test showed significant difference compared to Ctrl, *p < 0.05, **p < 0.01, and ***p < 0.001 and compared to DTX.Ctrl, ^†p < 0.05. C Representative immunofluorescence images of the Ki67 (pink) and DAPI (blue) (×400 magnification, scale bar = 69.3 μm). Quantitative analysis of the percentage of Ki67 immunoreactive area. Data represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05 and **p < 0.01. D Representative immunofluorescence images of the p-p65 (pink) and DAPI (blue) staining from representative images of the p-p65 (red) and DAPI (blue) immunofluorescence in the lesion area (×400 magnification, scale bar = 69.3 μm). Quantitative analysis of the percentage of p-p65 immunoreactive area. Data represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test showed significant difference compared to Ctrl, ***p < 0.001 and compared to DTX.Ctrl, ^†p < 0.05.