Blocking PPARγ interaction facilitates Nur77 interdiction of fatty acid uptake and suppresses breast cancer progression

Abstract

Nuclear receptor Nur77 participates in multiple metabolic regulations and plays paradoxical roles in tumorigeneses. Herein, we demonstrated that the knockout of Nur77 stimulated mammary tumor development in two mouse models, which would be reversed by a specific reexpression of Nur77 in mammary tissues. Mechanistically, Nur77 interacted and recruited corepressors, the SWI/SNF complex, to the promoters of CD36 and FABP4 to suppress their transcriptions, which hampered the fatty acid uptake, leading to the inhibition of cell proliferation. Peroxisome proliferator-activated receptor-γ (PPARγ) played an antagonistic role in this process through binding to Nur77 to facilitate ubiquitin ligase Trim13-mediated ubiquitination and degradation of Nur77. Cocrystallographic and functional analysis revealed that Csn-B, a Nur77-targeting compound, promoted the formation of Nur77 homodimer to prevent PPARγ binding by steric hindrance, thereby strengthening the Nur77's inhibitory role in breast cancer. Therefore, our study reveals a regulatory function of Nur77 in breast cancer via impeding fatty acid uptake.

Keywords: breast cancer; cytosporone-B; fatty acid uptake; nuclear receptors Nur77 and PPARγ; ubiquitination.

Fig. 1.

Nur77 plays an inhibitory effect on breast cancer progression. (A, C, and E, Upper), Kaplan–Meier plots for mammary tumor-free survival of WT (n = 35) and Nur77 KO (n = 29) female mice in MMTV-PyMT mouse model (A), WT (n = 25) and Nur77 KO (n = 24) female mice in MPA/DMBA mouse model (C), or PyMT-KO;LSL-Nur77 (n = 22) and PyMT-KO;LSL-Nur77;MMTV-Cre (n = 19) female mice that reexpressing Nur77 in mammary tissue of Nur77 knockout MMTV-PyMT mouse model (E). (Lower) Representative images of mammary glands and the total tumor weights in MMTV-PyMT (12 wk; n = 17 for WT, n = 15 for KO), MPA/DMBA (13 wk; n = 25 for WT, n = 24 for KO), and PyMT-KO;LSL-Nur77;MMTV-Cre female mouse models (13 wk; n = 19 for PyMT-KO;LSL-Nur77, n = 18 for PyMT-KO;LSL-Nur77;MMTV-Cre). (B, D, and F) Expression of Nur77 in mammary tissue from normal mice and tumor tissues from the above three mouse models, detected by immunohistochemistry. Data are presented as mean ± SEM **P < 0.01, ***P < 0.001.

Fig. 2.

Blocking lipid uptake by Nur77 contributes to growth inhibition of breast cancer cells. (A) Evaluation of the proliferative status by Ki67 IHC staining in mammary tumors from MMTV-PyMT (8 wk), MPA/DMBA (13 wk), and PyMT-KO;LSL-Nur77;MMTV-Cre (13 wk) mouse models. (B) Lipid droplets indicated by Bodipy 493/503 in Ki67⁺ cells. Tumor samples were from PyMT mouse model. (C, D, F, and G) Comparison of cell proliferation (C and F) and lipid-droplet contents (D and G) in primary mammary tumor cells from WT or Nur77 KO female mice in PyMT mouse model (n = 3). Cells were cultured in the medium containing 0.5% lipoprotein-deficient serum (LPDS) with or without adipocyte coculture (C and D), or lipid mixture addition (F and G) for the indicated times (C and F) or 24 h (D and G). (E and H) Analysis of lipid content (E, Left, and H) and fatty acid uptake (E, Center and Right) in primary cells from mammary tumors of PyMT mice. Bodipy FL C16 or [9,10-³H] palmitate acid was employed to evaluate fatty acid uptake. Coculture conditions were the same as above. DAPI was used to stain the nucleus. Data are presented as the mean ± SEM of two or three independent experiments. **P < 0.01, ***P < 0.001, ns: not significant.

Fig. 3.

Nur77 down-regulates CD36 and FABP4 to block fatty acid absorption. (A) The expression of genes involved in lipids metabolism. GSEA of lipid and organic acids transport-related (Upper and Middle) and lipids metabolism-related gene signatures (Lower) in PyMT-WT versus PyMT-KO primary tumor cells. Heatmap illustrates the most obvious genes inhibited by Nur77 in each gene set. FDR, false-discovery rate; NES, normalized enrichment score. (B) The expression levels of different genes in Nur77 knockdown MCF-7 cells. (C and D) Detection of fatty acid uptake by Bodipy FL C16 (C, Upper Left) or [9,10-³H] palmitate acid (C, Upper Right), lipid droplets accumulation (C, Lower), and cell proliferation (D) in MCF-7 cells. CD36 and FABP4 were separately or simultaneously knocked down as indicated, and Nur77 was then knocked down in the cells. Cells were cultured in medium with lipid mixture (D). (E) The mRNA (Left) and protein (Right) levels of CD36 or FABP4 in primary cells from mammary tumors of PyMT mice. (F and G) The protein expression levels of CD36 and FABP4 in mammary tumor samples of PyMT mice, detected by Western blotting (F, n = 6) and IHC (G). Tubulin was used to determine the loading of total proteins. All data, except A, E (Right), F, and G, are presented as the mean ± SEM of two or three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

Fig. 4.

Nur77 recruits corepressors to inhibit gene expression of CD36 and FABP4. (A and C) Effect of Nur77 on transcription activities of different truncations (A) and mutations (C) of CD36 and FABP4 promoters, detected by luciferase assay. (B) The occupations of Nur77 on the different NBRE-L sequences in the promoters of CD36 and FABP4, detected by ChIP-qPCR assay. (D) The interaction of Nur77 with different corepressors, detected by Co-IP assay. (E and F) The occupations of different corepressors on the different NBRE-L sequences in the promoters of CD36 (E) and FABP4 (F) in control or Nur77 knockdown MCF-7 cells, detected by ChIP-qPCR assay. (G) Histone acetylation levels at the NBRE-L sequences in the promoters of CD36 (Upper) and FABP4 (Lower) in control or Nur77 knockdown MCF-7 cells, detected by ChIP-qPCR assay. (H and I) Effects of different corepressors on Nur77-inhibited promoter activities (H) and gene-expression levels (I) of CD36 and FABP4. Different corepressors were first knocked down, and then Nur77 was transfected into MCF-7 cells. All data, except D, are presented as the mean ± SEM of two or three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

Fig. 5.

PPARγ induces Nur77 degradation through ubiquitination pathway. (A) The level of PPARγ in mammary tumor samples of PyMT mice detected by IHC. Samples were the same as those in Fig. 1B. (B) Endogenous Nur77 expression levels in control, PPARγ transfection (Left) or knockdown (Right) MCF-7 cells. (C) The stability of Nur77 detected in the endogenous (Upper) or transfection case (Lower). MCF-7 cells were transfected with different plasmids and treated with CHX (100 μg/mL) at indicated times. (D) The ubiquitination of endogenous Nur77 in control or PPARγ knockdown MCF-7 cells. (E) Comparison of PPARγ effect on the ubiquitination of Nur77 and Nur77^K536R. Different plasmids as indicated were transfected into MCF-7 cells. (F) Effect of PPARγ on the endogenous Nur77–Trim13 interaction, determined in PPARγ knockdown (Left) or overexpressing (Right) MCF-7 cells. (G and H) Effect of Trim13 on PPARγ-induced endogenous Nur77 ubiquitination (G) and CD36 and FABP4 expression levels (H). Trim13 was knocked down first, and PPARγ were transfected into MCF-7 cells. (I–K) Effects of PPARγ ΔDBD on Nur77–Trim13 interaction (I), Nur77 ubiquitination (J), and endogenous Nur77 expression level (K) in MCF-7 cells. PPARγ was used as a positive control. Tubulin was used to determine the loading of total proteins.

Fig. 6.

Csn-B inhibits fatty acid uptake through blocking PPARγ–Nur77 interaction. Csn-B at indicated concentrations or 10 μM was used to treat MCF-7 cells for 24 h, unless specially defined. (A) Structure of Csn-B. (B–E) Effects of Csn-B on lipid accumulation (B, Upper), fatty acid uptake (Lower), transcriptional activity (C, Left) and mRNA levels (Right) of CD36 and FABP4, lipid content (D), and cell proliferation (E) in control or Nur77 knockdown MCF-7 cells. Cells were cultured with lipid mixture (D and E). (F and G) Effects of Csn-B on fatty acids uptake (F) and cell proliferation (G) in primary tumor cells from mammary tumors of PyMT mice. Cells were cultured with lipid mixture (G). (H and I) Effects of Csn-B on the endogenous PPARγ–Nur77 interaction (H) and PPARγ-induced endogenous Nur77 ubiquitination (I). PPARγ was knocked down in MCF-7 cells. (J and K) Effect of Csn-B on expression levels of endogenous Nur77 (J), CD36 and FABP4 (K). Tubulin was used to determine the loading of total proteins. Data are presented as the mean ± SEM of two or three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

Fig. 7.

Csn-B functions by binding at the Nur77 dimer interface. (A) Csn-B bridges the two LBDs. The two Nur77 monomers, Mol I and Mol II, are with different conformations at the crystallographic asymmetric unit. The Nur77 residues involved in the interaction are labeled and surface changes around the binding site are colored in red for negative charges and in blue for positive charges. (B) Effects of Csn-B on the interaction of the Nur77–Nur77 homodimer and Nur77–PPARγ heterodimer. Plasmids of Nur77 with different tags and PPARγ were transfected into MCF-7 cells. (C) Csn-B promoted endogenous Nur77 dimerization in MCF-7 cells, detected by the cross-linking reactions. (D) Molecular docking shows the preferential PPARγ DBD (red) interaction with LBD Molecule I (cyan). The place of Mol II (gray) would be clash with PPARγ DBD, indicating that the formations of LBD homodimer and Nur77–PPARγ heterodimer are mutually exclusive. (E) Critical Nur77 residues for Csn-B binding. Residues D481, Q571, and R572 are at the Csn-B binding site (Left). Mutation of D481A and Q571A abolished hydrogen bonds between Csn-B and Nur77 while R572W (spheres in yellow) produced space constraints for Csn-B binding (Right). (F and G) Csn-B binds to Nur77 LBD (F, Left) but not LBD mutant (F, Right). Fluorescence spectra of His-LBD or LBD mutant was obtained in the absence or presence of increasing amounts of Csn-B. GST was used as inner filter controls (F). The ΔTm value of Csn-B binding to Nur77 LBD and LBD mutant with different Csn-B concentrations (0 to 1 mM) in a thermal-shift assay (G). (H) Effect of Csn-B on the Nur77 LBD dimerization. The in vitro cross-linking reactions were performed. (I and J) Effects of Csn-B on the interaction of PPARγ–Nur77 mutant (I), lipid droplet accumulation, and fatty acid uptake (J) in MCF-7 cells. Nur77 was knocked down first, and the siRNA-resistant form of Nur77 or Nur77 mutant was reintroduced into the cells. Cells were cultured in the medium with or with lipid mixture addition. Data are presented as the mean ± SEM of two or three independent experiments. ***P < 0.001, ns: not significant.

Fig. 8.

Roles of Csn-B in repression of breast cancer progression. (A–D) Effects of intravenous injection of Csn-B (10 mg/kg) on mammary tumor development in PyMT (A and B, female, 12 wk, n = 17 for WT, n = 17 for KO, n = 18 for WT-Csn-B, and n = 20 for KO-Csn-B) or MPA/DMPA mammary tumor models (C and D, female, 13 wk, n = 20 for WT, n = 21 for KO, n = 22 for WT-Csn-B, and n = 24 for KO-Csn-B). (E and F) Effects of Csn-B on the expression of Nur77 (E), CD36 and FABP4 (F) in mammary tumor tissues of PyMT or MPA/DMPA mouse models. Samples were from above. (G and H) Comparison of expression levels of Nur77 (n = 71), PPARγ (n = 72), CD36 (n = 87), and FABP4 (n = 83) between clinical carcinoma samples and their paired paracarcinoma samples (G), or different stages of clinical breast cancer samples (H, n = 23 for stage I, n = 75 for stage II, n = 30 for stage III). (I) Kaplan–Meier survival curve shows correlation between overall survival of breast cancer patients and Nur77, PPARγ, CD36, or FABP4 expression levels, respectively. Patients were classified into two group based on the median of the expression value of those genes in the entire population of patients, as defined in SI Appendix, Supplemental Materials and Methods. (J) A working model for this study. Data are presented as the mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.