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Loss of ELF5–FBXW7 stabilizes IFNGR1 to promote the growth and metastasis of triple-negative breast cancer through interferon-γ signalling

Nature Cell Biology volume 22, pages 591–602 (2020)Cite this article

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An Author Correction to this article was published on 04 August 2021

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Abstract

Triple-negative breast cancer (TNBC) is characterized by a high degree of immune infiltrate in the tumour microenvironment, which may influence the fate of TNBC cells. We reveal that loss of the tumour suppressive transcription factor Elf5 in TNBC cells activates intrinsic interferon-γ (IFN-γ) signalling, promoting tumour progression and metastasis. Mechanistically, we find that loss of the Elf5-regulated ubiquitin ligase FBXW7 ensures stabilization of its putative protein substrate IFN-γ receptor 1 (IFNGR1) at the protein level in TNBC. Elf5low tumours show enhanced IFN-γ signalling accompanied by an increase of immunosuppressive neutrophils within the tumour microenvironment and increased programmed death ligand 1 expression. Inactivation of either programmed death ligand 1 or IFNGR1 elicited a robust anti-tumour and/or anti-metastatic effect. A positive correlation between ELF5 and FBXW7 expression and a negative correlation between ELF5, FBXW7 and IFNGR1 expression in the tumours of patients with TNBC strongly suggest that this signalling axis could be exploited for patient stratification and immunotherapeutic treatment strategies for Elf5low patients with TNBC.

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Fig. 1: Loss of Elf5 increases tumour initiation, progression and metastasis in a basal/mesenchymal C3-T+ mouse model.
Fig. 2: Loss of Elf5 promotes IFN-γ signalling in C3-T+ Elf5+/− tumours.
Fig. 3: ELF5 regulates FBXW7-mediated destabilization of IFNGR1 in TNBC.
Fig. 4: Loss of Elf5 alters the immune landscape in C3-T+ tumours.
Fig. 5: Re-expression of Elf5 in EpRas TNBC cells decreases tumour growth and metastasis.
Fig. 6: Antibody-mediated and genetic inhibition of IFNGR1 in TNBC cells hampers tumour growth and metastasis.
Fig. 7: Antibody-mediated depletion of Ly6G+ neutrophils decreases tumour growth and metastasis in spontaneous and orthotopic TNBC models.
Fig. 8: Compared with normal breast cells, tumour cells have lower expression of the proteins ELF5 and FBXW7 and higher expression of IFNGR1.

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Data availability

The GSEA data can be accessed using the accession numbers GSE122090 and GSE122180. Previously published MDA-MB-231 GSEA data can be accessed at GSE32144. The data and/or reagents that support the findings of this study are available from the corresponding author upon reasonable request. Source data for Figs. 1–8 and Extended Data Figs. 1–5 and 7–10 are provided online. Each experiment was repeated independently with similar results.

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Acknowledgements

We thank L. King (University of Pennsylvania) for critical reading of the manuscript and helpful discussions. We thank A. Minn (University of Pennsylvania) for helpful discussions. We thank the Penn Vet Comparative Pathology Core for assistance with embedding, sectioning, consultation and slide evaluation. The Penn Vet Comparative Pathology Core is supported by the Abramson Cancer Center Support Grant (P30 CA016520). We thank Dr. Andres J.P. Klein-Szanto (Fox Chase Cancer Center) for helpful discussions and consultation on primary and metastatic tumour samples. We thank Y. Kang (Princeton University) for the HEK293T, EpRas, 4T1, LM2 and BT549 cell lines and lentiviral vector pLEX-hFL2iG. We thank S. Ran (Southern Illinois University) for the HCC1806 cell line. We thank the Eastern Division of the Cooperative Human Tissue Network at The University of Pennsylvania for providing human breast cancer fixed tissues from patients. We thank A. Welm (University of Utah) for the PDX tumour tissues. We thank the members of the Flow Cytometry Core at the Children’s Hospital of Philadelphia and University of Pennsylvania. We thank the Penn Vet Imaging Core for confocal microscopy. This work was supported by grants from the American Cancer Society, an NCI-K22 grant to R.C. (K22CA193661-01) and an NCI-R01 (R01 CA237243-01A1) grant to R.C.

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  1. These authors contributed equally: Sushil Kumar, Ratnesh Kumar Srivastava.

Authors and Affiliations

  1. Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Snahlata Singh, Sushil Kumar, Ratnesh Kumar Srivastava, Ajeya Nandi, Gatha Thacker, Hemma Murali, Sabrina Kim, Mary Baldeon, Mario Andres Blanco, Serge Y. Fuchs & Rumela Chakrabarti

  2. Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    John Tobias, Rizwan Saffie & Luca Busino

  3. Fels Institute for Cancer Research and Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA

    M. Raza Zaidi

  4. Department of Biochemistry, State University of New York at Buffalo, Buffalo, NY, USA

    Satrajit Sinha

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Contributions

S.Singh and R.C. designed all of the experiments. S.Singh, S.Kumar, R.K.S., A.N., H.M., G.T., S.Kim and M.B. performed all of the experiments. M.A.B. performed all of the clinical and statistical analyses using human breast cancer samples and datasets. J.T. performed the RNA-Seq analysis and generated the GSEA datasets. R.S. and L.B. provided experimental support in the FBXW7-related experiments. S.Sinha provided the Elf5+/− and Elf5–GFP mice. R.M.Z. and S.Y.F. provided the reagents and valuable insights into the work. S.Singh and R.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Rumela Chakrabarti.

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Extended data

Extended Data Fig. 1 C3-T+; Elf5+/- tumor cells show increased tumorigenesis in REAR mice.

a, mRNA expression of Elf5 in tumor cells from C3-T+; Elf5+/- (n = 10) and C3-T+; Elf5+/+ (n = 3) individual tumors. qPCR values were normalized to Gapdh. Experiments were performed thrice with qPCR in technical duplicate. b, H&E staining of tumors shows more mesenchymal cells in C3-T+; Elf5+/- tumors. Images are representative of two independent experiments. c, Quantification of mitotic index in tumor sections stained with H&E (right panel), n= 3, C3-T+; Elf5+/- and n=4, C3-T+; Elf5+/+ individual tumors were used. d, Schematic showing representation of experiment in REAR recipient mice. 225,000 cells/MFP of sorted tumor cells from C3-T+; Elf5+/+ and C3-T+; Elf5+/- tumors were injected into contralateral mammary fat pad of REAR mice REAR mice. C3-T+; Elf5+/- tumor cells formed e, tumors earlier, n=6 tumors/group f, g, with rapid growth and volume at sacrifice, n=5 tumors for C3-T+; Elf5+/- and n=4 tumors for C3-T+; Elf5+/+ group. Log-rank test was used for KM plots to calculate p-value, (C3-T+; Elf5+/+, n=6 tumors; C3-T+; Elf5+/-, n=6 tumors in (e). f, Two-way ANOVA test was used with Bonferroni post hoc test to calculate statistical significance. Data presented as mean ± SEM. h, i, Metastatic nodules visible in lungs of REAR mice injected with C3-T+; Elf5+/- tumor cells, n=6 mice/genotype, p=0.049. Experiment performed once. j, k, H&E images showed increased mesenchymal features and invasive structures in tumors formed from C3-T+; Elf5+/- tumor cells. Images are representative to two independent experiments. (a, c, i) Two-tailed student’s t test was used to calculate statistical significance. Data presented as mean ± SEM. Scale bars, (b) 40 µm, (h), 40 µm, (j) 60 µm, (k) 200 µm. **p < 0.01.

Source data

Extended Data Fig. 2 Hyperplastic mammary glands from C3-T+; Elf5+/- tumors show increased P-STAT1 and P-STAT3 protein levels and Elf5 is predominantly expressed in tumor cells.

a, b, IHC staining indicates increased staining of IFNGR1 and P-STAT1 protein in hyperplastic mammary glands harvested from C3-T+; Elf5+/- compared to C3-T+; Elf5+/+ mice. One representative image is shown from multiple experiments (n=5). Arrows in (b) indicate P-STAT1+ cells. c, d, IHC staining indicates increased staining of P-STAT1 (Ser727) and P-STAT3 (Tyr705) proteins in tumors harvested from C3-T+; Elf5+/- compared to C3-T+; Elf5+/+ mice. n=5 individual tumors were taken in each group and quantification was done using 16 random fields. Two-tailed Mann-Whitney U test was used to calculate statistical significance and data is represented as mean ± SEM in (c) and mean ± SD in (d). e, Immunofluorescence image showing expression of GFP (Elf5) in tumor cells and their absence in stromal cells in tumor tissues obtained from C3-T+; Elf5-GFP. Absence of GFP in stromal cells of C3-T+ was confirmed by immunofluorescence in n=5 tumors. f, Flow cytometry graph showing presence of Elf5 predominantly in epithelial cells in tumors obtained from C3-T+; Elf5-GFP, one representative image shown from multiple experiments (n=5). FOV; Field of view. Scale bar in (a, c, d, e) 40 µm, (b) 20 µm.

Source data

Extended Data Fig. 3 Elf5 negatively correlates with IFN-γ signaling in C3-T+ tumors.

a, Schematic diagram showing generation of C3-T+; Elf5-GFP mice. b, Immunofluorescence images showing Elf5 loss during progression from mammary gland (N) to hyperplasia (H) to tumors. K14 antibody was used to mark basal epithelial cells. Experiment was repeated twice with similar results. c, Histogram showing %GFP+ cells in epithelial enriched tumor cells in C3-T+; Elf5-GFP mice. Tumor cell enriched population was sorted using CD31, CD45 and Tert119 markers following published protocol37. Red line denotes WT tumor cells as control. Experiment was repeated thrice with similar results. d, qPCR analysis of Elf5 expression in GFP+ and GFP- tumor cells sorted from C3-T+; Elf5-GFP mice, n=3. qPCR values were normalized to the housekeeping gene Gapdh. Experiments were performed three times, each with qPCR in technical duplicate, and data presented as the mean ± SD. Two- tailed Student’s t test was used to calculate statistical significance. e-i, GSEA graphs showing high EMT signatures, (e); increased invasive signatures, (f); high expression of signatures associated with stemness, (g); and increased IFN-γ signaling in GFP- (Elf5-) tumor cells, (h-i). n=2 GFP+ and n=4 GFP- samples were used for RNA sequencing and GSEA analysis. Statistical significance was assessed by comparing the ES to enrichment results generated from 1000 random permutations of the gene set to obtain p values (nominal p value) j, Western blot showing high P-STAT1 and STAT1 in GFP- (Elf5-). Cell sorting was performed to isolate tumor cells and multiple independent tumors were pooled to make samples. This is a representative blot and experiment was repeated twice with n=9 tumors. k, Heat map showing increased ISG in GFP- (Elf5-) tumor cells. n=2 GFP+ and n=4 GFP- tumor cell population was used to generate heat map. Scale bar in (b), 20 µm.

Source data

Extended Data Fig. 4 FBXW7, a ubiquitin ligase is a direct target of ELF5.

a, b, GSEA of GFP+ (Elf5+) and GFP- (Elf5-) tumor cells show upregulation of genes involved in proteasomal and ubiquitin mediated degradation in GFP+ (Elf5+) tumor cells as compared to GFP- (Elf5-) tumor cells, n=2 GFP+ and n=4 GFP- individual samples. c, List of ubiquitin ligases differentially expressed in GFP+ (Elf5+) and GFP- (Elf5-) tumor cells. d-g, qPCR analysis shows (d) increased ELF5 (e) increased FBXW7 (f) increased VHL and (g) no change in IFNGR1 mRNA in ELF5 expressing LM2 (lung metastatic derivative of MDA-MB-231) cells, n= independent 4 samples. h, i, Data mining for our published microarray data16 shows (h) decreased FBXW7 (i) and no change in VHL in cells upon transduction of ELF5 plasmid encoding mutation in DNA binding domain of ELF5 in MDA-MB-231 cells16 (n=3 independent biological samples/group). j, Western blot showing high ELF5, high FBXW7 and low IFNGR1 proteins upon re-expression of ELF5 in LM2 cells. k, Western blot images showing positive correlation between FBXW7 and ELF5, and their negative correlation with IFNGR1 in TNBC cells (MX1, LM2, HCC1806, BT549). MCF7 cells was used as positive control. l-n, qPCR of EpRas cells (n=6 independent biological samples/group) with re-expression of ELF5 shows (l) high Elf5 (m) high Fbxw7 (n) and no change in Ifngr1 mRNA levels. o, Expression of ELF5 in EpRas cells increases FBXW7 and decreases IFNGR1 protein levels as shown by western blotting. qPCR values were normalized to the housekeeping gene Gapdh. Experiments were performed three times, each with qPCR in technical duplicate, and data presented as the mean ± SD. Two tailed Student’s t test was used to calculate statistical significance. Please see uncropped WB images in Source data file.

Source data

Extended Data Fig. 5 ELF5 binds to multiple genomic loci of FBXW7 in normal mammary epithelial cells and ELF5 KD MX1 cells show increased expression of IFNGR1.

a, In silico ChIP-seq analysis showing multiple binding sites of ELF5 on genomic loci of FBXW7. Lac1 denotes mammary gland obtained from lactating mouse and P13 denotes mammary gland obtained from pregnant mouse at day 13. Black vertical lines show binding sites. b, FACS analysis showing increase in IFNGR1 protein expression upon stable knockdown of ELF5 in MX1 TNBC cells using multiple shRNAs. Data is representative of two independent experiments. c, d IHC images showing FoxP3 staining in periphery and core of tumor sections obtained from C3-T+; ELF5+/+ and C3-T+; ELF5+/- mice. n=3 samples were used. e, Contour plots and f, g, scatter plots showing increased Ly6G+ neutrophil population in tumors from C3-T+; Elf5+/- mice as compared to C3-T+; Elf5+/+ tumors, n=3, C3-T+; Elf5+/+ tumors and n=5, C3-T+; Elf5+/- individual tumors. h, Suppression of T-cell proliferation confirms that increased myeloid cells in tumors are Gr1+ myeloid cells. Tumor CD45+CD11b+Gr1+ neutrophils were used. Tumor neutrophils (Gr1+ myeloid cells) were enriched using Gr1 enrichment kit following manufacture’s protocol. Data is representative of two independent experiments. (f, g) Mann-Whitney U test was used to calculate statistical significance. Data presented as mean ± SEM. Scale bar, 200 μM.

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Extended Data Fig. 6 Flow cytometry gates showing different immune populations.

All the gates were drawn according to published protocol from Dr. Vonderheide group59. (a, b) Different myeloid and lymphoid populations are shown in a and b respectively.

Extended Data Fig. 7 Alteration of Elf5 in tumor cells alters TME in REAR background.

IHC analysis shows a, b, increased myeloid cells in C3-T+; Elf5+/- (n=8 FOV) tumors as compared to C3-T+; Elf5+/+ (n=6 FOV) c, d, decreased number of macrophages in C3-T+; Elf5+/- tumors (n=9 FOV) as compared to C3-T+; Elf5+/+ tumors (n=7 FOV), e, f, increased number of Foxp3+ cells in C3-T+; Elf5+/- tumors. (n=10 FOV for C3-T+; Elf5+/+ and n=11 FOV for C3-T+; Elf5+/-), n=3 individual tumors were used in a-f. g, j, increased Gr1+ myeloid cells (n=5 tumors/group) and Ly6G+ neutrophils (CD45+CD11b+Gr1+) (n=5 tumors/group) and decreased k, cytotoxic T-cells (CD45+CD8+) population in C3-T+; Elf5+/- tumors as compared to C3-T+; Elf5+/+ tumors (n=5 tumors/group) h, Macrophages (CD45+F4/80+) (n=5 C3-T+; Elf5+/+ tumors and n=6 C3-T+; Elf5+/- tumors) and i, Regulatory T-cells (CD45+CD3+FoxP3+) (n=3 tumors/group) were observed. IHC image showing increased FBXW7 (n=6 tumors for control and n=7 tumors for Elf5-OE from n=3 independent tumors). (l), decreased IFNGR1 (n=10 FOV for control and n=7 FOV for Elf5-OE from n=3 independent tumors) m, in Elf5-OE EpRas tumors. FACS plots showing no change in n, macrophages, o, CD4+ T-cells, p, increased Ly6C+ neutrophils in Elf5-OE tumors (n=6, control and n=4, Elf5-OE individual tumors). q FACS plots showing decreased number of Gr1+ myeloid cells in lungs of EpRas Elf5-OE tumor bearing mice (n=3 individual tumors/group). (a-j and n-q) Two-tailed student’s t test was used to compute p-value. (k, l, m) Mann-Whitney U test was used to calculate p value. Data are presented as the mean ± SEM. FOV; Field of view. (b, d, f, l, m) Boxplot data represent median, interquartile range, and spikes to upper and lower adjacent values. FOV; Field of view. (a, c, e, l, m) Images are representative of minimum three independent experiments, Scale bars, 40 µm.

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Extended Data Fig. 8 IFN-γ signaling imparts more tumorigenic and metastatic potential.

IFN-γ treatment upregulates a, STAT1 and P-STAT1 (Data representative of minimum of three independent experiments) b, mRNA expression of IFN-γ target genes. qPCR values were normalized to Gapdh. Experiments were performed twice in technical duplicate, and data presented as mean ± SD. Two-sided Student’s t test used to calculate p-value. c, FACS analysis show upregulation of PD-L1 but not CTLA4 in IFN-γ-treated EpRas cells. Data representative of two independent experiments. d, e, IFN-γ treated EpRas cells form larger and more invasive tumors, n=5 tumors/group. Inset shows higher magnification images. Data represented as mean ± SD. f, g, Small metastatic nodules (black arrows) in lungs of mice injected with IFN-γ treated EpRas cells. g, GFP labelled IFN-γ treated EpRas cells show micro metastatic foci in lungs of mice (n=4), while control untreated cell injected mice show no micro metastatic foci (n=4). EpRas 250,000 cells/MFP were injected into mice for (g). Cells were labeled with GFP (lentiviral vector pLEX-hFL2iG was used) using standard method. White arrows show GFP+ micro metastatic foci in whole mount lung image. (e-g) Experiment was repeated twice with similar results. (h-l) IHC analysis shows high (h) P-STAT1+, (i) S100A8+ cells in IFN-γ treated EpRas tumors. (j) Decreased CD8+T-cells observed in IFN-γ treated tumors. (k, l) Increase in S100A8+ cells observed in IFN-γ treated EpRas lung sections (metastatic site) with quantification in the right panel. Boxplot data represent median, interquartile range, and spikes to upper and lower adjacent values. (h-l). Two-way ANOVA test was used with Bonferroni post hoc test to calculate p value in (d). Two-tailed Mann-Whitney U test was used to compute p-value for h, j, i, l and data is represented as mean ± SEM. n=10 random FOV were evaluated from 3 individual tumors (h, j) n=3 and 5 random FOV were evaluated (i), n=3 and 7 random FOV/group were used for quantification (l). (e-g) Representative images of minimum two independent experiments are presented. Scale bar, (e) 200µm, (f) 2mm, and (g) 500 μm and (h-k) 40 μm respectively. ***p < 0.001. FOV; Field of view.

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Extended Data Fig. 9 IFN-γ signaling renders more metastatic properties to EpRas cells via PD-1/PD-L1 signaling.

a, b, Tail vein (TV) injection of control and IFN-γ treated EpRas cells, n=7 mice/group. c, d, Lung images and metastatic nodule numbers in NSG mice injected with EpRas cells treated with IFN-γ via TV (n=6 mice, control and n=7 mice, IFN-γ treated). e, f, TV injection of control and IFN-γ treated EpRas cells in mice treated with anti-IgG (n=5 mice), anti-PD-1 (n=5 mice) and anti-PD-L1 (n=6 mice) antibodies. g, h, IHC in tumors from control cells compared to IFNGR1-KD tumor cells, n=3 individual tumors were used and 7 random FOV were used. i, FBXW7-KD tumors cells grew faster than control, n=5 tumors/group. k, Western blot showed decreased FBXW7 in EpRas cells transduced with FBXW7 shRNAs compared to control. (l) IF analysis shows increased expression of IFNGR1 in FBXW7-KD cells compared to control. Experiment was repeated twice (k, l). j, m, TV injection of control and FBXW7-KD EpRas cells shows increased number of lung metastasis in FBXW7-KD mice compared to control mice, n=3 CTRL, n=4 KD1, and n=4 KD2 mice were used. Two-tailed Mann-Whitney U test was used to compute p-value in (b, d) and data represented as mean ± SEM (b) or ±S.D (d). Boxplot represent median, interquartile range, and spikes to upper and lower adjacent values (f, h). One-way ANOVA test was used with Tukey post hoc test to compute p-values of multiple comparison data. Data is represented as mean ± SEM (f, h, j). Two-way ANOVA test was used with Bonferroni post hoc test to calculate p values, data is represented as mean ± SD (i). Scale bar (a, c, e, m) 2 mm, (g) 40 µm (l) 100 µm. **p < 0.01, ***p < 0.001. FOV; Field of view.

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Extended Data Fig. 10 High expression of ELF5/FBXW7 and low expression of IFNGR1 is protective in Mesenchymal, basal and immunomodulatory TNBC.

a–d, High expression of ELF5/FBXW7 and low expression of IFNGR1 is correlated with better Distant metastasis free survival (DMFS) in (a) Basal (n=24 for Elf5/FBXW7 and (n=32 for IFNGR1) (b) Immunomodulatory (n=47 patient samples for Elf5/FBXW7 and (n=36 patient samples for IFNGR1) (c) Mesenchymal TNBC subsets (n=57 for Elf5/FBXW7 and (n=57 patient samples for IFNGR1). (d) High expression of ELF5/FBXW7 and low expression of IFNGR1 is correlated with worse Distant metastasis free survival (DMFS) in Non-TNBC ER+ patients (n=437 patient samples for Elf5/FBXW7 and (n=1391 patient samples for IFNGR1). P-value was computed by Log Rank test. KM plotter database was used57. e, TNBC PDXs showing direct correlation between ELF5 and FBXW7, and indirect correlation with IFNGR1. 5 TNBC PDXs were used for this experiment. Two-tailed Mann-Whitney U test was used for statistical analysis and data is represented as mean ± SEM. (f-h) IHC images of TNBC PDXs showing direct correlation between ELF5 and FBXW7, and indirect correlation with IFNGR1. Scale bar, 40 µm.

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Singh, S., Kumar, S., Srivastava, R.K. et al. Loss of ELF5–FBXW7 stabilizes IFNGR1 to promote the growth and metastasis of triple-negative breast cancer through interferon-γ signalling. Nat Cell Biol 22, 591–602 (2020). https://doi.org/10.1038/s41556-020-0495-y

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