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Screening of organoids derived from patients with breast cancer implicates the repressor NCOR2 in cytotoxic stress response and antitumor immunity

Nature Cancer volume 3, pages 734–752 (2022)Cite this article

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Abstract

Resistance to antitumor treatment contributes to patient mortality. Functional proteomic screening of organoids derived from chemotherapy-treated patients with breast cancer identified nuclear receptor corepressor 2 (NCOR2) histone deacetylase as an inhibitor of cytotoxic stress response and antitumor immunity. High NCOR2 in the tumors of patients with breast cancer predicted chemotherapy refractoriness, tumor recurrence and poor prognosis. Molecular studies revealed that NCOR2 inhibits antitumor treatment by regulating histone deacetylase 3 (HDAC3) to repress interferon regulatory factor 1 (IRF-1)-dependent gene expression and interferon (IFN) signaling. Reducing NCOR2 or impeding its epigenetic activity by modifying its interaction with HDAC3 enhanced chemotherapy responsiveness and restored antitumor immunity. An adeno-associated viral NCOR2–HDAC3 competitor potentiated chemotherapy and immune checkpoint therapy in culture and in vivo by permitting transcription of IRF-1-regulated proapoptosis and inflammatory genes to increase IFN-γ signaling. The findings illustrate the utility of patient-derived organoids for drug discovery and suggest that targeting stress and inflammatory–repressor complexes such as NCOR2–HDAC3 could overcome treatment resistance and improve the outcome of patients with cancer.

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Fig. 1: Identification of NCOR2 as an IRF-1 stress-dependent death regulator.
Fig. 2: NCOR2 modulates the innate death resistance of breast cancers.
Fig. 3: NCOR2 expression correlates with chemoresistance in human breast cancer.
Fig. 4: The NCOR2 stress checkpoint is HDAC3-dependent.
Fig. 5: NCOR2 is a chromatin-mediated checkpoint of STAT1/IRF-1 stress signaling.
Fig. 6: Functionally blocking NCOR2 potentiates chemotherapy in breast tumors.
Fig. 7: NCOR2 deficiency or its functional inhibition creates a T cell-inflamed tumor microenvironment.
Fig. 8: Functional blocking of NCOR2 potentiates immune checkpoint therapy.

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Data availability

All data used to generate the results are found in the extended data and supplementary information. The RNA-seq data reported in Fig. 1 are available at the GEO under accession no. GSE183477. The raw and analyzed data from the microarray experiments are available at the GEO under accession nos. GSE138900 and GSE8346. Reanalyzed, previously published gene expression data are available under accession no. GSE6434 or can be requested from the authors, including the I-SPY 1 Trial Investigators or Siemens Healthcare Diagnostics Products GmbH. Source data have been provided as source data files. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank G. Timblin for constructive suggestions regarding the interpretation of the impact of NCOR2 on antitumor immunity, B. Welm (Huntsman Cancer Institute) and M. Lewis (Baylor College of Medicine) for the patient-derived organoids, M. Lazar (University of Pennsylvania) for the monoclonal anti-NCOR2-producing hybridoma cell line and pcDNA-NCOR2 shRNA and mutant NCOR2 expression constructs, J. Park (Sungkyunkwan University) for the pcDNA3-IRF-1 vector, C. Li for the statistical analysis and K. Tan for the breast cancer tissues (Tung’s Metro-Harbor Hospital). The cell culture, functional and molecular studies were supported by grants from the National Institutes of Health (nos. R35-CA242447-01A1, R01-CA192914 and CA222508-01 to V.M.W.), the Department of Defense (no. BC122990 to V.M.W.) and the Breast Cancer Research Foundation (no. A132292 to V.M.W.). The development of the DeCOR2 gene therapy was funded by TMU (nos. 107TMU-WFH-16 and 110-5433-001-400 to K.K.T.).

Author information

Author notes

  1. These authors contributed equally: Kelvin K. Tsai, Shenq-Shyang Huang.

  2. These authors jointly supervised this work: Kelvin K. Tsai, Valerie M. Weaver.

Authors and Affiliations

  1. Department of Surgery and Center for Bioengineering and Tissue Regeneration, University of California, San Francisco, CA, USA

    Kelvin K. Tsai, Jason J. Northey, Michael E. Werner, Jonathon N. Lakins & Valerie M. Weaver

  2. Department of Radiation Oncology, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA

    Kelvin K. Tsai & Valerie M. Weaver

  3. Department of Bioengineering and Therapeutic Sciences, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA

    Kelvin K. Tsai & Valerie M. Weaver

  4. Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

    Kelvin K. Tsai, Shenq-Shyang Huang, Wen-Ying Liao, Chung-Chi Hsu & Li-Hsin Cheng

  5. Department of Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

    Kelvin K. Tsai

  6. Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan

    Chih-Pin Chuu

  7. Department of Pathology and Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA

    Chandrima Chatterjee, Jonathon N. Lakins & Valerie M. Weaver

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Contributions

V.M.W. conceived the original idea for the study, acquired provisional funding and directed the proof-of-concept studies, which were executed by C.C. and J.N.L. Thereafter, V.M.W. and K.K.T. acquired sustained funding to execute the study and together designed and directed the investigative work. K.K.T. conceptualized the DeCOR2 gene therapy and acquired funding to support the proof-of-concept work. K.K.T., J.J.N., S-S.H., C-C.H., L-H.C., M.E.W., C.C. and J.N.L. conducted the molecular, biochemical and functional studies. C-P.C. carried out the MWA analysis. S-S.H. and C-C.H. performed the IHC analyses. K.K.T. analyzed the microarray data, directed the bioinformatics analysis and performed the clinical correlative analysis. J.J.N. and S-S.H. completed the patient-derived organoid studies. S-S.H., W-Y.L. and J.J.N. conducted the animal work. K.K.T. and V.M.W. jointly supervised the study and data analysis and together summarized and interpreted the data and cowrote the manuscript with input from the coauthors.

Corresponding authors

Correspondence to Kelvin K. Tsai or Valerie M. Weaver.

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Competing interests

K.K.T. is the named inventor on a US provisional patent application (application no. 63302570). The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 IRF-1 and its role in cytotoxic stress-induced cell death.

a, Pre-malignant (HMT3522 S-1) mammary epithelial cells, and breast cancer cell lines including HMT3522 T4-2, HCC-1954, BT-474, and T47D cells were treated with vehicle (Veh), doxorubicin (Dox; 1 μM) or paclitaxel (Pac; 500 nM) and their gene expression was profiled at early (8 hr) or late (24 hr) time points following treatment initiation. The blue dots represent each of 30 combinations of the different cell lines studied, the chemotherapeutic agents used, and the treatment time points examined. b, IRF1 as the conserved, top-ranked, transcription factor induced by chemotherapeutic agents in a. c, The transcript level of IRF1 (top) and the average transcript level of putative IRF1-target genes (bottom), in Veh-, Dox- or Pac-treated breast cancer cell lines (n = 2 independent experiments). d, Hierarchical clustering of putative IRF1-target genes that exhibited differential expression between the Veh- and Dox- or Pac-treated breast tumor cell lines. e, The IRF1-target genes up-regulated in chemotherapeutic agent-treated breast tumor cells in d. f, Knockdown (KD) of IRF1 expression in HMT-3522 T4-2 breast cancer cells using two different shRNAs (representative data of n = 2 independent experiments with similar results). g, The percent cell death in HMT-3522 T4-2 cells expressing a scrambled shRNA or an IRF1 shRNA treated with doxorubicin, paclitaxel, or TRAIL (n = 3 independent experiments). h, The percent cell death of P53-mutant T47D breast cancer cells with shRNA-mediated knockdown (KD) of IRF1 or a scrambled shRNA (control) treated with doxorubicin (n = 3-5 independent experiments, the exact n are provided in the numerical source data). i, Bioluminescence imaging (BLI) of flank regions of nude mice inoculated with scrambled control- or IRF1-shRNA (IRF1 KD) expressing HCC-1954 cells four weeks following systemic treatment with doxorubicin (n = 2-5 mice per group, the exact n are provided in the numerical source data). j, The rate of growth of scrambled shRNA- and IRF1-shRNA-infected HMT-3522 T4-2 cells (n = 3 independent experiments). k, Representative immunoblots of p53, p21, γH2AX, and total and phospho-ATM (S1981; p-ATM) in HMT-3522 T4-2 cells with KD of IRF1 expression or a scrambled shRNA (control) treated with doxorubicin (24 hr). l, IRF-1 contributes to cytotoxic stress-induced cell death independent of p53 status (n = 4 independent experiments). Data are presented as the mean ± s.e.m. (c,g−j,l). *P < 0.05; **P < 0.01; ***P < 0.001 versus control (g,h,i,j); *P < 0.05 versus control; †P < 0.05 versus P53 KD (l), two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 2 The protein domains mediating the IRF-1−NCOR2 interaction.

a, Co-immunoprecipitation (IP) of IRF-1 with NCOR2 in the nuclear lysates of HMT-3522 S-1 mammary epithelial cells, HMT-3522 T4-2 neoplastic mammary epithelial cells, HCC-1954, BT-474 breast tumor cell lines and in primary patient breast cancer NHRI-BC-008 cells. Cells were pre-treated with TRAIL (1 µg/ml) and the cell lysates were collected at 3 hours post-treatment. Histone 2B (H2B) was included as a loading control (representative data of n = 2 independent experiments with similar results). b, The functional domains of IRF-1 and the various truncated mutants. NLS, nuclear localization signal; a.a., amino acid. c, The N-terminal region (a.a. 1-185) as the NCOR2-binding region (NBD) of IRF1. MDA-MB-436 breast cancer cells were transfected with the V5-epitope-tagged truncated IRF1 mutants depicted in b and the nuclear lysate was subjected to co-IP (representative data of n = 2 independent experiments with similar results). d, The functional domains of NCOR2, its fragments, and a C-terminal truncated NCOR2 protein (NCOR2 Δa.a. 2079-2514). RD, repressor domain; DAD, deacetylase-activating domain; NLS, nuclear localization signal. RID, nuclear receptor interaction domain. e, The NCOR2 protein fragments NCOR2 (a.a. 701-1499) and NCOR2 (a.a. 1500-2078) do not interact with endogenous IRF-1. MDA-MB-436 cells were transfected with V5-epitope-tagged NCOR2 protein fragments and the nuclear lysate was subjected to co-IP using anti-V5 antibody or an isotype-matched IgG. Shown are representative IB of IRF-1 and V5 in the immunoprecipitated lysate, and in the total cellular lysate (input) (representative data of n = 2 independent experiments with similar results). f, The C-terminal region of NCOR2 (NCOR2 a.a. 2079-2514) interacts with IRF-1, STAT-1, and NF-κB p50 in MDA-MB-436 cells. Shown are IB of IRF-1, NF-κB p50, NF-κB p65, and STAT-1 in the immunoprecipitated lysate (representative data of n = 2 independent experiments with similar results). g, The C-terminal truncated NCOR2 (NCOR2 Δa.a. 2079-2514) fails to interact with IRF-1 and STAT-1 in MDA-MB-436 cells. Shown are IB of IRF-1 and STAT-1 in the immunoprecipitated lysate (representative data of n = 2 independent experiments with similar results).

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Extended Data Fig. 3 Knockdown of NCOR2 sensitizes breast cancer cells to cytotoxic stress.

a, Knockdown (KD) of NCOR2 expression in HMT-3522 T4-2 breast cancer cells using an shRNA vector (clone #60706) (representative data of n = 2 independent experiments with similar results). b, The transcriptional activity of IRF-1 in HMT-3522 T4-2 cells with KD of NCOR2 expression (n = 4 independent experiments). c, KD of NCOR2 expression rendered HMT-3522 T4-2 cells hypersensitive to paclitaxel (Pac), doxorubicin (Dox), or TRAIL (TRA)-induced apoptosis (n = 3 independent experiments). d, KD of NCOR2 expression rendered BT-474 or HCC-1954 spheroids hypersensitive to cytotoxic stress-induced death (n = 3 independent experiments). e, HCC-1954 spheroids with KD of NCOR2 expression are more sensitive to ionizing radiation (IR)-induced cell death (n = 3-4 independent experiments, the exact n are provided in the numerical source data). f, KD of NCOR2 expression rendered pancreatic ductal adenocarcinoma (PDAC) PANC-1 cells hypersensitive to TRAIL- or gemcitabine-induced death (n = 3 independent experiments). g, The percent cell death of vector- or NCOR2-overexpressed (OE) HMT-3522 T4-2 cells grown as a 2D cell monolayer on rBM-coated plates (left) or as single cells (middle) or three-dimensional (3D) organoids embedded within rBM (right) following exposure to increasing doses of TRA or Pac (n = 3 independent experiments). h, Immunofluorescence staining of γH2AX and NBS-1 in the vehicle (Veh)- or etoposide (1 μM × 24 hr)-treated HMT-3522 T4-2 cells with lentiviral-shRNA-mediated KD of NCOR2 expression or infected with a scrambled shRNA. Scale bar, 20 μm. i,j, The number of γH2AX nuclear foci (i) and the fluorescence intensity of nuclear NBS-1 staining per cell (j) in cells described and treated in h (n = 3 independent experiments). k, Immunofluorescence images in HMT-3522 T4-2 cells with shRNA-mediated KD of NCOR2 expression or infected with a scrambled shRNA stained with lamin B and a marker of histone trimethylation H3K9 (Me3-H3K9). Scale bar, 20 μm. l, Total nuclear acetylated (Ac) histone H3K9 (Ac-H3K9), Ac-H3K14, Me3-H3K9 and methyl-CpG binding protein 2 (MECP2) in HMT-3522 T4-2 cells with shRNA-mediated KD of NCOR2 expression or infected with a scrambled shRNA (representative data of n = 2 independent experiments with similar results). Data are presented as the mean ± s.e.m. (b,c−g,i,j). *P < 0.05; **P < 0.01; ***P < 0.001 versus control (b−f), vector (g) or vehicle (i,j), two-tailed unpaired Student’s t-test.

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Extended Data Fig. 4 Cytotoxic stress-induced nuclear translocation of NCOR2.

a, Left: Immunofluorescence staining of NCOR2 in vehicle- or TRAIL-treated primary breast cancer NHRI-BC-008 cells. Right: The percentage of cells displaying nuclear versus cytoplasmic localized NCOR2 following exposure to TRAIL (n = 4 independent experiments). Scale bar, 20 μm. b, The percentage of NHRI-BC-008 cells displaying nuclear versus cytoplasmic localized NCOR2 following exposure to paclitaxel (1 µM) (n = 4 independent experiments). c, Immunofluorescence staining of NCOR2, DAPI (cell nuclei), and the early apoptosis marker Annexin V in NHRI-BC-008 cells 1 hour following treatment with TRAIL (1 μg ml-1). Scale, 10 μm. Right: The percentage of cells with positive Annexin V staining in cells with mainly nuclear versus those with mainly cytoplasmic localized NCOR2 (n = 100 cells counted per experiment, n = 6 independent experiments). d, The TRAIL-induced nuclear versus cytoplasmic localization of NCOR2 in HMT-3522 T4-2 cells infected with and without the mutant Ran GTPase Q69L Ran (n = 3 independent experiments). Data indicate that the treatment-induced nuclear translocation of NCOR2 is Ran-dependent. e, Inhibiting NCOR2 nuclear translocation by expressing the Ran (Q69L) mutant sensitizes HMT-3522 T4-2 cell spheroids to TRAIL-induced apoptosis (n = 3 independent experiments). f, The location of the nuclear localization signal (NLS; amino acids 680-685) of NCOR2. g, Immunofluorescence images showing that the NLS-deficient NCOR2 mutant (NCOR2 ΔNLS) fails to translocate into the cell nucleus in response to TRAIL treatment. Scale bar, 20 μm. Right: Overexpression (OE) of NCOR2 ΔNLS hypersensitizes HMT-3522 T4-2 cell spheroids to TRAIL (n = 2-5 independent experiments, the exact n are provided in the numerical source data). Data are presented as the mean ± s.e.m. (a−e,g). *P < 0.05; **P < 0.01; ***P < 0.001 compared to vehicle (a,b,d), cytoplasmic NCOR2 (c), vector (e), or NCOR2 OE (g), two-tailed unpaired Student’s t-test.

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Extended Data Fig. 5 NCOR2 represses programmed cell death gene expression in breast cancer cells.

Bar graphs showing fold expression of TNFSF10 (encoding TRAIL), CASP1 (encoding caspase 1), CASP7 (encoding caspase 7), and IRF1 (encoding IRF-1) gene transcripts in triple-negative breast cancer MDA-MB-231 cells with and without stable overexpression (OE) of NCOR2 or an empty vector (Vector) following treatment with TRAIL (0.5 µg ml-1 × 3-12 hr), doxorubicin (1 µM × 24 hr), or paclitaxel (0.5 µM × 24 hr). The cells were co-treated with caspase inhibitors to avoid apoptosis and losing cells during RNA collection. Data are presented as the mean ± s.e.m. (n = 3 independent experiments). **P < 0.01; ***P < 0.001 compared to vector, two-tailed unpaired Student’s t-test.

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Extended Data Fig. 6 NCOR2 associates with the promoters of the genes in the STAT-1/IRF1 death signaling pathway.

a, Results of chromatin immunoprecipitation (ChIP) and ChIP-re-ChIP assays showing that NCOR2 and HDAC3 are concomitantly recruited to the IRF-1 binding site (IRF-E) at the TNFSF10, CASP1, CASP7 or STAT1 promoters in TRAIL (1 µg ml-1 × 3 hr)-treated HMT-3522 T4-2 cells overexpressing (OE) NCOR2 or an empty vector (Vector). Re-ChIP was then carried out using the precipitates from the first round of ChIP for the indicated genes using an anti-HDAC3 antibody (representative data of n = 2 independent experiments with similar results). b, ChIP assay data showing association of NCOR2 together with STAT-1 at the IRF1 promoter in TRAIL-treated HMT-3522 T4-2 cells with the OE of NCOR2 or an empty vector (Vector) using anti-NCOR2, STAT-1, acetylated histone H3 (Ac-H3), transcription factor IIB (TF IIB), RNA polymerase II (Pol II) antibodies or an isotype control IgG (representative data of n = 2 independent experiments with similar results). c, ChIP assay data showing molecular associations of NCOR2 at the promoters of TNFSF10, CASP1, CASP7, or STAT1 in TRAIL-treated HMT-3522 T4-2 cells with the OE of NCOR2 or an empty vector (Vector) using anti-NCOR2, HDAC3, Ac-H3, TF IIB, Pol II antibodies or an isotype control IgG (representative data of n = 2 independent experiments with similar results). d, Results of ChIP from HMT-3522 T4-2 cells with lentiviral-shRNA-mediated knockdown (KD) of NCOR2 expression showing attenuation of the association of HDAC3 with the IRF-E on the promoter of TNFSF10, CASP1, CASP7 or STAT1 following TRAIL treatment. Findings also showed that loss of NCOR2 simultaneously permitted histone hyperacetylation and enhanced the recruitment of the TF IIB and Pol II (representative data of n = 2 independent experiments with similar results).

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Extended Data Fig. 7 Overexpressing NCOR2 does not interfere with DNA-damage signaling nor grossly alter chromatin conformation.

a, p53, p21, and phosphorylated histone H2AX (γH2AX) in HMT-3522 T4-2 cells overexpressing NCOR2 (NCOR2 OE) compared to vector-infected cells treated with doxorubicin or vehicle (Veh) for 24 hr (representative data of n = 2 independent experiments with similar results). b, Left: HMT-3522 T4-2 cells with NCOR2 OE or infected with an empty vector (Vector) and treated with the DNA-damaging agent etoposide (5 μM × 2 hr) were stained for γH2AX and NBS-1. Scale, 20 μm. Right: The number of γH2AX nuclear foci (top; n = 2 independent experiments) and the fluorescence intensity of nuclear NBS-1 per cell (bottom; n = 3 independent experiments). c, RAD50, ATM, and phospho-ATM (S1981; p-ATM) in vehicle- or doxorubicin-treated HMT-3522 T4-2 cells with NCOR2 OE or infected with an empty vector and with or without shRNA-mediated knockdown (KD) of RAD50 expression (representative data of n = 2 independent experiments with similar results). Loss of RAD50 expression compromises DNA repair regardless of NCOR2 expression. d, Immunofluorescence staining of γH2AX and NBS1 in HMT-3522 T4-2 cells overexpressing NCOR2 or infected with an empty vector with KD of RAD50 expression and treatment with etoposide. Scale bar, 20 μm. Loss of RAD50 expression abrogates DNA double-strand break complex formation independent of cellular NCOR2 status. e, The number of γH2AX foci per cell nucleus (top; n = 2 independent experiments) and the fluorescence intensity of nuclear NBS-1 staining (bottom; n = 3 independent experiments) in the cells shown in d. f, The percent cell death of HMT-3522 T4-2 cell spheroids treated with doxorubicin when they overexpressed NCOR2 or an empty vector and when RAD50 is knocked down. The OE of NCOR2 was able to override the impact of the loss of RAD50 expression on cell death induction following doxorubicin treatment (n = 3 independent experiments). g, Total nuclear acetylated (Ac)-histone-3 lysine-9 (Ac-H3K9), Ac-H3K14, trimethylated H3K9 (Me3-H3K9), and methyl-CpG binding protein 2 (MECP2) in HMT-3522 T4-2 cells overexpressing NCOR2 compared to vector infected cells (representative data of n = 2 independent experiments with similar results). Data are presented as the mean ± s.e.m. (b,e,f). *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle (b,e); *P < 0.05 versus vector (f), two-tailed unpaired Student’s t-test.

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Supplementary information

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Tsai, K.K., Huang, SS., Northey, J.J. et al. Screening of organoids derived from patients with breast cancer implicates the repressor NCOR2 in cytotoxic stress response and antitumor immunity. Nat Cancer 3, 734–752 (2022). https://doi.org/10.1038/s43018-022-00375-0

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