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Astrocyte-induced Cdk5 expedites breast cancer brain metastasis by suppressing MHC-I expression to evade immune recognition

Nature Cell Biology volume 26, pages 1773–1789 (2024)Cite this article

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Abstract

Brain metastases (BrMs) evade the immune response to develop in the brain, yet the mechanisms of BrM immune evasion remains unclear. This study shows that brain astrocytes induce the overexpression of neuronal-specific cyclin-dependent kinase 5 (Cdk5) in breast cancer-derived BrMs, which facilitates BrM outgrowth in mice. Cdk5-overexpressing BrMs exhibit reduced expression and function of the class I major histocompatibility complex (MHC-I) and antigen-presentation pathway, which are restored by inhibiting Cdk5 genetically or pharmacologically, as evidenced by single-cell RNA sequencing and functional studies. Mechanistically, Cdk5 suppresses MHC-I expression on the cancer cell membrane through the Irf2bp1–Stat1–importin α–Nlrc5 pathway, enabling BrMs to avoid recognition by T cells. Treatment with roscovitine—a clinically applicable Cdk5 inhibitor—alone or combined with immune checkpoint inhibitors, significantly reduces BrM burden and increases tumour-infiltrating functional CD8+ lymphocytes in mice. Thus, astrocyte-induced Cdk5 overexpression endorses BrM immune evasion, whereas therapeutically targeting Cdk5 markedly improves the efficacy of immune checkpoint inhibitors and inhibits BrM growth.

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Fig. 1: CDK5 is overexpressed in human BrM and restricts antigen presentation and the T cell response.
Fig. 2: Cdk5 confers resistance to antigen-specific T cell killing in vitro.
Fig. 3: Cdk5 suppresses the expression of MHC-I and APP genes in breast cancer BrM.
Fig. 4: Cdk5 inhibits MHC-I expression through the Stat1–importin α–Nlrc5 pathway.
Fig. 5: Cdk5 inhibits Stat1 expression and Stat1pS727 phosphorylation by phosphorylating Irf2bp1 at S66.
Fig. 6: BrM-educated astrocytes promote Cdk5 production in BrM cells through emilin-1 secretion.
Fig. 7: Pharmacological inhibition of Cdk5 delays BrM growth in vivo through re-expression of MHC-I and an augmented T cell response.
Fig. 8: Pre-clinical trial of RSV + anti-PD-1 combination treatment to inhibit breast cancer BrM.

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Data availability

RNA-seq data that support the findings of this study have been deposited in the BioProject under accession code PRJNA731241. Previously published sequencing data that were re-analysed here are available under accession codes GSE52604, GSE125989, GSE164150, GSE52604, GSE43837, GSE186344, GSE19184, GSE148283 and PRJNA681304. Proteomic data have been deposited in ProteomeXchange with the primary accession code PXD053434. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank W. Xia, Z. Han, L. Li, S. Pan, L. Tan, L. Norberg, J. Ritchie, A. Yuzhalina, T. Locke and S. Bronson for their help. This work was supported by NIH grants R01CA184836 (D.Y.), R01CA208213 (D.Y.), R01CA231149 (D.Y.), METAvivor research grant numbers 56675 and 58284 (D.Y.), Congressionally Directed Medical Research Program - Breast Cancer Research Program Breakthrough Award Level 1 W81XWH-22-1-0064 (A.Y.) and NIH Cancer Center Support Grant P30 CA016672 to the MD Anderson Cancer Center (Functional Genomic Core, Flow Cytometry and Cellular Imaging Facility, Research Histology Core Laboratory, Characterized Cell Line Core Facility and Research Animal Support Facility–Houston). A.Y. is supported by the Odyssey Fellowship Program of the MD Anderson Cancer Center. A.F. is supported by the PCCSM program. D.Y. is the Hubert L. and Olive Stringer Distinguished Chair in Basic Science at the MD Anderson Cancer Center and is partially supported by Patel Memorial Breast Cancer Endowment Fund, and Ting Tsung and Wei Fong Chao Research Fund. Sequencing data were generated by the UTHealth Cancer Genomics Core (CPRIT RP180734). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Figures 2d, 4c, 5a, 6c,j, 8c, Extended Data Figs. 2h, 8h and Supplementary Fig. 4 were created with BioRender.com.

Author information

Author notes

  1. Frank J. Lowery

    Present address: Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

  2. Huihui Fan

    Present address: John P and Katherine G McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA

  3. Mien-Chie Hung

    Present address: Graduate Institute of Biomedical Sciences, Research Center for Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung City, Taiwan

Authors and Affiliations

  1. Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Arseniy E. Yuzhalin, Frank J. Lowery, Yohei Saito, Xiangliang Yuan, Jun Yao, Yimin Duan, Jingzhen Ding, Sunil Acharya, Chenyu Zhang, Abigail Fajardo, Hao-Nien Chen, Yongkun Wei, Yutong Sun, Lin Zhang, Yi Xiao, Ping Li, Mien-Chie Hung & Dihua Yu

  2. Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Philip L. Lorenzi

  3. Departments of Pathology and Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Jason T. Huse

  4. Center for Precision Health, McWilliams School of Biomedical Informatics, Houston, TX, USA

    Huihui Fan & Zhongming Zhao

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Contributions

A.Y. designed and performed experiments, conducted data analysis and interpretation, statistical analysis, procured partial funding and wrote the manuscript. F.L., Y.S., X.Y., Y.S., Y.D., S.A., J.D., L.Z., P.L., C.Z., A.F., H-N.C., Y.W. and X.Y. designed and performed experiments, conducted data analysis, statistical analysis and made intellectual contributions. J.Y., H.F., Y.X. and Z.Z. performed statistical and bioinformatics analyses. J.T.H. provided clinical samples. P.L.L. and M.C.H. conducted data analysis, provided methodology and intellectual contributions. D.Y. conceived and designed the overall study and experiments, performed data analysis and interpretation, made intellectual contributions, wrote and edited the manuscript, procured funding and supervised the study team.

Corresponding author

Correspondence to Dihua Yu.

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Extended data

Extended Data Fig. 1 Cdk5 is overexpressed in BrM and correlates with reduced overall survival from breast cancer but does not directly promote the proliferation of breast cancer BrM in vitro and in vivo.

A. CDKs expression in MDA-MB-231.Br3 BrM was normalized to their corresponding expression in MFP tumours, and ranked based on BrM-specific upregulation (N = 6 and 5, respectively). B. CDK5 expression in MFP tumours and BrMs from A. N = 5 and 6 biological replicates. Two-sided T-test. C. CDK5 expression in breast cancers and matched BrM (N = 13). Each dot indicates a patient. Paired two-sided T-test. D. Cdk immunoblotting in the indicated cell lines. E. Survival of mice bearing BrMs from 4T1.Br3.shCtl and 4T1.Br3.shCdk5#2 cells (N = 11 and 12 mice, respectively). Logrank test. F. Cdk5 immunoblotting in BrMs from EO771.Br3.shCtl and EO771.Br3.shCdk5 cells (N = 2 mice per group). G. p-Cdk5 staining in BrMs of mice ICA-injected with indicated cells. Scale bar = 50 μm. H, I Ki-67+ (H) and TUNEL+ (I) cells in BrMs of mice ICA-injected with indicated cells. Each dot indicates a lesion. Data pooled from N = 4 and 3 mice per group, respectively. 2-way ANOVA with Tukey's multiple comparisons test. J. Correlation analysis between CDK5 expression and DNA replication signature across human BrM datasets. K, L Proliferation of EO771.Br3 (K) and 4T1.Br3 (L) cells transfected with control or Cdk5 shRNA (N = 2 biological replicates). Each dot indicates the mean. M. Hard agar assay of indicated cells. N = 4 biological replicates. Two-sided T-test. N. 4-day BrM seeding experiment of EO771.Br3.GFP.shCtl and EO771.Br3.GFP.shCdk5 cells. Brains stained as indicated. Scale bar = 20 μm. O. Quantification of GFP+ cells in brains from N. 15 fields of view per mouse. Each dot indicates a mouse. Two-sided Mann–Whitney U Test. P. UMAP plot. Q, R. Expression of Mki67 (Q) and Cdk5 (R) in cancer cells sorted from BrMs from EO771.Br3.shCtl or EO771.Br3.shCdk5 cells (N = 3 mice per group). Each dot indicates a cell, violin shape indicates data distribution, red dot indicates the mean, bounds of the box indicate 1st and 3rd quartiles, whiskers indicate the range. Two-sided Wilcoxon rank-sum test.Throughout, results are shown as means ± SEM. N.S. – not significant.

Source data

Extended Data Fig. 2 Cdk5 promotes CD8+ T cell infiltration in mouse and human BrM and boosts the antigen-specific CD8+ T cell killing in vitro.

A. UMAP plots illustrate predicted immune populations in BrM from EO771.Br3.shCtl or EO771.Br3.shCdk5 cells (N = 3 mice per group). T cell clusters are circled in red. B. Major immune populations in BrM from EO771.Br3.shCtl or EO771.Br3.shCdk5 cells (N = 3 mice per group) based on scRNA-seq analysis. N = 4 technical replicates. C. Major T cell subsets in BrM from EO771.Br3.shCtl or EO771.Br3.shCdk5 cells (N = 3 mice per group) based on scRNA-seq analysis. N = 4 technical replicates. D. IHC profiling of major immune populations in BrM lesions of mice ICA-injected with EO771.Br3 or 4T1.Br3 cells transfected with control or Cdk5 shRNA (N = 3 mice per group). Cell number was normalized to BrM area. Each dot indicates a BrM lesion. Two-sided T-test. E. Representative IHC staining of CD8+ TILs in BrM lesions of mice ICA-injected with T11.Br1 cells transfected with control or Cdk5 shRNA (N = 3-5 mice per group). Scale bar = 100 μm. F. Two-sided Spearman’s correlation analysis of the expression of CDK5 and 3-gene TIL signature (CX3CR1, FGFBP2, FCGR3A) in 35 breast cancer BrM tissues from the GSE52604 human dataset. Each dot represents a patient. G. Spearman’s correlation analysis of the expression of CDK4/CDK6 and 12-gene TIL signature (CD8A, CCL2, CCL3, CCL4, CXCL9, CXCL10, ICOS, GZMK, IRF1, HLA.DMB, HLA.DOA, and HLA.DOB) in 35 breast cancer BrM tissues from the GSE52604 human dataset. Each dot represents a patient. H. Design of an experiment to test whether Cdk5 affects antigen-specific T cell killing of BrM cells in vitro. I. Quantification of remaining live cells 48 h after co-culture of indicated cancer cells with or without OT-I T cells (1:1 effector:target ratio). N = 3 biological replicates. Two-way ANOVA with Tukey’s post-hoc test. J. Representative images of culture wells from the experiment in J. Scale bar = 400 μm. Throughout, results are shown as means ± SEM. N.S. – not significant. TIL – tumour-infiltrating lymphocytes.

Source data

Extended Data Fig. 3 Cdk5 does not affect Pd-l1 in human or experimental BrM but negatively regulates APP and MHC-I gene expression in BrM both in vitro and in vivo.

A. Mean fluorescence intensity of Pd-l1 on the surface of indicated cells. One-way ANOVA with Dunnett’s post-hoc test. B. Immunoblotting for Pd-l1 in the indicated cell lines transfected with control or Cdk5 shRNA. C. mRNA expression of Cd274 in GFP+ single cells sorted from mice bearing BrM from GFP+ EO771.Br3.shCtl (N = 2,676 cells) or EO771.Br3.shCdk5 cells (N = 2,606 cells). Error bars indicate maximum and minimum values, box bounds indicate 1rd and 3rd quartiles, centre values indicate median, dots indicate outliers. Two-sided Wilcoxon rank-sum test. D. Two-sided Spearman's correlation analysis of the expression of CD274 and CDK5 in BrM patients from GSE164150, GSE52604 and Varešlija et al. human datasets. N = 47, 35, and 21 patients, respectively. Each dot represents a patient. E. Expression of indicated APP genes in GFP+ single cells sorted from mice bearing BrM from GFP+ EO771.Br3.shCtl (N = 2,676 cells) or EO771.Br3.shCdk5 cells (N = 2,606 cells). Each dot indicates a cell, violin shape indicates data distribution, red dot indicates the mean, bounds of the box indicate 1st and 3rd quartiles, whiskers indicate the range. Two-sided Wilcoxon rank-sum test. F. Two-sided Spearman's correlation analysis of the expression of human CDK5 and human HLA-A/B/C expression in mouse BrM lesions from MDA-MB-231.Br cells (GSE19184). Each dot represents a biological replicate. G. Two-sided Spearman's correlation analysis of the expression of CDK5 and HLA-A/B/C in single cancer cells from human breast cancer BrM lesions (GSE186344). Tables below display a cross-correlation (Spearman's) between HLA-A/B/C genes. Only samples with R2>0.5 for all cross-correlations were considered as passed the quality check. For A, results are shown as means ± SEM. N.S., not significant. QC – quality check, BrM – brain metastasis, Corr – correlation.

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Extended Data Fig. 4 Cdk5 knockdown promotes MHC-I re-expression in BrM cells, and it is dependent on Cdk5’s kinase activity.

A. Flow cytometry histograms showing surface expression of MHC-I in indicated cell lines. B. Mean fluorescence intensity of samples in A. C. Quantification of surface MHC-I staining intensity in cultured 4T1.Br3.shCtl and 4T1.Br3.shCdk5 #2 cells. Each dot indicates a field of view. N = 11 fields of view for 4T1.Br3.shCtl and 10 fields of view for 4T1.Br3.shCdk5 #2, pooled from at least 3 culture wells. Two-sided T-test. D. Representative MHC-I staining of 4T1.Br3.shCtl and 4T1.Br3.shCdk5#2 cells. Scale bar = 20 μm. E. mRNA expression of APP genes in 4T1.Br3.shCtl and 4T1.Br3.shCdk5#1 cells. N = 3 technical replicates. F. mRNA expression of APP genes in T11.Br1.shCtl and T11.Br1.shCdk5#2 cells. Und. – signal undetected. N = 3 technical replicates. G. Histograms showing surface expression of HLA-A/B/C in the indicated cell lines. H. mRNA expression of HLA-A/B/C genes, ERAP1, and CDK5 in the indicated cell lines. N = 3 biological replicates. Two-sided T-test. I. EO771.Br3 cells were treated with 7.5 μM RSV, harvested at indicated timepoints, and probed for indicated proteins. J. DKAT cells were transfected with an empty vector, CDK5-overexpressing vector (CDK5wt), and a vector containing dominant negative CDK5 mutation (CDK5kd), followed by probing for indicated proteins. Kinase activity of CDK5kd cells is impaired compared to CDK5wt cells. K. Flow cytometry histograms showing surface expression of MHC-I in DKAT cells transfected with vectors overexpressing wild-type CDK5 (CDK5wt), defective dominant negative CDK5 mutant (CDK5kd), or control (Vec. Ctl). L. Expression of HLA-A and HLA-B genes in MDA-BM-231.Br3 primary MFP breast tumours (N = 5 mice) and MDA-BM-231.Br3 BrM (N = 6 mice). Two-sided T-test. M. Expression of HLA-A and HLA-B genes in breast tumours and matched BrM (N = 21 patients) from the dataset of Varešlija et al. Paired two-sided T-test. N. Expression of HLA-A and HLA-B genes in breast tumours and matched BrM (N = 16 patients) from the GSE125989 dataset. Paired two-sided T-test. For L-N, results are shown as means (red line). For other panels, results are shown as means ± SEM. N.S. – not significant. BrM – brain metastasis.

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Extended Data Fig. 5 MHC-I overexpression diminishes experimental BrM in immunocompetent mice.

A. mRNA expression of H2-k1 in mouse primary MFP tumours and matched BrMs derived from EO771.Br3 cells (N = 2 biological replicates). B. Immunoblotting of indicated proteins in HEK923FT producer cell lines transfected with pLenti-C-Myc-DDK-P2A-Puro (Vec. Ctl) or H2-k1-C-Myc-DDK-P2A-Puro (H2-k1) vectors. C. Representative H&E staining scans of brains from mice ICA-injected using 4T1.Br3.Vec.Ctl or 4T1.Br3.H2-k1 cells. Metastatic lesions are outlined in yellow. Scale bar = 4 mm. D. Frequency of metastatic lesions (total) in immunocompetent mice bearing BrM from 4T1.Br3.Vec.Ctl or 4T1.Br3.H2-k1 cells. Each bar indicates an animal. E. Frequency of metastatic lesions (>100 μm2) in immunocompetent mice bearing BrM from 4T1.Br3.Vec.Ctl or 4T1.Br3.H2-k1 cells. Each bar indicates an animal. F. Representative IHC staining of CD8, CD4, GZMB and Ki-67 in immunocompetent mice bearing BrM from 4T1.Br3.Vec.Ctl or 4T1.Br3.H2-k1 cells (N = 4 mice per group). Scale bar = 100 μm. G. IHC quantification of CD8, CD4, GZMB and Ki-67 in immunocompetent mice bearing BrM from 4T1.Br3.Vec.Ctl or 4T1.Br3.H2-k1 cells (N = 4 mice per group). Cell number was normalized to the BrM area. Each dot indicates a BrM lesion. Two-sided Mann–Whitney U test. Throughout, results are shown as means ± SEM. BrM – brain metastasis, MFP – mammary fat pad.

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Extended Data Fig. 6 Cdk5 regulates the expression and nuclear localization of Stat1 and Nlrc5 to control the surface MHC-I expression in BrM-seeking cells.

A. mRNA expression of MHC-I-driving transcription factors in 4T1.Br3 and T11.Br1 cells transfected with control or Cdk5 shRNA. Cdk5 expression was probed as a control. N = 3 technical replicates. B. mRNA expression of Stat1 and Nlrc5 in EO771.Br3 cells transfected with control or Cdk5 shRNA. Cdk5 expression was probed as a control. N = 4 technical replicates except for Stat1 (N = 3 technical replicates). C. Stat1 expression in GFP+ cancer cells sorted from mice bearing BrM from EO771.Br3.shCtl or EO771.Br3.shCdk5 cells (N = 3 mice per group). Error bars indicate maximum and minimum values, box bounds indicate 1rd and 3rd quartiles, centre values indicate median, dots indicate outliers. Wilcoxon rank-sum test. D. Immunoblotting of indicated proteins in cytoplasm and nuclear fractions from EO771.Br3 cells transfected with control or Cdk5 shRNA. E. mRNA expression of Nlrc5 in 4T1.Br3.shCdk5 cells transfected with control or Stat1 shRNA. N = 3 technical replicates. F. Immunoblotting of indicated proteins in 4T1.Br3.shCdk5 cells transfected with control or Stat1 shRNA. G. Flow cytometry histograms showing surface expression of MHC-I in 4T1.Br3 cells transfected with shRNA targeting Cdk5, Cdk5+Stat1, or control. H. mRNA expression of Kpna2 (3 primer pairs) in EO771.Br3 and 4T1.Br3 cells transfected with control or Cdk5 shRNA. N = 3 biological replicates. Two-sided T-test. I. Immunoblotting of Nlrc5 and Importin ɑ in cytoplasm and nuclear fractions from 4T1.Br3.shCdk5 cells treated with vehicle or ivermectin for 24 h. J. IHC analyses of mouse BrM (N = 3 per group) from T11.Br1 cells transfected with control or Cdk5 shRNA. Representative staining images are shown. Scale bar = 100 μm. K. IHC quantification of staining in J. Each dot indicates a BrM lesion. Two-sided T-test. Throughout, results are shown as means ± SEM. N.S. - not significant.

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Extended Data Fig. 7 Cdk5 expression is induced by educated astrocytes but not cancer-associated fibroblasts or microglial cells.

A. In vitro cultured parental and paired BrM-seeking cells or patient-derived xenografts were probed for CDK5. B. Representative bright-field photograph of primary mouse astrocytes cultured in vitro. C. mRNA expression of the indicated genes in primary mouse astrocytes. N = 4 technical replicates for all genes except for Trem2, s100b, and Ndrg2 (N = 3 technical replicates). D-F. Indicated cell lines were cultured in CM from CAFs (D), BV-2 microglial cells (E) or primary mouse microglia (F) and probed for Cdk5 at indicated timepoints. G,H. EO771.GFP cells were directly co-cultured with CAFs (G), BV-2 microglial cells (G) or primary mouse microglia (H), GFP-sorted at indicated timepoints, and probed for Cdk5. I,J. 4T1 cells were treated as indicated followed by immunoblotting for Cdk5. K. Cytokine array of CM from primary mouse astrocytes, naïve or pre-educated by 4T1.Br3 CM. L. Pre-proteomics verification of educated astrocytes CM’s ability to induce Cdk5 in 4T1 cells by immunoblotting. M. Indicated cell lines were cultured in the presence of recombinant Versican at the indicated doses for 4 h or 8 h, followed by Cdk5 immunoblotting. N. 4T1 cell lysates were immunoprecipitated by Emilin-1 antibody or IgG, and immunoprecipitation efficiency was evaluated by Emilin-1 immunoblotting. PDX – patient-derived xenografts, CAF – cancer-associated fibroblast, CM – conditioned media, EV – extracellular vesicles, IgG – Immunoglobulin G.

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Extended Data Fig. 8 RSV penetrates the blood-brain barrier when administered in vivo and does not cause major toxicities, while inhibiting the growth of breast cancer BrM.

A. 4T1.Br3 cells were treated with 7.5 μM RSV, harvested at different timepoints, and probed for indicated proteins. B. 4T1.Br3 and T11.Br1 cells were treated with RSV or vehicle as indicated, harvested, and probed for indicated proteins. C. 4T1.Br3 cells were pre-treated with 7.5 μM RSV or vehicle for 10 days, followed by the addition of the activated CD8+ T cells (E:T = 10:1). Bars indicate a lactate dehydrogenase activity indicative of cell death, in supernatant samples collected 48 h after addition of T cells. Each dot indicates a biological replicate. Two-sided T-test. D. Mass spectrometry analysis of RSV standard and BrM sample isolated from mice bearing EO771.Br3 BrM. The table shows RSV concentration in BrM lesions. N = 3 biological replicates. E. Dynamics of mouse weight changes upon twice daily oral gavage of RSV (100 mg/kg) or vehicle. Two-way ANOVA. F. Top: Representative H&E images of livers of EO771.Br3 BrM-bearing mice after 2 weeks of twice daily oral gavage treatment with vehicle or RSV. Scale bar = 100 μm. Bottom: Serum concentration of AST, ALT and LDH in EO771.Br3 BrM-bearing mice after 2 weeks of twice daily oral gavage treatment with vehicle or RSV. Two-sided T-test. G. Representative H&E staining of brains from mice ICA-injected using 4T1.Br3 or T11 cells and treated with vehicle or RSV. BrMs are outlined in yellow. Scale bar = 3 mm. H. Model illustrating the role of Cdk5 in breast cancer BrMs. Communication between BrM cells and BrM-educated astrocytes leads to the secretion of Emilin-1 by astrocytes, which in turn induces overexpression of Cdk5 in BrMs. Through phosphorylation of Irf2bp1, Cdk5 restricts phosphorylation and expression of Stat1 and Nlrc5, leading to reduced MHC-I expression on the surface of BrM cells, which eventually leads to immune evasion followed by metastatic outgrowth in the brain. Targeting Cdk5 by small molecule inhibitor roscovitine may control the outgrowth of breast cancer BrM. For C, results are shown as means ± SEM. N.S – not significant, RSV – roscovitine, Veh – vehicle, ICA – internal carotid artery, BrM – brain metastasis, LDH – lactate dehydrogenase, AST – aspartate transaminase, ALT – alanine transaminase.

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Supplementary information

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Supplementary Data 1

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Supplementary Table 1

Supplementary Table 1. Antibodies used in this study. Supplementary Table 2. Primers used for quantitative PCR with reverse transcription.

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Yuzhalin, A.E., Lowery, F.J., Saito, Y. et al. Astrocyte-induced Cdk5 expedites breast cancer brain metastasis by suppressing MHC-I expression to evade immune recognition. Nat Cell Biol 26, 1773–1789 (2024). https://doi.org/10.1038/s41556-024-01509-5

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