Abstract
Breast cancer brain metastasis (BCBM) is a devastating disease. Radiation therapy remains the mainstay for treatment of this disease. Unfortunately, its efficacy is limited by the dose that can be safely applied. One promising approach to overcoming this limitation is to sensitize BCBMs to radiation by inhibiting their ability to repair DNA damage. Here, we report a DNA repair suppressor, leucine-rich repeat-containing protein 31 (LRRC31), that was identified through a genome-wide CRISPR screen. We found that overexpression of LRRC31 suppresses DNA repair and sensitizes BCBMs to radiation. Mechanistically, LRRC31 interacts with Ku70/Ku80 and the ataxia telangiectasia mutated and RAD3-related (ATR) at the protein level, resulting in inhibition of DNA-dependent protein kinase, catalytic subunit (DNA–PKcs) recruitment and activation, and disruption of the MutS homologue 2 (MSH2)–ATR module. We demonstrate that targeted delivery of the LRRC31 gene via nanoparticles improves the survival of tumour-bearing mice after irradiation. Collectively, our study suggests LRRC31 as a major DNA repair suppressor that can be targeted for cancer radiosensitizing therapy.
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Data availability
The cDNA array data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE117453. The data for human rectum adenocarcinoma and breast invasive carcinoma were derived from the TCGA Research Network (http://cancergenome.nih.gov). The dataset derived from this resource that supports the findings of this study is available in Source Data Extended Data Fig. 7. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank C. Ma, T. Qin and R. Zhang for assistance with surgical operations and The Youth Innovation Team of Shaanxi Universities for help. This work was supported by NIH grant NS095817 (J.Z.), the State of Connecticut (J.Z.), Projects of International Cooperation and Exchanges Natural Science Foundation of ShaanXi Province of China (Y.C., 2017KW-059) and the Scientific Research and Sharing Platform Construction Project of Shaanxi Province (Y.C., 2018PT-09).
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Contributions
J.Z. and Y.C. conceived the project. J.Z., Y.C. and H.Z. designed the experiments with the help of C. Huang, J.D., J.E.H., R.S.B., Y.C. and Y.Z. T.J., H.Z., X.G., C. Han, J.W., A.T.C., J.M., J.L., Z.C., X.J., H.L., Z.W. and Y.B. performed the experiments. Y.C. and J.Z. wrote the manuscript, with assistance from A.T.C. and M.B. All authors read and approved the final manuscript.
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Extended data
Extended Data Fig. 1 Characterize of lead candidate genes in vitro and in vivo.
a, Schematic of the CRISPR screen. b, Characterization of the proliferation of control or LRRC31-knockout 231BR cells with and without irradiation (6 Gy). c, Schematic diagram of characterization of LRRC31 in mice bearing intracranial 231BR tumors. Cells were engineered to express both luciferase and GFP. d, Changes of tumor volume versus time in mice received subcutaneous inoculation of control or LRRC31-knockout 231BR cells and treated with irradiation (10 Gy). e, f, qRT-PCR analysis of the expression of miR4796 and miR1287 in 231BR cells transduced with lentiviral vectors for expression of the candidate miRNAs or control vector. g, h, WB analysis of the expression of KATNA1 and MYBL2 in 231BR cells transduced with control vector or vectors for overexpression of the indicated gene. Blot is representative of two biologically independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 1. i-l, Clonogenic analysis of 231BR cells engineered for overexpression of miR4796 (i), miR1287 (j), KATNA1 (g) and MYBL2 (h) 7 days after irradiation. m, Characterization of the proliferation of control or LRRC31-overexpressed 231BR cells with and without irradiation (6 Gy). n, Changes of tumor volume versus time in mice received subcutaneous inoculation of control or LRRC31-overexpressed 231BR cells and treated with irradiation (10 Gy). For b, e, f, i-l, and m, data show the mean ± s.d. (n = 3 biologically independent experiments). For d and n, data show the mean ± s.d. (n = 3 animals). Statistical analysis was performed using the two-tailed, unpaired Student’s t-test.
Extended Data Fig. 2 Characterize of LRRC31 for its effects on tumor development in vivo, and on cell cycle, proliferation, and apoptosis in vitro.
Characterize of LRRC31 for its effects on tumor development in vivo, and on cell cycle, proliferation, and apoptosis in vitro. a, b, Representative images of tumors in the brain imaged by IVIS (a) and semi-quantification of the bioluminescence signal (b) in mice received intracranial inoculation of the indicated engineered cells with and without irradiation treatment (5 Gy×2). Data show the mean ± s.d. (n = 5 animals). c, Ex vivo imaging the brains isolated from mice received the indicated treatment. d-g, Characterization of the effects of LRRC31 overexpression on cell cycle determined by flow cytometry (d), proliferation determined based on BrdU staining (e), apoptosis determined based Annexin V staining (f), and Caspase-3 cleavage determined based on WB analysis (g) in the indicated cells with and without irradiation at 6 Gy. For d-f, data show the mean ± s.d. (n = 3 biologically independent experiments). Statistical analysis was performed using the two-tailed, unpaired Student’s t-test. Blot is representative of two biologically independent experiments with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 2.
Extended Data Fig. 3 Representative diagrams of flow cytometry analysis of the effects of LRRC31 overexpression on the NHEJ and HR pathways.
Representative diagrams of flow cytometry analysis of the effects of LRRC31 overexpression on the NHEJ and HR pathways. HEK293 cells were co-transfected with LRRC31- pcDNA3.1 plasmid or control vector and pEJ5-GFP or DR-GFP. After 24 hours, the cells were treated with or without irradiation at 4 Gy. After additional 24 hours, the expression of GFP in cells were quantified by flow cytometry. Example gating strategies were included. Three biologically independent experiments were performed. Data are presented in Fig. 2f,g in Main text.
Extended Data Fig. 4 Characterization of the interaction of LRRC31 with Ku70, Ku80 and MSH2 (a) and with DNA-PKcs and ATR (b).
HEK293 cells were co-transfected with Myc-tagged LRRC31 and Flag-tagged Ku70, Ku80, MSH2, DNA–PKcs, or ATR. Cell lysates were prepared and immunoprecipitated with anti-Flag or anti-Myc antibody. The precipitated proteins were then separated using 10% (a) and 6% gel (b) SDS-PAGE, and probed with anti-Myc or anti-Flag antibody. WCE, whole cell extract. Two biologically independent experiments were performed with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 4.
Extended Data Fig. 5 Characterization of the intracellular localization of LRRC31 with Ku70 and ATR as well as the effects of LRRC31 on DNA–PKcs recruitment.
a, b, Confocal analysis of the intracellular localization of LRRC31 and Ku70 in 231BR cells without (a) and with (b) overexpression of LRRC31. Irradiation was performed at 4 Gy. Scale bar: 10 µm. c, d, IP-WB analysis (c) and semi-quantification (d) of the effect of LRRC31 downregulation or overexpression on DNA–PKcs recruitment in 231BR cells. e, f, Confocal analysis of the intracellular localization of LRRC31 and ATR in 231BR cells without (e) and with (f) overexpression of LRRC31. Irradiation was performed at 4 Gy. Scale bar: 10 µm. g, h, IP-WB analysis (g) and semi-quantification (h) of the effect of LRRC31 downregulation or overexpression on DNA–PKcs recruitment in MCF7 cells. For all the studies, three biologically independent experiments were performed with similar results obtained. Data in c and f show the mean ± s.d. (n = 3 biologically independent experiments). Unprocessed immunoblots are shown in Source Data Extended Data Fig. 5.
Extended Data Fig. 6 Procedures for analysis of chromatin recruitment of DNA–PKcs by biochemical fractionation and Immunoblotting.
Detailed description of the procedures is provided in Methods.
Extended Data Fig. 7 Characterization of LRRC31 as a therapeutic target.
Characterization of LRRC31 as a therapeutic target. a, Analysis of the expression of LRRC31 in the indicated tumors in the TCGA database using Gene Expression Profiling Interactive Analysis (GEPIA). BRCA: breast invasive carcinoma; COAD: colon adenocarcinoma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; PAAD: pancreatic adenocarcinoma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma. b, Analysis of the correlation of patient survival with LRRC31 expression in BRCA patients using OncoLnc. Analysis was performed by comparing those patients with the expression level of LRRC31 among top 80th percentile (high, n = 805 biologically independent samples) with the rest (low, n = 200 biologically independent samples). c, Analysis of the correlation of patient survival with LRRC31 expression in READ patients. Analysis was performed by comparing those patients with the expression level of LRRC31 among top 80th percentile (high, n = 127 biologically independent samples) with the rest (low, n = 31 biologically independent samples). Statistical analyses for b and c were performed using the Log-rank (Mantel-Cox) test. d, Schematic diagram of characterization of LRRC31 NP-mediated gene therapy in mice bearing intracranial 231BR tumors. Cells were engineered to express both luciferase and GFP. e, f, Representative images of tumors in the brain imaged by IVIS (e) and semi-quantification of the bioluminescence signal (f) in tumor-bearing mice received intravenous administration of the indicated NPs following with and without irradiation treatment (5 Gy×2). Data in f show the mean ± s.d. (n = 5 animals). Statistical analysis was performed using the two-tailed, unpaired Student’s t-test. g, Ex vivo imaging the brains isolated from the mice received the indicated treatment. h-j, Representative images of H&E (h), Caspase-3(i), and Ki67 (j), staining of tumors isolated from mice received the indicated treatment. Scale bar: 100 µm. Three biologically independent experiments were performed with similar results obtained.
Extended Data Fig. 8 Validation of the biological effects of LRRC31 in 231BR cells transduced with doxycycline (DOX)-inducible lentiviral vector.
a, WB analysis of the expression of LRRC31 expression in 231BR cells that were transduced with control vector or DOX-inducible LRRC31 overexpression vector and treated with and without DOX (100 ng/ml). Blot is representative of two biologically independent experiments with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 8. b-d, DOX-induced overexpression of LRRC31 sensitized cells to irradiation (b), and inhibited cell proliferation (c) and DNA–PK activity (d). Data show the mean ± s.d. (n = 3 biologically independent experiments). Statistical analysis was performed using the two-tailed, unpaired Student’s t-test.
Extended Data Fig. 9 Unrooted phylogenetic tree based on LRRC31 mRNA sequences constructed by the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA7.
The analysis was performed according to a previously reported method (Kumar S, Stecher G, and Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets (2016) Molecular Biology and Evolution 33:1870-1874). Branch lengths are proportional to percentage sequence difference. Scale bar: 10% difference.
Extended Data Fig. 10 Validation of the selected LRRC31 antibody in 231BR cells with up- or down- regulation of LRRC31.
Representative fluorescence images of LRRC31 immunostaining in wild type (WT) 231BR cells, and 231BR cells with overexpression or knockout of LRRC31. Scale bar: 25 µm. Two biologically independent experiments were performed with similar results obtained.
Supplementary information
Supplementary Tables
Supplementary Table 1. List of genes identified from sgRNA sequencing; Supplementary Table 2. List of genes that are up- or downregulated by >1.5 fold identified by cDNA array. Supplementary Table 3. List of protein candidates identified by mass spectroscopy (MS). Supplementary Table 4. List of antibodies and plasmids used in this study. Supplementary Table 5. Sequences of siRNAs, sgRNAs and primers used in this study.
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Chen, Y., Jiang, T., Zhang, H. et al. LRRC31 inhibits DNA repair and sensitizes breast cancer brain metastasis to radiation therapy. Nat Cell Biol 22, 1276–1285 (2020). https://doi.org/10.1038/s41556-020-00586-6
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DOI: https://doi.org/10.1038/s41556-020-00586-6
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