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Long noncoding RNA LINP1 regulates repair of DNA double-strand breaks in triple-negative breast cancer

Nature Structural & Molecular Biology volume 23, pages 522–530 (2016)Cite this article

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Abstract

Long noncoding RNAs (lncRNAs) play critical roles during tumorigenesis by functioning as scaffolds that regulate protein-protein, protein-DNA or protein-RNA interactions. Using a clinically guided genetic screening approach, we identified lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1), which is overexpressed in human triple-negative breast cancer. We found that LINP1 enhances repair of DNA double-strand breaks by serving as a scaffold linking Ku80 and DNA-PKcs, thereby coordinating the NHEJ pathway. Importantly, blocking LINP1, which is regulated by p53 and epidermal growth factor receptor (EGFR) signaling, increases the sensitivity of the tumor-cell response to radiotherapy in breast cancer.

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Figure 1: Identification of the TNBC-associated lncRNA LINP1.
Figure 2: Expression and genomic alteration of LINP1 in breast cancer.
Figure 3: LINP1 RNA associates with Ku80 and DNA-PKcs.
Figure 4: LINP1 serves as a modular scaffold in the NHEJ pathway.
Figure 5: LINP1 is activated by the EGF signaling pathway.
Figure 6: LINP1 is repressed by the p53 signaling pathway.
Figure 7: Alteration of LINP1 modulates IR sensitivity.
Figure 8: Role of LINP1 in the NHEJ DNA-repair pathway.

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References

  1. Foulkes, W.D., Smith, I.E. & Reis-Filho, J.S. Triple-negative breast cancer. N. Engl. J. Med. 363, 1938–1948 (2010).

    Article  CAS  Google Scholar 

  2. Carey, L., Winer, E., Viale, G., Cameron, D. & Gianni, L. Triple-negative breast cancer: disease entity or title of convenience? Nat. Rev. Clin. Oncol. 7, 683–692 (2010).

    Article  Google Scholar 

  3. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012).

  4. Derrien, T. et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 22, 1775–1789 (2012).

    Article  CAS  Google Scholar 

  5. Mercer, T.R., Dinger, M.E. & Mattick, J.S. Long non-coding RNAs: insights into functions. Nat. Rev. Genet. 10, 155–159 (2009).

    Article  CAS  Google Scholar 

  6. Rinn, J.L. & Chang, H.Y. Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166 (2012).

    Article  CAS  Google Scholar 

  7. Guttman, M. & Rinn, J.L. Modular regulatory principles of large non-coding RNAs. Nature 482, 339–346 (2012).

    Article  CAS  Google Scholar 

  8. Batista, P.J. & Chang, H.Y. Long noncoding RNAs: cellular address codes in development and disease. Cell 152, 1298–1307 (2013).

    Article  CAS  Google Scholar 

  9. Ørom, U.A. & Shiekhattar, R. Long noncoding RNAs usher in a new era in the biology of enhancers. Cell 154, 1190–1193 (2013).

    Article  Google Scholar 

  10. Ulitsky, I. & Bartel, D.P. lincRNAs: genomics, evolution, and mechanisms. Cell 154, 26–46 (2013).

    Article  CAS  Google Scholar 

  11. Tay, Y., Rinn, J. & Pandolfi, P.P. The multilayered complexity of ceRNA crosstalk and competition. Nature 505, 344–352 (2014).

    Article  CAS  Google Scholar 

  12. Sahu, A., Singhal, U. & Chinnaiyan, A.M. Long noncoding RNAs in cancer: from function to translation. Trends Cancer 1, 93–109 (2015).

    Article  Google Scholar 

  13. Ling, H. et al. Junk DNA and the long non-coding RNA twist in cancer genetics. Oncogene 34, 5003–5011 (2015).

    Article  CAS  Google Scholar 

  14. Iyer, M.K. et al. The landscape of long noncoding RNAs in the human transcriptome. Nat. Genet. 47, 199–208 (2015).

    Article  CAS  Google Scholar 

  15. Yan, X. et al. Comprehensive genomic characterization of long non-coding RNAs across human cancers. Cancer Cell 28, 529–540 (2015).

    Article  CAS  Google Scholar 

  16. Gupta, R.A. et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464, 1071–1076 (2010).

    Article  CAS  Google Scholar 

  17. Tsai, M.C. et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010).

    Article  CAS  Google Scholar 

  18. Lin, A. et al. The LINK-A lncRNA activates normoxic HIF1α signalling in triple-negative breast cancer. Nat. Cell Biol. 18, 213–224 (2016).

    Article  CAS  Google Scholar 

  19. Helleday, T., Petermann, E., Lundin, C., Hodgson, B. & Sharma, R.A. DNA repair pathways as targets for cancer therapy. Nat. Rev. Cancer 8, 193–204 (2008).

    Article  CAS  Google Scholar 

  20. Jackson, S.P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    Article  CAS  Google Scholar 

  21. Lieber, M.R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79, 181–211 (2010).

    Article  CAS  Google Scholar 

  22. Lord, C.J. & Ashworth, A. The DNA damage response and cancer therapy. Nature 481, 287–294 (2012).

    Article  CAS  Google Scholar 

  23. Chiruvella, K.K., Liang, Z. & Wilson, T.E. Repair of double-strand breaks by end joining. Cold Spring Harb. Perspect. Biol. 5, a012757 (2013).

    Article  Google Scholar 

  24. Deriano, L. & Roth, D.B. Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage. Annu. Rev. Genet. 47, 433–455 (2013).

    Article  CAS  Google Scholar 

  25. Mehta, A. & Haber, J.E. Sources of DNA double-strand breaks and models of recombinational DNA repair. Cold Spring Harb. Perspect. Biol. 6, a016428 (2014).

    Article  Google Scholar 

  26. Goldstein, M. & Kastan, M.B. The DNA damage response: implications for tumor responses to radiation and chemotherapy. Annu. Rev. Med. 66, 129–143 (2015).

    Article  CAS  Google Scholar 

  27. Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).

    Article  CAS  Google Scholar 

  28. Simon, M.D. et al. The genomic binding sites of a noncoding RNA. Proc. Natl. Acad. Sci. USA 108, 20497–20502 (2011).

    Article  CAS  Google Scholar 

  29. Seluanov, A., Mittelman, D., Pereira-Smith, O.M., Wilson, J.H. & Gorbunova, V. DNA end joining becomes less efficient and more error-prone during cellular senescence. Proc. Natl. Acad. Sci. USA 101, 7624–7629 (2004).

    Article  CAS  Google Scholar 

  30. Ugalde, A.P. et al. Aging and chronic DNA damage response activate a regulatory pathway involving miR-29 and p53. EMBO J. 30, 2219–2232 (2011).

    Article  CAS  Google Scholar 

  31. Ting, N.S., Yu, Y., Pohorelic, B., Lees-Miller, S.P. & Beattie, T.L. Human Ku70/80 interacts directly with hTR, the RNA component of human telomerase. Nucleic Acids Res. 33, 2090–2098 (2005).

    Article  CAS  Google Scholar 

  32. Nyati, M.K., Morgan, M.A., Feng, F.Y. & Lawrence, T.S. Integration of EGFR inhibitors with radiochemotherapy. Nat. Rev. Cancer 6, 876–885 (2006).

    Article  CAS  Google Scholar 

  33. Chen, D.J. & Nirodi, C.S. The epidermal growth factor receptor: a role in repair of radiation-induced DNA damage. Clin. Cancer Res. 13, 6555–6560 (2007).

    Article  CAS  Google Scholar 

  34. Ule, J., Jensen, K., Mele, A. & Darnell, R.B. CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37, 376–386 (2005).

    Article  CAS  Google Scholar 

  35. Zhao, J. et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell 40, 939–953 (2010).

    Article  CAS  Google Scholar 

  36. Zhang, C. & Darnell, R.B. Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. Nat. Biotechnol. 29, 607–614 (2011).

    Article  CAS  Google Scholar 

  37. Méndez, J. & Stillman, B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000).

    Article  Google Scholar 

  38. Shang, Y., Hu, X., DiRenzo, J., Lazar, M.A. & Brown, M. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103, 843–852 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the TCGA and CCLE project teams. We thank Dr. B. Vogelstein (Johns Hopkins University) for providing HCT116 WT and HCT116 TP53 cell lines. This work was supported, in whole or in part, by the Basser Center for BRCA (L.Z.), the US National Institutes of Health (R01CA142776 to L.Z., R01CA190415 to L.Z., P50CA083638 to L.Z., P50CA174523 to L.Z., R01CA148759 to Q.H., and R01NS094533 to Y.F.), the Breast Cancer Alliance (L.Z. and C.V.D.), the Ovarian Cancer Research Fund (X.H.), the Foundation for Women's Cancer (X.H.), and the Marsha Rivkin Center for Ovarian Cancer Research (L.Z.). S.W. was supported by the China Scholarship Council.

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Author notes

  1. Youyou Zhang and Qun He: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Research on Reproduction & Women's Health, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Youyou Zhang, Qun He, Zhongyi Hu, Yi Feng, Lingling Fan, Zhaoqing Tang, Jiao Yuan, Weiwei Shan, Chunsheng Li, Xiaowen Hu & Lin Zhang

  2. Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Yi Feng & Chi V Dang

  3. Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Chunsheng Li, Xiaowen Hu, Janos L Tanyi & Lin Zhang

  4. Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Yi Fan

  5. Wistar Institute, Philadelphia, Pennsylvania, USA

    Qihong Huang

  6. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Kathleen Montone

  7. Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Chi V Dang

  8. Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Chi V Dang & Lin Zhang

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Contributions

Y.Z., C.V.D. and L.Z. conceptualized and designed the experiments. Y.Z., Q. He, L.F., W.S., and C.L. performed the molecular and cellular biology experiments. Z.H., Y. Fan, Z.T. and J.Y. performed bioinformatics analysis. Y.Z., Z.H., J.Y., X.H., J.L.T., Y.F., Q. Huang, and K.M. analyzed and interpreted data. Y.Z., Y. Feng, C.V.D., and L.Z. wrote the manuscript.

Corresponding authors

Correspondence to Chi V Dang or Lin Zhang.

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Integrated supplementary information

Supplementary Figure 1 Identification of the TNBC-associated lncRNA LINP1.

(a) A schematic diagram of the functional siRNA screen based upon a doxorubicin-induced apoptosis assay. (b) Upper panel: RNA-seq results of (MDA-MB-231, MDA-MB-468 and MCF7) from CCLE database. Lower panel: Histone marks of the LINP1 gene region in MDA-MB-231 (upper) and MCF7 (lower). The ChIP-seq of MDA-MB-231 and MCF7 were retrieved from GSE72141 and ENCODE, respectively. (c) Northern blot analysis of LINP1 expression in MDA-MB-231, MDA-MB-468, MCF10A, and MCF7 cells.

Supplementary Figure 2 Expression of LINP1 in normal human tissues.

(a) The normal corresponding tissue RNA-seq data were retrieved from the TCGA database. (b) LINP1 RNA expression in different subcellular fractions in MDA-MB-231 cells (no IR treatment). Red indicates nuclear fraction; blue indicates cytoplasmic fraction. The percentage was calculated by qRT-PCR. The nuclear RNA (U2 snRNA) and the cytoplasmic RNA (house-keeping RNA S14) were used as control.

Supplementary Figure 3 LINP1 RNA associates with Ku80 and DNA-PKcs.

(a) RNA pull-down assay in combination with MS analysis was used to identify binding proteins of LINP1. Left, mass spectroscopy identifies peptides mapped to Ku80 and DNA-PKcs, respectively. Right, peptide coverage of these two proteins; blue indicates the covered regions. (b) Deletion mapping of Ku80 and DNA-PKcs -binding domains in LINP1. Left panel: the schematic diagram of full-length and deleted fragments of LINP1. Right panel: quantitative data of the binding intensity between LINP1-Ku80 (left) or LINP1-DNA-PKcs (right).

Supplementary Figure 4 LINP1 serves as a modular scaffold in the NHEJ pathway.

(a) Western analysis of γH2AX in the control and LINP1 knockdown MDA-MB-231 cells at different time points (0, 0.5, 4 and 24 hours) after IR treatment. (b) Levels of Ku80 and DNA-PKcs in the complex pulled down by DNA-PKcs (upper panel) and Ku80 (low panel) specific antibodies, in control and LINP1 knockdown cells. 10 Gy of irradiation was used for all experiments. Uncropped images of gels are shown in Supplementary Data Set 2.

Supplementary Figure 5 ChIP–seq analysis of the LINP1 promoter region.

ChIP-seq analysis of the LINP1 promoter region in MDA-MB-468 and MCF7 cells. The ChIP-seq data were retrieved from the ENCODE project.

Supplementary Figure 6 p53 regulates miR-29 expression in cancer cell lines.

miR-29 expression in cells of different TP53 status, in response to nutlin-3a treatment. MCF10 and HCT116, TP53 WT cells; MDA-MB-231, TP53 mutant; HCT116 with TP53 deletion, TP53 null. Error bars indicate SD; two-tailed Student's t-test; ∗ indicates p-value<0.05; n=3 independent technical replicates.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 742 kb)

Supplementary Table 1

The list of lncRNAs that were significantly enriched in expression in TNBC/basal tumors (XLSX 30 kb)

Supplementary Table 2

The list of siRNA sequences for the lncRNAs (XLSX 12 kb)

Supplementary Table 3

Mass spectrometry (MS) results of identified proteins by RNA pulldown (XLSX 10 kb)

Supplementary Data Set 1

The breast cancer specimens from TCGA (XLSX 9 kb)

Supplementary Data Set 2

Raw gel images from all figures (PDF 5087 kb)

Supplementary Data Set 3

Oligos and primers used in this study (XLSX 9 kb)

Supplementary Data Set 4

Antibody information (XLSX 10 kb)

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Zhang, Y., He, Q., Hu, Z. et al. Long noncoding RNA LINP1 regulates repair of DNA double-strand breaks in triple-negative breast cancer. Nat Struct Mol Biol 23, 522–530 (2016). https://doi.org/10.1038/nsmb.3211

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