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The LINK-A lncRNA activates normoxic HIF1α signalling in triple-negative breast cancer

Nature Cell Biology volume 18, pages 213–224 (2016)Cite this article

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Abstract

Although long non-coding RNAs (lncRNAs) predominately reside in the nucleus and exert their functions in many biological processes, their potential involvement in cytoplasmic signal transduction remains unexplored. Here, we identify a cytoplasmic lncRNA, LINK-A (long intergenic non-coding RNA for kinase activation), which mediates HB-EGF-triggered, EGFR:GPNMB heterodimer-dependent HIF1α phosphorylation at Tyr 565 and Ser 797 by BRK and LRRK2, respectively. These events cause HIF1α stabilization, HIF1α–p300 interaction, and activation of HIF1α transcriptional programs under normoxic conditions. Mechanistically, LINK-A facilitates the recruitment of BRK to the EGFR:GPNMB complex and BRK kinase activation. The BRK-dependent HIF1α Tyr 565 phosphorylation interferes with Pro 564 hydroxylation, leading to normoxic HIF1α stabilization. Both LINK-A expression and LINK-A-dependent signalling pathway activation correlate with triple-negative breast cancer (TNBC), promoting breast cancer glycolysis reprogramming and tumorigenesis. Our findings illustrate the magnitude and diversity of cytoplasmic lncRNAs in signal transduction and highlight the important roles of lncRNAs in cancer.

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Figure 1: LINK-A is a TNBC-upregulated cytoplasmic lncRNA with prognostic value.
Figure 2: LINK-A is involved in an HB-EGF-triggered, EGFR:GPNMB-mediated signalling pathway.
Figure 3: LINK-A mediates recruitment of BRK to GPNMB for kinase activation.
Figure 4: LINK-A-dependent BRK phosphorylation of HIF1α at Tyr 565 antagonizes HIF1α Pro 564 hydroxylation.
Figure 5: LINK-A-recruited LRRK2 phosphorylates HIF1α at Ser 797, enhances HIF1α transcriptional activity and promotes tumour growth.
Figure 6: The LINK-A-dependent normoxic HIF1α signalling pathway correlates with TNBC.

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Acknowledgements

We thank S. Kopetz for providing cetuximab and J. Chen for providing SFB-tagged expression vector. We thank D. Aten for assistance with figure presentation. This work was supported by the NIH R00 award (R00DK094981), UT Startup and UT STARS grants to C.Lin, and the NIH R00 award (R00CA166527), CPRIT award (R1218), UT Startup and UT STARS grants to L.Y.

Author information

Author notes

  1. Aifu Lin, Chunlai Li and Zhen Xing: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Oncology, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA

    Aifu Lin, Chunlai Li, Zhen Xing, Qingsong Hu, Ke Liang, Shouyu Wang, Yanyan Zhang, Yongkun Wei, Peter K. Park, Mien-Chie Hung, Chunru Lin & Liuqing Yang

  2. Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston McGroven Medical School, Houston, Texas 77030, USA

    Leng Han

  3. Department of Epidemiology and Biostatistics and Ministry of Education (MOE), School of Public Health, Nanjing Medical University, 210029, China

    Cheng Wang & Zhibin Hu

  4. Department of System Biology, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA

    David H. Hawke & Han Liang

  5. Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA

    Guolin Ma & Yubin Zhou

  6. Department of Molecular Cell Biology and Toxicology, School of Public Health, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China

    Jianwei Zhou

  7. Department of Oncology, Yixing People’s Hospital, 75 Zhenguan Road, Yixing 214200, China

    Yan Zhou

  8. Department of Surgery, Division of Surgical Science, Duke University, School of Medicine, Durham, North Carolina 27710, USA

    Jeffery R. Marks

  9. Department of Bioinformatics and Computational Biology, Division of Quantitative Sciences, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA

    Han Liang

  10. The Graduate School of Biomedical Sciences, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA

    Mien-Chie Hung, Chunru Lin & Liuqing Yang

  11. Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University, Taichung 404, Taiwan

    Mien-Chie Hung

  12. Center for RNA Interference and Non-Coding RNAs, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA

    Liuqing Yang

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  1. Aifu Lin
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Contributions

C.Lin, L.Y. and A.L. designed the research, and A.L., C.Li and Z.X. performed most of the experiments, with participation of K.L., S.W., Q.H., Y.Zhang, G.M. and Yubin Z. D.H.H. executed mass spectrometry analysis. Clinical specimens were ascertained and processed by S.W., J.Z., Yan Z. and J.R.M. The histological staining and corresponding analysis were performed by K.L. and Y.W. P.K.P. helped with manuscript preparation. TCGA data and microarray data analysis was performed by C.W., Z.H., L.H. and H.L. M.-C.H. provided reagents and conceptual advice L.Y., C.Lin and A.L. wrote the manuscript.

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Correspondence to Chunru Lin or Liuqing Yang.

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Integrated supplementary information

Supplementary Figure 1 Characterization of LINK-A protein-coding potential, subcellular localization, and LINK-A-protein interactions.

(a) Northern blot detection of Beta-Actin and LINK-A in MDA-MB-231 cells with indicated treatment. (b) 5′ and 3′ RACE-PCR of LINK-A in MDA-MB-231 cells. (c) In vitro translation of LINK-A sense or antisense transcript. Luciferase (Luc) is used as a positive control. Endogenous biotinylated protein in rabbit reticulocyte lysate was indicated with an asterisk. (d) qRT-PCR analyses of LINK-A expression level in various breast normal and cancer cell lines. (e and f) RNAScope® analysis of the indicated lncRNAs in breast cancer tissues (e) or indicated breast cancer cell lines (f). Scale bars, 100 μm. (g) RNAScope® detection of plasma membrane localization of LINK-A in MDA-MB-468 cell fractionations. Scale bars, 100 μm. (h and i) Cytoplasmic and nuclear RNA were fractionated and detected by qPCR. GAPDH and BCAR4 were used as cytoplasmic and nuclear markers respectively. (j) Denaturing agarose gel electrophoresis of in vitro transcribed biotinylated LINK-A sense and antisense transcripts. (k–n) RNA pulldown followed by IB detection of proteins retrieved by in vitro transcribed biotinylated LINK-A from MDA-MB-231 cell lysates (k) or from recombinant proteins (l–n). Streptavidin-HRP indicated the presence of equal amount of biotinylated RNA transcripts (l–n). (o) Streptavidin pulldown followed by IB detection using biotinylated LINK-A and cell lysates extracted from MDA-MB-231 cells transfected with indicated expression vectors. Streptavidin-HRP indicated the presence of equal amount of biotinylated RNA transcripts. (p and q) RIP-qPCR detection of indicated RNAs retrieved by FLAG-tag (p) or Myc-tag (q) in MDA-MB-231 cells transfected with indicated expression vectors. (r) Graphic illustration of predicted LINK-A secondary structure and the binding sites of LINK-A corresponding to BRK and LRRK2 binding. For panels d,h,i,p,q, error bars, s.e.m., n = 3 independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001, two-tailed paired Student’s t-test).

Supplementary Figure 2 Characterization of HB-EGF-induced phosphorylation of GPNMB, BRK and HIF1α and domain mapping of EGFR-GPNMB interaction.

(a) Annotated MS/MS spectrum assigned to GPNMB peptide sequence: EYNPIENSPGNVVR, Y2-Phospho (79.96633 Da) double charge, monoisotopic m/z: 834.37469 Da (−0.22 mmu/−0.26 ppm), MH+: 1667.74211 Da, RT: 2.88 min, mascot (v1.30); ionScore:58, exp value:4.7E-004. (b) Annotated MS/MS spectrum assigned to BRK peptide sequence: EDVYLSHDHNIPYK, Y13-Phospho (79.96633 Da) double charge, monoisotopic m/z: 905.39587 Da (+0.04 mmu/+0.04 ppm), MH+: 1809.78447 Da, RT: 1.97 min, identified with: Mascot (v1.30); ionScore:35, exp value:9.9E-002. (c) Annotated MS/MS spectrum assigned to HIF1α peptide sequence: NPFSTQDTDLDLEMLAPYIPMDDDFQLR, Y18-Phospho (79.96633 Da), charge: +3, monoisotopic m/z: 1127.49597 Da (−0.22 mmu/−0.19 ppm), MH+: 3380.47336 Da, RT: 29.88 min, identified with: Mascot (v1.30); ionScore:55, exp value:2.5E-003. (d) Annotated MS/MS spectrum assigned to HIF1α peptide sequence: LLGQSMDESGLPQLTSYDCEVNAPIQGSR, S16-Phospho (79.96633 Da), charge: +3, monoisotopic m/z: 1063.48022 Da (−0.53 mmu/−0.5 ppm), MH+: 3188.42612 Da, RT: 18.35 min, identified with: mascot (v1.30); ionScore:50, exp value:8.2E-003. Data were acquired from analysis of the tryptic digest by high-sensitivity LCMS/MS on an Orbitrap Elite high-resolution mass spectrometer. (e–i) IB detection of phospho-GPNMB (Tyr525) (e), phospho-BRK (Tyr351) (f), phospho-HIF1α (Tyr565) (g), phospho-HIF1α (Ser797) (h), and GAPDH (i) in lysates extracted from MDA-MB-231 cells treated with or without HB-EGF using antibodies pre-incubated with indicated blocking peptides. Antibodies generated from two independent rabbits were tested and the highlighted (red) one was used in this study. (j)IB detection using indicated antibodies in MDA-MB-231 cells treated with indicated ligands. (k)IP followed by IB detection using indicated antibodies in MDA-MB-231 cells transfected with indicated siRNAs followed by HB-EGF treatment. (l) qRT-PCR analyses of LINK-A expression level in MDA-MB-231 (left panel) and in MDA-MB-468 (right panel) cells transfected with control siRNA or LINK-A siRNA smart pool. Error bars, s.e.m., n = 3 independent experiments (*p < 0.05, two-tailed paired Student’s t-test). (m) His-tag pulldown followed by IB detection using His-tagged GPNMB intracellular domain (ICD) and GST-tagged EGFR intracellular domain (ICD), kinase domain (KD), C-terminal domain (CTD). (n,o) IP followed by IB detection using indicated antibodies in MDA-MB-231 and MDA-MB-468 cells transfected with indicated siRNAs treated with Cetuximab (20 μg ml−1) for 4 h followed by HB-EGF treatment for 30 min.

Supplementary Figure 3 Characterization of HB-EGF-triggered, LINK-A-dependent BRK recruitment and activation.

(a) Immuno-RNA FISH assay using RNA FISH probes against LINK-A (upper panel) or Actin mRNA (lower panel) and antibody against EGFR in MDA-MB-231 cells treated with HB-EGF. Scale bars, 20 μm. (b) RIP-qPCR detection of indicated RNAs retrieved by EGFR-, GPNMB- or BRK- specific antibodies in MDA-MB-231 cells treated with or without HB-EGF. (c) Immunofluorescence imaging using antibodies as indicated in MDA-MB-231 cells harboring control (left panel) or LINK-A shRNA (right panel) followed by HB-EGF stimulation. Scale bars, 20 μm. (d) qRT-PCR analyses of LINK-A expression level in MDA-MB-231 cells transfected with indicated LNAs. (e) qRT-PCR analysis of LINK-A expression level in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of indicated rescue plasmids and HB-EGF treatment. For panels b,d,e, error bars, s.e.m., n = 3 independent experiments (*p < 0.05 and ***p < 0.001, two-tailed paired Student’s t-test).

Supplementary Figure 4 Examination of LINK-A-regulated HIF1α Tyr565 phosphorylation, Pro564 hydroxylation and protein half-life.

(a) Conservation of HIF1α pYXXM motif containing phosphorylated tyrosine between species. (b) qRT-PCR analysis of HIF1α expression level in MDA-MB-231 cells treated with HB-EGF at indicated time points. Error bars, s.e.m. of three independent experiments. Error bars, s.e.m., n = 3 independent experiments (n.s., p > 0.05, two-tailed paired Student’s t-test). (c) IB detection of indicated phospho-proteins in MDA-MB-231 cells transfected with control or LINK-A siRNA followed by hypoxia treatment for 4 h. (d) IB detection using indicated antibodies in MDA-MB-468 cells transfected with control or LINK-A siRNAs followed by MG-132 and further HB-EGF treatment. (e–j) In vitro hydroxylation assay with unphosphorylated (e–g) or Tyr565 phosphorylated (h–j) HIF1α peptide showing the PHD1-dependent HIF1α hydroxylation at Pro564 in the absence or presence of the PHD inhibitor, DMOG. The resultant peptides were subjected to LC-MS analysis. The peptides with correspondent modifications were shown. (k,l) Upper panel: IB detection using indicated antibodies in MDA-MB-231 (k) or MDA-MB-468 (l) cells transfected with control or LINK-A siRNAs followed by HB-EGF and cycloheximide (CHX, 100 μg ml−1) treatment at indicated time point. Lower panel: quantification of HIF1α protein levels in k and l. (m) IB detection using indicated antibodies in MDA-MB-231 (left panel) or MDA-MB-468 (right panel) cells transfected with Myc-HIF1α WT or mutant followed by HB-EGF treatment. (n,o) Upper and middle panel: IB detection using indicated antibodies in MDA-MB-231 (n) or MDA-MB-468 (o) cells transfected with Myc-HIF1α WT or mutants followed by no treatment (upper panel) or HB-EGF treatment (middle panel) and further cycloheximide (CHX, 100 μg ml−1) treatment at indicated time point. Lower panel: quantification of HIF1α protein levels in n and o. For panels k,l,n and o, error bars, s.e.m., n = 3 independent experiments (*p < 0.05, two-tailed paired Student’s t-test).

Supplementary Figure 5 LINK-A enhances HIF1α transcriptional activity, breast cancer cell glucose metabolism, and tumor growth in vivo.

(a) Quantitative detection of LRRK2 kinase activity in the absence or presence of LINK-A or indicated deletion transcripts. Left panel: Relative Pi release monitored by OD 620 nm. Right panel: calculated specific kinase activity (pmol/min/μg) based on Pi measurement. (b) qRT-PCR detection of exogenous expressed LINK-A wild-type, ATG or TGA mutants. (c,d) Immunoblotting detection of BRK (c) and HIF1α (d) phosphorylation in MDA-MB-231 cells transfected with indicated LNA and expression vectors followed by HB-EGF stimulation. (e) qRT-PCR analysis of HIF1α target genes expression in MDA-MB-468 cells transfected with control or LINK-A siRNA followed by HB-EGF treatment. (f) qRT-PCR analyses of LINK-A expression level in MDA-MB-231 cells transfected with control shRNA or LINK-A shRNAs. (g–i) Lactate production (g and h) or glucose uptake (i) assay in MDA-MB-231 and MDA-MB-468 cells transfected with control or LINK-A siRNAs. (j) Cell proliferation rate was assessed by OD density (590 nm) in MDA-MB-231 cells transfected with LNAs as indicated. (k,l) Glucose uptake (k) or lactate production (l) was measured in MDA-MB-231 cells transfected with scramble or LINK-A LNAs. For panels a,b,e–l, error bars, s.e.m., n = 3 independent experiments (n.s., p > 0.05, *p < 0.05, and **p < 0.01, two-tailed paired Student’s t-test). (m) Measurement of tumor volume in mice that were subcutaneously injected with MDA-MB-231 cells harboring control or LINK-A shRNA at indicated post-injection time point. Data are mean ± s.e.m. n = 5 mice per group (**p < 0.01, two-tailed paired Student’s t-test).

Supplementary Figure 6 Correlation of LINK-A-mediated signalling pathway activation with TNBC.

(a) IHC staining of phospho-GPNMB (Tyr525), phospho-BRK (Tyr351), phospho-HIF1α (Tyr565) and phospho-HIF1α (Ser797) in human breast cancer tissues. Scale bars, 100 μm. (b) Oncomine boxed plot showing BRK and LRRK2 expression levels in human normal and breast cancer tissues. (c) Graphic illustration of functional roles of LINK-A in HB-EGF-triggered, EGFR: GPNMB receptor-dependent and BRK/LRRK2-mediated HIF1α signalling cascade.

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Lin, A., Li, C., Xing, Z. et al. The LINK-A lncRNA activates normoxic HIF1α signalling in triple-negative breast cancer. Nat Cell Biol 18, 213–224 (2016). https://doi.org/10.1038/ncb3295

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