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

Journal name:
Nature Cell Biology
Volume:
18,
Pages:
213–224
Year published:
DOI:
doi:10.1038/ncb3295
Received
Accepted
Published online

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.

At a glance

Figures

  1. LINK-A is a TNBC-upregulated cytoplasmic lncRNA with prognostic value.
    Figure 1: LINK-A is a TNBC-upregulated cytoplasmic lncRNA with prognostic value.

    (a,b) Scatter plots comparing LINK-A expression in breast tumour samples with different ER, PR and HER2 status including ER/PR/HER2 (n = 119), ER/PR/HER2+ (n = 30), ER+/PR+/HER2 (n = 482), and ER+/PR+/HER2+ (n = 80) (a), or in breast tumour tissue samples with different subtypes including basal (n = 139), HER2 (n = 67), LumA (n = 417), LumB (n = 191) and normal-like (n = 23) (b). Statistical significance was determined by two-way ANOVA. The boxes show the median and the interquartile range. The whiskers show the minimum and maximum. (c,d) RNAScope detection of LINK-A expression in human breast cancer and adjacent normal tissues (training (c) and validation (d) set, respectively). Left panel in c: representative images (scale bars, 100μm); d and right panel in c: statistical analysis. Training set: TNBC (n = 10), ER/PR/HER2+ (n = 7), ER+/PR+/HER2 (n = 18), and ER+/PR+/HER2+ (n = 2); validation set: ER/PR/HER2 (n = 38), ER/PR/HER2+ (n = 2), ER+/PR+/HER2 (n = 6), ER+/PR+/HER2+ (n = 9) and normal tissue (n = 20) (median, two-way ANOVA). Horizontal black lines represent median. Coloured error bars represent 95% quantile. (e) Recurrence-free survival analysis of LINK-A status in breast cancer patients detected by qRT–PCR (n = 123 patients, Gehan–Breslow test). (f) A list of the top LINK-A-associated proteins identified by RNA pulldown and MS analysis in MDA-MB-231 cells. (g) RIP–qPCR detection of indicated RNAs retrieved by BRK-, LRRK2- or eIF4B-specific antibodies in MDA-MB-231 cells. Error bars, s.e.m., n = 3 independent experiments (*P < 0.05, two-tailed paired Students t-test). (h,i) In vitro RNA–protein binding assay showing the interaction of biotinylated LINK-A with wild-type (WT) FLAG-tagged BRK and a deletion mutant (h), or WT Myc-tagged LRRK2 and a deletion mutant (i). Dot-blot of RNA–protein binding samples indicates equal RNA transcript present in the assay. Bottom panel: graphic illustration of BRK or LRRK2 domain deletion mutants. IB, immunoblot. (j) Upper panel: In vitro RNA–protein binding followed by dot-blot assays using biotinylated LINK-A sense (sen.) or antisense (as.) transcripts and GST-tagged, bacterially expressed BRK or LRRK2 proteins. The hybridized RNA fragments were detected by streptavidin–HRP. Bottom panel: graphic illustration of LINK-A probes. (k) Immunoblot detection of proteins retrieved by in-vitro-transcribed biotinylated LINK-A full-length (FL) or deletion mutants expressed in MDA-MB-231 cells. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

  2. LINK-A is involved in an HB-EGF-triggered, EGFR:GPNMB-mediated signalling pathway.
    Figure 2: LINK-A is involved in an HB-EGF-triggered, EGFR:GPNMB-mediated signalling pathway.

    (a) Summary of the phosphorylation sites of the indicated proteins identified from RNA pulldown followed by MS analysis. (b) Immunoprecipitation (IP) followed by immunoblot (IB) detection of the indicated proteins in MDA-MB-231 cells treated with the indicated growth factors for 30 min. (c) Immunoblot detection using the indicated antibodies in MDA-MB-231 cells stimulated with vehicle, EGF or HB-EGF followed by DTSSP chemical crosslinking (1mM, 30min). (d,e) His tag (d) or FLAG tag (e) pulldown followed by immunoblot detection using the indicated antibodies in MDA-MB-231 cells transfected with the indicated expression vectors followed by HB-EGF stimulation. ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain. (f) Immunoprecipitation followed by immunoblot detection of GPNMB, BRK and HIF1α phosphorylation in MDA-MB-231 cells treated with the indicated growth factors. (g) In vitro kinase assay using the indicated recombinant proteins, followed by Coomassie blue staining (CBB), and immunoblot detection using the indicated antibodies. (h,i) Immunoprecipitation followed by immunblot detection using the indicated antibodies in cells transfected with the indicated expression vectors followed by HB-EGF stimulation. Left panel (i): graphic illustration of BRK domain deletion mutants. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

  3. LINK-A mediates recruitment of BRK to GPNMB for kinase activation.
    Figure 3: LINK-A mediates recruitment of BRK to GPNMB for kinase activation.

    (a) Immunofluorescence detection using the indicated antibodies in MDA-MB-231 cells harbouring control (upper panel) or LINK-A shRNA, followed by HB-EGF stimulation (upper panel). Scale bars, 20μm. (b) Graphic illustration of the BRK– and LRRK2–LINK-A interactions (upper panel) and the corresponding deletion abolishing these interactions (lower panel). (c,d) Immunofluorescence imaging (c, scale bars, 20μm) or immunoblot (IB) detection (d) was performed using the indicated antibodies in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of the indicated rescue plasmids with HB-EGF stimulation. The dotted line on the blots of d indicates the position where the images of single blots were vertically cropped to juxtapose non-adjacent lanes. (e) In vitro kinase assay using recombinant BRK and in vitro-transcribed RNA transcripts as indicated in the presence or absence of [32P]ATP. The dot-blot indicates equal RNA transcript present in the assay. (f) Quantification of BRK kinase activity in the presence of the indicated in vitro-transcribed RNA transcripts using HIF1α peptide (amino acids 557–566) as the substrate. Upper panel: release of free phosphate ion (Pi) amount measured at OD620nm; lower panel: calculation of BRK kinase activity (pmolmin−1μg−1). Error bars, s.e.m., n = 3 independent experiments (*P < 0.05, two-tailed paired Students t-test). (g) Immunblot detection of BRK using the indicated antibodies in the presence of the indicated lncRNA transcripts with or without caspase-1 digestion. Left panel: graphic illustration of caspase-1-mediated BRK cleavage in the absence or presence of lncRNA. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

  4. LINK-A-dependent BRK phosphorylation of HIF1[alpha] at Tyr 565 antagonizes HIF1[alpha] Pro 564 hydroxylation.
    Figure 4: LINK-A-dependent BRK phosphorylation of HIF1α at Tyr 565 antagonizes HIF1α Pro 564 hydroxylation.

    (a) In vitro phosphorylation assay using recombinant proteins (WT or mutants as indicated). IB, immunblot. (b) In vitro kinase assay using bacterially expressed GST-tagged BRK WT or mutant and His-tagged HIF1α. (cf) Immunoblot detection using the indicated antibodies in MDA-MB-231 (ce) or MDA-MB-468 (f) cells treated with HB-EGF at the indicated time point (c) or transfected with the indicated siRNAs followed by HB-EGF treatment (df). (gi) Immunoblot detection using the indicated antibodies in MDA-MB-231 cells treated with MG-132 followed by HB-EGF treatment at the indicated time (g) or in cells transfected with the indicated siRNAs followed by MG-132 and HB-EGF treatment (h,i). (j) LC–MS sequencing of the HIF1α peptide (557–566) in an in vitro hydroxylation assay. The total peptide numbers of HIF1α proline non-hydroxylated versus proline hydroxylated (P-OH) under the indicated conditions are shown. The peptide number of hydroxylated WT peptide is indicated in red. (k) His-tag pulldown followed by immunoblot detection of HIF1α phosphorylation and hydroxylation (WT versus Y565F) in an in vitro kinase assay (1°) followed by in vitro hydroxylation assay (2°). (l) Immunoprecipitation (IP) followed by immunoblot detection of HIF1α phosphorylation and hydroxylation (WT versus Y565F) in MDA-MB-231 cells transfected with the indicated plasmids and treated with MG-132 followed by HB-EGF treatment. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

  5. LINK-A-recruited LRRK2 phosphorylates HIF1[alpha] at Ser 797, enhances HIF1[alpha] transcriptional activity and promotes tumour growth.
    Figure 5: LINK-A-recruited LRRK2 phosphorylates HIF1α at Ser 797, enhances HIF1α transcriptional activity and promotes tumour growth.

    (a,b) Immunoprecipitation (IP) followed by immunoblot (IB) detection using the indicated antibodies in MDA-MB-231 cells transfected with the indicated siRNAs (a) or plasmids (b), and treated with MG-132 followed by HB-EGF treatment. (c) Immunoblot detection using the indicated antibodies in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of the indicated rescue plasmids and HB-EGF stimulation. The dotted line indicates the position where the images of single blots were vertically cropped to juxtapose non-adjacent lanes. (d) HIF1α ChIP-seq analysis showing the top enriched HIF-binding consensus motifs. (e) HIF1α ChIP-seq analysis showing signalling pathways in MDA-MB-231 cells treated with HB-EGF. (f,g) ChIP–qPCR detection of HIF1α occupancy on indicated target gene promoters (f) and qRT–PCR analysis of HIF1α target genes expression (g) in MDA-MB-231 cells transfected with control or LINK-A siRNA followed by HB-EGF treatment. (h) Colony formation assay in MDA-MB-231 cells transduced with control and LINK-A shRNAs. Scale bars, 200μm. For fh, error bars, s.e.m.; n = 3 independent experiments (*P < 0.05 and **P < 0.01, two-tailed paired Students t-test). (i,j) In vivo analyses of tumour growth (i) or weight (j) in mice that were subcutaneously injected with MDA-MB-231 cells harbouring control or LINK-A shRNA. Data are mean ± s.e.m., n = 5 mice per group (**P < 0.01, two-tailed paired Students t-test). Unprocessed original scans of blots are shown in Supplementary Fig. 7.

  6. The LINK-A-dependent normoxic HIF1[alpha] signalling pathway correlates with TNBC.
    Figure 6: The LINK-A-dependent normoxic HIF1α signalling pathway correlates with TNBC.

    (ac) Immunohistochemical staining using antibodies against phospho-BRK (Tyr 351) (a), phospho-HIF1α (Tyr 565) (b) or phospho-GPNMB (Tyr 525) (c) in human breast cancer tissues. Upper panel: representative images (scale bars, 100μm; lower panel: statistics analysis based on non-TNBC tissues (n = 5) versus TNBC tissues (n = 40) and non-metastasis (TnN0M0) TNBC (n = 27) versus metastasis (TnN > 0M ≥ 0) breast tissues (n = 13) (median, two-way ANOVA). (df) Upper panel: statistical analysis of immunohistochemical staining using antibodies against phospho-BRK (Tyr 351) (d), phospho-HIF1α (Tyr 565) (e) or phospho-GPNMB (Tyr 525) (f) in human breast cancer tissues including TNBC (n = 10), ER/PR/HER2+ (n = 7), ER+/PR+/HER2 (n = 18), and ER+/PR+/HER2+ (n = 2) (median, two-way ANOVA). Lower panel: Pearsons correlation analysis comparing staining density between LINK-A expression and phospho-BRK (Tyr 351) (d), phospho-HIF1α (Tyr 565) (e) or phospho-GPNMB (Tyr 525) (f) within the TNBC group (n = 10 tissue samples, Fishers exact test). (gi) Kaplan–Meier survival analysis of phosphor-BRK (Tyr 351) (g), phosphor-HIF1α (Tyr 565) (h) and phospho-GPNMB (Tyr 525) (i) status in breast cancer patients (n = 160, Gehan–Breslow test).

  7. Characterization of LINK-A protein-coding potential, subcellular localization, and LINK-A-protein interactions.
    Supplementary Fig. 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, 100μm. (g) RNAScope® detection of plasma membrane localization of LINK-A in MDA-MB-468 cell fractionations. Scale bars, 100μm. (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. (kn) 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 (ln). Streptavidin-HRP indicated the presence of equal amount of biotinylated RNA transcripts (ln). (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 Students t-test).

  8. Characterization of HB-EGF-induced phosphorylation of GPNMB, BRK and HIF1[alpha] and domain mapping of EGFR-GPNMB interaction.
    Supplementary Fig. 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 (79.96633Da) double charge, monoisotopic m/z: 834.37469Da (−0.22 mmu/−0.26 ppm), MH+: 1667.74211Da, 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 (79.96633Da) double charge, monoisotopic m/z: 905.39587Da (+0.04mmu/+0.04 ppm), MH+: 1809.78447Da, 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 (79.96633Da), charge: +3, monoisotopic m/z: 1127.49597Da (−0.22 mmu/−0.19 ppm), MH+: 3380.47336Da, 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 (79.96633Da), charge: +3, monoisotopic m/z: 1063.48022Da (−0.53 mmu/−0.5 ppm), MH+: 3188.42612Da, 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. (ei) 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 Students 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 (20μgml−1) for 4h followed by HB-EGF treatment for 30min.

  9. Characterization of HB-EGF-triggered, LINK-A-dependent BRK recruitment and activation.
    Supplementary Fig. 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, 20μm. (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, 20μm. (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 Students t-test).

  10. Examination of LINK-A-regulated HIF1[alpha] Tyr565 phosphorylation, Pro564 hydroxylation and protein half-life.
    Supplementary Fig. 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 Students 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 4h. (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. (ej) In vitro hydroxylation assay with unphosphorylated (eg) or Tyr565 phosphorylated (hj) 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, 100μgml−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, 100μgml−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 Students t-test).

  11. LINK-A enhances HIF1[alpha] transcriptional activity, breast cancer cell glucose metabolism, and tumor growth in vivo.
    Supplementary Fig. 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 620nm. 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. (gi) 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 (590nm) 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,el, error bars, s.e.m., n = 3 independent experiments (n.s., p > 0.05, *p < 0.05, and **p < 0.01, two-tailed paired Students 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 Students t-test).

  12. Correlation of LINK-A-mediated signalling pathway activation with TNBC.
    Supplementary Fig. 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, 100μm. (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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Author information

  1. These authors contributed equally to this work.

    • Aifu Lin,
    • Chunlai Li &
    • Zhen Xing

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 Peoples 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

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Characterization of LINK-A protein-coding potential, subcellular localization, and LINK-A-protein interactions. (808 KB)

    (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, 100μm. (g) RNAScope® detection of plasma membrane localization of LINK-A in MDA-MB-468 cell fractionations. Scale bars, 100μm. (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. (kn) 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 (ln). Streptavidin-HRP indicated the presence of equal amount of biotinylated RNA transcripts (ln). (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 Students t-test).

  2. Supplementary Figure 2: Characterization of HB-EGF-induced phosphorylation of GPNMB, BRK and HIF1α and domain mapping of EGFR-GPNMB interaction. (846 KB)

    (a) Annotated MS/MS spectrum assigned to GPNMB peptide sequence: EYNPIENSPGNVVR, Y2-Phospho (79.96633Da) double charge, monoisotopic m/z: 834.37469Da (−0.22 mmu/−0.26 ppm), MH+: 1667.74211Da, 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 (79.96633Da) double charge, monoisotopic m/z: 905.39587Da (+0.04mmu/+0.04 ppm), MH+: 1809.78447Da, 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 (79.96633Da), charge: +3, monoisotopic m/z: 1127.49597Da (−0.22 mmu/−0.19 ppm), MH+: 3380.47336Da, 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 (79.96633Da), charge: +3, monoisotopic m/z: 1063.48022Da (−0.53 mmu/−0.5 ppm), MH+: 3188.42612Da, 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. (ei) 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 Students 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 (20μgml−1) for 4h followed by HB-EGF treatment for 30min.

  3. Supplementary Figure 3: Characterization of HB-EGF-triggered, LINK-A-dependent BRK recruitment and activation. (998 KB)

    (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, 20μm. (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, 20μm. (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 Students t-test).

  4. Supplementary Figure 4: Examination of LINK-A-regulated HIF1α Tyr565 phosphorylation, Pro564 hydroxylation and protein half-life. (715 KB)

    (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 Students 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 4h. (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. (ej) In vitro hydroxylation assay with unphosphorylated (eg) or Tyr565 phosphorylated (hj) 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, 100μgml−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, 100μgml−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 Students t-test).

  5. Supplementary Figure 5: LINK-A enhances HIF1α transcriptional activity, breast cancer cell glucose metabolism, and tumor growth in vivo. (561 KB)

    (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 620nm. 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. (gi) 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 (590nm) 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,el, error bars, s.e.m., n = 3 independent experiments (n.s., p > 0.05, *p < 0.05, and **p < 0.01, two-tailed paired Students 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 Students t-test).

  6. Supplementary Figure 6: Correlation of LINK-A-mediated signalling pathway activation with TNBC. (960 KB)

    (a) IHC staining of phospho-GPNMB (Tyr525), phospho-BRK (Tyr351), phospho-HIF1α (Tyr565) and phospho-HIF1α (Ser797) in human breast cancer tissues. Scale bars, 100μm. (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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