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
- Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012).
- Triple-negative breast cancer. N. Engl. J. Med. 363, 1938–1948 (2010). , &
- Triple-negative breast cancer: an unmet medical need. Oncologist 16, 1–11 (2011). &
- The emergence of lncRNAs in cancer biology. Cancer Discov. 1, 391–407 (2011). &
- Long noncoding RNAs and the genetics of cancer. Br. J. Cancer 108, 2419–2425 (2013). , , &
- The hallmarks of cancer: a long non-coding RNA point of view. RNA Biol. 9, 703–719 (2012). &
- Epigenetic regulation by long noncoding RNAs. Science 338, 1435–1439 (2012).
- Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166 (2012). &
- Long noncoding RNAs: cellular address codes in development and disease. Cell 152, 1298–1307 (2013). &
- The role of long noncoding RNAs in the epigenetic control of gene expression. ChemMedChem 9, 505–510 (2014). , , &
- Long noncoding RNAs as enhancers of gene expression. Cold Spring Harb. Symp. Quant. Biol. 75, 325–331 (2010). , , &
- Evolution and functions of long noncoding RNAs. Cell 136, 629–641 (2009). , &
- Transcriptional regulatory functions of nuclear long noncoding RNAs. Trends Genet. 30, 348–355 (2014). &
- The long arm of long noncoding RNAs: roles as sensors regulating gene transcriptional programs. Cold Spring Harb. Perspect. Biol. 3, a003756 (2011). , , &
- lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature 500, 598–602 (2013). et al.
- lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell 159, 1110–1125 (2014). et al.
- A long noncoding RNA activated by TGF-β promotes the invasion-metastasis cascade in hepatocellular carcinoma. Cancer Cell 25, 666–681 (2014). et al.
- The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex. Nat. Genet. 45, 1392–1398 (2013). et al.
- Genome-wide mapping and characterization of notch-regulated long noncoding RNAs in acute leukemia. Cell 158, 593–606 (2014). et al.
- Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464, 1071–1076 (2010). et al.
- Structure and function of long noncoding RNAs in epigenetic regulation. Nat. Struct. Mol. Biol. 20, 300–307 (2013). &
- Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491, 454–457 (2012). et al.
- The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science 344, 310–313 (2014). et al.
- Posttranscriptional gene regulation by long noncoding RNA. J. Mol. Biol. 425, 3723–3730 (2013). , &
- Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406 (2012). et al.
- Metabolic enzymes that bind RNA: yet another level of cellular regulatory network? Acta Biochim. Pol. 53, 11–32 (2006).
- RNA-binding proteins: modular design for efficient function. Nat. Rev. Mol. Cell Biol. 8, 479–490 (2007). , &
- Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721–732 (2003).
- Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nat. Rev. Cancer 8, 705–713 (2008).
- Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation. Proc. Natl Acad. Sci. USA 108, 16369–16374 (2011). et al.
- HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464–468 (2001). et al.
- Structure of an HIF-1α–pVHL complex: hydroxyproline recognition in signaling. Science 296, 1886–1889 (2002). et al.
- Normoxic stabilization of hypoxia-inducible factor-1α by modulation of the labile iron pool in differentiating U937 macrophages: effect of natural resistance-associated macrophage protein 1. Cancer Res. 66, 2600–2607 (2006). , , &
- Functional regulation of HIF-1α under normoxia—is there more than post-translational regulation? J. Cell. Physiol. 227, 514–524 (2012). , &
- Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod. Pathol. 19, 264–271 (2006). et al.
- Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists. Mod. Pathol. 24, 157–167 (2011). et al.
- Scan of human genome reveals no new loci under ancient balancing selection. Genetics 173, 2165–2177 (2006). et al.
- Breast tumor kinase (Brk/PTK6) is a mediator of hypoxia-associated breast cancer progression. Cancer Res. 73, 5810–5820 (2013). et al.
- Constitutive activation of breast tumor kinase accelerates cell migration and tumor growth in vivo. Oncogenesis 1, e11 (2012). , &
- Higher frequency of certain cancers in LRRK2 G2019S mutation carriers with Parkinson disease: a pooled analysis. JAMA Neurol. 72, 58–65 (2015). et al.
- Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson’s disease. Cell 157, 472–485 (2014). et al.
- Glycoprotein nonmetastatic B is an independent prognostic indicator of recurrence and a novel therapeutic target in breast cancer. Clin. Cancer Res. 16, 2147–2156 (2010). et al.
- Targeting EGFR in triple negative breast cancer. J. Cancer 2, 324–328 (2011). &
- GPNMB cooperates with neuropilin-1 to promote mammary tumor growth and engages integrin α5β1 for efficient breast cancer metastasis. Oncogene 34, 5494–5504 (2015). et al.
- Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation. Mol. Cell. Biol. 21, 6387–6394 (2001). , , , &
- Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition. J. Biol. Chem. 277, 34634–34641 (2002). &
- Brk, a breast tumor-derived non-receptor protein-tyrosine kinase, sensitizes mammary epithelial cells to epidermal growth factor. J. Biol. Chem. 271, 30956–30963 (1996). et al.
- Building a better understanding of the intracellular tyrosine kinase PTK6—BRK by BRK. Biochim. Biophys. Acta 1806, 66–73 (2010). &
- FIH-1: a novel protein that interacts with HIF-1α and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 15, 2675–2686 (2001). , &
- Emerging evidence for functional peptides encoded by short open reading frames. Nat. Rev. Genet. 15, 193–204 (2014). &
- Long non-coding RNAs as a source of new peptides. eLife 3, e03523 (2014). , , &
- A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell 160, 595–606 (2015). et al.
- HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J. 15, 2445–2453 (2001). et al.
- Hypoxia-inducible factor 1α promotes primary tumor growth and tumor-initiating cell activity in breast cancer. Breast Cancer Res. 14, R6 (2012). et al.
- Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling. Mol. Biol. Cell 13, 2547–2557 (2002). , , , &
- MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res. 38, e178 (2010). et al.
- Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008). , , , &
- The Pan-Cancer analysis of pseudogene expression reveals biologically and clinically relevant tumour subtypes. Nat. Commun. 5, 3963 (2014). et al.
- The soft agar colony formation assay. J. Vis. Exp. 92, e51998 (2014). et al.
- Genome-wide identification of hypoxia-inducible factor binding sites and target genes by a probabilistic model integrating transcription-profiling data and in silico binding site prediction. Nucleic Acids Res. 38, 2332–2345 (2010). , , , &
- High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq. Blood 117, e207–e217 (2011). et al.
- An integrative genomics approach identifies hypoxia inducible factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res. 37, 4587–4602 (2009). et al.
- 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, 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. (846 KB)
(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. (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, 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. (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 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. (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 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. (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, 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.
- Supplementary Information (10 MB)