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. Now, researchers from MD Anderson Cancer Center have identified 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. These findings illustrate the magnitude and diversity of cytoplasmic lncRNAs in signal transduction and highlight the important roles of lncRNAs in cancer.
LINK-A mediates recruitment of BRK to GPNMB for kinase activation
(a) Immunofluorescence detection using indicated antibodies in MDA-MB-231 cells harboring control (upper panel) or LINK-A shRNA, followed by HB-EGF stimulation (upper panel). Scale bars, 20µm. (b) Graphic illustration of the BRK-, LRRK2-LINK-A interactions (upper panel) and corresponding deletion abolishing these interactions (lower panel). (c and d) Immunofluorescence imaging (c, Scale bars, 20µm) or IB detection (d) was performed using indicated antibodies in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of indicated rescue plasmids with HB-EGF stimulation. (e) In vitro kinase assay using recombinant BRK and in vitro transcribed RNA transcripts as indicated in the presence or absence of [32P]-ATP. Dot-blot indicates equal RNA transcript present in the assay. (f) Quantification analysis of BRK kinase activity in the presence of indicated in vitro transcribed RNA transcripts using HIF1α peptide (a.a. 557–566) as substrate. Upper panel: release of free Pi amount measured at OD620; lower panel: calculation of BRK kinase activity (pmol/min/µg). Error bars, S.E.M., n=3 independent experiments (*p<0.05, two-tailed paired Student’s t-test) (g) IB detection of BRK using indicated antibodies in the presence of 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.