Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3 or PIP3) mediates signalling pathways as a second messenger in response to extracellular signals. Although primordial functions of phospholipids and RNAs have been hypothesized in the ‘RNA world’, physiological RNA–phospholipid interactions and their involvement in essential cellular processes have remained a mystery. We explicate the contribution of lipid-binding long non-coding RNAs (lncRNAs) in cancer cells. Among them, long intergenic non-coding RNA for kinase activation (LINK-A) directly interacts with the AKT pleckstrin homology domain and PIP3 at the single-nucleotide level, facilitating AKT–PIP3 interaction and consequent enzymatic activation. LINK-A-dependent AKT hyperactivation leads to tumorigenesis and resistance to AKT inhibitors. Genomic deletions of the LINK-A PIP3-binding motif dramatically sensitized breast cancer cells to AKT inhibitors. Furthermore, meta-analysis showed the correlation between LINK-A expression and incidence of a single nucleotide polymorphism (rs12095274: A > G), AKT phosphorylation status, and poor outcomes for breast and lung cancer patients. PIP3-binding lncRNA modulates AKT activation with broad clinical implications.
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We are grateful to P. Marshall at Southern University at New Orleans and J. King at University of North Texas, Center for Human Identification for Barocycler usage, and E. Lauwers and P. Verstreken at the VIB Center for the Biology of Disease, Belgium for discussing the procedures of PIP3 vesicle generation. We thank G. Wan, Pennsylvania State University, for providing the MS2-24x-pCDNA construct. We thank J. Chen and L. Li from the Gene Editing/Cellular Model Core Facility of MD Anderson Cancer Center, for assistance with CRISPR–Cas9-mediated gene editing. We thank B.-F. Pan from the Proteomics and Metabolomics Core Facility of MD Anderson Cancer Center for assistance with mass spectrometry analysis. We thank L. Zheng, Department of Biochemistry & Molecular Biology, University of Texas, Health Science Center at Houston, for consulting of structural analysis. We thank D. Aten for assistance with figure presentation. This work was supported by NIH grant (R01GM112003) to Yan Z., 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.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Lipid binding affinity (Enrichment, lipid/total, L/T%) of TNBC-upregulated lncRNAs were calculated using normalized density. (b) Fold change of lncRNAs expression in tumor tissues compared to normal breast tissue. (c and d) Lipid-coated beads pull-down followed by RT–qPCR detection of LINK-A-lipid (c) or GAPDH-lipid (d) interactions. (e) Fluorescence spectra of BODIPY FL-PIP3 (donor) in the presence of Alexa-555-Strep alone (black line) or with Alexa-555-Strep-biotin-RP11.38310.5 (red line). (f) Graphic illustration of Alpha assay using DIG-LINK-A and Biotin-PIP3. (g) Competition binding Alpha assay to determine Kd for interaction between Biotin-PIP3 and DIG-LINK-A, in the presence of unlabeled full-length LINK-A (left), PC-binding motif (middle) or PIP3-binding motif (right) titrated from 0.4 mM to 0.05 nM (mean ± s.e.m. were derived from n = 3 independent experiments). For c and d, mean ± s.e.m. were derived from n = 3 independent experiments (*P < 0.05, two-tailed paired Student’s t-test).
Supplementary Figure 2 Determination of LINK-A-PIP3 and LINK-A-Ins (1,3,4,5)P4 interactions by giant unilamellar vesicles and MS2-TRAP.
(a and b) Fluorescence imaging of interaction between DOPC lipid vesicles (visualized by Nile Red) and indicated lncRNA (visualized by YOYO-1) (a) or YOYO-1 dye only (b). Left panel: Representative images. Scale bars, 50 nm. Middle and right panels: fluorescence intensities along the diagonal bars (middle panel) and intensity correlation between two channels (right panel) were shown. (c) Overlap coefficient between channel 1 (Nile Red) and channel 2 (YOYO-1) was calculated based on the number of giant lipid vesicles, LINK-A (n = 34 lipid vesicles), BCAR4 (n = 19 lipid vesicles), Lnc-131 (n = 21 lipid vesicles), and H19 (n = 7 lipid vesicles) (median, one-way ANOVA, ***P < 0.001). (d and g) Relative expression level of MS2-tagged full-length LINK-A (d) or ΔPIP3 deletion mutant (g) in MDA-MB-231 cells was detected by RT–qPCR. (e and h) ELISA assay detecting the conversion of PIP2 to PIP3 by immunoprecipitated PI3K p110α from cells pretreated with DMSO, PI(1,4,5,6)P4 or PI(1,3,4,5,6)P5 (100 μM, 2 h) (e) or transfected with MS2-tagged full-length LINK-A or ΔPIP3 deletion mutant (h). (f and i) Immunoblotting detection of immunoprecipitated PI3K p110α from cells pretreated with DMSO, PI(1,4,5,6)P4 or PI(1,3,4,5,6)P5 (100 μM, 2 h) (f) or transfected with MS2-tagged full-length LINK-A or ΔPIP3 deletion mutant (i). For d, e, g and h, mean ± s.e.m. were derived from n = 3 independent experiments (n.s. P > 0.05 and ***P < 0.001, two-tailed paired Student’s t-test). Statistics source data for a are in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.
Supplementary Figure 3 Determination of LINK-A-PIP3 and LINK-A-Ins (1,3,4,5)P4 interactions by RIP and Alpha assay respectively.
(a) Saturation curve used to determine Kd of the interactions between Biotin-Ins (1,3,4,5)P4 and Digoxigenin-labeled full-length LINK-A (left panel), ΔPC LINK-A (middle panel), or ΔPIP3 LINK-A (right panel) in Alpha format (mean ± s.e.m. were derived from n = 3 independent experiments). (b) Competition binding assay to determine Kd for the interactions between biotin- Ins(1,3,4,5)P4 and Digoxigenin-labeled LINK-A in the presence of unlabeled Ins(1,3,4,5)P4 as competitor (mean ± s.e.m. were derived from n = 3 independent experiments). (c and d) Immunoblotting detection (c) or RIP-qPCR detection of indicated RNAs retrieved by PIP3-specific antibody (d) in MDA-MB-231 cells treated with DMSO or LY294002. For a, b and d, mean ± s.e.m. were derived from n = 3 independent experiments (*P < 0.05 and ***P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.
Supplementary Figure 4 Characterization of LINK-A subcellular localization, knockdown efficiency and effect on AKT kinase activity in vitro.
(a) Immunoblotting of membrane and cytoplasmic fractionations from MDA-MB-231 treated with EGF (10 ng ml−1) for 30 min using indicated antibodies. (b) RT–qPCR detection of LINK-A expression in membrane and cytoplasmic fractionations, B2M was used as a cytoplasmic RNA control. (c) Determination of copy number of LINK-A in MDA-MB-231 cells. (d) RT–qPCR analyses of LINK-A knockdown efficiency in MDA-MB-231 cells transfected with indicated LNAs. (e) RT–qPCR analyses of LINK-A expression level in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of indicated rescue plasmids and EGF stimulation. (f) RIP-qPCR detection of indicated RNAs retrieved by PIP3-specific antibody in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of indicated rescue plasmids with or without EGF treatment. (g) Quantification of AKT kinase activity in the presence of control or PIP3 polyPIPosomes, with or without full-length LINK-A, ΔPIP3 or ΔPC deletion transcripts. For b–g, mean ± s.e.m. were derived from n = 3 independent experiments (n.s. P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.
(a) Graphic illustration of predicted LINK-A secondary structure and the stem loops corresponding to protein (black dot line circle) and lipid (red dot line circle) binding. (b) Secondary structure of LINK-A (nt. 1,081-1,140) harboring a stem-loop corresponding to PIP3 binding (red dot line circle). (c) List of DIG-labeled wild-type LINK-A and mutant oligonucleotides used in Alpha assay. (d) RNA agarose gel of in vitro transcribed biotinylated LINK-A (wild-type and single nucleotide mutants). (e) Relative retrieval level of MS2-tagged full-length LINK-A or indicated mutants in MS2-TRAP assay detected by RT–qPCR. For e, mean ± s.e.m. were derived from n = 3 independent experiments (n.s. P > 0.05, two-tailed paired Student’s t-test). Unprocessed original scans of all blots and gels with size marker are shown in Supplementary Fig. 9.
Supplementary Figure 6 Determination of LINK-A copy number and characterization of functional sufficiency of LINK-A copy number.
(a) Generation of a standard curve for calculating LINK-A copy number. In vitro transcribed LINK-A (a range of amounts from 10 copies to 106 copies) was used to generate cDNAs and generated cDNAs were used in real-time PCR. The resultant CT values decreased linearly with increasing LINK-A copy number, indicating sensitive detection from a wide range of template amounts. (b–d) Determination of LINK-A copy number (b), AKT/GSK-3β activation (c) and cell proliferation (d) in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of indicated rescue plasmids and EGF stimulation. (e–g) LINK-A copy number (e), AKT/GSK-3β activation (f) and cell proliferation (g) were determined by RT–qPCR, immunoblotting and cell proliferation assay in MCF-10A cells stably expressing full-length LINK-A or ΔPIP3 deletion mutant, with or without EGF stimulation. (h and i) RT–qPCR determination of LINK-A copy number (h) and quantification of cellular PIP3 in DLD-1 PIK3CA+/− cells delivered with PIP3 and indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. (j) RT–qPCR determination of LINK-A copy number in DLD-1 PIK3CA+/+ cells delivered with indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. For b, d, e, g, h, i and j, mean ± s.e.m. were derived from n = 3 independent experiments (n.s. P > 0.05, **P < 0.01 and ***P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.
Supplementary Figure 7 Generation of LINK-A PIP3-binding motif knockout cell line by CRISPR/Cas9 gene editing.
(a) gRNA sequences targeting LINK-A PIP3-binding motif. (b) PCR analysis of targeted locus showing the band corresponding to the genomic deletion in colonies #1, 3 and 6. (c and d) Genotyping PCR results showing the deletion of LINK-A PIP3 binding region in colonies #3 (c) and #6 (d) of MDA-MB-231 cells. Unprocessed original scans of all gels with size marker are shown in Supplementary Fig. 9.
Supplementary Figure 8 Identification of LINK-A SNP and its correlation with LINK-A expression level and phospho-AKT status in breast cancer tissues.
(a) SNPs within LINK-A gene locus significantly associated with survival of breast cancer in TCGA samples (n = 712 breast tumors; **P < 0.01, two-tailed Wilcoxon test). (b) SNPs within LINK-A gene locus significantly associated with survival of lung cancer in TCGA samples (LUAD + LUSC) (n = 793 lung tumors; *P < 0.05, two-tailed Wilcoxon test). (c) SNPs within LINK-A gene locus significantly associated with lung cancer compared to normal lung tissues (n = 2,332 lung tumors versus n = 3,077 normal lung tissues; *P < 0.05 and **P < 0.01, two-tailed Wilcoxon test). (d) SNPs within LINK-A gene locus significantly associated with gastric cancer () compared to normal gastric tissues (n = 1,006 gastric tumors versus n = 2,273 normal gastric tissues; *P < 0.05 and **P < 0.01, two-tailed Wilcoxon test). (e) Breast cancer tissue microarrays were subjected to RNAscope to detect LINK-A expression. Left panel: representative images. Scale bars, 200 μm. Right panel: statistical analysis (n = 3 independent tissue microarrays with 3, 20, 6 normal breast tissues and 37, 20, 35 breast tumors). (f) Kaplan-Meier survival analysis of LINK-A low and high breast cancer patients (n = 84 and 66 patients respectively, log rank test). (g) Immunohistochemical staining of indicated phospho-AKT in breast cancer tissues. Scale bars, 200 μm. (h) RT–qPCR analyses of LINK-A expression level in MDA-MB-231 (left panel) and MDA-MB-468 (right panel) cells transfected with control or LINK-A siRNA. (i) RT–qPCR analyses of LINK-A expression level in MDA-MB-231 cells harboring control or LINK-A shRNA. (j) Cell proliferation rate was assessed by OD density (590 nm) in MDA-MB-231 cells harboring control or LINK-A shRNA. (k) Cell apoptosis rate was assessed by FACS in MDA-MB-231 cells transfected with control or LINK-A siRNA. For e, h, i, j and k, mean ± s.e.m. were derived from n = 3 independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed paired Student’s t-test). Statistics source data for e, f and k are in Supplementary Table 6.
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Lin, A., Hu, Q., Li, C. et al. The LINK-A lncRNA interacts with PtdIns(3,4,5)P3 to hyperactivate AKT and confer resistance to AKT inhibitors. Nat Cell Biol 19, 238–251 (2017). https://doi.org/10.1038/ncb3473
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