Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The LINK-A lncRNA interacts with PtdIns(3,4,5)P3 to hyperactivate AKT and confer resistance to AKT inhibitors

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Global identification of lipid-interacting lncRNAs and characterization of LINK-A–lipid interaction.
Figure 2: Characterization of LINK-A–PIP3 interaction.
Figure 3: LINK-A enhances AKT–PIP3 interaction and AKT kinase activation.
Figure 4: Molecular mechanisms of LINK-A–PIP3–AKT interaction.
Figure 5: Functional involvement of LINK-A–PIP3 interaction in mediating AKT activation.
Figure 6: LINK-A confers resistance to AKT inhibitors.
Figure 7: Depletion of LINK-A sensitizes cancer cells to treatment of AKT inhibitors.
Figure 8: Genetic variation of LINK-A correlates with breast cancer risk.

References

  1. 1

    Wymann, M. P. & Schneiter, R. Lipid signalling in disease. Nat. Rev. Mol. Cell Biol. 9, 162–176 (2008).

    CAS  PubMed  Google Scholar 

  2. 2

    Mayer, I. A. & Arteaga, C. L. The PI3K/AKT pathway as a target for cancer treatment. Annu. Rev. Med. 67, 11–28 (2016).

    CAS  PubMed  Google Scholar 

  3. 3

    Ono, Y. et al. Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proc. Natl Acad. Sci. USA 86, 4868–4871 (1989).

    CAS  PubMed  Google Scholar 

  4. 4

    Park, W. S. et al. Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging. Mol. Cell 30, 381–392 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Gaullier, J. M. et al. FYVE fingers bind PtdIns(3)P. Nature 394, 432–433 (1998).

    CAS  PubMed  Google Scholar 

  6. 6

    Franke, T. F., Kaplan, D. R., Cantley, L. C. & Toker, A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275, 665–668 (1997).

    CAS  PubMed  Google Scholar 

  7. 7

    Le Good, J. A. et al. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281, 2042–2045 (1998).

    PubMed  Google Scholar 

  8. 8

    Hendriks, R. W., Yuvaraj, S. & Kil, L. P. Targeting Bruton’s tyrosine kinase in B cell malignancies. Nat. Rev. Cancer 14, 219–232 (2014).

    CAS  PubMed  Google Scholar 

  9. 9

    Vivanco, I. & Sawyers, C. L. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat. Rev. Cancer 2, 489–501 (2002).

    CAS  PubMed  Google Scholar 

  10. 10

    Luo, J., Manning, B. D. & Cantley, L. C. Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell 4, 257–262 (2003).

    CAS  PubMed  Google Scholar 

  11. 11

    Manning, B. D. & Cantley, L. C. AKT/PKB signaling: navigating downstream. Cell 129, 1261–1274 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Stokoe, D. et al. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277, 567–570 (1997).

    CAS  PubMed  Google Scholar 

  13. 13

    Yap, T. A. et al. First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors. J. Clin. Oncol. 29, 4688–4695 (2011).

    CAS  PubMed  Google Scholar 

  14. 14

    Wisinski, K. B. et al. Phase I study of an AKT inhibitor (MK-2206) combined with lapatinib in adult solid tumors followed by dose expansion in advanced HER2+ breast cancer. Clin. Cancer Res. 22, 2659–2667 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Hudis, C. et al. A phase 1 study evaluating the combination of an allosteric AKT inhibitor (MK-2206) and trastuzumab in patients with HER2-positive solid tumors. Breast Cancer Res. 15, R110 (2013).

    PubMed  PubMed Central  Google Scholar 

  16. 16

    Qi, L. et al. PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells. Cancer Cell Int. 15, 91 (2015).

    PubMed  PubMed Central  Google Scholar 

  17. 17

    Stottrup, C., Tsang, T. & Chin, Y. R. Upregulation of AKT3 confers resistance to the AKT inhibitor MK2206 in breast cancer. Mol. Cancer Ther. 15, 1964–1974 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Xing, Z. et al. lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell 159, 1110–1125 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Lin, A. et al. The LINK-A lncRNA activates normoxic HIF1α signalling in triple-negative breast cancer. Nat. Cell Biol. 18, 213–224 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Wang, P. et al. The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science 344, 310–313 (2014).

    CAS  Google Scholar 

  21. 21

    Liu, B. et al. A cytoplasmic NF-κB interacting long noncoding RNA blocks IκB phosphorylation and suppresses breast cancer metastasis. Cancer Cell 27, 370–381 (2015).

    CAS  Google Scholar 

  22. 22

    Arun, G., Akhade, V. S., Donakonda, S. & Rao, M. R. mrhl RNA, a long noncoding RNA, negatively regulates Wnt signaling through its protein partner Ddx5/p68 in mouse spermatogonial cells. Mol. Cell Biol. 32, 3140–3152 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Batey, R. T., Rambo, R. P., Lucast, L., Rha, B. & Doudna, J. A. Crystal structure of the ribonucleoprotein core of the signal recognition particle. Science 287, 1232–1239 (2000).

    CAS  PubMed  Google Scholar 

  24. 24

    MacIntosh, G. C., Bariola, P. A., Newbigin, E. & Green, P. J. Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: unexpected functions for ancient enzymes? Proc. Natl Acad. Sci. USA 98, 1018–1023 (2001).

    CAS  PubMed  Google Scholar 

  25. 25

    Mindaye, S. T., Ra, M., Lo Surdo, J., Bauer, S. R. & Alterman, M. A. Improved proteomic profiling of the cell surface of culture-expanded human bone marrow multipotent stromal cells. J. Proteomics 78, 1–14 (2013).

    CAS  PubMed  Google Scholar 

  26. 26

    Gross, V. et al. Tissue fractionation by hydrostatic pressure cycling technology: the unified sample preparation technique for systems biology studies. J. Biomol. Tech. 19, 189–199 (2008).

    PubMed  PubMed Central  Google Scholar 

  27. 27

    Olszowy, P. P., Burns, A. & Ciborowski, P. S. Pressure-assisted sample preparation for proteomic analysis. Anal. Biochem. 438, 67–72 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Kim, Y., Lichtenbergova, L., Snitko, Y. & Cho, W. A phospholipase A2 kinetic and binding assay using phospholipid-coated hydrophobic beads. Anal. Biochem. 250, 109–116 (1997).

    CAS  PubMed  Google Scholar 

  29. 29

    Bubb, K. L. et al. Scan of human genome reveals no new Loci under ancient balancing selection. Genetics 173, 2165–2177 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Perez, Y. et al. Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism. Sci. Rep. 3, 1295 (2013).

    PubMed  PubMed Central  Google Scholar 

  31. 31

    Huranova, M. et al. In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer. RNA 15, 2063–2071 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Frech, M. et al. High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity. J. Biol. Chem 272, 8474–8481 (1997).

    CAS  PubMed  Google Scholar 

  33. 33

    Ferguson, K. M., Lemmon, M. A., Schlessinger, J. & Sigler, P. B. Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell 83, 1037–1046 (1995).

    CAS  PubMed  Google Scholar 

  34. 34

    Ke, J. et al. Structural basis for RNA recognition by a dimeric PPR-protein complex. Nat. Struct. Mol. Biol. 20, 1377–1382 (2013).

    CAS  PubMed  Google Scholar 

  35. 35

    Pedram Fatemi, R. et al. Screening for small-molecule modulators of long noncoding RNA-protein interactions using αscreen. J. Biomol. Screen. 20, 1132–1141 (2015).

    PubMed  PubMed Central  Google Scholar 

  36. 36

    Khuong, T. M. et al. Synaptic PI(3,4,5)P3 is required for Syntaxin1A clustering and neurotransmitter release. Neuron 77, 1097–1108 (2013).

    CAS  PubMed  Google Scholar 

  37. 37

    Janas, T. & Yarus, M. Visualization of membrane RNAs. RNA 9, 1353–1361 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Janas, T., Janas, T. & Yarus, M. Specific RNA binding to ordered phospholipid bilayers. Nucleic Acids Res. 34, 2128–2136 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Wang, J. & Richards, D. A. Segregation of PIP2 and PIP3 into distinct nanoscale regions within the plasma membrane. Biol. Open 1, 857–862 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Yoon, J. H., Srikantan, S. & Gorospe, M. MS2-TRAP (MS2-tagged RNA affinity purification): tagging RNA to identify associated miRNAs. Methods 58, 81–87 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Han, C. et al. The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression. Cell Rep. 8, 1447–1460 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Costa, C. et al. Measurement of PIP3 levels reveals an unexpected role for p110β in early adaptive responses to p110α-specific inhibitors in luminal breast cancer. Cancer Cell 27, 97–108 (2015).

    CAS  PubMed  Google Scholar 

  43. 43

    Yarden, Y. & Shilo, B. Z. SnapShot: EGFR signaling pathway. Cell 131, 1018.e1–1018.e2 (2007).

    Google Scholar 

  44. 44

    Citri, A. & Yarden, Y. EGF-ERBB signalling: towards the systems level. Nat. Rev. Mol. Cell Biol. 7, 505–516 (2006).

    CAS  PubMed  Google Scholar 

  45. 45

    Fruman, D. A. & Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Disc. 13, 140–156 (2014).

    CAS  Google Scholar 

  46. 46

    Altomare, D. A. & Testa, J. R. Perturbations of the AKT signaling pathway in human cancer. Oncogene 24, 7455–7464 (2005).

    CAS  PubMed  Google Scholar 

  47. 47

    Jones, S., Daley, D. T., Luscombe, N. N., Berman, H. M. & Thornton, J. M. Protein-RNA interactions: a structural analysis. Nucleic Acids Res. 29, 943–954 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Gosai, S. J. et al. Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus. Mol. Cell 57, 376–388 (2015).

    CAS  PubMed  Google Scholar 

  49. 49

    Thomas, C. C., Deak, M., Alessi, D. R. & van Aalten, D. M. High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate. Curr. Biol. 12, 1256–1262 (2002).

    CAS  PubMed  Google Scholar 

  50. 50

    Milburn, C. C. et al. Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change. Biochem. J. 375, 531–538 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Panchenko, A. R., Luthey-Schulten, Z. & Wolynes, P. G. Foldons, protein structural modules, and exons. Proc. Natl Acad. Sci. USA 93, 2008–2013 (1996).

    CAS  PubMed  Google Scholar 

  52. 52

    Feng, Y. et al. Global analysis of protein structural changes in complex proteomes. Nat. Biotechnol. 32, 1036–1044 (2014).

    CAS  PubMed  Google Scholar 

  53. 53

    Liu, F. & Fitzgerald, M. C. Large-scale analysis of breast cancer-related conformational changes in proteins using limited proteolysis. J. Proteome Res. 15, 4666–4674 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Gambhir, A. et al. Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins. Biophys. J. 86, 2188–2207 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Gills, J. J. & Dennis, P. A. Perifosine: update on a novel Akt inhibitor. Curr. Oncol. Rep. 11, 102–110 (2009).

    CAS  PubMed  Google Scholar 

  56. 56

    Ahad, A. M. et al. Development of sulfonamide AKT PH domain inhibitors. Bioorg. Med. Chem. 19, 2046–2054 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Yang, L. et al. Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res. 64, 4394–4399 (2004).

    CAS  PubMed  Google Scholar 

  58. 58

    Hirai, H. et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. Mol. Cancer Ther. 9, 1956–1967 (2010).

    CAS  PubMed  Google Scholar 

  59. 59

    Zanoni, M. et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci. Rep. 6, 19103 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Lemmon, M. A., Ferguson, K. M., O’Brien, R., Sigler, P. B. & Schlessinger, J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proc. Natl Acad. Sci. USA 92, 10472–10476 (1995).

    CAS  PubMed  Google Scholar 

  61. 61

    Laddha, S. V., Ganesan, S., Chan, C. S. & White, E. Mutational landscape of the essential autophagy gene BECN1 in human cancers. Mol. Cancer Res. 12, 485–490 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Gui, Y. et al. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat. Genet. 43, 875–878 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

A.L., Q.H. and C.Li devised and performed most experiments. Z.X., K.L. and S.W. helped with biochemistry and lipid studies. G.M. and Yubin Z. performed FRET assays. Y.Y. generated the CRISPR–Cas9 KO cell line. D.H.H. performed mass spectrometry analysis for the LiP assay. J.Z., Yan Z. and J.R.M. provided clinical specimens and assisted with pathological analyses. The histological staining was performed by K.L. TCGA and bioinformatics data analysis were performed by C.W., Z.H., H.S., J.Y., J.L., L.H. and H.L. P.K.P. helped with manuscript preparation. M.-C.H. contributed to discussion and data interpretation. L.Y. and C.Lin initiated and supervised the project and wrote the paper with input from all authors.

Corresponding authors

Correspondence to Chunru Lin or Liuqing Yang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of lipid-lncRNA interaction.

(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 bg, 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.

Supplementary Figure 5 Secondary structural modeling of LINK-A-protein and –lipid interactions.

(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. (bd) 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. (eg) 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.

Supplementary information

Supplementary Information

Supplementary Information (PDF 8115 kb)

Supplementary Table 1

Supplementary Information (XLSX 957 kb)

Supplementary Table 2

Supplementary Information (XLSX 16 kb)

Supplementary Table 3

Supplementary Information (XLSX 51 kb)

Supplementary Table 4

Supplementary Information (XLSX 10 kb)

Supplementary Table 5

Supplementary Information (XLSX 12 kb)

Supplementary Table 6

Supplementary Information (XLSX 32 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing