cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth
Zirong Chen, Jian-Liang Li, Shuibin Lin, Chunxia Cao, Nicholas T. Gimbrone, Rongqiang Yang, Dongtao A. Fu, Miranda B. Carper, Eric B. Haura, Matthew B. Schabath, Jianrong Lu, Antonio L. Amelio, W. Douglas Cress, Frederic J. Kaye, Lizi Wu
Zirong Chen, Jian-Liang Li, Shuibin Lin, Chunxia Cao, Nicholas T. Gimbrone, Rongqiang Yang, Dongtao A. Fu, Miranda B. Carper, Eric B. Haura, Matthew B. Schabath, Jianrong Lu, Antonio L. Amelio, W. Douglas Cress, Frederic J. Kaye, Lizi Wu
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Research Article Oncology

cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth

Abstract

The LKB1 tumor suppressor gene is frequently mutated and inactivated in non–small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP–responsive element–binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC.

Authors

Zirong Chen, Jian-Liang Li, Shuibin Lin, Chunxia Cao, Nicholas T. Gimbrone, Rongqiang Yang, Dongtao A. Fu, Miranda B. Carper, Eric B. Haura, Matthew B. Schabath, Jianrong Lu, Antonio L. Amelio, W. Douglas Cress, Frederic J. Kaye, Lizi Wu

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Figure 6

LINC00473 is associated with NONO protein and stimulates CRTC-NONO interaction.

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LINC00473 is associated with NONO protein and stimulates CRTC-NONO inter...
(A) Coomassie blue staining of the LINC00473-associated proteins by RNA pull-down in A549 cells. (B) Specific association of LINC00473 RNA with NONO protein was validated through RNA pull-down followed by Western blot analysis. LINC00473 antisense and MEG RNA were used as controls. (C) Immunoprecipitation of endogenous NONO protein was validated via Western blotting (HC, heavy chain). (D) LINC00473 was significantly enriched in NONO immunoprecipitates relative to the IgG control by qRT-PCR assay. ASNS was used as a negative control (n = 3, ***P < 0.0001). (E) Depletion of LINC00473 caused reduced CRTC1-NONO interaction. A549 cells, after transduction with LINC00473 shRNA (shLINC00473-2 and -4) or the scramble shRNA lentiviruses (Ctl) for 72 hours, were cotransfected with Gal4-NONO, pSG5-luc (a firefly luciferase reporter containing Gal4-binding sites), pEF-RL (Renilla luciferase), as well as vector control or CRTC1. The luciferase activity was measured 24 hours after transfection (n = 3, *P < 0.05). (F) Overexpression of LINC00473 enhanced CRTC1-NONO interaction. HEK293T cells were transfected with Gal4-NONO, CRTC1, pSG5-luc, or pEF-RL in the presence of vector or LINC00473 construct. The luciferase activity was determined at 24 hours after transfection (n = 3, ***P < 0.0001). (G) Relative expression levels of LKB1-regulated genes in shLINC00473- versus shCtl-expressing A549 cells and in LKB1 versus vector control A549 cells showed that LINC00473 depletion attenuated some common target gene expression induced by LKB1 loss. See also Supplemental Figure 13.