Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, Nuclear LUCAT1 (NLUCAT1), which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-κB and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $2.38 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The experimental data from microarray and RNA sequencing have been deposited in the NCBI Gene Expression Omnibus (GEO) database under SuperSerie GSE117049 containing 5 distinct datasets.
Chang MY, Mentzer SJ, Colson YL, Linden PA, Jaklitsch MT, Lipsitz SR, et al. Factors predicting poor survival after resection of stage IA non-small cell lung cancer. J Thorac Cardiovasc Surg. 2007;134:850–6.
Ou SH, Zell JA, Ziogas A, Anton-Culver H. Prognostic factors for survival of stage I nonsmall cell lung cancer patients: a population-based analysis of 19,702 stage I patients in the California Cancer Registry from 1989 to 2003. Cancer. 2007;110:1532–41.
Pouyssegur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006;441:437–43.
Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug resistance: an evolving paradigm. Nat Rev Cancer. 2013;13:714–26.
Akagi I, Okayama H, Schetter AJ, Robles AI, Kohno T, Bowman ED, et al. Combination of protein coding and non-coding gene expression as a robust prognostic classifier in stage I lung adenocarcinoma. Cancer Res. 2013;73:3821–32.
Ilie M, Mazure NM, Hofman V, Ammadi RE, Ortholan C, Bonnetaud C, et al. High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer. Br J Cancer. 2010;102:1627–35.
Gee HE, Ivan C, Calin GA, Ivan M. HypoxamiRs and cancer: from biology to targeted therapy. Antioxid Redox Signal. 2014;21:1220–38.
Bertero T, Rezzonico R, Pottier N, Mari B. Impact of microRNAs in the cellular response to hypoxia. Int Rev Cell Mol Biol. 2017;333:91–158.
Puissegur MP, Mazure NM, Bertero T, Pradelli L, Grosso S, Robbe-Sermesant K, et al. miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity. Cell Death Differ. 2011;18:465–78.
Grosso S, Doyen J, Parks SK, Bertero T, Paye A, Cardinaud B, et al. MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines. Cell Death Dis. 2013;4:e544.
Guttman M, Rinn JL. Modular regulatory principles of large non-coding RNAs. Nature. 2012;482:339–46.
Ulitsky I, Bartel DP. lincRNAs: genomics, evolution, and mechanisms. Cell. 2013;154:26–46.
Quinn JJ, Chang HY. Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet. 2016;17:47–62.
Du Z, Fei T, Verhaak RG, Su Z, Zhang Y, Brown M, et al. Integrative genomic analyses reveal clinically relevant long noncoding RNAs in human cancer. Nat Struct Mol Biol. 2013;20:908–13.
Schmitt AM, Chang HY. Long noncoding RNAs in cancer pathways. Cancer Cell. 2016;29:452–63.
Yang F, Huo XS, Yuan SX, Zhang L, Zhou WP, Wang F, et al. Repression of the long noncoding RNA-LET by histone deacetylase 3 contributes to hypoxia-mediated metastasis. Mol Cell. 2013;49:1083–96.
Ferdin J, Nishida N, Wu X, Nicoloso MS, Shah MY, Devlin C, et al. HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts. Cell Death Differ. 2013;20:1675–87.
Yang F, Zhang H, Mei Y, Wu M. Reciprocal regulation of HIF-1alpha and lincRNA-p21 modulates the Warburg effect. Mol Cell. 2014;53:88–100.
Lin A, Li C, Xing Z, Hu Q, Liang K, Han L, et al. The LINK-A lncRNA activates normoxic HIF1alpha signalling in triple-negative breast cancer. Nat Cell Biol. 2016;18:213–24.
Chang YN, Zhang K, Hu ZM, Qi HX, Shi ZM, Han XH, et al. Hypoxia-regulated lncRNAs in cancer. Gene. 2016;575:1–8.
Schmidt LH, Spieker T, Koschmieder S, Schaffers S, Humberg J, Jungen D, et al. The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth. J Thorac Oncol. 2011;6:1984–92.
Loewen G, Jayawickramarajah J, Zhuo Y, Shan B. Functions of lncRNA HOTAIR in lung cancer. J Hematol Oncol. 2014;7:90.
Dong J, Xu J, Wang X, Jin B. Influence of the interaction between long noncoding RNAs and hypoxia on tumorigenesis. Tumour Biol. 2016;37:1379–85.
Buffa FM, Harris AL, West CM, Miller CJ. Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene. Br J Cancer. 2010;102:428–35.
Thai P, Statt S, Chen CH, Liang E, Campbell C, Wu R. Characterization of a novel long non-coding RNA, SCAL1, induced by cigarette smoke and elevated in lung cancer cell lines. Am J Respir Cell Mol Biol. 2013;49:204–11.
Volders PJ, Helsens K, Wang X, Menten B, Martens L, Gevaert K, et al. LNCipedia: a database for annotated human lncRNA transcript sequences and structures. Nucleic Acids Res. 2013;41:D246–D251.
Clark MB, Johnston RL, Inostroza-Ponta M, Fox AH, Fortini E, Moscato P, et al. Genome-wide analysis of long noncoding RNA stability. Genome Res. 2012;22:885–98.
Tani H, Mizutani R, Salam KA, Tano K, Ijiri K, Wakamatsu A, et al. Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Res. 2012;22:947–56.
Fitzpatrick SF, Tambuwala MM, Bruning U, Schaible B, Scholz CC, Byrne A, et al. An intact canonical NF-kappaB pathway is required for inflammatory gene expression in response to hypoxia. J Immunol. 2011;186:1091–6.
Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS, Nizet V, et al. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature. 2008;453:807–11.
Toth RK, Warfel NA. Strange bedfellows: nuclear factor, erythroid 2-like 2 (Nrf2) and hypoxia-inducible factor 1 (HIF-1) in tumor hypoxia. Antioxidants (Basel). 2017;6:E27.
Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF, McIntyre KW, et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J Biol Chem. 2003;278:1450–6.
Choi YM, Kim HK, Shim W, Anwar MA, Kwon JW, Kwon HK, et al. Mechanism of cisplatin-induced cytotoxicity is correlated to impaired metabolism due to mitochondrial ROS generation. PLoS ONE. 2015;10:e0135083.
Marullo R, Werner E, Degtyareva N, Moore B, Altavilla G, Ramalingam SS, et al. Cisplatin induces a mitochondrial-ROS response that contributes to cytotoxicity depending on mitochondrial redox status and bioenergetic functions. PLoS ONE. 2013;8:e81162.
Shelton P, Jaiswal AK. The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J. 2013;27:414–23.
Singh A, Rangasamy T, Thimmulappa RK, Lee H, Osburn WO, Brigelius-Flohe R, et al. Glutathione peroxidase 2, the major cigarette smoke-inducible isoform of GPX in lungs, is regulated by Nrf2. Am J Respir Cell Mol Biol. 2006;35:639–50.
Liu X, Xavier C, Jann J, Wu H. Salvianolic acid B (Sal B) protects retinal pigment epithelial cells from oxidative stress-induced cell death by activating glutaredoxin 1 (Grx1). Int J Mol Sci. 2016;17:E1835.
Duong HQ, You KS, Oh S, Kwak SJ, Seong YS. Silencing of NRF2 reduces the expression of ALDH1A1 and ALDH3A1 and sensitizes to 5-FU in pancreatic cancer cells. Antioxidants (Basel). 2017;6:E52.
Woolbright BL, Choudhary D, Mikhalyuk A, Trammel C, Shanmugam S, Abbott E, et al. The role of pyruvate dehydrogenase kinase-4 (PDK4) in bladder cancer and chemoresistance. Mol Cancer Ther. 2018;17:2004–12.
Zhang Y, Zhang Y, Geng L, Yi H, Huo W, Talmon G, et al. Transforming growth factor beta mediates drug resistance by regulating the expression of pyruvate dehydrogenase kinase 4 in colorectal cancer. J Biol Chem. 2016;291:17405–16.
Wu X, Tudoran OM, Calin GA, Ivan M. The many faces of long noncoding RNAs in cancer. Antioxid Redox Signal. 2017;29:922–35.
Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2011;25:1915–27.
Hangauer MJ, Vaughn IW, McManus MT. Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs. PLoS Genet. 2013;9:e1003569.
Iyer MK, Niknafs YS, Malik R, Singhal U, Sahu A, Hosono Y, et al. The landscape of long noncoding RNAs in the human transcriptome. Nat Genet. 2015;47:199–208.
Fang S, Zhang L, Guo J, Niu Y, Wu Y, Li H, et al. NONCODEV5: a comprehensive annotation database for long non-coding RNAs. Nucleic Acids Res. 2018;46:D308–D314.
Harrow J, Frankish A, Gonzalez JM, Tapanari E, Diekhans M, Kokocinski F, et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 2012;22:1760–74.
Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012;22:1775–89.
Volders PJ, Verheggen K, Menschaert G, Vandepoele K, Martens L, Vandesompele J, et al. An update on LNCipedia: a database for annotated human lncRNA sequences. Nucleic Acids Res. 2015;43:4363–4.
Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med. 2010;49:1603–16.
Tafani M, Sansone L, Limana F, Arcangeli T, De Santis E, Polese M, et al. The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression. Oxid Med Cell Longev. 2016;2016:3907147.
Eltzschig HK, Carmeliet P. Hypoxia and inflammation. N Engl J Med. 2011;364:656–65.
Gorlach A, Bonello S. The cross-talk between NF-kappaB and HIF-1: further evidence for a significant liaison. Biochem J. 2008;412:e17–19.
Oliver KM, Taylor CT, Cummins EP. Hypoxia. Regulation of NFkappaB signalling during inflammation: the role of hydroxylases. Arthritis Res Ther. 2009;11:215.
Patten DA, Lafleur VN, Robitaille GA, Chan DA, Giaccia AJ, Richard DE. Hypoxia-inducible factor-1 activation in nonhypoxic conditions: the essential role of mitochondrial-derived reactive oxygen species. Mol Biol Cell. 2010;21:3247–57.
Nakajima S, Kitamura M. Bidirectional regulation of NF-kappaB by reactive oxygen species: a role of unfolded protein response. Free Radic Biol Med. 2013;65:162–74.
Buelna-Chontal M, Zazueta C. Redox activation of Nrf2 & NF-kappaB: a double end sword? Cell Signal. 2013;25:2548–57.
Ashouri A, Sayin VI, Van den Eynden J, Singh SX, Papagiannakopoulos T, Larsson E. Pan-cancer transcriptomic analysis associates long non-coding RNAs with key mutational driver events. Nat Commun. 2016;7:13197.
White NM, Cabanski CR, Silva-Fisher JM, Dang HX, Govindan R, Maher CA. Transcriptome sequencing reveals altered long intergenic non-coding RNAs in lung cancer. Genome Biol. 2014;15:429.
Galluzzi L, Vitale I, Michels J, Brenner C, Szabadkai G, Harel-Bellan A, et al. Systems biology of cisplatin resistance: past, present and future. Cell Death Dis. 2014;5:e1257.
Zheng ZG, Xu H, Suo SS, Xu XL, Ni MW, Gu LH, et al. The essential role of H19 contributing to cisplatin resistance by regulating glutathione metabolism in high-grade serous ovarian cancer. Sci Rep. 2016;6:26093.
Sun Y, Jin SD, Zhu Q, Han L, Feng J, Lu XY, et al. Long non-coding RNA LUCAT1 is associated with poor prognosis in human non-small lung cancer and regulates cell proliferation via epigenetically repressing p21 and p57 expression. Oncotarget. 2017;8:28297–311.
Heery R, Finn SP, Cuffe S, Gray SG. Long non-coding RNAs: key regulators of epithelial-mesenchymal transition, tumour drug resistance and cancer stem cells. Cancers (Basel). 2017;9:38.
Hu Y, Zhu QN, Deng JL, Li ZX, Wang G, Zhu YS. Emerging role of long non-coding RNAs in cisplatin resistance. Onco Targets Ther. 2018;11:3185–94.
Yan W, Chen X. GPX2, a direct target ofp63, inhibits oxidative stress-induced apoptosis in a p53-dependent manner. J Biol Chem. 2006;281:7856–62.
Naiki T, Naiki-Ito A, Asamoto M, Kawai N, Tozawa K, Etani T, et al. GPX2 overexpression is involved in cell proliferation and prognosis of castration-resistant prostate cancer. Carcinogenesis. 2014;35:1962–7.
Liu X, Jann J, Xavier C, Wu H. Glutaredoxin 1 (Grx1) protects human retinal pigment epithelial cells from oxidative damage by preventing AKT glutathionylation. Invest Ophthalmol Vis Sci. 2015;56:2821–32.
Lassen N, Pappa A, Black WJ, Jester JV, Day BJ, Min E, et al. Antioxidant function of corneal ALDH3A1 in cultured stromal fibroblasts. Free Radic Biol Med. 2006;41:1459–69.
Muzio G, Maggiora M, Paiuzzi E, Oraldi M, Canuto RA. Aldehyde dehydrogenases and cell proliferation. Free Radic Biol Med. 2012;52:735–46.
Jang JH, Bruse S, Liu Y, Duffy V, Zhang C, Oyamada N, et al. Aldehyde dehydrogenase 3A1 protects airway epithelial cells from cigarette smoke-induced DNA damage and cytotoxicity. Free Radic Biol Med. 2014;68:80–6.
Leclerc D, Pham DN, Levesque N, Truongcao M, Foulkes WD, Sapienza C, et al. Oncogenic role of PDK4 in human colon cancer cells. Br J Cancer. 2017;116:930–6.
Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci USA. 2009;106:11667–72.
Davidovich C, Zheng L, Goodrich KJ, Cech TR. Promiscuous RNA binding by Polycomb repressive complex 2. Nat Struct Mol Biol. 2013;20:1250–7.
Blanco MR, Guttman M. Re-evaluating the foundations of lncRNA-Polycomb function. EMBO J. 2017;36:964–6.
Portoso M, Ragazzini R, Brencic Z, Moiani A, Michaud A, Vassilev I, et al. PRC2 is dispensable for HOTAIR-mediated transcriptional repression. EMBO J. 2017;36:981–94.
Engreitz JM, Haines JE, Perez EM, Munson G, Chen J, Kane M, et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature. 2016;539:452–5.
Li W, Notani D, Rosenfeld MG. Enhancers as non-coding RNA transcription units: recent insights and future perspectives. Nat Rev Genet. 2016;17:207–23.
Lino Cardenas CL, Henaoui IS, Courcot E, Roderburg C, Cauffiez C, Aubert S, et al. miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1. PLoS Genet. 2013;9:e1003291.
Lugthart S, Cheok MH, den Boer ML, Yang W, Holleman A, Cheng C, et al. Identification of genes associated with chemotherapy crossresistance and treatment response in childhood acute lymphoblastic leukemia. Cancer Cell. 2005;7:375–86.
Bertero T, Gastaldi C, Bourget-Ponzio I, Mari B, Meneguzzi G, Barbry P, et al. CDC25A targeting by miR-483-3p decreases CCND-CDK4/6 assembly and contributes to cell cycle arrest. Cell Death Differ. 2013;20:800–11.
Wang Y, Zhu W, Levy DE. Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR. Methods. 2006;39:356–62.
Gastaldi C, Bertero T, Xu N, Bourget-Ponzio I, Lebrigand K, Fourre S, et al. miR-193b/365a cluster controls progression of epidermal squamous cell carcinoma. Carcinogenesis. 2014;35:1110–20.
We gratefully acknowledge the outstanding technical support of the UCA GenomiX platform and MICA imaging facility of the University Côte d’Azur, and the staff from the CHU Nice Biobank. This work was supported by Plan Cancer 2018 « ARN non-codants en cancérologie: du fondamental au translationnel » (number 18CN045), Cancéropole PACA, Fondation ARC pour la Recherche sur le Cancer, Fondation Unice (AIR project), Ligue contre le cancer (comité départemental du Nord), and the French Government (Agence Nationale de Recherche, ANR) through the Investments for the Future LABEX SIGNALIFE (ANR-11-LABX-0028-01) and FRANCE GENOMIQUE (ANR-10-INBS-09-03 and ANR-10-INBS-09-02). LML was a recipient of the Fondation pour la Recherche Médicale.
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The original online version of this article was revised: The term “LUCAT1” was missing in the abstract.
About this article
Cite this article
Moreno Leon, L., Gautier, M., Allan, R. et al. The nuclear hypoxia-regulated NLUCAT1 long non-coding RNA contributes to an aggressive phenotype in lung adenocarcinoma through regulation of oxidative stress. Oncogene 38, 7146–7165 (2019). https://doi.org/10.1038/s41388-019-0935-y