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Aberrant RSPO3-LGR4 signaling in Keap1-deficient lung adenocarcinomas promotes tumor aggressiveness

Oncogene volume 34, pages 4692–4701 (2015)Cite this article

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Abstract

The four R-spondins (RSPO1–4) and their three related receptors LGR4, 5 and 6 (LGR4–6) have emerged as a major ligand-receptor system with critical roles in development and stem cell survival through modulation of Wnt signaling. Recurrent, gain-of-expression gene fusions of RSPO2 (to EIF3E) and RSPO3 (to PTPRK) occur in a subset of human colorectal cancer. However, the exact roles and mechanisms of the RSPO-LGR system in oncogenesis remain largely unknown. We found that RSPO3 is aberrantly expressed at high levels in approximately half of Keap1-mutated lung adenocarcinomas (ADs). This high RSPO3 expression is driven by a combination of demethylation of its own promoter region and deficiency in Keap1 instead of gene fusion as in colon cancer. Patients with RSPO3-high tumors (~9%, 36/412) displayed much poorer survival than the rest of the cohort (median survival of 28 vs 163 months, log-rank test P<0.0001). Knockdown (KD) of RSPO3, LGR4 or their signaling mediator IQGAP1 in lung cancer cell lines with Keap1 deficiency and high RSPO3-LGR4 expression led to reduction in cell proliferation and migration in vitro, and KD of LGR4 or IQGAP1 resulted in decrease in tumor growth and metastasis in vivo. These findings suggest that aberrant RSPO3-LGR4 signaling potentially acts as a driving mechanism in the aggressiveness of Keap1-deficient lung ADs.

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References

  1. de Lau WB, Snel B, Clevers HC . The R-spondin protein family. Genome Biol 2012; 13: 242.

    Article  CAS  Google Scholar 

  2. Bell SM, Schreiner CM, Wert SE, Mucenski ML, Scott WJ, Whitsett JA . R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis. Development 2008; 135: 1049–1058.

    Article  CAS  Google Scholar 

  3. Yamada W, Nagao K, Horikoshi K, Fujikura A, Ikeda E, Inagaki Y et al. Craniofacial malformation in R-spondin2 knockout mice. Biochem Biophys Res Commun 2009; 381: 453–458.

    Article  CAS  Google Scholar 

  4. Carmon KS, Gong X, Lin Q, Thomas A, Liu Q . R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci USA 2011; 108: 11452–11457.

    Article  CAS  Google Scholar 

  5. de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 2011; 476: 293–297.

    Article  CAS  Google Scholar 

  6. Glinka A, Dolde C, Kirsch N, Huang YL, Kazanskaya O, Ingelfinger D et al. LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and Wnt/PCP signalling. EMBO Rep 2011; 12: 1055–1061.

    Article  CAS  Google Scholar 

  7. McDonald T, Wang R, Bailey W, Xie G, Chen F, Caskey CT et al. Identification and cloning of an orphan G protein-coupled receptor of the glycoprotein hormone receptor subfamily. Biochem Biophys Res Commun 1998; 247: 266–270.

    Article  CAS  Google Scholar 

  8. Hsu SY, Kudo M, Chen T, Nakabayashi K, Bhalla A, van der Spek PJ et al. The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (LGR): identification of LGR6 and LGR7 and the signaling mechanism for LGR7. Mol Endocrinol 2000; 14: 1257–1271.

    Article  CAS  Google Scholar 

  9. Hsu SY, Liang SG, Hsueh AJ . Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol 1998; 12: 1830–1845.

    Article  CAS  Google Scholar 

  10. Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 2012; 485: 195–200.

    Article  CAS  Google Scholar 

  11. Carmon KS, Gong X, Yi J, Thomas A, Liu Q . RSPO-LGR4 functions via IQGAP1 to potentiate Wnt signaling. Proc Natl Acad Sci USA 2014; 111: E1221–E1229.

    Article  CAS  Google Scholar 

  12. Noritake J, Watanabe T, Sato K, Wang S, Kaibuchi K . IQGAP1: a key regulator of adhesion and migration. J Cell Sci 2005; 118: 2085–2092.

    Article  CAS  Google Scholar 

  13. White CD, Erdemir HH, Sacks DB . IQGAP1 and its binding proteins control diverse biological functions. Cell Signal 2012; 24: 826–834.

    Article  CAS  Google Scholar 

  14. Seshagiri S, Stawiski EW, Durinck S, Modrusan Z, Storm EE, Conboy CB et al. Recurrent R-spondin fusions in colon cancer. Nature 2012; 488: 660–664.

    Article  CAS  Google Scholar 

  15. Lowther W, Wiley K, Smith GH, Callahan R . A new common integration site, Int7, for the mouse mammary tumor virus in mouse mammary tumors identifies a gene whose product has furin-like and thrombospondin-like sequences. J Virol 2005; 79: 10093–10096.

    Article  CAS  Google Scholar 

  16. Theodorou V, Kimm MA, Boer M, Wessels L, Theelen W, Jonkers J et al. MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer. Nat Genet 2007; 39: 759–769.

    Article  CAS  Google Scholar 

  17. Starr TK, Allaei R, Silverstein KA, Staggs RA, Sarver AL, Bergemann TL et al. A transposon-based genetic screen in mice identifies genes altered in colorectal cancer. Science 2009; 323: 1747–1750.

    Article  CAS  Google Scholar 

  18. Weng J, Luo J, Cheng X, Jin C, Zhou X, Qu J et al. Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2. Proc Natl Acad Sci USA 2008; 105: 6081–6086.

    Article  CAS  Google Scholar 

  19. Mendive F, Laurent P, Van Schoore G, Skarnes W, Pochet R, Vassart G . Defective postnatal development of the male reproductive tract in LGR4 knockout mice. Dev Biol 2006; 290: 421–434.

    Article  CAS  Google Scholar 

  20. Kato S, Matsubara M, Matsuo T, Mohri Y, Kazama I, Hatano R et al. Leucine-rich repeat-containing G protein-coupled receptor-4 (LGR4, Gpr48) is essential for renal development in mice. Nephron Exp Nephrol 2006; 104: e63–e75.

    Article  CAS  Google Scholar 

  21. Gugger M, White R, Song S, Waser B, Cescato R, Riviere P et al. GPR87 is an overexpressed G-protein coupled receptor in squamous cell carcinoma of the lung. Dis Markers 2008; 24: 41–50.

    Article  CAS  Google Scholar 

  22. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 2013; 6: pl1.

    Article  Google Scholar 

  23. Li B, Dewey CN . RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 2011; 12: 323.

    Article  CAS  Google Scholar 

  24. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012; 483: 603–607.

    Article  CAS  Google Scholar 

  25. Yi J, Xiong W, Gong X, Bellister S, Ellis LM, Liu Q . Analysis of LGR4 receptor distribution in human and mouse tissues. PLoS ONE 2013; 8: e78144.

    Article  CAS  Google Scholar 

  26. Bild AH, Yao G, Chang JT, Wang Q, Potti A, Chasse D et al. Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 2006; 439: 353–357.

    Article  CAS  Google Scholar 

  27. Okayama H, Kohno T, Ishii Y, Shimada Y, Shiraishi K, Iwakawa R et al. Identification of genes upregulated in ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas. Cancer Res 2012; 72: 100–111.

    Article  CAS  Google Scholar 

  28. Network TCGA. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012; 487: 330–337.

    Article  Google Scholar 

  29. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO et al. Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med 2006; 3: e420.

    Article  Google Scholar 

  30. Hayes JD, McMahon M . NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem Sci 2009; 34: 176–188.

    Article  CAS  Google Scholar 

  31. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 2012; 150: 1107–1120.

    Article  CAS  Google Scholar 

  32. Solis LM, Behrens C, Dong W, Suraokar M, Ozburn NC, Moran CA et al. Nrf2 and Keap1 abnormalities in non-small cell lung carcinoma and association with clinicopathologic features. Clin Cancer Res 2010; 16: 3743–3753.

    Article  CAS  Google Scholar 

  33. Takahashi T, Sonobe M, Menju T, Nakayama E, Mino N, Iwakiri S et al. Mutations in Keap1 are a potential prognostic factor in resected non-small cell lung cancer. J Surg Oncol 2010; 101: 500–506.

    CAS  PubMed  Google Scholar 

  34. Singh A, Happel C, Manna SK, Acquaah-Mensah G, Carrerero J, Kumar S et al. Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis. J Clin Invest 2013; 123: 2921–2934.

    Article  CAS  Google Scholar 

  35. Malhotra D, Portales-Casamar E, Singh A, Srivastava S, Arenillas D, Happel C et al. Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis. Nucleic Acids Res 2010; 38: 5718–5734.

    Article  CAS  Google Scholar 

  36. Ke N, Wang X, Xu X, Abassi YA . The xCELLigence system for real-time and label-free monitoring of cell viability. Methods Mol Biol 2011; 740: 33–43.

    Article  CAS  Google Scholar 

  37. Johnson M, Sharma M, Henderson BR . IQGAP1 regulation and roles in cancer. Cell Signal 2009; 21: 1471–1478.

    Article  CAS  Google Scholar 

  38. Thiery JP, Acloque H, Huang RY, Nieto MA . Epithelial-mesenchymal transitions in development and disease. Cell 2009; 139: 871–890.

    Article  CAS  Google Scholar 

  39. Jenkins DE, Oei Y, Hornig YS, Yu SF, Dusich J, Purchio T et al. Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clin Exp Metastasis 2003; 20: 733–744.

    Article  CAS  Google Scholar 

  40. Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C et al. Mutational landscape and significance across 12 major cancer types. Nature 2013; 502: 333–339.

    Article  CAS  Google Scholar 

  41. Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, Golub TR et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 2014; 505: 495–501.

    Article  CAS  Google Scholar 

  42. Mazieres J, He B, You L, Xu Z, Jablons DM . Wnt signaling in lung cancer. Cancer Lett 2005; 222: 1–10.

    Article  CAS  Google Scholar 

  43. Shinmura K, Kahyo T, Kato H, Igarashi H, Matsuura S, Nakamura S et al. RSPO fusion transcripts in colorectal cancer in Japanese population. Mol Biol Rep 2014; 41 (8): 5375–5384.

    Article  CAS  Google Scholar 

  44. Nam JS, Turcotte TJ, Yoon JK . Dynamic expression of R-spondin family genes in mouse development. Gene Expr Patterns 2007; 7: 306–312.

    Article  CAS  Google Scholar 

  45. Gattelli A, Zimberlin MN, Meiss RP, Castilla LH, Kordon EC . Selection of early-occurring mutations dictates hormone-independent progression in mouse mammary tumor lines. J Virol 2006; 80: 11409–11415.

    Article  CAS  Google Scholar 

  46. Nguyen DX, Chiang AC, Zhang XH, Kim JY, Kris MG, Ladanyi M et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell 2009; 138: 51–62.

    Article  CAS  Google Scholar 

  47. Pacheco-Pinedo EC, Durham AC, Stewart KM, Goss AM, Lu MM, Demayo FJ et al. Wnt/beta-catenin signaling accelerates mouse lung tumorigenesis by imposing an embryonic distal progenitor phenotype on lung epithelium. J Clin Invest 2011; 121: 1935–1945.

    Article  CAS  Google Scholar 

  48. Chien AJ, Conrad WH, Moon RT . A Wnt survival guide: from flies to human disease. J Invest Dermatol 2009; 129: 1614–1627.

    Article  CAS  Google Scholar 

  49. Hayashi H, Nabeshima K, Aoki M, Hamasaki M, Enatsu S, Yamauchi Y et al. Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis-critical role in tumor invasion. Int J Cancer 2010; 126: 2563–2574.

    CAS  PubMed  Google Scholar 

  50. Liu Y, Lin D, Xiao T, Ma Y, Hu Z, Zheng H et al. An immunohistochemical analysis-based decision tree model for estimating the risk of lymphatic metastasis in pN0 squamous cell carcinomas of the lung. Histopathology 2011; 59: 882–891.

    Article  Google Scholar 

  51. Miyoshi T, Shirakusa T, Ishikawa Y, Iwasaki A, Shiraishi T, Makimoto Y et al. Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern. Pathol Int 2005; 55: 419–424.

    Article  Google Scholar 

  52. Fujita PA, Rhead B, Zweig AS, Hinrichs AS, Karolchik D, Cline MS et al. The UCSC Genome Browser database: update 2011. Nucleic Acids Res 2011; 39: D876–D882.

    Article  CAS  Google Scholar 

  53. Kim D, Salzberg SL . TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol 2011; 12: R72.

    Article  CAS  Google Scholar 

  54. Furukawa M, Xiong Y . BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase. Mol Cell Biol 2005; 25: 162–171.

    Article  CAS  Google Scholar 

  55. Nakatani Y, Ogryzko V . Immunoaffinity purification of mammalian protein complexes. Methods Enzymol 2003; 370: 430–444.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank TCGA for generating the genomics data, collecting the clinical information and making them available for analysis. We also thank cBioportal for providing data analysis and visualization tools and downloading capability. We thank Dr B Fang at the University of Texas MD Anderson Cancer for the cell lines A549, H460 and H2009 cells. This work was supported in part by the Cancer Prevention and Research Institute of Texas (CPRIT, RP100678), the US National Institute of Health-NIH (R01GM102485), the Texas Emerging Technology Fund and the Janice D. Gordon endowment for bowel cancer research (to QJL), by NIH (R00LM009837, TL1TR000371) and CPRIT (R1006; to JTC) and by the Texas Emerging Technology Fund (to ZA).

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Authors and Affiliations

  1. Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA

    X Gong, J Yi, K S Carmon, C A Crumbley, W Xiong, A Thomas, X Fan, Z An, J T Chang & Q J Liu

  2. Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, TX, USA

    X Gong, J Yi, K S Carmon, C A Crumbley, W Xiong, A Thomas, X Fan, Z An & Q J Liu

  3. Divison of Oncology, University of Texas Health Science Center at Houston, Houston, TX, USA

    S Guo

  4. Center for Clinical and Translational Sciences, University of Texas Health Science Center at Houston, Houston, TX, USA

    J T Chang

  5. Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX, USA

    J T Chang

  6. School of Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX, USA

    J T Chang

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  1. X Gong
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Gong, X., Yi, J., Carmon, K. et al. Aberrant RSPO3-LGR4 signaling in Keap1-deficient lung adenocarcinomas promotes tumor aggressiveness. Oncogene 34, 4692–4701 (2015). https://doi.org/10.1038/onc.2014.417

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