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Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

Oncogene (2018) | Download Citation


It remains unclear whether PAX6 acts as a crucial transcription factor for lung cancer stem cell (CSC) traits. We demonstrate that PAX6 acts as an oncogene responsible for induction of cancer stemness properties in lung adenocarcinoma (LUAD). Mechanistically, PAX6 promotes GLI transcription, resulting in SOX2 upregulation directly by the binding of GLI to the proximal promoter region of the SOX2 gene. The overexpressed SOX2 enhances the expression of key pluripotent factors (OCT4 and NANOG) and suppresses differentiation lineage factors (HOPX and NKX2-1), driving cancer cells toward a stem-like state. In contrast, in the differentiated non-CSCs, PAX6 is transcriptionally silenced by its promoter methylation. In human lung cancer tissues, the positive linear correlations of PAX6 expression with GLI and SOX2 expression and its negative correlations with HOPX and NKX2-1 expression were observed. Therapeutically, the blockade of the PAX6-GLI-SOX2 signaling axis elicits a long-lasting therapeutic efficacy by limiting CSC expansion following chemotherapy. Furthermore, a methylation panel including the PAX6 gene yielded a sensitivity of 79.1% and specificity of 83.3% for cancer detection using serum DNA from stage IA LUAD. Our findings provide a rationale for targeting the PAX6-GLI-SOX2 signaling axis with chemotherapy as an effective therapeutic strategy and support the clinical utility of PAX6 gene promoter methylation as a biomarker for early lung cancer detection.

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  1. 1.

    Eramo A, Haas TL, De Maria R. Lung cancer stem cells: tools and targets to fight lung cancer. Oncogene. 2010;29:4625–35.

  2. 2.

    Beck B, Blanpain C. Unravelling cancer stem cell potential. Nat Rev Cancer. 2013;13:727–38.

  3. 3.

    Asami M, Pilz GA, Ninkovic J, Godinho L, Schroeder T, Huttner WB, et al. The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex. Development. 2011;138:5067–78.

  4. 4.

    Sansom SN, Griffiths DS, Faedo A, Kleinjan DJ, Ruan Y, Smith J, et al. The level of the transcription factor Pax6 is essential for controlling the balance between neural stem cell self-renewal and neurogenesis. PLoS Genet. 2009;5:e1000511.

  5. 5.

    Lang D, Powell SK, Plummer RS, Young KP, Ruggeri BA. PAX genes: roles in development, pathophysiology, and cancer. Biochem Pharmacol. 2007;73:1–14.

  6. 6.

    Marquardt T, Ashery-Padan R, Andrejewski N, Scardigli R, Guillemot F, Gruss P. Pax6 is required for the multipotent state of retinal progenitor cells. Cell. 2001;105:43–55.

  7. 7.

    Yamaoka T, Yano M, Yamada T, Matsushita T, Moritani M, Ii S, et al. Diabetes and pancreatic tumours in transgenic mice expressing Pa x 6. Diabetologia. 2000;43:332–9.

  8. 8.

    Osumi N, Shinohara H, Numayama-Tsuruta K, Maekawa M. Concise review: Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. Stem Cells. 2008;26:1663–72.

  9. 9.

    Hu B, Wang Q, Wang YA, Hua S, Sauve CG, Ong D, et al. Epigenetic activation of WNT5A drives glioblastoma stem. Cell Differ Invasive Growth Cell. 2016;167:e18.

  10. 10.

    Shyr CR, Tsai MY, Yeh S, Kang HY, Chang YC, Wong PL, et al. Tumor suppressor PAX6 functions as androgen receptor co-repressor to inhibit prostate cancer growth. Prostate. 2010;70:190–9.

  11. 11.

    Mayes DA, Hu Y, Teng Y, Siegel E, Wu X, Panda K, et al. PAX6 suppresses the invasiveness of glioblastoma cells and the expression of the matrix metalloproteinase-2 gene. Cancer Res. 2006;66:9809–17.

  12. 12.

    Lang D, Mascarenhas JB, Powell SK, Halegoua J, Nelson M, Ruggeri BA. PAX6 is expressed in pancreatic adenocarcinoma and is downregulated during induction of terminal differentiation. Mol Carcinog. 2008;47:148–56.

  13. 13.

    Mascarenhas JB, Young KP, Littlejohn EL, Yoo BK, Salgia R, Lang D. PAX6 is expressed in pancreatic cancer and actively participates in cancer progression through activation of the MET tyrosine kinase receptor gene. J Biol Chem. 2009;284:27524–32.

  14. 14.

    Zhao X, Yue W, Zhang L, Ma L, Jia W, Qian Z, et al. Downregulation of PAX6 by shRNA inhibits proliferation and cell cycle progression of human non-small cell lung cancer cell lines. PLoS ONE. 2014;9:e85738.

  15. 15.

    Shiraishi M, Sekiguchi A, Terry MJ, Oates AJ, Miyamoto Y, Chuu YH, et al. A comprehensive catalog of CpG islands methylated in human lung adenocarcinomas for the identification of tumor suppressor genes. Oncogene. 2002;21:3804–13.

  16. 16.

    Rauch TA, Wang Z, Wu X, Kernstine KH, Riggs AD, Pfeifer GP. DNA methylation biomarkers for lung cancer. Tumour Biol. 2012;33:287–96.

  17. 17.

    Belinsky SA. Gene-promoter hypermethylation as a biomarker in lung cancer. Nat Rev Cancer. 2004;4:707–17.

  18. 18.

    Leon G, MacDonagh L, Finn SP, Cuffe S, Barr MP. Cancer stem cells in drug resistant lung cancer: targeting cell surface markers and signaling pathways. Pharmacol Ther. 2016;158:71–90.

  19. 19.

    Ooki A, Maleki Z, Tsay JJ, Goparaju C, Brait M, Turaga N, et al. A panel of novel detection and prognostic methylated DNA markers in primary non-small cell lung cancer and serum DNA. Clin Cancer Res. 2017;23:7141–52.

  20. 20.

    Takebe N, Miele L, Harris PJ, Jeong W, Bando H, Kahn M, et al. Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. Nat Rev Clin Oncol. 2015;12:445–64.

  21. 21.

    Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, et al. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol. 2007;9:625–35.

  22. 22.

    Justilien V, Fields AP. Molecular pathways: novel approaches for improved therapeutic targeting of Hedgehog signaling in cancer stem cells. Clin Cancer Res. 2015;21:505–13.

  23. 23.

    Ooki A, Del Carmen Rodriguez Pena M, Marchionni L, Dinalankara W, Begum A, Hahn NM, et al. YAP1 and COX2 coordinately regulate urothelial cancer stem-like cells. Cancer Res. 2018;78:168–81.

  24. 24.

    Boumahdi S, Driessens G, Lapouge G, Rorive S, Nassar D, Le Mercier M, et al. SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma. Nature. 2014;511:246–50.

  25. 25.

    Santini R, Pietrobono S, Pandolfi S, Montagnani V, D’Amico M, Penachioni JY, et al. SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells. Oncogene. 2014;33:4697–708.

  26. 26.

    Bora-Singhal N, Perumal D, Nguyen J, Chellappan S. Gli1-mediated regulation of Sox2 facilitates self-renewal of stem-like cells and confers resistance to EGFR inhibitors in non-small cell lung cancer. Neoplasia. 2015;17:538–51.

  27. 27.

    Infante P, Alfonsi R, Botta B, Mori M, Di Marcotullio L. Targeting GLI factors to inhibit the Hedgehog pathway. Trends Pharmacol Sci. 2015;36:547–58.

  28. 28.

    Stolzenburg S, Rots MG, Beltran AS, Rivenbark AG, Yuan X, Qian H, et al. Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer. Nucleic Acids Res. 2012;40:6725–40.

  29. 29.

    Cheung WK, Zhao M, Liu Z, Stevens LE, Cao PD, Fang JE, et al. Control of alveolar differentiation by the lineage transcription factors GATA6 and HOPX inhibits lung adenocarcinoma metastasis. Cancer Cell. 2013;23:725–38.

  30. 30.

    Watanabe H, Meyerson M. Hopping between differentiation states in lung adenocarcinoma. Cancer Cell. 2013;23:707–9.

  31. 31.

    Liu YP, Yang CJ, Huang MS, Yeh CT, Wu AT, Lee YC, et al. Cisplatin selects for multidrug-resistant CD133 + cells in lung adenocarcinoma by activating Notch signaling. Cancer Res. 2013;73:406–16.

  32. 32.

    Holland PW, Booth HA, Bruford EA. Classification and nomenclature of all human homeobox genes. BMC Biol. 2007;5:47.

  33. 33.

    Greenberg AK, Lu F, Goldberg JD, Eylers E, Tsay JC, Yie TA, et al. CT scan screening for lung cancer: risk factors for nodules and malignancy in a high-risk urban cohort. PLoS ONE. 2012;7:e39403.

  34. 34.

    Nam HS. Malignant pleural effusion: medical approaches for diagnosis and management. Tuberc Respir Dis. 2014;76:211–7.

  35. 35.

    Kamachi Y, Uchikawa M, Tanouchi A, Sekido R, Kondoh H. Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development. Genes Dev. 2001;15:1272–86.

  36. 36.

    Maeda Y, Dave V, Whitsett JA. Transcriptional control of lung morphogenesis. Physiol Rev. 2007;87:219–44.

  37. 37.

    Yin Z, Gonzales L, Kolla V, Rath N, Zhang Y, Lu MM, et al. Hop functions downstream of Nkx2.1 and GATA6 to mediate HDAC-dependent negative regulation of pulmonary gene expression. Am J Physiol Lung Cell Mol Physiol. 2006;291:L191–9.

  38. 38.

    Shen Y, Chow J, Wang Z, Fan G. Abnormal CpG island methylation occurs during in vitro differentiation of human embryonic stem cells. Hum Mol Genet. 2006;15:2623–35.

  39. 39.

    Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature. 2007;447:425–32.

  40. 40.

    Po A, Silvano M, Miele E, Capalbo C, Eramo A, Salvati V, et al. Noncanonical GLI1 signaling promotes stemness features and in vivo growth in lung adenocarcinoma. Oncogene. 2017;36:4641–52.

  41. 41.

    Coutinho P, Pavlou S, Bhatia S, Chalmers KJ, Kleinjan DA, van Heyningen V. Discovery and assessment of conserved Pax6 target genes and enhancers. Genome Res. 2011;21:1349–59.

  42. 42.

    Aberle DR, Adams AM, Berg CD, Black WC, Clapp JD, Fagerstrom RM, et al. Reduced lung-cancer mortality with low-dose computed tomographic screening. N Engl J Med. 2011;365:395–409.

  43. 43.

    Tammemagi MC, Katki HA, Hocking WG, Church TR, Caporaso N, Kvale PA, et al. Selection criteria for lung-cancer screening. N Engl J Med. 2013;368:728–36.

  44. 44.

    Blanchon T, Brechot JM, Grenier PA, Ferretti GR, Lemarie E, Milleron B, et al. Baseline results of the Depiscan study: a French randomized pilot trial of lung cancer screening comparing low-dose CT scan (LDCT) and chest X-ray (CXR). Lung Cancer. 2007;58:50–8.

  45. 45.

    Begum S, Brait M, Dasgupta S, Ostrow KL, Zahurak M, Carvalho AL, et al. An epigenetic marker panel for detection of lung cancer using cell-free serum DNA. Clin Cancer Res. 2011;17:4494–503.

  46. 46.

    The Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543–50.

  47. 47.

    Ooki A, Yamashita K, Kikuchi S, Sakuramoto S, Katada N, Kokubo K, et al. Potential utility of HOP homeobox gene promoter methylation as a marker of tumor aggressiveness in gastric cancer. Oncogene. 2010;29:3263–75.

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This work was funded by the Flight Attendant Medical Research Institute Clinical Innovative Award 103015 (M.O. Hoque), NCI R01CA206027 (M.O. Hoque), the Career Development award from SPORE in Cervical Cancer Grants P50 CA098252 (MOH).

Author information


  1. Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    • Akira Ooki
    •  & Mohammad O. Hoque
  2. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    • Wikum Dinalankara
    • , Luigi Marchionni
    •  & Mohammad O. Hoque
  3. Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, New York University School of Medicine, New York, NY, 10016, USA

    • Jun-Chieh J. Tsay
    •  & William N. Rom
  4. Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Langone Medical Center, New York University of Medicine, New York, NY, 10016, USA

    • Chandra Goparaju
    •  & Harvey I. Pass
  5. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA

    • Zahra Maleki
  6. Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA

    • Mohammad O. Hoque


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The authors declare that they have no conflict of interest.

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Correspondence to Mohammad O. Hoque.

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