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Cellular Oncology

, Volume 38, Issue 3, pp 205–214 | Cite as

ECRG4 acts as a tumor suppressor and as a determinant of chemotherapy resistance in human nasopharyngeal carcinoma

  • Yanjie YouEmail author
  • Wenjun Yang
  • Xin Qin
  • Fei Wang
  • Haijun Li
  • Canfeng Lin
  • Wenmei Li
  • Cunguo Gu
  • Yinpo Zhang
  • Yonggang Ran
Original Paper

Abstract

Background

Human nasopharyngeal carcinoma (NPC) is a malignant type of cancer with an increasing incidence. As yet, however, molecular biomarkers with a strong diagnostic impact and a major therapeutic promise have remained elusive. Here, we identified the esophageal carcinoma related gene 4 (ECRG4) as a novel candidate tumor suppressor gene and a promising therapeutic target for NPC.

Methods

RT-PCR, Western blotting, methylation-specific PCR and bisulfite sequencing were performed to assess the expression and methylation status of the ECRG4 gene in primary NPC samples, NPC-derived cell lines and patient-derived peripheral blood samples. The NPC-derived cell line CNE1 was selected for treatment with a methylation inhibitor to restore ECRG4 expression. In addition, cell proliferation, invasion and colony formation assays were performed to assess the inhibitory effects of exogenous ECRG4 expression in CNE1 cells.

Results

Down-regulated ECRG4 expression was found to occur in 82.5 % (33/40) of the primary NPC biopsies tested. This down-regulation was significantly correlated with its tumor-specific promoter methylation status (72.5 %, 29/40) and was also observed in the matching peripheral blood samples from the NPC patients (57.5 %, 23/40). Pharmacologic demethylation through 5-aza-dC treatment led to gene reactivation in ECRG4 methylated and silenced NPC cell lines. Moreover, exogenous expression of ECRG4 in the CNE1 cell line strongly inhibited its growth and invasive capacities, as well as its enhanced chemosensitivity to cisplatin through autophagy induction.

Conclusion

Our data suggest that methylation-mediated suppression of the ECRG4 gene occurs frequently in NPC and that restoration of its expression may have therapeutic benefits.

Keywords

ECRG4 Tumor suppressor gene DNA methylation Nasopharyngeal carcinoma Cancer surveillance 

Notes

Acknowledgments

We thank Mr. Zhen Zhang (Department of Biochemistry and Molecular Biology, University of Kansas Medical Center) for his careful reading of this manuscript and kind suggestions. This work was supported by the Science and Technology Planning Project of Henan Province, China (142102310464), the 2015 Annual Natural Science Foundation of Luohe Medical College (Y.-J. You), the Natural Science Foundation of Hubei Province (2014CFC1154), and the Foundation of Medical College of Hubei University of Arts and Science (YXKY 201402).

Conflicts of Interest

The authors have no conflict of interest.

References

  1. 1.
    H. Li, Y. You, C. Lin, M. Zheng, C. Hong, J. Chen, D. Li, W.W. Au, Z. Chen, XRCC1 codon 399Gln polymorphism is associated with radiotherapy-induced acute dermatitis and mucositis in nasopharyngeal carcinoma patients. Radiat Oncol. 8, 31 (2013)CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    G. Sanguineti, F.B. Geara, A.S. Garden, S.L. Tucker, K.K. Ang, W.H. Morrison, L.J. Peters, Carcinoma of the nasopharynx treated by radiotherapy alone: determinants of local and regional control. Int J Radiat Oncol. Biol. Phys. 37, 985–996 (1997)CrossRefPubMedGoogle Scholar
  3. 3.
    Y. Ran, S. Wu, Y. You, Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy. J Biochem. 149, 49–54 (2011)CrossRefPubMedGoogle Scholar
  4. 4.
    Y. You, W. Yang, Z. Wang, H. Zhu, H. Li, C. Lin, Y. Ran, Promoter hypermethylation contributes to the frequent suppression of the CDK10 gene in human nasopharyngeal carcinomas. Cell Oncol. 36, 323–331 (2013)Google Scholar
  5. 5.
    A. Geurts van Kessel. The cancer genome: from structure to function. Cell Oncol. 37, 155–165 (2014)Google Scholar
  6. 6.
    B. Ramaswamy, S. Majumder, S.K. Roy, H. Ghoshal, J. Kutay, J. Datta, M. Younes, C.L. Shapiro, T. Motiwala, S.T. Jacob, Estrogen-mediated suppression of the gene encoding protein tyrosine phosphatase PTPRO in human breast cancer: mechanism and role in tamoxifen sensitivity. Mol Endocrinol. 23, 176–187 (2009)CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    T. Nozoe, T. Oyama, M. Takenoyama, T. Hanagiri, K. Sugio, K. Yasumoto, Significance of immunohistochemical expression of estrogen receptors alpha and beta in squamous cell carcinoma of the esophagus. Clin Cancer Res. 13, 4046–4050 (2007)CrossRefPubMedGoogle Scholar
  8. 8.
    Z. Li, X. Zou, L. Xie, H. Dong, Y. Chen, Q. Liu, X. Wu, D. Zhou, D. Tan, H. Zhang, Prognostic importance and therapeutic implications of PAK1, a drugable protein kinase, in gastroesophageal junction adenocarcinoma. PLoS One. 8, e80665 (2013)CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    A. Baird, J. Lee, S. Podvin, A. Kurabi, X. Dang, R. Coimbra, T. Costantini, V. Bansal, B.P. Eliceiri, Esophageal cancer-related gene 4 at the interface of injury, inflammation, infection, and malignancy. Gastrointest Cancer. 2014, 131–142 (2014)CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    T. Su, H. Liu, S. Lu, Cloning and identification of cDNA fragments related to human esophageal cancer. China J Oncol. 20, 254–257 (1998)Google Scholar
  11. 11.
    L.W. Li, X.Y. Yu, Y. Yang, C.P. Zhang, L.P. Guo, S.H. Lu, Expression of esophageal cancer related gene 4 (ECRG4), a novel tumor suppressor gene, in esophageal cancer and its inhibitory effect on the tumor growth in vitro and in vivo. Int J Cancer. 125, 1505–1513 (2009)CrossRefPubMedGoogle Scholar
  12. 12.
    T. Xu, D. Xiao, X. Zhang, ECRG4 inhibits growth and invasiveness of squamous cell carcinoma of the head and neck in vitro and in vivo. Oncol Lett. 5, 1921–1926 (2013)PubMedCentralPubMedGoogle Scholar
  13. 13.
    A. Kurabi, K. Pak, X. Dang, R. Coimbra, B.P. Eliceiri, A.F. Ryan, A. Baird, Ecrg4 attenuates the inflammatory proliferative response of mucosal epithelial cells to infection. PLoS One. 8, e61394 (2013)CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    A.M. Gonzalez, S. Podvin, S.Y. Lin, M.C. Miller, H. Botfield, W.E. Leadbeater, A. Roberton, X. Dang, S.E. Knowling, E. Cardenas-Galindo, J.E. Donahue, E.G. Stopa, C.E. Johanson, R. Coimbra, B.P. Eliceiri, A. Baird, Ecrg4 expression and its product augurin in the choroid plexus: impact on fetal brain development, cerebrospinal fluid homeostasis and neuroprogenitor cell response to CNS injury. Fluids Barriers CNS. 8, 6 (2011)CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    R. Sabatier, P. Finetti, J. Adelaide, A. Guille, J.P. Borg, M. Chaffanet, L. Lane, D. Birnbaum, F. Bertucci, Down-regulation of ECRG4, a candidate tumor suppressor gene, in human breast cancer. PLoS One. 6, e27656 (2011)CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    S. Götze, V. Feldhaus, T. Traska, M. Wolter, G. Reifenberger, A. Tannapfel, C. Kuhnen, D. Martin, O. Müller, S. Sievers, ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma. BMC Cancer. 9, 447 (2009)CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Y. Mori, H. Ishiguro, Y. Kuwabara, M. Kimura, A. Mitsui, H. Kurehara, R. Mori, K. Tomoda, R. Ogawa, T. Katada, K. Harata, Y. Fujii, Expression of ECRG4 is an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma. Oncol Rep. 18, 981–985 (2007)PubMedGoogle Scholar
  18. 18.
    W. Li, X. Liu, B. Zhang, D. Qi, L. Zhang, Y. Jin, H. Yang, Overexpression of candidate tumor suppressor ECRG4 inhibits glioma proliferation and invasion. J Exp Clin Cancer Res. 29, 89 (2010)CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    C.P. Jiang, B.H. Wu, B.Q. Wang, M.Y. Fu, M. Yang, Y. Zhou, F. Liu, Overexpression of ECRG4 enhances chemosensitivity to 5-fluorouracil in the human gastric cancer SGC-7901 cell line. Tumour Biol. 34, 2269–2273 (2013)CrossRefPubMedGoogle Scholar
  20. 20.
    Y. You, Y. Chen, X. Zheng, J. Meltzer, H. Zhang, Aberrant methylation of the PTPRO gene in peripheral blood as a potential biomarker in esophageal squamous cell carcinoma patients. Cancer Lett. 315, 138–144 (2012)CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Y. You, J. Liu, Z. Wang, Y. Zhang, Y. Ran, X. Guo, H. Liu, H. Wang, The enhancement of radio-sensitivity in human esophageal squamous cell carcinoma cells by zoledronic acid and its potential mechanism. Cytotechnology. 66, 17–25 (2014)CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    A. Coker-Gurkan, E.D. Arisan, P. Obakan, E. Guvenir, N.P. Unsal, Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 coloncancer cells. Exp Cell Res. 328, 87–98 (2014)CrossRefPubMedGoogle Scholar
  23. 23.
    P. Ulivi, R. Silvestrini, Role of quantitative and qualitative characteristics of free circulating DNA in the management of patients with non-small cell lung cancer. Cell Oncol. 36, 439–448 (2013)Google Scholar
  24. 24.
    Y.B. Wang, C.F. Ba, Promoter methylation of esophageal cancer-related gene 4 in gastric cancer tissue and its clinical significance. Hepatogastroenterology. 59, 1696–1698 (2012)CrossRefPubMedGoogle Scholar
  25. 25.
    D.K. Vanaja, M. Ehrich, D. Van den Boom, J.C. Cheville, R.J. Karnes, D.J. Tindall, C.R. Cantor, C.Y. Young, Hypermethylation of genes for diagnosis and risk stratification of prostate cancer. Cancer Invest. 27, 549–560 (2009)CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Y. Kujuro, N. Suzuki, T. Kondo, Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed by aged CNS precursor cells. Proc Natl Acad Sci U S A. 107, 8259–8264 (2010)CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    X. Dang, S. Podvin, R. Coimbra, B. Eliceiri, A. Baird, Cell-specific processing and release of the hormone-like precursor and candidate tumor suppressor gene product, Ecrg4. Cell Tissue Res. 348, 505–514 (2012)CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    S.Y. Low, B.S. Tan, H.L. Choo, K.H. Tiong, A.S. Khoo, C.O. Leong, Suppression of BCL-2 synergizes cisplatin sensitivity in nasopharyngeal carcinoma cells. Cancer Lett. 314(2), 166–175 (2012)CrossRefPubMedGoogle Scholar
  29. 29.
    J. Wang, H. Wang, L. Zhao, S. Fan, Z. Yang, F. Gao, L. Chen, G.G. Xiao, J. Molnár, Q. Wang, Down-regulation of P-glycoprotein is associated with resistance to cisplatin and VP-16 in human lung cancer cell lines. Anticancer Res. 30, 3593–3598 (2010)PubMedGoogle Scholar
  30. 30.
    Z.H. Siddik, Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene. 22, 7265–7279 (2003)CrossRefPubMedGoogle Scholar
  31. 31.
    X. Wang, J.R. Masters, Y.C. Wong, A.K. Lo, S.W. Tsao, Mechanism of differential sensitivity to cisplatin in nasopharyngeal carcinoma cells. Anticancer Res. 21, 403–408 (2001)PubMedGoogle Scholar
  32. 32.
    I.A. Voutsadakis, The chemosensitivity of testicular germ cell tumors. Cell Oncol. 37, 79–94 (2014)Google Scholar
  33. 33.
    T.R. O'Donovan, G.C. O'Sullivan, S.L. McKenna, Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics. Autophagy. 7, 509–524 (2011)CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    M.J. Nyhan, T.R. O'Donovan, B. Elzinga, L.C. Crowley, G.C. O'Sullivan, S.L. McKenna, The BH3 mimetic HA14-1 enhances 5-fluorouracil-induced autophagy and type II cell death in oesophageal cancer cells. Br J Cancer. 106, 711–718 (2012)Google Scholar
  35. 35.
    J.M. Yuk, D.M. Shin, K.S. Song, K. Lim, K.H. Kim, S.H. Lee, J.M. Kim, J.S. Lee, T.H. Paik, J.S. Kim, E.K. Jo, Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy. Autophagy. 6, 46–60 (2010)CrossRefPubMedGoogle Scholar
  36. 36.
    Y. Wei, T. Kadia, W. Tong, M. Zhang, Y. Jia, H. Yang, Y. Hu, F.P. Tambaro, J. Viallet, S. O'Brien, G. Garcia-Manero, The combination of a histone deacetylase inhibitor with the Bcl-2 homology domain-3 mimetic GX15-070 has synergistic antileukemia activity by activating both apoptosis and autophagy. Clin. Cancer Res. 16, 3923–3932 (2010)CrossRefPubMedCentralPubMedGoogle Scholar
  37. 37.
    B. Sirichanchuen, T. Pengsuparp, P. Chanvorachote, Long-term cisplatin exposure impairs autophagy and causes cisplatin resistance in human lung cancer cells. Mol. Cell. Biochem. 364, 11–18 (2012)CrossRefPubMedGoogle Scholar
  38. 38.
    W. Hu, S.S. Chen, J.L. Zhang, X.E. Lou, H.J. Zhou, Dihydroartemisinin induces autophagy by suppressing NF-κB activation. Cancer Lett. 343, 239–248 (2014)CrossRefPubMedGoogle Scholar
  39. 39.
    J. Pan, C. Cheng, S. Verstovsek, Q. Chen, Y. Jin, Q. Cao, The BH3-mimetic GX15-070 induces autophagy, potentiates the cytotoxicity of carboplatin and 5-fluorouracil in esophageal carcinoma cells. Cancer Lett. 293, 167–174 (2010)CrossRefPubMedGoogle Scholar
  40. 40.
    Y. Sun, J.H. Liu, L. Jin, Y.X. Sui, L. Lai, Y. Yang, Inhibition of Beclin 1 expression enhances cisplatin-induced apoptosis through a mitochondrial-dependent pathway in human ovarian cancer SKOV3/DDP cells. Oncol. Res. 21, 261–269 (2014)CrossRefPubMedGoogle Scholar
  41. 41.
    X.L. Guo, D. Li, F. Hu, J.R. Song, S.S. Zhang, W.J. Deng, K. Sun, Q.D. Zhao, X.Q. Xie, Y.J. Song, M.C. Wu, L.X. Wei, Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells. Cancer Lett. 320, 171–179 (2012)CrossRefPubMedGoogle Scholar
  42. 42.
    S. Daido, A. Yamamoto, K. Fujiwara, R. Sawaya, S. Kondo, Y. Kondo, Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy. Cancer Res. 65, 4368–4375 (2005)CrossRefPubMedGoogle Scholar
  43. 43.
    C.E. Zois, M.I. Koukourakis. Radiation-induced autophagy in normal and cancer cells: towards novel cytoprotection and radio-sensitization policies? Autophagy 5, 442–450 (2009)
  44. 44.
    A. Apel, I. Herr, H. Schwarz, H.P. Rodemann, A. Mayer, Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer Res. 68, 1485–1494 (2008)CrossRefPubMedGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2015

Authors and Affiliations

  • Yanjie You
    • 1
    • 2
    Email author
  • Wenjun Yang
    • 3
  • Xin Qin
    • 4
  • Fei Wang
    • 2
    • 5
  • Haijun Li
    • 6
  • Canfeng Lin
    • 7
  • Wenmei Li
    • 1
  • Cunguo Gu
    • 1
  • Yinpo Zhang
    • 8
  • Yonggang Ran
    • 9
  1. 1.Department of PharmacyLuohe Medical CollegeLuoheChina
  2. 2.Luohe Key Laboratory of Medical BioengineeringLuoheChina
  3. 3.Key Laboratory of Preservation and Maintenance (Ministry of Education), Medical Oncology Department of General HospitalNingxia Medical UniversityYinchuanChina
  4. 4.Medical CollegeHubei University of Arts and ScienceXiangyangChina
  5. 5.Bioengineering LaboratoryLuohe Medical CollegeLuoheChina
  6. 6.Department of Radiation Oncologythe Second People’s Hospital of Neijiang CityNeijaingChina
  7. 7.Department of Radiation OncologyShantou Central HospitalShantouChina
  8. 8.Department of PathologyLuohe Medical CollegeLuoheChina
  9. 9.Department of Teaching and TrainingBethune Military Medical NCO Academy of PLAShijiazhuangChina

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