Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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RNA Interference-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth, Induces Apoptosis, and Increases the Chemosensitivity to 5-Fluorouracil in Renal Cancer Caki-1 Cells

  • Wang, Peng (The Second Department of Urology, Shengjing Hospital of China Medical University) ;
  • Yin, Bo (The Second Department of Urology, Shengjing Hospital of China Medical University) ;
  • Shan, Liping (The Second Department of Urology, Shengjing Hospital of China Medical University) ;
  • Zhang, Hui (The Second Department of Urology, Shengjing Hospital of China Medical University) ;
  • Cui, Jun (The Second Department of Urology, Shengjing Hospital of China Medical University) ;
  • Zhang, Mo (The Second Department of Urology, Shengjing Hospital of China Medical University) ;
  • Song, Yongsheng (The Second Department of Urology, Shengjing Hospital of China Medical University)
  • Received : 2014.04.07
  • Accepted : 2014.09.18
  • Published : 2014.12.31
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Abstract

Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the antiapoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADPribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.

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References

  1. Chen, W., Ke, Z., Shi, H., Yang, S., and Wang, L. (2010). Overexpression of AEG-1 in renal cell carcinoma and its correlation with tumor nuclear grade and progression. Neoplasma 57, 522-529. https://doi.org/10.4149/neo_2010_06_522
  2. Deng, H., Zhou, Z., Tu, W., Xia, Y., Huang, H., and Tian, D. (2014). Knockdown of astrocyte elevated gene-1 inhibits growth through suppression of IL-6 secretion in HepG2 human hepatoma cells. Oncol. Lett. 7, 101-106. https://doi.org/10.3892/ol.2013.1645
  3. Duman-Scheel, M., Weng, L., Xin, S., and Du, W. (2002). Hedgehog regulates cell growth and proliferation by inducing cyclin D and cyclin E. Nature 417, 299-304. https://doi.org/10.1038/417299a
  4. Dutcher, J.P. (2013). Recent developments in the treatment of renal cell carcinoma. Ther. Adv. Urol. 5, 338-353. https://doi.org/10.1177/1756287213505672
  5. Elstein, K.H., and Zucker, R.M. (1994). Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations. Exp. Cell Res. 211, 322-331. https://doi.org/10.1006/excr.1994.1094
  6. Emdad, L., Lee, S.G., Su, Z.Z., Jeon, H.Y., Boukerche, H., Sarkar, D., and Fisher, P.B. (2009). Astrocyte elevated gene-1 (AEG-1) functions as an oncogene and regulates angiogenesis. Proc. Natl. Acad. Sci. USA 106, 21300-21305. https://doi.org/10.1073/pnas.0910936106
  7. Emdad, L., Sarkar, D., Lee, S.G., Su, Z.Z., Yoo, B.K., Dash, R., Yacoub, A., Fuller, C.E., Shah, K., Dent, P., et al. (2010). Astrocyte elevated gene-1: a novel target for human glioma therapy. Mol. Cancer Ther. 9, 79-88.
  8. Erdem, H., Oktay, M., Yildirim, U., Uzunlar, A.K., and Kayikci, M.A. (2013). Expression of AEG-1 and p53 and their clinicopathological significance in malignant lesions of renal cell carcinomas: a microarray study. Pol. J. Pathol. 64, 28-32.
  9. Gnosa, S., Shen, Y.M., Wang, C.J., Zhang, H., Stratmann, J., Arbman, G., and Sun, X.F. (2012). Expression of AEG-1 mRNA and protein in colorectal cancer patients and colon cancer cell lines. J. Transl. Med. 10, 109. https://doi.org/10.1186/1479-5876-10-109
  10. Gnosa, S., Zhang, H., Brodin, V.P., Carstensen, J., Adell, G., and Sun, X.F. (2014). AEG-1 expression is an independent prognostic factor in rectal cancer patients with preoperative radiotherapy: a study in a Swedish clinical trial. Br. J. Cancer 111, 166-173. https://doi.org/10.1038/bjc.2014.250
  11. Gupta, K., Miller, J.D., Li, J.Z., Russell, M.W., and Charbonneau, C. (2008). Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): a literature review. Cancer Treat. Rev. 34, 193-205. https://doi.org/10.1016/j.ctrv.2007.12.001
  12. Huang, K., Li, L.A., Meng, Y., You, Y., Fu, X., and Song, L. (2013a). High expression of astrocyte elevated gene-1 (AEG-1) is associated with progression of cervical intraepithelial neoplasia and unfavorable prognosis in cervical cancer. World J. Surg. Oncol. 11, 297. https://doi.org/10.1186/1477-7819-11-297
  13. Huang, W., Yang, L., Liang, S., Liu, D., Chen, X., Ma, Z., Zhai, S., Li, P., and Wang, X. (2013b). AEG-1 is a target of perifosine and is over-expressed in gastric dysplasia and cancers. Dig. Dis. Sci. 58, 2873-2880. https://doi.org/10.1007/s10620-013-2735-5
  14. Huang, S., Wu, B., Li, D., Zhou, W., Deng, G., Zhang, K., and Li, Y. (2014). Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells. Biochem. Biophys. Res. Commun. 444, 338-345. https://doi.org/10.1016/j.bbrc.2014.01.046
  15. Jiang, T., Zhu, A., Zhu, Y., and Piao, D. (2012). Clinical implications of AEG-1 in liver metastasis of colorectal cancer. Med. Oncol. 29, 2858-2863. https://doi.org/10.1007/s12032-012-0186-z
  16. Kang, D.C., Su, Z.Z., Sarkar, D., Emdad, L., Volsky, D.J., and Fisher, P.B. (2005). Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1. Gene 353, 8-15. https://doi.org/10.1016/j.gene.2005.04.006
  17. Ke, Z.F., Mao, X., Zeng, C., He, S., Li, S., and Wang, L.T. (2013). AEG-1 expression characteristics in human non-small cell lung cancer and its relationship with apoptosis. Med. Oncol. 30, 383. https://doi.org/10.1007/s12032-012-0383-9
  18. Leibovich, B.C., Lohse, C.M., Crispen, P.L., Boorjian, S.A., Thompson, R.H., Blute, M.L., and Cheville, J.C. (2010). Histological subtype is an independent predictor of outcome for patients with renal cell carcinoma. J. Urol. 183, 1309-1315. https://doi.org/10.1016/j.juro.2009.12.035
  19. Li, J., Yang, L., Song, L., Xiong, H., Wang, L., Yan, X., Yuan, J., Wu, J., and Li, M. (2009). Astrocyte elevated gene-1 is a proliferation promoter in breast cancer via suppressing transcriptional factor FOXO1. Oncogene 28, 3188-3196. https://doi.org/10.1038/onc.2009.171
  20. Li, C., Liu, J., Lu, R., Yu, G., Wang, X., Zhao, Y., Song, H., Lin, P., Sun, X., Yu, X., et al. (2011). AEG -1 overexpression: a novel indicator for peritoneal dissemination and lymph node metastasis in epithelial ovarian cancers. Int. J. Gynecol. Cancer 21, 602-608. https://doi.org/10.1097/IGC.0b013e3182145561
  21. Li, C., Li, Y., Wang, X., Wang, Z., Cai, J., Wang, L., Zhao, Y., Song, H., Meng, X., Ning, X., et al. (2012). Elevated expression of astrocyte elevated gene-1 (AEG-1) is correlated with cisplatin-based chemoresistance and shortened outcome in patients with stages III-IV serous ovarian carcinoma. Histopathology 60, 953-963. https://doi.org/10.1111/j.1365-2559.2012.04182.x
  22. Liu, H., Song, X., Liu, C., Xie, L., Wei, L., and Sun, R. (2009). Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells. J. Exp. Clin. Cancer Res. 28, 19. https://doi.org/10.1186/1756-9966-28-19
  23. Liu, L., Wu, J., Ying, Z., Chen, B., Han, A., Liang, Y., Song, L., Yuan, J., Li, J., and Li, M. (2010). Astrocyte elevated gene-1 upregulates matrix metalloproteinase-9 and induces human glioma invasion. Cancer Res. 70, 3750-3759. https://doi.org/10.1158/0008-5472.CAN-09-3838
  24. Liu, H.Y., Liu, C.X., Han, B., Zhang, X.Y., and Sun, R.P. (2012). AEG-1 is associated with clinical outcome in neuroblastoma patients. Cancer Biomark 11, 115-121. https://doi.org/10.3233/CBM-2012-0268
  25. Moroy, T., and Geisen, C. (2004). Cyclin E. Int. J. Biochem. Cell Biol. 36, 1424-1439. https://doi.org/10.1016/j.biocel.2003.12.005
  26. Nikpour, M., Emadi-Baygi, M., Fischer, U., Niegisch, G., Schulz, W.A., and Nikpour, P. (2014). MTDH/AEG-1 contributes to central features of the neoplastic phenotype in bladder cancer. Urol. Oncol. 32, 670-677. https://doi.org/10.1016/j.urolonc.2013.11.005
  27. Riccardi, C., and Nicoletti, I. (2006). Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1, 1458-1461. https://doi.org/10.1038/nprot.2006.238
  28. Song, H., Li, C., Lu, R., Zhang, Y., and Geng, J. (2010). Expression of astrocyte elevated gene-1: a novel marker of the pathogenesis, progression, and poor prognosis for endometrial cancer. Int. J. Gynecol. Cancer 20, 1188-1196. https://doi.org/10.1111/IGC.0b013e3181ef8e21
  29. Su, Z.Z., Chen, Y., Kang, D.C., Chao, W., Simm, M., Volsky, D.J., and Fisher, P.B. (2003). Customized rapid subtraction hybridization (RaSH) gene microarrays identify overlapping expression changes in human fetal astrocytes resulting from human immunodeficiency virus-1 infection or tumor necrosis factor-alpha treatment. Gene 306, 67-78. https://doi.org/10.1016/S0378-1119(03)00404-9
  30. Sun, W., Fan, Y.Z., Xi, H., Lu, X.S., Ye, C., and Zhang, J.T. (2011). Astrocyte elevated gene-1 overexpression in human primary gallbladder carcinomas: an unfavorable and independent prognostic factor. Oncol. Rep. 26, 1133-1142.
  31. Sutherland, H.G., Lam, Y.W., Briers, S., Lamond, A.I., and Bickmore, W.A. (2004). 3D3/lyric: a novel transmembrane protein of the endoplasmic reticulum and nuclear envelope, which is also present in the nucleolus. Exp. Cell Res. 294, 94-105. https://doi.org/10.1016/j.yexcr.2003.11.020
  32. Xia, Z., Zhang, N., Jin, H., Yu, Z., Xu, G., and Huang, Z. (2010). Clinical significance of astrocyte elevated gene-1 expression in human oligodendrogliomas. Clin. Neurol. Neurosurg. 112, 413-419. https://doi.org/10.1016/j.clineuro.2010.02.007
  33. Yoo, B.K., Emdad, L., Su, Z.Z., Villanueva, A., Chiang, D.Y., Mukhopadhyay, N.D., Mills, A.S., Waxman, S., Fisher, R.A., Llovet, J.M., et al. (2009a). Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression. J. Clin. Invest. 119, 465-477. https://doi.org/10.1172/JCI36460
  34. Yoo, B.K., Gredler, R., Vozhilla, N., Su, Z.Z., Chen, D., Forcier, T., Shah, K., Saxena, U., Hansen, U., Fisher, P.B., et al. (2009b). Identification of genes conferring resistance to 5-fluorouracil. Proc. Natl. Acad. Sci. USA 106, 12938-12943. https://doi.org/10.1073/pnas.0901451106
  35. Yoo, B.K., Chen, D., Su, Z.Z., Gredler, R., Yoo, J., Shah, K., Fisher, P.B., and Sarkar, D. (2010). Molecular mechanism of chemoresistance by astrocyte elevated gene-1. Cancer Res. 70, 3249-3258. https://doi.org/10.1158/0008-5472.CAN-09-4009
  36. Yu, C., Chen, K., Zheng, H., Guo, X., Jia, W., Li, M., Zeng, M., Li, J., and Song, L. (2009). Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis. Carcinogenesis 30, 894-901. https://doi.org/10.1093/carcin/bgp064
  37. Zhang, C.F., Xia, Y.H., Zheng, Q.F., Li, Z.J., Guo, X.H., Zhou, H.C., Zhang, L.L., Dong, L.P., Han, Y., Liu, Z.E., et al. (2013a). Effect of silencing AEG-1 with small interfering RNA on the proliferation and cell cycle of gastric carcinoma SGC-7901 cells. Zhonghua Zhong Liu Za Zhi 35, 22-27.
  38. Zhang, N., Wang, X., Huo, Q., Li, X., Wang, H., Schneider, P., Hu, G., and Yang, Q. (2013b). The oncogene metadherin modulates the apoptotic pathway based on the tumor necrosis factor superfamily member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in breast cancer. J. Biol. Chem. 288, 9396-9407. https://doi.org/10.1074/jbc.M112.395913

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