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Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling

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Abstract

Background

Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR.

Methods

EGFR expression was assessed using Western blotting, immunofluorescence and capacitance assays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stably transfected with a CUG2 expression vector (A549-CUG2; BEAS-CUG2) or an empty control vector (A549-Vec; BEAS-Vec). After siRNA-mediated EGFR, Stat1 and HDAC4 silencing, antioxidant and multidrug resistance protein and mRNA levels were assessed using Western blotting and RT-PCR. In addition, the respective cells were treated with doxorubicin after which apoptosis and reactive oxygen species (ROS) levels were measured. Stat1 acetylation was assessed by immunoprecipitation.

Results

We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFR silencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR.

Conclusion

Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.

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References

  1. S. Lee, J. Gang, S.B. Jeon, S.H. Choo, B. Lee, Y.G. Kim, Y.S. Lee, J. Jung, S.Y. Song, S.S. Koh, Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 (CUG2). Biochem Biophys Res Commun 360, 633–639 (2007)

    Article  CAS  PubMed  Google Scholar 

  2. T. Hori, M. Amano, A. Suzuki, C.B. Backer, J.P. Welburn, Y. Dong, B.F. McEwen, W.H. Shang, E. Suzuki, K. Okawa, I.M. Cheeseman, T. Fukagawa, CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Cell 135, 1039–1052 (2008)

    Article  CAS  PubMed  Google Scholar 

  3. H. Kim, M. Lee, S. Lee, B. Park, W. Koh, D.J. Lee, D.S. Lim, S. Lee, Cancer-upregulated gene 2 (CUG2), a new component of centromere complex, is required for kinetochore function. Mol Cell 27, 697–701 (2009)

    Article  CAS  Google Scholar 

  4. E.H. Park, I.R. Cho, R. Srisuttee, H.J. Min, M.J. Oh, Y.J. Jeong, B.H. Jhun, R.N. Johnston, S. Lee, S.S. Koh, Y.H. Chung, CUG2, a novel oncogene confers reoviral replication through Ras and p38 signaling pathway. Cancer Gene Ther 17, 307–314 (2010)

    Article  CAS  PubMed  Google Scholar 

  5. W. Malilas, S.S. Koh, R. Srisuttee, W. Boonying, I.R. Cho, C.S. Jeong, R.N. Johnston, Y.H. Chung, Cancer upregulated gene 2, a novel oncogene, confers resistance to oncolytic vesicular stomatitis virus through STAT1-OASL2 signaling. Cancer Gene Ther 20, 125–132 (2013)

    Article  CAS  PubMed  Google Scholar 

  6. W. Malilas, S.S. Koh, S. Kim, R. Srisuttee, I.R. Cho, J. Moon, H.S. Yoo, S. Oh, R.N. Johnston, Y.H. Chung, Cancer upregulated gene 2, a novel oncogene, enhances migration and drug resistance of colon cancer cells via STAT1 activation. Int J Oncol 43, 1111–1116 (2013)

    Article  CAS  PubMed  Google Scholar 

  7. S. Kaowinn, J. Kim, J. Lee, D.H. Shin, C.D. Kang, D.K. Kim, S. Lee, M.K. Kang, S.S. Koh, S.J. Kim, Y.H. Chung, Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-beta signaling. Oncotarget 8, 5092–5110 (2017)

    PubMed  Google Scholar 

  8. K.J. Wilson, J.L. Gilmore, J. Foley, M.A. Lemmon, D.J. Riese 2nd, Functional selectivity of EGF family peptide growth factors: Implications for cancer. Pharmacol Ther 122, 1–8 (2009)

    Article  CAS  PubMed  Google Scholar 

  9. N. Normanno, A. De Luca, C. Bianco, L. Strizzi, M. Mancino, M.R. Maiello, A. Carotenuto, G. De Feo, F. Caponigro, D.S. Salomon, Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366, 2–16 (2006)

    Article  CAS  PubMed  Google Scholar 

  10. M.A. Lemmon, J. Schlessinger, Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Y. Zhen, L. Guanghui, Z. Xiefu, Knockdown of EGFR inhibits growth and invasion of gastric cancer cells. Cancer Gene Ther 21, 491–497 (2014)

    Article  CAS  PubMed  Google Scholar 

  12. Y. Yarden, M.X. Sliwkowski, Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2, 127–137 (2001)

    Article  CAS  PubMed  Google Scholar 

  13. R.J. Silva-Oliveira, V.A. Silva, O. Martinho, A. Cruvinel-Carloni, M.E. Melendez, M.N. Rosa, F.E. de Paula, L. de Souza Viana, A.L. Carvalho, R.M. Reis, Cytotoxicity of allitinib, an irreversible anti-EGFR agent, in a large panel of human cancer-derived cell lines: KRAS mutation status as a predictive biomarker. Cell Oncol 39, 253–263 (2016)

    Article  CAS  Google Scholar 

  14. C. Gridelli, F. De Marinis, M. Di Maio, D. Cortinovis, F. Cappuzzo, T. Mok, Gefitinib as first-line treatment for patients with advanced non-small-cell lung cancer with activating epidermal growth factor receptor mutation: Review of the evidence. Lung Cancer 71, 249–257 (2011)

    Article  CAS  PubMed  Google Scholar 

  15. M. Murphy, B. Stordal, Erlotinib or gefitinib for the treatment of relapsed platinum pretreated non-small cell lung cancer and ovarian cancer: A systematic review. Drug Resist Updat 14, 177–190 (2011)

    Article  CAS  PubMed  Google Scholar 

  16. R. Bianco, T. Gelardi, V. Damiano, F. Ciardiello, G. Tortora, Rational bases for the development of EGFR inhibitors for cancer treatment. Int J Biochem Cell Biol 39, 1416–1431 (2007)

    Article  CAS  PubMed  Google Scholar 

  17. S.N. Bichev, D.M. Marinova, Y.G. Slavova, A.S. Savov, Epidermal growth factor receptor mutations in east European non-small cell lung cancer patients. Cell Oncol 38, 145–153 (2015)

    Article  CAS  Google Scholar 

  18. Y.X. Bao, X.D. Zhao, H.B. Deng, C.L. Lu, Y. Guo, X. Lu, L.L. Deng, Schedule-dependent cytotoxicity of sunitinib and TRAIL in human non-small cell lung cancer cells with or without EGFR and KRAS mutations. Cell Oncol 39, 343–352 (2016)

    Article  CAS  Google Scholar 

  19. M.J. Sotelo, B. Garcia-Paredes, C. Aguado, J. Sastre, E. Diaz-Rubio, Role of cetuximab in first-line treatment of metastatic colorectal cancer. World J Gastroenterol 20, 4208–4219 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. J.E. Frampton, Cetuximab: A review of its use in squamous cell carcinoma of the head and neck. Drugs 70, 1987–2010 (2010)

    Article  CAS  PubMed  Google Scholar 

  21. X.J. Yang, E. Seto, HATs and HDACs: From structure, function and regulation to novel strategies for therapy and prevention. Oncogene 26, 5310–5318 (2007)

    Article  CAS  PubMed  Google Scholar 

  22. A. Ferraro, Altered primary chromatin structures and their implications in cancer development. Cell Oncol 39, 195–210 (2016)

    Article  Google Scholar 

  23. H. Geng, C.T. Harvey, J. Pittsenbarger, Q. Liu, T.M. Beer, C. Xue, D.Z. Qian, HDAC4 protein regulates HIF1α protein lysine acetylation and cancer cell response to hypoxia. J Biol Chem 286, 38095–38102 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. M.M. Mihaylova, D.S. Vasquez, K. Ravnskjaer, P.D. Denechaud, R.T. Yu, J.G. Alvarez, M. Downes, R.M. Evans, M. Montminy, R.J. Shaw, Class IIa histone deacetylases are hormone-activated regulators of FOXO and mammalian glucose homeostasis. Cell 145, 607–621 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. E.A. Stronach, A. Alfraidi, N. Rama, C. Datler, J.B. Studd, R. Agarwal, T.G. Guney, C. Gourley, B.T. Hennessy, G.B. Mills, A. Mai, R. Brown, R. Dina, H. Gabra, HDAC4-regulated STAT1 activation mediates platinum resistance in ovarian cancer. Cancer Res 71, 4412–4422 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Y. Sun, Y.E. Chin, E. Weisiger, C. Malter, I. Tawara, T. Toubai, E. Gatza, P. Mascagni, C.A. Dinarello, P. Reddy, Cutting edge: Negative regulation of dendritic cells through acetylation of the nonhistone protein STAT-3. J Immunol 182, 5899–5903 (2009)

    Article  CAS  PubMed  Google Scholar 

  27. Z.L. Yuan, Y.J. Guan, D. Chatterjee, Y.E. Chin, Stat3 dimerization regulated by reversible acetylation of a single lysine residue. Science 307, 269–273 (2005)

    Article  CAS  PubMed  Google Scholar 

  28. Z.H. Kang, C.Y. Wang, W.L. Zhang, J.T. Zhang, C.H. Yuan, P.W. Zhao, Y.Y. Lin, S. Hong, C.Y. Li, L. Wang, Histone deacetylase HDAC4 promotes gastric cancer SGC-7901 cells progression via p21 repression. PLoS One 9, e98894 (2014)

    Article  PubMed  PubMed Central  Google Scholar 

  29. A.J. Wilson, D.S. Byun, S. Nasser, L.B. Murray, K. Ayyanar, D. Arango, M. Figueroa, A. Melnick, G.D. Kao, L.H. Augenlicht, J.M. Mariadason, HDAC4 promotes growth of colon cancer cells via repression of p21. Mol Biol Cell 19, 4062–4075 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. D.M. Shin, Y.C. Shin, J.H. Lee, T.H. Kim, D.W. Han, J.M. Kim, H.K. Kim, K. Kim, Y.H. Hwang, Highly sensitive detection of epidermal growth factor receptor expression levels using a capacitance sensor. Sensors Actuators B Chem 209, 438–443 (2015)

    Article  CAS  Google Scholar 

  31. O.H. Kramer, T. Heinzel, Phosphorylation-acetylation switch in the regulation of STAT1 signaling. Mol Cell Endocrinol 315, 40–48 (2010)

    Article  PubMed  Google Scholar 

  32. S. Zhuang, Regulation of STAT signaling by acetylation. Cell Signal 25, 1924–1931 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. D. Zhao, B. Zhai, C. He, G. Tan, X. Jiang, S. Pan, X. Dong, Z. Wei, L. Ma, H. Qiao, H. Jiang, X. Sun, Upregulation of HIF-2alpha induced by sorafenib contributes to the resistance by activating the TGF-alpha/EGFR pathway in hepatocellular carcinoma cells. Cell Signal 26, 1030–1039 (2014)

    Article  CAS  PubMed  Google Scholar 

  34. N. Ungerleider, C. Han, J. Zhang, L. Yao and T. Wu, TGFbeta signaling confers sorafenib resistance via induction of multiple RTKs in hepatocellular carcinoma cells. Mol Carcinog 56, 1302-1311 (2017)

  35. R. Kurimoto, S. Iwasawa, T. Ebata, T. Ishiwata, I. Sekine, Y. Tada, K. Tatsumi, S. Koide, A. Iwama, Y. Takiguchi, Drug resistance originating from a TGF-beta/FGF-2-driven epithelial-to-mesenchymal transition and its reversion in human lung adenocarcinoma cell lines harboring an EGFR mutation. Int J Oncol 48, 1825–1836 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. S.L. Yu, D.C. Lee, J.W. Son, C.G. Park, H.Y. Lee, J. Kang, Histone deacetylase 4 mediates SMAD family member 4 deacetylation and induces 5-fluorouracil resistance in breast cancer cells. Oncol Rep 30, 1293–1300 (2013)

    Article  CAS  PubMed  Google Scholar 

  37. N.N. Khodarev, P. Roach, S.P. Pitroda, D.W. Golden, M. Bhayani, M.Y. Shao, T.E. Darga, M.G. Beveridge, R.F. Sood, H.G. Sutton, M.A. Beckett, H.J. Mauceri, M.C. Posner, R.R. Weichselbaum, STAT1 pathway mediates amplification of metastatic potential and resistance to therapy. PLoS One 4, e5821 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

  38. D. Roberts, J. Schick, S. Conway, S. Biade, P.B. Laub, J.P. Stevenson, T.C. Hamilton, P.J. O'Dwyer, S.W. Johnson, Identification of genes associated with platinum drug sensitivity and resistance in human ovarian cancer cells. Br J Cancer 92, 1149–1158 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. M. Fryknas, S. Dhar, F. Oberg, L. Rickardson, M. Rydaker, H. Goransson, M. Gustafsson, U. Pettersson, P. Nygren, R. Larsson, A. Isaksson, STAT1 signaling is associated with acquired crossresistance to doxorubicin and radiation in myeloma cell lines. Int J Cancer 120, 189–195 (2007)

    Article  PubMed  Google Scholar 

  40. L. Rickardson, M. Fryknas, S. Dhar, H. Lovborg, J. Gullbo, M. Rydaker, P. Nygren, M.G. Gustafsson, R. Larsson, A. Isaksson, Identification of molecular mechanisms for cellular drug resistance by combining drug activity and gene expression profiles. Br J Cancer 93, 483–492 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. H.M. Chang, M. Paulson, M. Holko, C.M. Rice, B.R. Williams, I. Marie, D.E. Levy, Induction of interferon-stimulated gene expression and antiviral responses require protein deacetylase activity. Proc Natl Acad Sci U S A 101, 9578–9583 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. L. Klampfer, J. Huang, L.A. Swaby, L. Augenlicht, Requirement of histone deacetylase activity for signaling by STAT1. J Biol Chem 279, 30358–30368 (2004)

    Article  CAS  PubMed  Google Scholar 

  43. J. Vlasakova, Z. Novakova, L. Rossmeislova, M. Kahle, P. Hozak, Z. Hodny, Histone deacetylase inhibitors suppress IFNalpha-induced up-regulation of promyelocytic leukemia protein. Blood 109, 1373–1380 (2007)

    Article  CAS  PubMed  Google Scholar 

  44. I. Nusinzon, C.M. Horvath, Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone deacetylase 1. Proc Natl Acad Sci U S A 100, 14742–14747 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported by the Basic Research Program of the National Research Foundation, funded by the Korean government (NRF-2014R A1A2053750), (MSIP) (NRF- 2015R1A2A1A15056030), and (2015R1A5A7036513).

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Authors and Affiliations

  1. BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, 46241, Republic of Korea

    Sirichat Kaowinn, Seung Won Jun, Chang Seok Kim & Young-Hwa Chung

  2. Department of Nanoenergy Engineering, Pusan National University, Busan, 46241, Republic of Korea

    Dong-Myeong Shin & Yoon-Hwae Hwang

  3. Department of Optics & Mechatronics Engineering, Convergence Research Center of 3D Laser-Aided Innovative Manufacturing Technology, Pusan National University, Busan, 46241, Republic of Korea

    Kyujung Kim & Bosung Shin

  4. Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand

    Chutima Kaewpiboon

  5. Department of Biological Sciences, Dong-A University, Busan, 49315, Republic of Korea

    Hyeon Hee Jeong & Sang Seok Koh

  6. Department of Toxicology, University Medical Center, 55131, Mainz, Germany

    Oliver H. Krämer

  7. Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, T2N4N1, Canada

    Randal N. Johnston

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  1. Sirichat Kaowinn
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Contributions

S.K., R.N. J. and Y-H.C. conceived and designed the experiments. S.K., S.W.J., D.-M.S., C.K. and H.H.J. performed the experiments. Y.-H. H., K.K., B.S., C.S.K., S.S.K. and O.H.K contributed reagents and materials. S.K., R.N. J. and Y-H.C. wrote the paper.

Corresponding author

Correspondence to Young-Hwa Chung.

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Kaowinn, S., Jun, S.W., Kim, C.S. et al. Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling. Cell Oncol. 40, 549–561 (2017). https://doi.org/10.1007/s13402-017-0343-7

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