Histone Deacetylase-3/CAGE Axis Targets EGFR Signaling and Regulates the Response to Anti-Cancer Drugs

  • Kim, Hyuna (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Kim, Youngmi (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Goh, Hyeonjung (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Jeoung, Dooil (Department of Biochemistry, College of Natural Sciences, Kangwon National University)
  • Received : 2015.09.15
  • Accepted : 2015.11.30
  • Published : 2016.03.31


We have previously reported the role of miR-326-HDAC3 loop in anti-cancer drug-resistance. CAGE, a cancer/testis antigen, regulates the response to anti-cancer drug-resistance by forming a negative feedback loop with miR-200b. Studies investigating the relationship between CAGE and HDAC3 revealed that HDAC3 negatively regulated the expression of CAGE. ChIP assays demonstrated the binding of HDAC3 to the promoter sequences of CAGE. However, CAGE did not affect the expression of HDAC3. We also found that EGFR signaling regulated the expressions of HDAC3 and CAGE. Anti-cancer drug-resistant cancer cell lines show an increased expression of $pEGFR^{Y845}$. HDAC3 was found to negatively regulate the expression of $pEGFR^{Y845}$. CAGE showed an interaction and co-localization with EGFR. It was seen that miR-326, a negative regulator of HDAC3, regulated the expression of CAGE, $pEGFR^{Y845}$, and the interaction between CAGE and EGFR. miR-326 inhibitor induced the binding of HDAC3 to the promoter sequences in anti-cancer drug-resistant $Malme3M^R$ cells, decreasing the tumorigenic potential of $Malme3M^R$ cells in a manner associated with its effect on the expression of HDAC3, CAGE and $pEGFR^{Y845}$. The down-regulation of HDAC3 enhanced the tumorigenic, angiogenic and invasion potential of the anti-cancer drug-sensitive Malme3M cells in CAGE-dependent manner. Studies revealed that $PKC{\delta}$ was responsible for the increased expression of $pEGFR^{Y845}$ and CAGE in $Malme3M^R$ cells. CAGE showed an interaction with $PKC{\delta}$ in $Malme3M^R$ cells. Our results show that HDAC3-CAGE axis can be employed as a target for overcoming resistance to EGFR inhibitors.


Supported by : National Research Foundation, Ministry for Health and Welfare, Kangwon National University


  1. Abera, M.B., and Kazanietz, M.G. (2015). Protein kinase C$\alpha$ mediates erlotinib resistance in lung cancer cells. Mol. Pharmacol. 87, 832-841.
  2. Barter, M.J., Pybus, L., Litherland, G.J., Rowan, A.D., Clark, I.M., Edwards, D.R., Cawston, T.E., and Young, D.A. (2010). HDACmediated control of ERK- and PI3K-dependent TGF-$\beta$-induced extracellular matrix-regulating genes. Matrix Biol. 29, 602-612.
  3. Bhaskara, S., Knutson, S.K., Jiang, G., Chandrasekharan, M.B., Wilson, A.J., Zheng, S., Yenamandra, A., Locke, K., Yuan, J.L., Bonine-Summers, A.R.,et al. (2010). Hdac3 is essential for the maintenance of chromatin structure and genome stability. Cancer Cell 18, 436-447.
  4. Boyer, A.P., Collier, T.S., Vidavsky, I., and Bose, R. (2013). Quantitative proteomics with siRNA screening identifies novel mechanisms of trastuzumab resistance in HER2 amplified breast cancers. Mol. Cell. Proteomics 12, 180-193.
  5. Brodie, S.A., Li, G., El-Kommos, A., Kang, H., Ramalingam, S.S., Behera, M., Gandhi, K., Kowalski, J., Sica, G.L., Khuri, F.R., Vertino, P.M., and Brandes, J.C. (2014). Class I HDACs are mediators of smoke carcinogen-induced stabilization of DNMT1 and serve as promising targets for chemoprevention of lung cancer. Cancer Prev. Res. (Phila). 7, 351-361.
  6. Cho, B., Lim, Y., Lee, D.Y., Park, S.Y., Lee, H., Kim, W.H., Yang, H., Bang, Y.J., and Jeoung, D.I. (2002). Identification and characterization of a novel cancer/testis antigen gene CAGE. Biochem. Biophys. Res. Commun. 292, 715-726.
  7. Cho, B., Lee, H., Jeong, S., Bang, Y.J., Lee, H.J., Hwang, K.S., Kim, H.Y., Lee, Y.S., Kang, G.H., and Jeoung, D.I. (2003). Promoter hypomethylation of a novel cancer/testis antigen gene CAGE is correlated with its aberrant expression and is seen in premalignant stage of gastric carcinoma. Biochem. Biophys. Res. Commun. 307, 52-63.
  8. Chou, C.W., Wu, M.S., Huang, W.C., and Chen, C.C. (2011). HDAC inhibition decreases the expression of EGFR in colorectal cancer cells. PLoS One 6, e18087.
  9. Dhar, S.S., Alam, H., Li, N., Wagner, K.W., Chung, J., Ahn, Y.W., and Lee MG. (2014). Transcriptional repression of histone deacetylase 3 by the histone demethylase KDM2A is coupled to tumorigenicity of lung cancer cells. J. Biol. Chem. 289, 7483-7496.
  10. Dong, P., Xu, Z., Jia, N., Li, D., and Feng, Y. (2009). Elevated expression of p53 gain-of-function mutation R175H in endometrial cancer cells can increase the invasive phenotypes by activation of the EGFR/PI3K/AKT pathway. Mol. Cancer 8, 103.
  11. El-Khoury, V., Breuzard, G., Fourre, N., and Dufer, J. (2007). The histone deacetylase inhibitor trichostatin A downregulates human MDR1 (ABCB1). gene expression by a transcriptiondependent mechanism in a drug-resistant small cell lung carcinoma cell line model. Br. J. Cancer 97, 562-573.
  12. Feng, L., Pan, M., Sun, J., Lu, H., Shen, Q., Zhang, S., Jiang, T., Liu, L., Jin, W., Chen, Y., et al. (2013). Histone deacetylase 3 inhibits expression of PUMA in gastric cancer cells. J. Mol. Med (Berl). 91, 49-58.
  13. Gao, P., Yang, X., Xue, Y.W., Zhang, X.F., Wang, Y., Liu, W.J., and Wu, X.J. (2009). Promoter methylation of glutathione Stransferase pi1 and multidrug resistance gene 1 in bronchioloalveolar carcinoma and its correlation with DNA methyltransferase 1 expression. Cancer 115, 3222-3232.
  14. Garcia-Recio, S., Pastor-Arroyo, E.M., Marin-Aguilera, M., Almendro, V., and Gascon, P. (2015). The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation. PLoS One 10, e0129661.
  15. Gilbert, R.E., Huang, Q., Thai, K., Advani, S.L., Lee, K., Yuen, D.A., Connelly, K.A., and Advani, A. (2011). Histone deacetylase inhibition attenuates diabetes-associated kidney growth: potential role for epigenetic modification of the epidermal growth factor receptor. Kidney Int. 79, 1312-1321.
  16. Huang, S., Benavente, S., Armstrong, E.A., Li, C., Wheeler, D.L., and Harari, P.M. (2011). p53 modulates acquired resistance to EGFR inhibitors and radiation. Cancer Res. 71, 7071-70709.
  17. Itamochi, H., Kato, M., Nishimura, M., Oishi, T., Shimada, M., Sato, S., Naniwa, J., Sato, S., Nonaka, M., Kudoh, A., et al. (2012). Establishment and characterization of a novel ovarian serous adenocarcinoma cell line, TU-OS-4, that overexpresses EGFR and HER2. Hum. Cell 25, 111-115.
  18. Iwata, T., Fujita, T., Hirao, N., Matsuzaki, Y., Okada, T., Mochimaru, H., Susumu, N., Matsumoto, E., Sugano, K., Yamashita, N., et al. (2005). Frequent immune responses to a cancer/testis antigen, CAGE, in patients with microsatellite instability-positive endometrial cancer. Clin. Cancer Res. 11, 3949-3957.
  19. Kim, Y., Park, H., Park, D., Lee, Y.S., Choe, J., Hahn, J.H., Lee, H., Kim, Y.M., and Jeoung, D. (2010). Cancer/testis antigen CAGE exerts negative regulation on p53 expression through HDAC2 and confers resistance to anti-cancer drugs. J. Biol. Chem. 285, 25957-25968.
  20. Kim, Y., Park, D., Kim, H., Choi, M., Lee, H., Lee, Y.S., Choe, J., Kim, Y.M., and Jeoung, D. (2013). miR-200b and cancer/testis antigen CAGE form a feedback loop to regulate the invasion and tumorigenic and angiogenic responses of a cancer cell line to microtubule-targeting drugs. J. Biol. Chem. 288, 36502-36518.
  21. Kim, Y., Kim, H., Park, H., Park, D., Lee, H., Lee, Y.S., Choe, J., Kim, Y.M., and Jeoung, D. (2014). miR-326-histone deacetylase-3 feedback loop regulates the invasion and tumorigenic and angiogenic response to anti-cancer drugs. J. Biol. Chem. 289, 28019-28039.
  22. Kim, Y., Kim, H., and Jeoung, D. (2015a). Tubulin beta3 serves as a target of HDAC3 and mediates resistance to microtubuletargeting drugs. Mol. Cells 38, 705-714.
  23. Kim, Y., Kim, H., Park, D., and Jeoung, D. (2015b). miR-335 targets SIAH2 and confers sensitivity to anti-cancer drugs by increasing the expression of HDAC3. Mol. Cells 38, 562-572.
  24. Kuang, Y.H., Shen, T., Chen, X., Sodani, K., Hopper-Borge, E., Tiwari, A.K., Lee, J.W., Fu, L.W., and Chen, Z.S. (2010). Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10).-mediated multidrug resistance. Biochem. Pharmacol. 79, 154-161.
  25. Kusne, Y., Carrera-Silva, E.A., Perry, A.S., Rushing, E.J., Mandell, E.K., Dietrich, J.D., Errasti, A.E., Gibbs, D., Berens, M.E., Loftus, J.C., et al. (2014). Targeting aPKC disables oncogenic signaling by both the EGFR and the proinflammatory cytokine TNF$\alpha$ in glioblastoma. Sci. Signal. 7, ra75.
  26. Lee, C.H., Hung, H.W., Hung, P.H., and Shieh, Y.S. (2010). Epidermal growth factor receptor regulates beta-catenin location, stability, and transcriptional activity in oral cancer. Mol. Cancer 9, 64.
  27. Li, Y., Wang, J., Gao, X., Han, W., Zheng, Y., Xu, H., Zhang, C., He, Q., Zhang, L., Li, Z., and Zhou, D. (2014). c-Met targeting enhances the effect of irradiation and chemical agents against malignant colon cells harboring a KRAS mutation. PLoS One 9, e113186.
  28. Liu, R., Gu, J., Jiang, P., Zheng, Y., Liu, X., Jiang, X., Huang, E., Xiong, S., Xu, F., Liu G, et al. (2015). DNMT1-MicroRNA126 Epigenetic Circuit Contributes to Esophageal Squamous Cell Carcinoma Growth via ADAM9-EGFR-AKT Signaling. Clin. Cancer Res. 21, 854-863.
  29. Longworth, M.S., and Laimins, L.A. (2006). Histone deacetylase 3 localizes to the plasma membrane and is a substrate of Src. Oncogene 25, 4495-4500.
  30. Lundh, M., Christensen, D.P., Damgaard Nielsen, M., Richardson, S.J., Dahllof, M.S., Skovgaard, T., Berthelsen, J., Dinarello, C.A., Stevenazzi, A., Mascagni, P., et al. (2012). Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children. Diabetologia 55, 2421-2431.
  31. Matteucci, E., Ridolfi, E., Maroni, P., Bendinelli, P., and Desiderio, M.A. (2007). c-Src/histone deacetylase 3 interaction is crucial for hepatocyte growth factor dependent decrease of CXCR4 expression in highly invasive breast tumor cells. Mol. Cancer Res. 5, 833-845.
  32. Milane, L., Duan, Z., and Amiji, M. (2011). Therapeutic efficacy and safety of paclitaxel/lonidamine loaded EGFR-targeted nanoparticles for the treatment of multi-drug resistant cancer. PLoS One 6, e24075.
  33. Park, D., Park, H., Kim, Y., Kim, H., and Jeoung, D. (2014a). HDAC3 acts as a negative regulator of angiogenesis. BMB Rep. 47,227-232.
  34. Park, H., Kim, Y., Park, D., and Jeoung D. (2014b). Nuclear localization signal domain of HDAC3 is necessary and sufficient for the expression regulation of MDR1. BMB Rep. 47, 342-347.
  35. Por, E., Byun, H.J., Lee, E.J., Lim, J.H., Jung, S.Y., Park, I., Kim, Y.M., and Jeoung, D.I, and Lee, H. (2010). The cancer/testis antigen CAGE with oncogenic potential stimulates cell proliferation by up-regulating cyclins D1 and E in an AP-1- and E2Fdependent manner. J. Biol. Chem. 285, 14475-14485.
  36. Robertson, E.D., Weir, L., Romanowska, M., Leigh, I.M., and Panteleyev, A.A. (2012). ARNT controls the expression of epidermal differentiation genes through HDAC- and EGFR-dependent pathways. J. Cell Sci. 125, 3320-3332.
  37. Samarakoon, R., Dobberfuhl, A.D., Cooley, C., Overstreet, J.M., Patel, S., Goldschmeding, R., Meldrum, K.K., and Higgins, P.J. (2013). Induction of renal fibrotic genes by TGF-$\beta$1 requires EGFR activation, p53 and reactive oxygen species.Cell Signal 25, 2198-2209.
  38. Shi, Z., Tiwari, A.K., Shukla, S., Robey, R.W., Kim, I.W., Parmar, S., Bates, S.E., Si, Q.S., Goldblatt, C.S., Abraham, I., et al. (2009). Inhibiting the function of ABCB1 and ABCG2 by the EGFR tyrosine kinase inhibitor AG1478. Biochem. Pharmacol. 77, 781-793.
  39. Steinway, S.N., Dang, H., You, H., Rountree, C.B., and Ding, W. (2015). The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma. PLoS One 10, e0128159.
  40. Suzuki, A., Sanda, N., Miyawaki, Y., Fujimori, Y., Yamada, T., Takagi, A., Murate, T., Saito, H., and Kojima, T. (2010). Down-regulation of PROS1 gene expression by 17beta-estradiol via estrogen receptor alpha (ERalpha).-Sp1 interaction recruiting receptorinteracting protein 140 and the corepressor-HDAC3 complex. J. Biol. Chem. 285, 13444-13453.
  41. Tang, J., Yan, Y., Zhao, T.C., Gong, R., Bayliss, G., Yan, H., and Zhuang, S. (2014). Class I HDAC activity is required for renal protection and regeneration after acute kidney injury. Am. J. Physiol. Renal Physiol. 307, 303-316.
  42. To, K.K., Polgar, O., Huff, L.M., Morisaki, K., and Bates, S.E. (2008). Histone modifications at the ABCG2 promoter following treatment with histone deacetylase inhibitor mirror those in multidrugresistant cells. Mol. Cancer Res. 6, 151-164.
  43. Van Cutsem, E., Kohne, C.H., Hitre, E., Zaluski, J., Chang Chien, C.R., Makhson, A., D'Haens, G., Pintér, T., Lim, R., Bodoky, G., et al. (2009). Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N. Engl. J. Med. 360, 1408-1417.
  44. Walther, A., Johnstone, E., Swanton, C., Midgley, R., Tomlinson, I., and Kerr, D. (2009). Genetic prognostic and predictive markers in colorectal cancer. Nature reviews Cancer 9, 489-499.
  45. Weichert, W., Roske, A., Gekeler, V., Beckers, T., Ebert, M.P., Pross, M., Dietel, M., Denkert, C., and Rocken, C. (2008a). Association of patterns of class I histone deacetylase expression with patient prognosis in gastric cancer: a retrospective analysis. Lancet Oncol. 9, 139-148.
  46. Weichert, W., Roske, A., Gekeler, V, Beckers T, Stephan C, Jung K, Fritzsche FR, Niesporek S, Denkert C, Dietel M, et al. (2008b). Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy. Br. J. Cancer 98, 604-610.
  47. Wilson, A. J., Byun, D. S., Popova, N., Murray, L.B., L'Italien, K., Sowa, Y., Arango, D., Velcich, A., Augenlicht, L.H., and Mariadason J. M. (2006). Histone deacetylase 3 (HDAC3). and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J. Biol. Chem. 281, 13548-13558
  48. Xiang, Y., Ma, N., Wang, D., Zhang, Y., Zhou, J., Wu, G., Zhao, R., Huang, H., Wang, X., Qiao, Y., et al. (2014). MiR-152 and miR-185 co-contribute to ovarian cancer cells cisplatin sensitivity by targeting DNMT1 directly: a novel epigenetic therapy independent of decitabine. Oncogene 33, 378-386.
  49. Xu, Y., Jiang, Z., Yin, P., Li, Q., and Liu, J. (2012). Role for Class I histone deacetylases in multidrug resistance. Exp. Cell Res. 318, 177-186.
  50. Yoon, S., Han, E., Choi, Y.C., Kee, H., Jeong, Y., Yoon, J., and Baek, K. (2014). Inhibition of cell proliferation and migration by miR-509-3p that targets CDK2, Rac1, and PIK3C2A. Mol. Cells 37, 314-321.
  51. Zhu, J., Shimizu, E., Zhang, X., Partridge, N.C., and Qin, L. (2011). EGFR signaling suppresses osteoblast differentiation and inhibits expression of master osteoblastic transcription factors Runx2 and Osterix. J. Cell. Biochem. 112, 1749-1760.

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