The Role of Nuclear Receptor Subfamily 1 Group H Member 4 (NR1H4) in Colon Cancer Cell Survival through the Regulation of c-Myc Stability

  • Lee, Yun Jeong (Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, National Cancer Center) ;
  • Lee, Eun-Young (Division of Translational Science, Research Institute, National Cancer Center) ;
  • Choi, Bo Hee (Division of Translational Science, Research Institute, National Cancer Center) ;
  • Jang, Hyonchol (Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, National Cancer Center) ;
  • Myung, Jae-Kyung (Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, National Cancer Center) ;
  • You, Hye Jin (Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, National Cancer Center)
  • Received : 2020.02.06
  • Accepted : 2020.03.25
  • Published : 2020.05.31


Nuclear receptor subfamily group H member 4 (NR1H4), also known as farnesoid X receptor, has been implicated in several cellular processes in the liver and intestine. Preclinical and clinical studies have suggested a role of NR1H4 in colon cancer development; however, how NR1H4 regulates colon cancer cell growth and survival remains unclear. We generated NR1H4 knockout (KO) colon cancer cells using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (CAS9) technology and explored the effects of NR1H4 KO in colon cancer cell proliferation, survival, and apoptosis. Interestingly, NR1H4 KO cells showed impaired cell proliferation, reduced colony formation, and increased apoptotic cell death compared to control colon cancer cells. We identified MYC as an important mediator of the signaling pathway alterations induced by NR1H4 KO. NR1H4 silencing in colon cancer cells resulted in reduced MYC protein levels, while NR1H4 activation using an NR1H4 ligand, chenodeoxycholic acid, resulted in time- and dose-dependent MYC induction. Moreover, NR1H4 KO enhanced the anti-cancer effects of doxorubicin and cisplatin, supporting the role of MYC in the enhanced apoptosis observed in NR1H4 KO cells. Taken together, our findings suggest that modulating NR1H4 activity in colon cancer cells might be a promising alternative approach to treat cancer using MYC-targeting agents.


  1. Altman, B.J., Hsieh, A.L., Sengupta, A., Krishnanaiah, S.Y., Stine, Z.E., Walton, Z.E., Gouw, A.M., Venkataraman, A., Li, B., Goraksha-Hicks, P., et al. (2015). MYC disrupts the circadian clock and metabolism in cancer cells. Cell Metab. 22, 1009-1019.
  2. Bailey, A.M., Zhan, L., Maru, D., Shureiqi, I., Pickering, C.R., Kiriakova, G., Izzo, J., He, N., Wei, C., Baladandayuthapani, V., et al. (2014). FXR silencing in human colon cancer by DNA methylation and KRAS signaling. Am. J. Physiol. Gastrointest. Liver Physiol. 306, G48-G58.
  3. Bonamy, C., Sechet, E., Amiot, A., Alam, A., Mourez, M., Fraisse, L., Sansonetti, P.J., and Sperandio, B. (2018). Expression of the human antimicrobial peptide beta-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells. Sci. Rep. 8, 18043.
  4. Cancer Genome Atlas Network. (2012). Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330-337.
  5. Cao, Z., Fan-Minogue, H., Bellovin, D.I., Yevtodiyenko, A., Arzeno, J., Yang, Q., Gambhir, S.S., and Felsher, D.W. (2011). MYC phosphorylation, activation, and tumorigenic potential in hepatocellular carcinoma are regulated by HMG-CoA reductase. Cancer Res. 71, 2286-2297.
  6. Chen, H., Liu, H., and Qing, G. (2018). Targeting oncogenic Myc as a strategy for cancer treatment. Signal Transduct. Target. Ther. 3, 5.
  7. Conacci-Sorrell, M., McFerrin, L., and Eisenman, R.N. (2014). An overview of MYC and its interactome. Cold Spring Harb. Perspect. Med. 4, a014357.
  8. Dang, C.V. (2012). MYC on the path to cancer. Cell 149, 22-35.
  9. Date, Y. and Ito, K. (2020). Oncogenic RUNX3: a link between p53 deficiency and MYC dysregulation. Mol. Cells 43, 176-181.
  10. de Aguiar Vallim, T.Q., Tarling, E.J., and Edwards, P.A. (2013). Pleiotropic roles of bile acids in metabolism. Cell Metab. 17, 657-669.
  11. DeBerardinis, R.J. and Chandel, N.S. (2016). Fundamentals of cancer metabolism. Sci. Adv. 2, e1600200.
  12. Degirolamo, C., Modica, S., Palasciano, G., and Moschetta, A. (2011). Bile acids and colon cancer: solving the puzzle with nuclear receptors. Trends Mol. Med. 17, 564-572.
  13. Frenzel, A., Zirath, H., Vita, M., Albihn, A., and Henriksson, M.A. (2011). Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression. PLoS One 6, e27988.
  14. Fu, T., Coulter, S., Yoshihara, E., Oh, T.G., Fang, S., Cayabyab, F., Zhu, Q., Zhang, T., Leblanc, M., Liu, S., et al. (2019). FXR regulates intestinal cancer stem cell proliferation. Cell 176, 1098-1112.e18.
  15. Garcia-Gutierrez, L., Delgado, M.D., and Leon, J. (2019). MYC oncogene contributions to release of cell cycle brakes. Genes (Basel) 10, 244.
  16. Gomez-Ospina, N., Potter, C.J., Xiao, R., Manickam, K., Kim, M.S., Kim, K.H., Shneider, B.L., Picarsic, J.L., Jacobson, T.A., Zhang, J., et al. (2016). Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis. Nat. Commun. 7, 10713.
  17. Guinney, J., Dienstmann, R., Wang, X., de Reynies, A., Schlicker, A., Soneson, C., Marisa, L., Roepman, P., Nyamundanda, G., Angelino, P., et al. (2015). The consensus molecular subtypes of colorectal cancer. Nat. Med. 21, 1350-1356.
  18. Houlston, R.S. (2001). What we could do now: molecular pathology of colorectal cancer. Mol. Pathol. 54, 206-214.
  19. Hsieh, A.L., Walton, Z.E., Altman, B.J., Stine, Z.E., and Dang, C.V. (2015). MYC and metabolism on the path to cancer. Semin. Cell Dev. Biol. 43, 11-21.
  20. Jo, M.J., Paek, A.R., Choi, J.S., Ok, C.Y., Jeong, K.C., Lim, J.H., Kim, S.H., and You, H.J. (2015). Regulation of cancer cell death by a novel compound, C604, in a c-Myc-overexpressing cellular environment. Eur. J. Pharmacol. 769, 257-265.
  21. Kazi, A., Xiang, S., Yang, H., Delitto, D., Trevino, J., Jiang, R.H.Y., Ayaz, M., Lawrence, H.R., Kennedy, P., and Sebti, S.M. (2018). GSK3 suppression upregulates beta-catenin and c-Myc to abrogate KRas-dependent tumors. Nat. Commun. 9, 5154.
  22. Klag, T., Thomas, M., Ehmann, D., Courth, L., Mailander-Sanchez, D., Weiss, T.S., Dayoub, R., Abshagen, K., Vollmar, B., Thasler, W.E., et al. (2018). Beta-defensin 1 is prominent in the liver and induced during cholestasis by bilirubin and bile acids via farnesoid X receptor and constitutive androstane receptor. Front. Immunol. 9, 1735.
  23. Kong, B., Zhu, Y., Li, G., Williams, J.A., Buckley, K., Tawfik, O., Luyendyk, J.P., and Guo, G.L. (2016). Mice with hepatocyte-specific FXR deficiency are resistant to spontaneous but susceptible to cholic acid-induced hepatocarcinogenesis. Am. J. Physiol. Gastrointest. Liver Physiol. 310, G295-G302.
  24. Kuipers, E.J., Grady, W.M., Lieberman, D., Seufferlein, T., Sung, J.J., Boelens, P.G., van de Velde, C.J., and Watanabe, T. (2015). Colorectal cancer. Nat. Rev. Dis. Primers 1, 15065.
  25. Lajczak, N.K., Saint-Criq, V., O'Dwyer, A.M., Perino, A., Adorini, L., Schoonjans, K., and Keely, S.J. (2017). Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human beta-defensin-1 and -2 secretion by colonic epithelial cells. FASEB J. 31, 3848-3857.
  26. Leonetti, C., Biroccio, A., Candiloro, A., Citro, G., Fornari, C., Mottolese, M., Del Bufalo, D., and Zupi, G. (1999). Increase of cisplatin sensitivity by c-myc antisense oligodeoxynucleotides in a human metastatic melanoma inherently resistant to cisplatin. Clin. Cancer Res. 5, 2588-2595.
  27. Luengo, A., Gui, D.Y., and Vander Heiden, M.G. (2017). Targeting metabolism for cancer therapy. Cell Chem. Biol. 24, 1161-1180.
  28. Maran, R.R., Thomas, A., Roth, M., Sheng, Z., Esterly, N., Pinson, D., Gao, X., Zhang, Y., Ganapathy, V., Gonzalez, F.J., et al. (2009). Farnesoid X receptor deficiency in mice leads to increased intestinal epithelial cell proliferation and tumor development. J. Pharmacol. Exp. Ther. 328, 469-477.
  29. Nagarajan, A., Malvi, P., and Wajapeyee, N. (2016). Oncogene-directed alterations in cancer cell metabolism. Trends Cancer 2, 365-377.
  30. Okita, A., Takahashi, S., Ouchi, K., Inoue, M., Watanabe, M., Endo, M., Honda, H., Yamada, Y., and Ishioka, C. (2018). Consensus molecular subtypes classification of colorectal cancer as a predictive factor for chemotherapeutic efficacy against metastatic colorectal cancer. Oncotarget 9, 18698-18711.
  31. Okuyama, H., Endo, H., Akashika, T., Kato, K., and Inoue, M. (2010). Downregulation of c-MYC protein levels contributes to cancer cell survival under dual deficiency of oxygen and glucose. Cancer Res. 70, 10213-10223.
  32. Ortmayr, K., Dubuis, S., and Zampieri, M. (2019). Metabolic profiling of cancer cells reveals genome-wide crosstalk between transcriptional regulators and metabolism. Nat. Commun. 10, 1841.
  33. Rahl, P.B., Lin, C.Y., Seila, A.C., Flynn, R.A., McCuine, S., Burge, C.B., Sharp, P.A., and Young, R.A. (2010). c-Myc regulates transcriptional pause release. Cell 141, 432-445.
  34. Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281-2308.
  35. Sarosiek, K.A., Fraser, C., Muthalagu, N., Bhola, P.D., Chang, W., McBrayer, S.K., Cantlon, A., Fisch, S., Golomb-Mello, G., Ryan, J.A., et al. (2017). Developmental regulation of mitochondrial apoptosis by c-Myc governs age- and tissue-specific sensitivity to cancer therapeutics. Cancer Cell 31, 142-156.
  36. Satoh, K., Yachida, S., Sugimoto, M., Oshima, M., Nakagawa, T., Akamoto, S., Tabata, S., Saitoh, K., Kato, K., Sato, S., et al. (2017). Global metabolic reprogramming of colorectal cancer occurs at adenoma stage and is induced by MYC. Proc. Natl. Acad. Sci. U. S. A. 114, E7697-E7706.
  37. Sears, R., Nuckolls, F., Haura, E., Taya, Y., Tamai, K., and Nevins, J.R. (2000). Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 14, 2501-2514.
  38. Smith, D.R., Myint, T., and Goh, H.S. (1993). Over-expression of the c-myc proto-oncogene in colorectal carcinoma. Br. J. Cancer 68, 407-413.
  39. Soucek, L., Whitfield, J., Martins, C.P., Finch, A.J., Murphy, D.J., Sodir, N.M., Karnezis, A.N., Swigart, L.B., Nasi, S., and Evan, G.I. (2008). Modelling Myc inhibition as a cancer therapy. Nature 455, 679-683.
  40. Stine, Z.E., Walton, Z.E., Altman, B.J., Hsieh, A.L., and Dang, C.V. (2015). MYC, metabolism, and cancer. Cancer Discov. 5, 1024-1039.
  41. Sveen, A., Bruun, J., Eide, P.W., Eilertsen, I.A., Ramirez, L., Murumagi, A., Arjama, M., Danielsen, S.A., Kryeziu, K., Elez, E., et al. (2018). Colorectal cancer consensus molecular subtypes translated to preclinical models uncover potentially targetable cancer cell dependencies. Clin. Cancer Res. 24, 794-806.
  42. Takahashi, S., Tanaka, N., Fukami, T., Xie, C., Yagai, T., Kim, D., Velenosi, T.J., Yan, T., Krausz, K.W., Levi, M., et al. (2018). Role of farnesoid X receptor and bile acids in hepatic tumor development. Hepatol. Commun. 2, 1567-1582.