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Enhancement of Apo2L/TRAIL signaling pathway receptors by the activation of Klotho gene with CRISPR/Cas9 in Caco-2 colon cancer cells


Human Klotho gene has many known functions such as anti-aging and anti-tumor, and decreased expression of this gene causes malignant formations in most types of cancer, including colon cancer. Interacting with TRAIL death receptors (DR4 and DR5) induces an apoptotic effect in cancer treatments by reducing the proliferation of cancer cells. The present study aimed to investigate downstream effect of overexpression of Klotho gene, which is known to have an antitumor effect on resistant human colon cancer cells, by examining its action on TRAIL death and decoy (DcR1 and DcR2) receptors for the first time. For this purpose, upregulation of human Klotho gene was achieved with CRISPR/Cas9-mediated system in resistant human colon cancer Caco-2 cells. To determine the effect of upregulation of Klotho gene on cancer cells evaluations with flow cytometry, WST-8, qRT-PCR, ELISA, and immunohistochemical analysis were performed. Then, Klotho gene was knocked out and its apoptotic effect was tested to find out whether it is due to overexpression of Klotho gene or not. Our results indicate that overexpression of Klotho gene in Caco-2 cells via CRISPR/Cas9-sensitized TRAIL death receptor DR4 suppresses the proliferation of cells by leading to apoptosis. Thus, this study conducted on apoptosis-resistant colon cancer cells may bring new insights about the role of Klotho gene in colon cancer.

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  1. 1.

    Li X-X, Huang L-Y, Peng J-J, Liang L, Shi D-B, Zheng H-T, Cai S-J. Klotho suppresses growth and invasion of colon cancer cells through inhibition of IGF1R-mediated PI3K/AKT pathway. Int J Oncol. 2014;45(2):611–8.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Tang X, Wang Y, Fan Z, Ji G, Wang M, Lin J, Huang S. Klotho: a tumor suppressor and modulator of the Wnt/β-catenin pathway in human hepatocellular carcinoma. Lab Invest. 2016;96(2):197–205.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Pan J, Zhong J, Gan LH, Chen SJ, Jin HC, Wang X, Wang L. Klotho, an anti-senescence related gene, is frequently inactivated through promoter hypermethylation in colorectal cancer. Tumour Biol. 2011;32(4):729–35.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Shu G, Xie B, Ren F, Liu D-C, Zhou J, Li Q, Chen J, Yuan L, Zhou J. Restoration of klotho expression induces apoptosis and autophagy in hepatocellular carcinoma cells. Cell Oncol. 2013;6(2):121–9.

    CAS  Article  Google Scholar 

  5. 5.

    Wang Y, Chen L, Huang G, He D, He J, Xu W, Zou C, Zong F, Li Y, Chen B. Klotho sensitizes human lung cancer cell line to cisplatin via PI3k/Akt pathway. PLoS ONE. 2013;8(2): e57391.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Ibi T, Usuda J, Inoue T, Sato A, Takegahara K. Klotho expression is correlated to molecules associated with epithelial-mesenchymal transition in lung squamous cell carcinoma. Oncol Lett. 2017;14(5):5526–32.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Wolf I, Levanon-Cohen S, Bose S, Ligumsky H, Sredni B, Kanety H, Kuro-o M, Karlan B, Kaufman B, Koeffler H. Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer. Oncogene. 2008;27(56):7094–105.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Chen B, Wang X, Zhao W, Wu J. Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549. J Exp Clin Cancer Res. 2010;29(1):1–7.

    CAS  Article  Google Scholar 

  9. 9.

    Zhu Y, Xu L, Zhang J, Xu W, Liu Y, Yin H, Lv T, An H, Liu L, He H. Klotho suppresses tumor progression via inhibiting PI 3 K/A kt/GSK 3β/Snail signaling in renal cell carcinoma. Cancer Sci. 2013;104(6):663–71.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006;444(7120):770–4.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Razzaque MS. The FGF23–Klotho axis: endocrine regulation of phosphate homeostasis. Nat Rev Endocrinol. 2009;5(11):611–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Kuro-o M. Klotho. Pflüg Arch Eur J Physiol. 2010;459(2):333–43.

    CAS  Article  Google Scholar 

  13. 13.

    Xuan NT, Van Hai N. Changes in expression of klotho affect physiological processes, diseases, and cancer. Iran J Basic Med Sci. 2018;21(1):3.

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Behera R, Kaur A, Webster MR, Kim S, Ndoye A, Kugel CH, Alicea GM, Wang J, Ghosh K, Cheng P. Inhibition of age-related therapy resistance in melanoma by rosiglitazone-mediated induction of Klotho. Clin Cancer Res. 2017;23(12):3181–90.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Ligumsky H, Rubinek T, Merenbakh-Lamin K, Yeheskel A, Sertchook R, Shahmoon S, Aviel-Ronen S, Wolf I. Tumor suppressor activity of Klotho in breast cancer is revealed by structure–function analysis. Mol Cancer Res. 2015;13(10):1398–407.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Zhou X, Fang X, Jiang Y, Geng L, Li X, Li Y, Lu K, Li P, Lv X, Wang X. Klotho, an anti-aging gene, acts as a tumor suppressor and inhibitor of IGF-1R signaling in diffuse large B cell lymphoma. J Hematol Oncol. 2017;10(1):1–11.

    CAS  Article  Google Scholar 

  17. 17.

    Camilli TC, Xu M, O’Connell MP, Chien B, Frank BP, Subaran S, Indig FE, Morin PJ, Hewitt SM, Weeraratna AT. Loss of Klotho during melanoma progression leads to increased filamin cleavage, increased Wnt5A expression, and enhanced melanoma cell motility. Pigment Cell Melanoma Res. 2011;24(1):175–86.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    de Miguel D, Lemke J, Anel A, Walczak H, Martinez-Lostao L. Onto better TRAILs for cancer treatment. Cell Death Differ. 2016;23(5):733–47.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem. 1996;271(22):12687–90.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Kretz A-L, Von Karstedt S, Hillenbrand A, Henne-Bruns D, Knippschild U, Trauzold A, Lemke J. Should we keep walking along the trail for pancreatic cancer treatment? Revisiting TNF-related apoptosis-inducing ligand for anticancer therapy. Cancers. 2018;10(3):77.

    CAS  Article  PubMed Central  Google Scholar 

  21. 21.

    Zhang B, Liu B, Chen D, Setroikromo R, Haisma HJ, Quax W. Histone deacetylase inhibitors sensitize TRAIL-induced apoptosis in colon cancer cells. Cancers. 2019;11(5):645.

    CAS  Article  PubMed Central  Google Scholar 

  22. 22.

    Zhang Z, Li Z, Wu X, Zhang C-F, Calway T, He T-C, Du W, Chen J, Wang C-Z, Yuan C-S. TRAIL pathway is associated with inhibition of colon cancer by protopanaxadiol. J Pharmacol Sci. 2015;127(1):83–91.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Segawa K, Kurata S, Yanagihashi Y, Brummelkamp TR, Matsuda F, Nagata S. Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure. Science. 2014;344(6188):1164–8.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Dai X, Zhang J, Arfuso F, Chinnathambi A, Zayed M, Alharbi SA, Kumar AP, Ahn KS, Sethi G. Targeting TNF-related apoptosis-inducing ligand (TRAIL) receptor by natural products as a potential therapeutic approach for cancer therapy. Exp Biol Med (Maywood). 2015;240(6):760–73.

    CAS  Article  Google Scholar 

  25. 25.

    Nahacka Z, Svadlenka J, Peterka M, Ksandrova M, Benesova S, Neuzil J, Andera L. TRAIL induces apoptosis but not necroptosis in colorectal and pancreatic cancer cells preferentially via the TRAIL-R2/DR5 receptor. Biochim Biophys Acta Mol Cell Res. 2018;1865(3):522–31.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Thamkachy R, Kumar R, Rajasekharan K, Sengupta S. ERK mediated upregulation of death receptor 5 overcomes the lack of p53 functionality in the diaminothiazole DAT1 induced apoptosis in colon cancer models: efficiency of DAT1 in Ras-Raf mutated cells. Cancer Commun (Lond). 2018;38(1):43.

    Article  Google Scholar 

  27. 27.

    Qiao X, Wang X, Shang Y, Li Y, Chen S-Z. Azithromycin enhances anticancer activity of TRAIL by inhibiting autophagy and up-regulating the protein levels of DR4/5 in colon cancer cells in vitro and in vivo. Cancer Commun (Lond). 2018;38(1):43.

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Gravett AM, Dalgleish AG, Copier J. In vitro culture with gemcitabine augments death receptor and NKG2D ligand expression on tumour cells. Sci Rep. 2019;9(1):1–9.

    CAS  Article  Google Scholar 

  29. 29.

    Lee J, Jeong D-J, Kim J, Lee S, Park J-H, Chang B, Jung S-I, Yi L, Han Y, Yang Y. The anti-aging gene KLOTHO is a novel target for epigenetic silencing in human cervical carcinoma. Mol Cancer. 2010;9(1):1–10.

    CAS  Article  Google Scholar 

  30. 30.

    Zhou Y, Zheng S, Luo Q, Huang X, Li Z. Hypermethylation of DcR1, DcR2, DR4, DR5 gene promoters and clinical significance in tongue carcinoma. Am J Otolaryngol. 2019;40(6): 102258.​2019.07.002.

    Article  PubMed  Google Scholar 

  31. 31.

    Gura T. How TRAIL kills cancer cells, but not normal cells. Science. 1997;277(5327):768–768.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Hersey P, Zhang XD. How melanoma cells evade trail-induced apoptosis. Nat Rev Cancer. 2001;1(2):142–50.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Kim K, Fisher MJ, Xu S-Q, El-Deiry WS. Molecular determinants of response to TRAIL in killing of normal and cancer cells. Cancer Res. 2000;6(2):335–46.

    CAS  Google Scholar 

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This study was supported by Eskişehir Osmangazi University, Scientific Research Projects (ESOGU-BAP, ESTEM project code: 2020/46014). The funders had no role in design of the study; in collection, analyses, or interpretation of data; in writing of the manuscript; or in decision to publish the results.

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Idea/concept: SG, OU, AES; design: OU; control/supervision: AES, TS S; literature review: SG, MNS, OU; writing the article: SG, OU, AES, MNS; critical review: AES, OU, SG. All authors have read and approved the final manuscript.

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Correspondence to Sibel Gunes.

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Gunes, S., Soykan, M.N., Sariboyaci, A.E. et al. Enhancement of Apo2L/TRAIL signaling pathway receptors by the activation of Klotho gene with CRISPR/Cas9 in Caco-2 colon cancer cells. Med Oncol 38, 146 (2021).

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  • Klotho
  • Colon cancer
  • Caco-2 cells
  • CRISPR/Cas9
  • TRAIL death receptor