Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Revealing the tumor suppressive sequence within KL1 domain of the hormone Klotho

Oncogene volume 43, pages 354–362 (2024)Cite this article

Subjects

Abstract

Klotho, a 1012 amino acid transmembrane protein, is a potent tumor suppressor in different cancer types. Klotho is composed of two internal repeats KL1 and KL2, and the tumor suppressor activity is primarily attributed to the KL1 domain. Despite its significant role in regulating various cancer-related pathways, the precise mechanism underlying its tumor suppressor activity remains unresolved. In this study, we aimed to identify the sequence responsible for the tumor suppressor function of Klotho and gain insights into its mechanism of action. To accomplish this, we generated expression vectors of truncated KL1 at the C and N-terminal regions and evaluated their ability to inhibit the colony formation of several cancer cell lines. Our findings demonstrated that truncated KL1 1–340 (KL340) effectively inhibited colony formation similar to KL1, while truncated KL1 1–320 (KL320) lost this activity. Furthermore, this correlated with the inhibitory effect of KL1 and KL340 on the Wnt/β-catenin pathway, whereas KL320 had no effect. Transcriptomic analysis of MCF-7 cells expressing the constructs revealed enriched pathways associated with tumor suppressor activity in KL1 and KL340. Interestingly, the α-fold predictor tool highlighted distinct differences in the α and β sheets of the TIM barrel fold of the truncated Klotho constructs, adding to our understanding of their structural variations. In summary, this study identified the 340 N-terminal amino acids as the sequence that possesses Klotho’s tumor suppressor activity and reveals a critical role in the 320–340 sequence for this function. It also provides a foundation for the development of Klotho-based therapeutic approaches for cancer treatment.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic illustration of Klotho constructs and their expression.
Fig. 2: KL340 inhibits colony formation of pancreatic, breast and colon cancer cells similar to KL1.
Fig. 3: KL340 effectively inhibits Wnt3a expression and signaling.
Fig. 4: Transcriptomic analysis reveals unique pathways shared by KL1 and KL340.
Fig. 5: Structural modeling and multiple sequence alignment highlight a role for 320–340 amino acids.

Similar content being viewed by others

Data availability

RNA-seq data were deposited in the NCBI sequence read archive (SRA) under accession number PRJNA1032761.

References

  1. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, et al. Mutation of the mouse klotho gene leads to a syndrome resembling aging. Nature. 1997;390:45–51. https://doi.org/10.1038/36285.

  2. Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, et al. Suppression of aging in mice by the hormone Klotho. Science. 2005;309:1829–33.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Nabeshima YI. Klotho: a fundamental regulator of aging. Ageing Res Rev. 2002;1:627–38.

    Article  PubMed  CAS  Google Scholar 

  4. Kim J-H, Hwang K-H, Park K-S, Kong ID, Cha S-K. Biological role of anti-aging protein Klotho. J Lifestyle Med. 2015;5:1–6. https://doi.org/10.15280/jlm.2015.5.1.1.

  5. Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, et al. Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane. FEBS Lett. 2004;565:143–7.

    PubMed  CAS  Google Scholar 

  6. Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-O M, Nabeshima YI. Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem Biophys Res Commun. 1998;242:626–30. https://doi.org/10.1006/bbrc.1997.8019.

  7. Liu H, Fergusson MM, Castilho RM, Liu J, Cao L, Chen J, et al. Augmented Wnt signaling in a mammalian model of accelerated aging. Science. 2007;317:803–6. https://doi.org/10.1126/science.1143578.

  8. Chen CD, Podvin S, Gillespie E, Leeman SE, Abraham CR. Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17. Proc Natl Acad Sci USA. 2007;104:19796–801. https://doi.org/10.1073/pnas.0709805104.

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Chen G, Liu Y, Goetz R, Fu L, Jayaraman S, Hu MC, et al. α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling. Nature. 2018;553:461–6. https://doi.org/10.1038/nature25451.

  11. Abramovitz L, Rubinek T, Ligumsky H, Bose S, Barshack I, Avivi C, et al. KL1 internal repeat mediates klotho tumor suppressor activities and inhibits bFGF and IGF-I signaling in pancreatic cancer. Clin Cancer Res. 2011;17:4254–66. https://doi.org/10.1158/1078-0432.CCR-10-2749.

  12. Cha S-K, Hu M-C, Kurosu H, Kuro-o M, Moe O, Huang C-L. Regulation of renal outer medullary potassium channel and renal K(+) excretion by Klotho. Mol Pharm. 2009;76:38–46.

    Article  CAS  Google Scholar 

  13. Cha SK, Ortega B, Kurosu H, Rosenblatt KP, Kuro-o M, Huang CL. Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1. Proc Natl Acad Sci USA. 2008;105:9805–10. https://doi.org/10.1073/pnas.0803223105.

  14. Chang Q, Hoefs S, van der Kemp AW, Topala CN, Bindels RJ, Hoenderop JG. The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science. 2005;310:490–3.

    Article  PubMed  CAS  Google Scholar 

  15. Ligumsky H, Rubinek T, Merenbakh-Lamin K, Yeheskel A, Sertchook R, Shahmoon S, et al. Tumor suppressor activity of klotho in breast cancer is revealed by structure-function analysis. Mol Cancer Res. 2015;13:1398–407. https://doi.org/10.1158/1541-7786.MCR-15-0141.

  16. Rubinek T, Wolf I. The role of Alpha-Klotho as a universal tumor suppressor. Vitam Horm. 2016;101:197–214. https://doi.org/10.1016/bs.vh.2016.03.001.

  17. Imura A, Tsuji Y, Murata M, Maeda R, Kubota K, Iwano A, et al. Α-Klotho as a regulator of calcium homeostasis. Science. 2007;316:1615–8.

    Article  PubMed  CAS  Google Scholar 

  18. Shmulevich R, Nissim TB-K, Wolf I, Merenbakh-Lamin K, Fishman D, Sekler I, et al. Klotho rewires cellular metabolism of breast cancer cells through alteration of calcium shuttling and mitochondrial activity. Oncogene. 2020;39:4636–49.

    Article  PubMed  CAS  Google Scholar 

  19. Wolf I, Levanon-Cohen S, Bose S, Ligumsky H, Sredni B, Kanety H, et al. Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer. Oncogene. 2008;27:7094–105. https://doi.org/10.1038/onc.2008.292.

  20. Liu F, Wu S, Ren H, Gu J. Klotho suppresses RIG-I-mediated senescence-associated inflammation. Nat Cell Biol. 2011;13:254–62. https://doi.org/10.1038/ncb2167.

  21. Chen B, Ma X, Liu S, Zhao W, Wu J. Inhibition of lung cancer cells growth, motility and induction of apoptosis by Klotho, a novel secreted Wnt antagonist, in a dose-dependent manner. Cancer Biol Ther. 2012;13:1221–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. 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;29:1–7.

    Article  Google Scholar 

  23. Chen X, Tan H, Xu J, Tian Y, Yuan Q, Zuo Y, et al. Klotho-derived peptide 6 ameliorates diabetic kidney disease by targeting Wnt/β-catenin signaling. Kidney Int. 2022;102:506–20.

    Article  PubMed  CAS  Google Scholar 

  24. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461–73.

    Article  PubMed  CAS  Google Scholar 

  25. Ligumsky H, Merenbakh-Lamin K, Keren-Khadmy N, Wolf I, Rubinek T. The role of α-klotho in human cancer: molecular and clinical aspects. Oncogene. 2022;41:4487–97.

    Article  PubMed  CAS  Google Scholar 

  26. Tang X, Wang Y, Fan Z, Ji G, Wang M, Lin J, et al. Klotho: a tumor suppressor and modulator of the Wnt/β-catenin pathway in human hepatocellular carcinoma. Lab Investig. 2016;96:197–205.

    Article  PubMed  CAS  Google Scholar 

  27. Arbel Rubinstein T, Shahmoon S, Zigmond E, Etan T, Merenbakh-Lamin K, Pasmanik-Chor M, et al. Klotho suppresses colorectal cancer through modulation of the unfolded protein response. Oncogene. 2019;38:794–807.

    Article  PubMed  CAS  Google Scholar 

  28. Khorsandi L, Orazizadeh M, Niazvand F, Abbaspour MR, Mansouri E, Khodadadi A. Quercetin induces apoptosis and necroptosis in MCF-7 breast cancer cells. Bratisl Lek Listy. 2017;118:123–8.

    PubMed  CAS  Google Scholar 

  29. Gong Y, Fan Z, Luo G, Yang C, Huang Q, Fan K, et al. The role of necroptosis in cancer biology and therapy. Mol Cancer. 2019;18:1–17.

    Article  CAS  Google Scholar 

  30. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell. 2003;114:181–90.

    Article  PubMed  CAS  Google Scholar 

  31. Izquierdo MC, Perez-Gomez MV, Sanchez-Niño MD, Sanz AB, Ruiz-Andres O, Poveda J, et al. Klotho, phosphate and inflammation/ageing in chronic kidney disease. Nephrol Dial Transplant. 2012;27:6–10.

    Article  Google Scholar 

  32. Martin M. CUTADAPT removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17. https://doi.org/10.14806/ej.17.1.200.

  33. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.

    Article  PubMed  CAS  Google Scholar 

  34. Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J, Wilm A, et al. The nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol. 2020;38:276–8.

    Article  PubMed  CAS  Google Scholar 

  35. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14:417–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32:3047–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21.

    Article  Google Scholar 

  38. Yu G, Wang LG, Han Y, He QY. ClusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods. 2022;19:679–82.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Madeira F, Pearce M, Tivey ARN, Basutkar P, Lee J, Edbali O, et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 2022;50:W276–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014;42:320–4.

    Article  Google Scholar 

  43. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 2010;38:695–9.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Oncology Division, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

    Marana Abboud, Keren Merenbakh-Lamin, Shira Ben-Neriah, Hagai Ligumsky, Sarai Bronfeld, Noa Keren-Khadmy, Ido Wolf & Tami Rubinek

  2. Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Marana Abboud, Hadas Volkov, Sarai Bronfeld, Moshe Giladi, Noam Shomron, Ido Wolf & Tami Rubinek

  3. Edmond J. Safra Center for Bioinformatics at Tel-Aviv University, Tel Aviv, Israel

    Hadas Volkov & Noam Shomron

  4. Internal Medicine Division, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

    Moshe Giladi

Authors
  1. Marana Abboud
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Keren Merenbakh-Lamin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hadas Volkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shira Ben-Neriah
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hagai Ligumsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sarai Bronfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Noa Keren-Khadmy
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Moshe Giladi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Noam Shomron
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ido Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Tami Rubinek
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

IW, TR and HL conceptualized the project. IW, TR and KM-L supervised the project. IW, TR, KM-L and MA designed the experiments.

Corresponding authors

Correspondence to Marana Abboud or Tami Rubinek.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Abboud, M., Merenbakh-Lamin, K., Volkov, H. et al. Revealing the tumor suppressive sequence within KL1 domain of the hormone Klotho. Oncogene 43, 354–362 (2024). https://doi.org/10.1038/s41388-023-02904-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-023-02904-2

Search

Advanced search

Quick links