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:

Pentapeptide PYRAE triggers ER stress-mediated apoptosis of breast cancer cells in mice by targeting RHBDF1-BiP interaction

Acta Pharmacologica Sinica volume 45, pages 378–390 (2024)Cite this article

Abstract

Reinforced cellular responses to endoplasmic reticulum (ER) stress are caused by a variety of pathological conditions including cancers. Human rhomboid family-1 protein (RHBDF1), a multiple transmembrane protein located mainly on the ER, has been shown to promote cancer development, while the binding immunoglobulin protein (BiP) is a key regulator of cellular unfolded protein response (UPR) for the maintenance of ER protein homeostasis. In this study, we investigated the role of RHBDF1 in maintaining ER protein homeostasis in breast cancer cells. We showed that deleting or silencing RHBDF1 in breast cancer cell lines MCF-7 and MDA-MB-231 caused marked aggregation of unfolded proteins in proximity to the ER. We demonstrated that RHBDF1 directly interacted with BiP, and this interaction had a stabilizing effect on the BiP protein. Based on the primary structural motifs of RHBDF1 involved in BiP binding, we found a pentapeptide (PE5) targeted BiP and inhibited BiP ATPase activity. SPR assay revealed a binding affinity of PE5 toward BiP (Kd = 57.7 μM). PE5 (50, 100, 200 μM) dose-dependently promoted ER protein aggregation and ER stress-mediated cell apoptosis in MCF-7 and MDA-MB-231 cells. In mouse 4T1 breast cancer xenograft model, injection of PE5 (10 mg/kg, s.c., every 2 days for 2 weeks) significantly inhibited the tumor growth with markedly increased ER stress and apoptosis-related proteins in tumor tissues. Our results suggest that the ability of RHBDF1 to maintain BiP protein stability is critical to ER protein homeostasis in breast cancer cells, and that the pentapeptide PE5 may serve as a scaffold for the development of a new class of anti-BiP inhibitors.

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: Loss of RHBDF1 causes ER protein aggregation in breast cancer cells.
Fig. 2: RHBDF1 is critical for maintaining BiP protein stability in breast cancer cells.
Fig. 3: RHBDF1 directly interacts with the N-terminal domain of BiP at its multitransmembrane domain.
Fig. 4: PE5 targets BiP to inhibit its ATPase activity.
Fig. 5: PE5 induces ER protein aggregation and ER stress-mediated apoptosis in breast cancer cells.
Fig. 6: PE5 inhibits breast cancer development in mice.
Fig. 7: Schematic representation depicting RHBDF1-BiP interaction in the ER.

Similar content being viewed by others

Data availability

All data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011;334:1081–6.

    Article  PubMed  CAS  Google Scholar 

  2. Wang M, Kaufman RJ. The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat Rev Cancer. 2014;14:581–97.

    Article  PubMed  CAS  Google Scholar 

  3. Harris IS, Endress JE, Coloff JL, Selfors LM, McBrayer SK, Rosenbluth JM, et al. Deubiquitinases maintain protein homeostasis and survival of cancer cells upon glutathione depletion. Cell Metab. 2019;29:1166–81.e6

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Zheng S, Wang X, Liu H, Zhao D, Lin Q, Jiang Q, et al. iASPP suppression mediates terminal UPR and improves BRAF-inhibitor sensitivity of colon cancers. Cell Death Differ. 2023;30:327–40.

    Article  PubMed  CAS  Google Scholar 

  5. Chen X, Cubillos-Ruiz JR. Endoplasmic reticulum stress signals in the tumour and its microenvironment. Nat Rev Cancer. 2021;21:71–88.

    Article  PubMed  CAS  Google Scholar 

  6. Ghemrawi R, Khair M. Endoplasmic reticulum stress and unfolded protein response in neurodegenerative diseases. Int J Mol Sci. 2020;21:6127.

  7. Marciniak SJ, Chambers JE, Ron D. Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov. 2022;21:115–40.

    Article  PubMed  CAS  Google Scholar 

  8. Cubillos-Ruiz JR, Bettigole SE, Glimcher LH. Tumorigenic and immunosuppressive effects of endoplasmic reticulum stress in cancer. Cell. 2017;168:692–706.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Long F, Yang D, Wang J, Wang Q, Ni T, Wei G, et al. SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation. Acta Pharm Sin B. 2021;11:1261–73.

    Article  PubMed  CAS  Google Scholar 

  10. Wang J, Lee J, Liem D, Ping P. HSPA5 Gene encoding Hsp70 chaperone BiP in the endoplasmic reticulum. Gene. 2017;618:14–23.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000;2:326–32.

    Article  PubMed  CAS  Google Scholar 

  12. Lee AS. GRP78 induction in cancer: therapeutic and prognostic implications. Cancer Res. 2007;67:3496–9.

    Article  PubMed  CAS  Google Scholar 

  13. Lee AS. Glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential. Nat Rev Cancer. 2014;14:263–76.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Pobre KFR, Poet GJ, Hendershot LM. The endoplasmic reticulum (ER) chaperone BiP is a master regulator of ER functions: Getting by with a little help from ERdj friends. J Biol Chem. 2019;294:2098–108.

    Article  PubMed  CAS  Google Scholar 

  15. Zhu S, Zhang Q, Sun X, Zeh HJ 3rd, Lotze MT, Kang R, et al. HSPA5 regulates ferroptotic cell death in cancer cells. Cancer Res. 2017;77:2064–77.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Wu Z, Xu Z, Zhou X, Li H, Zhao L, Lv Y, et al. sGRP78 enhances selective autophagy of monomeric TLR4 to regulate myeloid cell death. Cell Death Dis. 2022;13:587.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Cerezo M, Benhida R, Rocchi S. Targeting BIP to induce endoplasmic reticulum stress and cancer cell death. Oncoscience. 2016;3:306–7.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Yang W, Xiu Z, He Y, Huang W, Li Y, Sun T. Bip inhibition in glioma stem cells promotes radiation-induced immunogenic cell death. Cell Death Dis. 2020;11:786.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Kung HC, Yu J. Targeted therapy for pancreatic ductal adenocarcinoma: mechanisms and clinical study. MedComm (2020). 2023;4:e216.

    PubMed  CAS  Google Scholar 

  20. Gifford JB, Hill R. GRP78 influences chemoresistance and prognosis in cancer. Curr Drug Targets. 2018;19:701–8.

    Article  PubMed  CAS  Google Scholar 

  21. Kwon D, Koh J, Kim S, Go H, Min HS, Kim YA, et al. Overexpression of endoplasmic reticulum stress-related proteins, XBP1s and GRP78, predicts poor prognosis in pulmonary adenocarcinoma. Lung Cancer. 2018;122:131–7.

    Article  PubMed  Google Scholar 

  22. Cerezo M, Lehraiki A, Millet A, Rouaud F, Plaisant M, Jaune E, et al. Compounds triggering ER stress exert anti-melanoma effects and overcome BRAF inhibitor resistance. Cancer Cell. 2016;29:805–19.

    Article  PubMed  CAS  Google Scholar 

  23. Yan Z, Zou H, Tian F, Grandis JR, Mixson AJ, Lu PY, et al. Human rhomboid family-1 gene silencing causes apoptosis or autophagy to epithelial cancer cells and inhibits xenograft tumor growth. Mol Cancer Ther. 2008;7:1355–64.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Urban S, Lee JR, Freeman M. Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell. 2001;107:173–82.

    Article  PubMed  CAS  Google Scholar 

  25. Zettl M, Adrain C, Strisovsky K, Lastun V, Freeman M. Rhomboid family pseudoproteases use the ER quality control machinery to regulate intercellular signaling. Cell. 2011;145:79–91.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Dulloo I, Muliyil S, Freeman M. The molecular, cellular and pathophysiological roles of iRhom pseudoproteases. Open Biol. 2019;9:190003.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Freeman M. Rhomboids, signalling and cell biology. Biochem Soc Trans. 2016;44:945–50.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Dusterhoft S, Babendreyer A, Giese AA, Flasshove C, Ludwig A. Status update on iRhom and ADAM17: it’s still complicated. Biochim Biophys Acta Mol Cell Res. 2019;1866:1567–83.

    Article  PubMed  Google Scholar 

  29. Zhou Z, Liu F, Zhang ZS, Shu F, Zheng Y, Fu L, et al. Human rhomboid family-1 suppresses oxygen-independent degradation of hypoxia-inducible factor-1alpha in breast cancer. Cancer Res. 2014;74:2719–30.

    Article  PubMed  CAS  Google Scholar 

  30. Xu Q, Chen C, Liu B, Lin Y, Zheng P, Zhou D, et al. Association of iRhom1 and iRhom2 expression with prognosis in patients with cervical cancer and possible signaling pathways. Oncol Rep. 2020;43:41–54.

    PubMed  CAS  Google Scholar 

  31. Song W, Liu W, Zhao H, Li S, Guan X, Ying J, et al. Rhomboid domain containing 1 promotes colorectal cancer growth through activation of the EGFR signalling pathway. Nat Commun. 2015;6:8022.

    Article  PubMed  CAS  Google Scholar 

  32. Li X, Maretzky T, Weskamp G, Monette S, Qing X, Issuree PD, et al. iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling. Proc Natl Acad Sci USA. 2015;112:6080–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Zhao Y, Davila EM, Li X, Tang B, Rabinowitsch AI, Perez-Aguilar JM, et al. Identification of molecular determinants in iRhoms1 and 2 that contribute to the substrate selectivity of stimulated ADAM17. Int J Mol Sci. 2022;23:12796.

  34. Yuan H, Wei R, Xiao Y, Song Y, Wang J, Yu H, et al. RHBDF1 regulates APC-mediated stimulation of the epithelial-to-mesenchymal transition and proliferation of colorectal cancer cells in part via the Wnt/beta-catenin signalling pathway. Exp Cell Res. 2018;368:24–36.

    Article  PubMed  CAS  Google Scholar 

  35. Li J, Bai TR, Gao S, Zhou Z, Peng XM, Zhang LS, et al. Human rhomboid family-1 modulates clathrin coated vesicle-dependent pro-transforming growth factor alpha membrane trafficking to promote breast cancer progression. EBioMedicine. 2018;36:229–40.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Gao S, Zhang LS, Wang L, Xiao NN, Long H, Yin YL, et al. RHBDF1 promotes AP-1-activated endothelial-mesenchymal transition in tumor fibrotic stroma formation. Signal Transduct Target Ther. 2021;6:273.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Peng XM, Gao S, Deng HT, Cai HX, Zhou Z, Xiang R, et al. Perturbation of epithelial apicobasal polarity by rhomboid family-1 gene overexpression. FASEB J. 2018;32:5577–86.

    Article  PubMed  CAS  Google Scholar 

  38. Oikonomidi I, Burbridge E, Cavadas M, Sullivan G, Collis B, Naegele H, et al. iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE. Elife. 2018;7:e35032.

  39. Mahoney DJ, Lefebvre C, Allan K, Brun J, Sanaei CA, Baird S, et al. Virus-tumor interactome screen reveals ER stress response can reprogram resistant cancers for oncolytic virus-triggered caspase-2 cell death. Cancer Cell. 2011;20:443–56.

    Article  PubMed  CAS  Google Scholar 

  40. Voronin MV, Abramova EV, Verbovaya ER, Vakhitova YV, Seredenin SB. Chaperone-dependent mechanisms as a pharmacological target for neuroprotection. Int J Mol Sci. 2023;24:823.

  41. Du T, Li H, Fan Y, Yuan L, Guo X, Zhu Q, et al. The deubiquitylase OTUD3 stabilizes GRP78 and promotes lung tumorigenesis. Nat Commun. 2019;10:2914.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Chang YW, Tseng CF, Wang MY, Chang WC, Lee CC, Chen LT, et al. Deacetylation of HSPA5 by HDAC6 leads to GP78-mediated HSPA5 ubiquitination at K447 and suppresses metastasis of breast cancer. Oncogene. 2016;35:1517–28.

    Article  PubMed  CAS  Google Scholar 

  43. Kahveci-Turkoz S, Blasius K, Wozniak J, Rinkens C, Seifert A, Kasparek P, et al. A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity. Cell Mol Life Sci. 2023;80:135.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Dusterhoft S, Kahveci-Turkoz S, Wozniak J, Seifert A, Kasparek P, Ohm H, et al. The iRhom homology domain is indispensable for ADAM17-mediated TNFalpha and EGF receptor ligand release. Cell Mol Life Sci. 2021;78:5015–40.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Ticha A, Collis B, Strisovsky K. The Rhomboid Superfamily: structural mechanisms and chemical biology opportunities. Trends Biochem Sci. 2018;43:726–39.

    Article  PubMed  CAS  Google Scholar 

  46. Luo H, Wang L, Zhang D, Sun Y, Wang S, Song S, et al. HA15 inhibits binding immunoglobulin protein and enhances the efficacy of radiation therapy in esophageal squamous cell carcinoma. Cancer Sci. 2023;114:1697–709.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Sadeghipour MM, Torabizadeh SA, Karimabad MN. The Glucose-Regulated Protein78 (GRP78) in the unfolded protein response (UPR) pathway: a potential therapeutic target for breast cancer. Anticancer Agents Med Chem. 2023;23:505–24.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grants 81972687 to ZSZ, 82073064 and 81874167 to LYL), Tianjin Science and Technology Program Project (21JCYBJC00170 to ZSZ), the Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00020 to LYL), and the Ministry of Education 111 Project B20016.

Author information

Authors and Affiliations

  1. State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research, Nankai University, and the Haihe Laboratory of Cell Ecosystem, Tianjin, 300350, China

    SungJu Ryu, Hui Long, Xin-ling Zheng, Yuan-yuan Song, Yan Wang, Yu-jie Zhou, Xiao-jing Quan, Lu-yuan Li & Zhi-song Zhang

  2. Institute of Microbiology, State Academy of Sciences, Pyongyang, Democratic People’s Republic of Korea

    SungJu Ryu

Authors
  1. SungJu Ryu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hui Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xin-ling Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuan-yuan Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yu-jie Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiao-jing Quan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lu-yuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhi-song Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

ZSZ and LYL conceived the study and wrote the manuscript with input from other authors. SJR performed most of the biochemical and cellular experiments. HL, XLZ and YYS provided technical assistance. YW, YJZ, and XJQ contribute to data analysis manuscript preparation.

Corresponding authors

Correspondence to Lu-yuan Li or Zhi-song Zhang.

Ethics declarations

Competing interests

The authors declare no competing interests.

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

Ryu, S., Long, H., Zheng, Xl. et al. Pentapeptide PYRAE triggers ER stress-mediated apoptosis of breast cancer cells in mice by targeting RHBDF1-BiP interaction. Acta Pharmacol Sin 45, 378–390 (2024). https://doi.org/10.1038/s41401-023-01163-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41401-023-01163-x

Keywords

Search

Advanced search

Quick links