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:

A critical ETV4/Twist1/Vimentin axis in Ha-RAS-induced aggressive breast cancer

Cancer Gene Therapy volume 29, pages 1590–1599 (2022)Cite this article

Subjects

Abstract

RAS oncogenes are major drivers of diverse types of cancer. However, they are largely not druggable, and therefore targeting critical downstream pathways and dependencies is an attractive approach. We have isolated a tumorigenic cell line (FE1.2), which exhibits mesenchymal characteristics, after inoculating Ha-Ras-expressing retrovirus into mammary glands of rats, and subsequently isolated a non-aggressive revertant cell line (FC5). This revertant has lost the rat Ha-Ras driver and showed a more epithelial morphology, slower proliferation in culture, and reduced tumorigenicity in vivo. Re-expression of human Ha-RAS in these cells (FC5-RAS) reinduced mesenchymal morphology, higher proliferation rate, and tumorigenicity that was still significantly milder than parental FE1.2 cells. RNA-seq analysis of FC5-RAS vs FC5-Vector cells identified multiple genes whose expressions were regulated by Ha-RAS. This analysis also identified many genes including those controlling cell growth whose expression was altered by loss of HA-Ras in FC5 cells but remained unchanged upon reintroduction of Ha-RAS. These results suggest that targeting the Ha-Ras driver oncogene induces partial tumor regression, but it still denotes strong efficacy for cancer therapy. Among the RAS-responsive genes, we identified Twist1 as a critical mediator of epithelial-to-mesenchymal transition through the direct transcriptional regulation of vimentin. Mechanistically, we show that Twist1 is induced by the ETS gene, ETV4, downstream of Ha-RAS, and that inhibition of ETV4 suppressed the growth of breast cancer cells driven by the Ha-RAS pathway. Targeting the ETV4/Twist1/Vimentin axis may therefore offer a therapeutic modality for breast tumors driven by the Ha-RAS pathway.

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: Re-expression of Ha-RAS in FC5 cells does not restore full tumorigenicity in culture or in vivo.
Fig. 2: Proviral integration silences the expression of the surrounding genes.
Fig. 3: Ha-RAS re-expression in FC5 cells induces or suppresses transcription of multiple genes.
Fig. 4: Ha-RAS restoration increases the expression of several genes.
Fig. 5: Knockdown of Ha-ras in FE1.2 cells results in changes in gene expression resembling FC5.
Fig. 6: Expression of Vimentin is regulated at the transcriptional level by Twist1.
Fig. 7: Ha-Ras controls Twist1 expression by transcriptional induction of ETV4.

Similar content being viewed by others

Data availability

All datasets presented in this study are included in the article/Supplementary Material.

References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49.

    Article  PubMed  Google Scholar 

  2. Zacksenhaus E, Liu JC, Jiang Z, Yao Y, Xia L, Shrestha M, et al. Transcription factors in breast cancer-lessons from recent genomic analyses and therapeutic implications. Adv Protein Chem Struct Biol. 2017;107:223–73.

    Article  CAS  PubMed  Google Scholar 

  3. Drilon A, Hu ZI, Lai GGY, Tan DSW. Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes. Nat Rev Clin Oncol. 2018;15:150.

    Article  PubMed  Google Scholar 

  4. Guo Y, Cao R, Zhang X, Huang L, Sun L, Zhao J, et al. Recent progress in rare oncogenic drivers and targeted therapy for non-small cell lung cancer. Onco Targets Ther. 2019;12:10343–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Siena S, Sartore-Bianchi A, Marsoni S, Hurwitz HI, McCall SJ, Penault-Llorca F, et al. Targeting the human epidermal growth factor receptor 2 (HER2) oncogene in colorectal cancer. Ann Oncol. 2018;29:1108–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Meng D, Carvajal RD. KIT as an oncogenic driver in melanoma: an update on clinical development. Am J Clin Dermatol. 2019;20:315–23.

    Article  PubMed  Google Scholar 

  7. Roman M, Baraibar I, Lopez I, Nadal E, Rolfo C, Vicent S, et al. KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target. Mol Cancer. 2018;17:33.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Takashima A, Faller DV. Targeting the RAS oncogene. Expert Opin Ther Targets. 2013;17:507–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Holderfield M, Deuker MM, McCormick F, McMahon M. Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nat Rev Cancer. 2014;14:455–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dankner M, Rose AAN, Rajkumar S, Siegel PM, Watson IR. Classifying BRAF alterations in cancer: new rational therapeutic strategies for actionable mutations. Oncogene. 2018;37:3183–99.

    Article  CAS  PubMed  Google Scholar 

  11. Samatar AA, Poulikakos PI. Targeting RAS-ERK signalling in cancer: promises and challenges. Nat Rev Drug Discov. 2014;13:928–42.

    Article  CAS  PubMed  Google Scholar 

  12. Felipe Lima J, Nofech-Mozes S, Bayani J, Bartlett JM. EMT in breast carcinoma—a review. J Clin Med. 2016;5:65.

  13. Vuoriluoto K, Haugen H, Kiviluoto S, Mpindi JP, Nevo J, Gjerdrum C, et al. Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene. 2011;30:1436–48.

    Article  CAS  PubMed  Google Scholar 

  14. Khan MA, Chen HC, Zhang D, Fu J. Twist: a molecular target in cancer therapeutics. Tumour Biol. 2013;34:2497–506.

    Article  CAS  PubMed  Google Scholar 

  15. Li YJ, Song R, Korkola JE, Archer MC, Ben-David Y. Cyclin D1 is necessary but not sufficient for anchorage-independent growth of rat mammary tumor cells and is associated with resistance of the Copenhagen rat to mammary carcinogenesis. Oncogene. 2003;22:3452–62.

    Article  CAS  PubMed  Google Scholar 

  16. Müller M, Beck IM, Gadesmann J, Karschuk N, Paschen A, Proksch E, et al. MMP19 is upregulated during melanoma progression and increases invasion of melanoma cells. Mod Pathol. 2010;23:511–21.

    Article  PubMed  Google Scholar 

  17. Meng Y, Wang L, Chen D, Chang Y, Zhang M, Xu JJ, et al. LAPTM4B: an oncogene in various solid tumors and its functions. Oncogene. 2016;35:6359–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhong FJ, Sun B, Cao MM, Xu C, Li YM, Yang LY. STMN2 mediates nuclear translocation of Smad2/3 and enhances TGFbeta signaling by destabilizing microtubules to promote epithelial-mesenchymal transition in hepatocellular carcinoma. Cancer Lett. 2021;506:128–41.

    Article  CAS  PubMed  Google Scholar 

  19. Sugimoto M, Kohashi K, Itsumi M, Shiota M, Abe T, Yamada Y, et al. Epithelial to mesenchymal transition in clear cell renal cell carcinoma with rhabdoid features. Pathobiology. 2016;83:277–86.

    Article  CAS  PubMed  Google Scholar 

  20. Taube JH, Herschkowitz JI, Komurov K, Zhou AY, Gupta S, Yang J, et al. Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc Natl Acad Sci USA. 2010;107:15449–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Meng J, Chen S, Han JX, Qian B, Wang XR, Zhong WL, et al. Twist1 regulates vimentin through Cul2 circular RNA to promote EMT in hepatocellular carcinoma. Cancer Res. 2018;78:4150–62.

    Article  CAS  PubMed  Google Scholar 

  22. Baker R, Kent CV, Silbermann RA, Hassell JA, Young LJ, Howe LR. Pea3 transcription factors and wnt1-induced mouse mammary neoplasia. PLoS ONE. 2010;5:e8854.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Liu CY, Yu T, Huang Y, Cui L, Hong W. ETS (E26 transformation-specific) up-regulation of the transcriptional co-activator TAZ promotes cell migration and metastasis in prostate cancer. J Biol Chem. 2017;292:9420–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Eckert LB, Repasky GA, Ulku AS, McFall A, Zhou H, Sartor CI, et al. Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis. Cancer Res. 2004;64:4585–92.

    Article  CAS  PubMed  Google Scholar 

  25. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012;72:2457–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Klock G, Storch S, Rickert J, Gutacker C, Koch-Brandt C. Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis. J Cell Physiol. 1998;177:593–605.

    Article  CAS  PubMed  Google Scholar 

  27. Zhang D, Sun B, Zhao X, Cui Y, Xu S, Dong X, et al. Secreted CLU is associated with the initiation of triple-negative breast cancer. Cancer Biol Ther. 2012;13:321–9.

    Article  CAS  PubMed  Google Scholar 

  28. Loomans HA, Andl CD. Intertwining of activin A and TGFbeta signaling: dual roles in cancer progression and cancer cell invasion. Cancers (Basel). 2014;7:70–91.

    Article  Google Scholar 

  29. Benatar T, Yang W, Amemiya Y, Evdokimova V, Kahn H, Holloway C, et al. IGFBP7 reduces breast tumor growth by induction of senescence and apoptosis pathways. Breast Cancer Res Treat. 2012;133:563–73.

    Article  CAS  PubMed  Google Scholar 

  30. Chang Y, Zuka M, Perez-Pinera P, Astudillo A, Mortimer J, Berenson JR, et al. Secretion of pleiotrophin stimulates breast cancer progression through remodeling of the tumor microenvironment. Proc Natl Acad Sci USA. 2007;104:10888–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Li Q, Zhu CC, Ni B, Zhang ZZ, Jiang SH, Hu LP, et al. Lysyl oxidase promotes liver metastasis of gastric cancer via facilitating the reciprocal interactions between tumor cells and cancer associated fibroblasts. EBioMedicine. 2019;49:157–71.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Luo XG, Zhang CL, Zhao WW, Liu ZP, Liu L, Mu A, et al. Histone methyltransferase SMYD3 promotes MRTF-A-mediated transactivation of MYL9 and migration of MCF-7 breast cancer cells. Cancer Lett. 2014;344:129–37.

    Article  CAS  PubMed  Google Scholar 

  33. Yang B, Huang J, Xiang T, Yin X, Luo X, Huang J, et al. Chrysin inhibits metastatic potential of human triple-negative breast cancer cells by modulating matrix metalloproteinase-10, epithelial to mesenchymal transition, and PI3K/Akt signaling pathway. J Appl Toxicol. 2014;34:105–12.

    Article  PubMed  Google Scholar 

  34. Margheri F, Serrati S, Lapucci A, Anastasia C, Giusti B, Pucci M, et al. Systemic sclerosis-endothelial cell antiangiogenic pentraxin 3 and matrix metalloprotease 12 control human breast cancer tumor vascularization and development in mice. Neoplasia. 2009;11:1106–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ye T, Liang Y, Zhang D, Zhang X. MicroRNA-16-1-3p represses breast tumor growth and metastasis by inhibiting PGK1-mediated Warburg effect. Front Cell Dev Biol. 2020;8:615154.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Chen X, Zhao M, Huang J, Li Y, Wang S, Harrington CA, et al. microRNA-130a suppresses breast cancer cell migration and invasion by targeting FOSL1 and upregulating ZO-1. J Cell Biochem. 2018;119:4945–56.

    Article  CAS  PubMed  Google Scholar 

  37. Wei M, Yu H, Cai C, Gao R, Liu X, Zhu H. MiR-3194-3p inhibits breast cancer progression by targeting Aquaporin1. Front Oncol. 2020;10:1513.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Yu G, Herazo-Maya JD, Nukui T, Romkes M, Parwani A, Juan-Guardela BM, et al. Matrix metalloproteinase-19 promotes metastatic behavior in vitro and is associated with increased mortality in non-small cell lung cancer. Am J Respir Crit Care Med. 2014;190:780–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Li Y, Zou L, Li Q, Haibe-Kains B, Tian R, Li Y, et al. Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer. Nat Med. 2010;16:214–8.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Barsyte-Lovejoy D, Lau SK, Boutros PC, Khosravi F, Jurisica I, Andrulis IL, et al. The c-Myc oncogene directly induces the H19 noncoding RNA by allele-specific binding to potentiate tumorigenesis. Cancer Res. 2006;66:5330–7.

    Article  CAS  PubMed  Google Scholar 

  41. Huang C, Ye Z, Wan J, Liang J, Liu M, Xu X, et al. Secreted frizzled-related protein 2 is associated with disease progression and poor prognosis in breast cancer. Dis Markers. 2019;2019:6149381.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Xu XL, Yu J, Zhang HY, Sun MH, Gu J, Du X, et al. Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis. World J Gastroenterol. 2004;10:3441–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Narayanaswamy PB, Baral TK, Haller H, Dumler I, Acharya K, Kiyan Y. Transcriptomic pathway analysis of urokinase receptor silenced breast cancer cells: a microarray study. Oncotarget. 2017;8:101572–90.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Kim MS, Lee WS, Jin W. TrkB promotes breast cancer metastasis via suppression of Runx3 and Keap1 expression. Mol Cells. 2016;39:258–65.

    Article  CAS  PubMed  Google Scholar 

  45. Boo LM, Lin HH, Chung V, Zhou B, Louie SG, O’Reilly MA, et al. High mobility group A2 potentiates genotoxic stress in part through the modulation of basal and DNA damage-dependent phosphatidylinositol 3-kinase-related protein kinase activation. Cancer Res. 2005;65:6622–30.

    Article  CAS  PubMed  Google Scholar 

  46. Ghafouri-Fard S, Esmaeili M, Taheri M. H19 lncRNA: roles in tumorigenesis. Biomed Pharmacother. 2020;123:109774.

    Article  CAS  PubMed  Google Scholar 

  47. Zhou T, Lin W, Zhu Q, Renaud H, Liu X, Li R, et al. The role of PEG3 in the occurrence and prognosis of colon cancer. Onco Targets Ther. 2019;12:6001–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Baines KJ, Rampersaud AM, Hillier DM, Jeyarajah MJ, Grafham GK, Eastabrook G, et al. Antiviral inflammation during early pregnancy reduces placental and fetal growth trajectories. J Immunol. 2020;204:694–706.

    Article  CAS  PubMed  Google Scholar 

  49. Chaffer CL, San Juan BP, Lim E, Weinberg RA. EMT, cell plasticity and metastasis. Cancer Metastasis Rev. 2016;35:645–54.

    Article  PubMed  Google Scholar 

  50. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004;117:927–39.

    Article  CAS  PubMed  Google Scholar 

  51. de Launoit Y, Chotteau-Lelievre A, Beaudoin C, Coutte L, Netzer S, Brenner C, et al. The PEA3 group of ETS-related transcription factors. Role in breast cancer metastasis. Adv Exp Med Biol. 2000;480:107–16.

    Article  PubMed  Google Scholar 

  52. Xia L, Xiao X, Liu WL, Song Y, Liu TJJ, Li YJ, et al. Coactosin-like protein CLP/Cotl1 suppresses breast cancer growth through activation of IL-24/PERP and inhibition of non-canonical TGFbeta signaling. Oncogene. 2018;37:323–31.

    Article  CAS  PubMed  Google Scholar 

  53. Song J, Yuan C, Yang J, Liu T, Yao Y, Xiao X, et al. Novel flavagline-like compounds with potent Fli-1 inhibitory activity suppress diverse types of leukemia. FEBS J. 2018;285:4631–45.

    Article  CAS  PubMed  Google Scholar 

  54. Haeri M, Li Y, Li Y, Li Q, Spaner DE, Ben-David Y. Insertional activation of myb by F-MuLV in SCID mice induces myeloid leukemia. Int J Oncol. 2013;43:169–76.

    Article  CAS  PubMed  Google Scholar 

  55. Liu T, Xia L, Yao Y, Yan C, Fan Y, Gajendran B, et al. Identification of diterpenoid compounds that interfere with Fli-1 DNA binding to suppress leukemogenesis. Cell Death Dis. 2019;10:117.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Li YJ, Zhao X, Vecchiarelli-Federico LM, Li Y, Datti A, Cheng Y, et al. Drug-mediated inhibition of Fli-1 for the treatment of leukemia. Blood Cancer J. 2012;2:e54.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Wang C, Song J, Liu W, Yao Y, Kapranov P, Sample KM, et al. FLI1 promotes protein translation via the transcriptional regulation of MKNK1 expression. Int J Oncol. 2020;56:430–8.

    CAS  PubMed  Google Scholar 

  58. Rodriguez-Viciana P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, et al. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell. 1997;89:457–67.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This study was supported by research grants from the Natural National Science Foundation of China (21867009, U1812403), the Science and Technology Department of Guizhou Province innovation and project grants (QKHPTRC [2019]5627), and the 100 Leading Talents of Guizhou Province to YBD and the Science and Technology Department of Guizhou Province (QKHZC[2019]2762) to YL.

Author information

Authors and Affiliations

  1. State Key Laboratory for Functions and Applications of Medicinal Plants/College of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China

    Wuling Liu, Babu Gajendran, Klarke M. Sample, Chunlin Wang, Anling Hu, Beiling Chen, Yanmei Li & Yaacov Ben-David

  2. The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academic of Sciences, Guiyang, Guizhou, China

    Wuling Liu, Babu Gajendran, Klarke M. Sample, Chunlin Wang, Anling Hu, Beiling Chen, Yanmei Li & Yaacov Ben-David

  3. School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang, Guizhou, China

    Babu Gajendran

  4. Department of Medicine, University of Toronto, Toronto, ON, Canada

    Eldad Zacksenhaus

  5. Division of Advanced Diagnostics, Toronto General Research Institute—University Health Network, Toronto, ON, Canada

    Eldad Zacksenhaus

Authors
  1. Wuling Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Babu Gajendran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Klarke M. Sample
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chunlin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anling Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Beiling Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yanmei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Eldad Zacksenhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yaacov Ben-David
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

WL, BG, CW, AH, BC, YL, and EZ contributed to the conception, design of the study, as well as data acquisition and interpretation. KMS and BG were involved in the data and statistical analysis. WL drafted the manuscript. YBD and EZ reviewed the manuscript critically. YBD supervised, conceived, and designed the study. All authors contributed to the interpretation of the findings, reviewed, edited, and approved the final manuscript.

Corresponding author

Correspondence to Yaacov Ben-David.

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

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, W., Gajendran, B., Sample, K.M. et al. A critical ETV4/Twist1/Vimentin axis in Ha-RAS-induced aggressive breast cancer. Cancer Gene Ther 29, 1590–1599 (2022). https://doi.org/10.1038/s41417-022-00471-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41417-022-00471-4

Search

Advanced search

Quick links