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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

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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.

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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.

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Data availability

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

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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.

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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

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  1. Wuling Liu
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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.

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Correspondence to Yaacov Ben-David.

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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

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