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Cellular and Molecular Biology

EEF1A2 promotes HIF1A mediated breast cancer angiogenesis in normoxia and participates in a positive feedback loop with HIF1A in hypoxia

British Journal of Cancer volume 130, pages 184–200 (2024)Cite this article

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Abstract

Background

The eukaryotic elongation factor, EEF1A2, has been identified as an oncogene in various solid tumors. Here, we have identified a novel function of EEF1A2 in angiogenesis.

Methods

Chick chorioallantoic membrane, tubulogenesis, aortic ring, Matrigel plug, and skin wound healing assays established EEF1A2’s role in angiogenesis.

Result

Higher EEF1A2 levels in breast cancer cells enhanced cell growth, movement, blood vessel function, and tubule formation in HUVECs, as confirmed by ex-ovo and in-vivo tests. The overexpression of EEF1A2 could be counteracted by Plitidepsin. Under normoxic conditions, EEF1A2 triggered HIF1A expression via ERK-Myc and mTOR signaling in TNBC and ER/PR positive cells. Hypoxia induced the expression of EEF1A2, leading to a positive feedback loop between EEF1A2 and HIF1A. Luciferase assay and EMSA confirmed HIF1A binding on the EEF1A2 promoter, which induced its transcription. RT-PCR and polysome profiling validated that EEF1A2 affected VEGF transcription and translation positively. This led to increased VEGF release from breast cancer cells, activating ERK and PI3K-AKT signaling in endothelial cells. Breast cancer tissues with elevated EEF1A2 showed higher microvessel density.

Conclusion

EEF1A2 exhibits angiogenic potential in both normoxic and hypoxic conditions, underscoring its dual role in promoting EMT and angiogenesis, rendering it a promising target for cancer therapy.

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Fig. 1: Effect of EEF1A2 perturbation in breast cancer cells on proliferation, migration, and tubule formation of HUVECs.
Fig. 2: Effect of EEF1A2 expression in breast cancer cells on angiogenesis in ex-ovo and in-vivo model systems.
Fig. 3: Paracrine Effect of EEF1A2 perturbation in cancer cell lines on VEGF-HIF1A, PI3k/AKT, and ERK signaling in HUVECs.
Fig. 4: HIF1A induces EEF1A2 expression irrespective molecular subtypes of cancer cells.
Fig. 5: HIF1A directly binds to EEF1A2 promoter region.
Fig. 6: Effect of EEF1A2 expression modulation on ERK/Myc signaling in TNBC and mTOR signaling in ER/PR+ breast cancer cell lines, and paracrine influence on endothelial cells.
Fig. 7: Effect of EEF1A2 levels and its pharmacological inhibitor on tumor progression in BALB/c mice.
Fig. 8: Effect of EEF1A2 expression and its association with MVD (CD31) in mouse model and breast cancer patient samples.

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All data and materials related to this research are available in the manuscript or supplementary information.

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Acknowledgements

We thank Dr. Saurabh Chawla, NISER, Bhubaneswar, India for helping with animal experiments. We thank Dr. Anup Kumar Ram and Dr. Pankaj V Alone for guidance in polysome analysis. We thank Ananya Palo, Soham Choudhury, Deeptima Jaiswar, and Talina Mohapatra for measuring absorbance of polysome fractions. We thank Ruchika Rai for helping in power analysis. We thank Shubhanjali and Anamika Singh for the bioinformatic data analysis. This work was supported by intramural funding from the National Institute of Science Education and Research (NISER), Department of Atomic Energy (DAE), Government of India (GOI). SAP, MKH, and MN received fellowships from NISER, DAE, GOI.

Funding

We thank National Institute of Science Education and Research (Department of Atomic Energy), the Government of India (GOI) for providing infrastructure, and intramural support.

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Authors and Affiliations

  1. National Institute of Science Education and Research, School of Biological Sciences, Bhubaneswar, 752050, Odisha, India

    Saket Awadhesbhai Patel, Md. Khurshidul Hassan, Monali Naik & Manjusha Dixit

  2. Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai, 400094, India

    Saket Awadhesbhai Patel, Md. Khurshidul Hassan, Monali Naik & Manjusha Dixit

  3. Apollo Hospitals, Plot No. 251,Old Sainik School Road, Bhubaneswar, 750015, Odisha, India

    Nachiketa Mohapatra

  4. CSIR-Central Leather Research Institute, Sardar Patel Road, Adyar, Chennai, 600020, India

    Poornima Balan & Purna Sai Korrapati

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  1. Saket Awadhesbhai Patel
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Contributions

SAP performed majority of the in vivo experiments; wound healing assay, matrigel plug assay, tumor development in mice, IHC of 92 samples and mice tissue, represented in Figs. 2c–f, 7a–h, 8a–f, Supplementary Fig. 9A–E and in vitro experiments; Western blot, EMSA, Luciferase, polysome assay, qRT-PCR, tubulogenesis, proliferation assay, ELISA, represented in Figs. 1a, c, d, 3a–n, 4a–i, 5a–j, 6a–n, and Supplementary Figs. 1C–G, 2A, B, 3A–D, 4B, 6A–L, 7A–F, 8A–L, data curation, formal analysis, and writing original draft and editing; MKH performed the in vitro experiments of Figs. 1b, c, 3a, 8e, Supplementary Fig. 1A, 1B, 1H, 1I, 5A-D, and performed IHC of 96 samples (Fig. 8e), wrote the original draft partly; MN performed the CAM assay (Fig. 2a) and Matrigel Plug assay (Supplementary Fig. 4A, B, D); NM analysed and scored patient IHC samples, PB performed and analysed the experiment given in Fig. 2b; PSK supervised the experiments carried out for generating Fig. 2b; MD conceptualized the whole project, designed experiments, planned and guided the research, formal analysis, supervision, funding acquisition, resources, review and editing the manuscript.

Corresponding author

Correspondence to Manjusha Dixit.

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The authors declare no competing interests.

Ethics approval and consent to participate

The animal experiment was approved by Institutional Animal Ethics Committee, NISER, India (protocol-NISER/SBS/AH184), (protocol-NISER/SBS/AH/224), and (protocol-NISER/SBS/AH/170) and conducted at NISER animal house facility. Human samples-based study was approved by the Institutional Ethics Committee, NISER, Bhubaneswar, India (protocol-NISER/IEC/2016–02).

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Patel, S.A., Hassan, M.K., Naik, M. et al. EEF1A2 promotes HIF1A mediated breast cancer angiogenesis in normoxia and participates in a positive feedback loop with HIF1A in hypoxia. Br J Cancer 130, 184–200 (2024). https://doi.org/10.1038/s41416-023-02509-2

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