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Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors

Oncogene volume 40, pages 5142–5152 (2021)Cite this article

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Abstract

Angiogenesis is essential for the sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Therefore, understanding the mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Moreover, phosphorylation of the analogous site in HIF-2α (S435) stabilizes the protein through the same mechanism, indicating post-translational modification within the oxygen-dependent degradation domain as a mechanism of regulating the HIF-α subunits. In vitro and in vivo models demonstrate that expression of PIM1 is sufficient to stabilize HIF-1α and HIF-2α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner. CRISPR mutants of HIF-1α (Thr455D) promoted increased tumor growth, proliferation, and angiogenesis. Moreover, HIF-1α-T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

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Fig. 1: PIM1 correlates with angiogenesis in human cancer samples.
Fig. 2: PIM1 induces angiogenesis in vivo and in vitro.
Fig. 3: PIM1 is sufficient to stabilize HIF-1α and activate HIF-1 in normoxia.
Fig. 4: PIM1 phosphorylates HIF-1α at Thr455.
Fig. 5: Phosphorylation of HIF-1α at Thr455 disrupts PHD2 binding and increases HIF-1α stability.
Fig. 6: Phosphorylation of HIF-2α at S435 increases protein stability.
Fig. 7: HIF-1α-T455D CRISPR mutants increase tumor growth and are resistant to PIM inhibition.

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Acknowledgements

We would like to thank the following shared resources at the University of Arizona Cancer Center for their help and support: EMSR, TACMASR, Gene Editing, and Biostatistics. We would also like to thank Dr. Dan Buster for performing in vitro kinase assays and Brenda Baggett and Dr. Marty Pagel for assisting with in vivo imaging. Studies were supported by funding from the National Cancer Institute (T32CA009213) on behalf of ALC, American Cancer Society (RSG-16-159-01-CDD) to NAW, and Department of Defense PCRP (W81XWH-19-1-0455) to NAW. A Cancer Center Support grant from the National Institute of Health (P30CA023074) also supported this research.

Author contributions

Study concept and design: NAW, ALC. Acquisition of data: ALC, SSC, RKT, CCJ, AGS, ANC, PRL, CKM, AEC, NAW. Analysis and presentation of data: NAW, ALC, PRL. Material support: CKM, AEC. Study supervision: NAW. Funding: NAW.C

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Authors and Affiliations

  1. Cancer Biology Graduate Interdisciplinary Program, The University of Arizona, Tucson, AZ, USA

    Andrea L. Casillas, Amber N. Clements & Corbin C. Jensen

  2. The University of Arizona Cancer Center, Tucson, AZ, USA

    Shailender S. Chauhan, Rachel K. Toth, Cindy K. Miranti, Anne E. Cress & Noel A. Warfel

  3. Salk Institute for Biological Studies, La Jolla, CA, USA

    Alva G. Sainz

  4. Department of Medicine, The University of Arizona, Tucson, AZ, USA

    Paul R. Langlais

  5. Department of Cellular and Molecular Medicine, The University of Arizona, Tucson, AZ, USA

    Cindy K. Miranti, Anne E. Cress & Noel A. Warfel

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  1. Andrea L. Casillas
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Casillas, A.L., Chauhan, S.S., Toth, R.K. et al. Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors. Oncogene 40, 5142–5152 (2021). https://doi.org/10.1038/s41388-021-01915-1

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