Ways into Understanding HIF Inhibition

Abstract

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein-protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

Keywords: FRET; HIF; PHD; cancer; hypoxia; hypoxia-inducible factor; inhibitor; pVHL; visualization.

Figure 1

Oxygen dependent hypoxia-inducible factor (HIF) regulation and deregulation of HIF in tumor environment. Normoxic oxygen conditions lead to proteasomal degradation of the hypoxia-inducible factor (HIF) protein and prevent target gene transcription (upper left); Decreased oxygen availability deactivates the hydroxylation of the HIF protein by cofactors, factor-inhibiting HIF (FIH) and prolyl hydroxylases (PHD), which enables HIF protein accumulation and translocation into the nucleus. HIF-α and -β subunits dimerize and form a transcription complex with the cofactors CBP/p300. Complex binding to the hypoxia responsive elements (HRE) on the promoter region of HIF target genes leads to their transcription (upper right); Under pathological conditions, hypoxia can be accompanied by increased reactive oxygen species (ROS) and impaired von Hippel–Lindau protein (pVHL) function, promoting HIF induction to an excessive extent, e.g., driving tumorigenesis under normoxic, as well as hypoxic condition (lower left and right). OH, hydroxylation; Ub, ubiquitination.

Figure 2

Inhibitors of the HIF pathway and intervention nodes summarized in this review. HIF inhibitors are broadly classed into their mode of action, targeting different levels of the HIF pathway. Starting from blockage of HIF mRNA expression to, finally, HIF target gene transcription and inhibition of the downstream genes, such as VEGF itself. For detailed information see Section 2.

Figure 3

Methods to unravel effects on HIF protein stability, interaction, and function in vitro and in vivo. (a) Interaction of protein (complexes) is detectable by proximity-depended resonance energy transfer such as fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). Protein localization and proximity are directly imaged in vitro or in vivo and recorded with high spatiotemporal resolution; (b) Transcriptional activity of transcription factor is assessed by chromatin immunoprecipitation (ChIP). Precise antibody recognition and accuracy is mandatory for qualitative evaluation of changes upon any perturbation; (c) Nanobodies (Nb), shown in relation to conventionally used IgG1, can be functionalized and used as intravital tracers. Due to their small size, penetration depth of cells and tissue is superior. Stoichiometric labeling of Nb enables quantitative imaging; (d) Nb tracer addressing interaction partners allow in vivo FRET/BRET independent of genetic manipulation, giving rise to a multitude of new life cell/intravital interaction studies.