The Biological Role of Factor-Inhibiting Hypoxia- Inducible Factor

Factor Inhibiting Hypoxia-inducible Factor (FIH) is an asparaginyl hydroxylase which regulates the transcription factor Hypoxia-Inducible Factor (HIF), via hydroxylation of a conserved asparagine residue of the HIF-alpha subunits (two isoforms of which are well established, HIF-1 and HIF-2alpha – HIF-3alpha is less well characterized currently). Other targets of FIH have also been reported. Little is known about both FIH expression and function. This thesis will investigate the expression of FIH in rodents, in cell lines and in renal cancer tissue samples. In addition, RNAi technology was used to study FIH function. Clear cell renal cell carcinoma (CCRCC) is commonly associated with inactivation of tumour suppressor von-Hippel Lindau protein (VHL) and constitutive activation of HIF. The main question I have addressed in this thesis is whether FIH decreases HIF activation in this setting. To address this I inhibited FIH using several approaches. Specifically, using hypoxia, dimethyloxalylglycine (DMOG) and RNA interference (RNAi). Each of these increased the expression of HIF target genes in two different CCRCC cell lines, RCC10 and RCC4. Investigating three different CCRCC cell lines, FIH inhibition decreased numbers of RCC4 and RCC10 cells growing in culture, which is likely to be due to an increase in expression of pro-apoptotic, FIH-regulated HIF target genes. Interestingly, 786-O cells exclusively express the HIF-2alpha isoform. Attenuation of FIH in this setting did not affect expression of HIF target genes, nor affect growth in culture. To determine if this was due to lack of FIH or may be due to lack of HIF-1alpha, I introduced HIF-1alpha via a viral vector. Following this, sensitivity to FIH was clearly demonstrated. This implies either specific ‘protection’ of HIF-2alpha in 786-O or more general FIH selectivity for the HIF- 1alpha isoform. My findings contrast with two reports suggesting that FIH expression is suppressed in CCRCC. My findings give insight into how FIH asparagine hydroxylation regulates HIF, in particular in renal cancer and makes this enzyme a potential target for therapeutic inhibition, in the majority of renal cancers

A CCR4 antagonist reverses the tumor-promoting microenvironment of renal cancer

CRUK programme grant C587/A16354 and a research grant from Affitech AS.The study was supported by Cancer Research UK (CRUK) programme grant C587/A16354 and a research grant from Affitech AS

Complement C1q is hydroxylated by collagen prolyl 4 hydroxylase and is sensitive to off-target inhibition by prolyl hydroxylase domain inhibitors that stabilize hypoxia-inducible factor

Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. C1q is composed of A, B, and C chains that require post-translational prolyl 4-hydroxylation of their N-terminal collagen-like domain to enable the formation of the functional triple helical multimers. The prolyl 4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognized prolyl 4-hydroxylases include collagen prolyl-4-hydroxylases (CP4H) and the more recently described prolyl hydroxylase domain (PHD) enzymes that act as oxygen sensors regulating hypoxia-inducible factor (HIF). We show that several small-molecule prolyl hydroxylase inhibitors that activate HIF also potently suppress C1q secretion by human macrophages. However, reducing oxygenation to a level that activates HIF does not compromise C1q hydroxylation. $\textit{In vitro}$ studies showed that a C1q A chain peptide is not a substrate for PHD2 but is a substrate for CP4H1. Circulating levels of C1q did not differ between wild-type mice or mice with genetic deficits in PHD enzymes, but were reduced by prolyl hydroxylase inhibitors. Thus, C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. Hence, reduction of C1q levels may be an important off-target side effect of small molecule PHD inhibitors developed as treatments for renal anemia.This work was supported by the Wellcome Trust and the NIHR Cambridge Biomedical Research Centre Senior Investigator Awards (to PHM, supporting SSH and NB), the Wellcome Trust Scientific strategic award [100140]. SK was supported by a British Heart Foundation grant awarded to PHM as well as a Kennedy Institute of Rheumatology trustees fund