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.