**8. Modulation of the HIF response during the pathogenesis of ccRCC**

Early evidence to support the pleiotropic nature of the HIF pathway in kidney cancer came from the observation that HIF-1α and HIF-2α have opposing actions on tumor growth in ccRCC xenograft models. Whilst HIF-2α promotes tumor growth, HIF-1α has the opposite effect and restricts tumor growth [46, 48, 50, 51]. Furthermore, expression of HIF-2α target genes in ccRCC tumors correlates with poor patient prognosis, whereas HIF-1α targets genes are associated with improved survival [91].

Commensurate with this, HIF isoform expression appears to switch from HIF-1α to HIF-2α during the development of kidney cancer [28, 112]. In renal tubule epithelial cells, including proximal tubular cells from which ccRCC is derived, HIF-1α mRNA is highly expressed, whereas HIF-2α mRNA is undetectable [28]. Conversely, HIF-2α mRNA (and protein) is highly expressed in ccRCC, possibly as a result of downregulation of DNMT3a and resultant promoter demethylation of the EPAS1 gene that encodes HIF-2α [113]. Furthermore, ccRCCs often downregulate HIF-1α through loss of copy number, deletion, truncation or transcript downregulation [25, 31, 32, 112, 114]. Given the tumor-suppressive function of HIF-1 and the oncogenic function of HIF-2, the shift from HIF-1α in the ccRCC cell of origin to dominant HIF-2α expression in overt ccRCC would favor a more oncogenic phenotype. However, even within the transcriptional repertoire of each isoform there are genes with heterogenous associations with prognosis, suggesting that other selective pressures, effective at the level of individual HIF target genes, may also be operating [91].

Indeed, suppression of individual HIF target genes with anti-tumorigenic properties has been reported in ccRCC. The pro-apoptotic gene BNIP3 is a canonical HIF target gene in many cell types. However, rather than being increased by constitutive HIF in ccRCC cells, its expression was found to be lower than in normal kidney cells. This is most likely as a result of epigenetic modification of the BNIP3 gene locus involving histone deacetylation [115].

In this respect, it is notable that of the many somatic mutations that co-occur with VHL inactivation in ccRCC, very few occur within HIF-target genes. However, to date, the majority of ccRCC sequencing efforts have focused on the coding genome or have targeted genomic regions of interest. Therefore, the majority of HIF binding sites (which are usually intergenic) have not been extensively examined and further studies may reveal somatic mutation of these sites in the future. However, epigenetic modifiers such as PBRM1, SETD2 and BAP1 are recurrently mutated in these tumors [25, 31, 32, 116–120]. PBRM1 encodes a subunit of the chromatin remodeling PBAF SWI/SNF complex; SETD2 encodes a histone methyltransferase; and BAP1 encodes a histone deubiquitinase. Interestingly, parallel evolution has been reported with respect to these mutations, whereby multiple mutations in the same gene are present in different cells of the same tumor [32]. This emphasizes their importance in driving ccRCC, as well as illustrating their temporal occurrence (i.e. subsequent to VHL mutation). Although the interaction between these ccRCC-associated somatic mutations and the HIF pathway remains unclear, PBRM1 inactivation enhances some aspects of the HIF response [121] and reduces the tumor-suppressor activity of HIF-1α, although the mechanisms are unknown [122]. Recurrent mutations are also found in genes within the PI3K/AKT/mTOR pathway, which is a master regulator of RNA translation. Expression of both HIF-1α and HIF-2α protein are differentially dependent on mTOR, with HIF-1α being regulated by both the mTORC1 and mTORC2 complexes, whilst HIF-2α is dependent solely on mTORC2 [123]. Therefore, HIF isoforms may be differentially affected by mutations in this pathway.

In addition, other oncogenic transcription factors activated in ccRCC may modulate the HIF response. For example, MYC activity is enhanced in ccRCC [124, 125] and synergizes preferentially with HIF-2, whilst antagonizing HIF-1 [102, 126]. In this way, MYC augments the switch from HIF-1 to the more oncogenic HIF-2 isoform. Importantly, MYC itself is a transcriptional target of HIF in ccRCC cells [127], providing a mechanism whereby stabilization of HIF following inactivation of VHL preferentially amplifies the HIF-2 transcriptional pathway in these cells.

#### **9. Variation in the HIF pathway pre-disposes to renal cancer**

As discussed above, genetic and epigenetic events occur somatically in ccRCC following VHL inactivation, allowing the HIF transcriptional output to adapt to a more oncogenic phenotype, thereby promoting tumor formation. However, differences in the HIF pathway that exist prior to VHL inactivation can also affect the ability of cells to form cancer. Indeed, it is highly likely that cell-type differences in the HIF pathway contribute to the tight tissue-specificity of VHL-associated cancer,

#### *HIF Pathways in Clear Cell Renal Cancer DOI: http://dx.doi.org/10.5772/intechopen.96539*

despite the almost universal operation of the VHL-HIF pathway in different mammalian cell types. Potentially, cell-type-specific components of the HIF pathway might favor tumorigenesis in permissive cell types, inhibit tumorigenesis in nonpermissive cell types, or a combination of both (**Figure 4**). The exact mechanism underlying this tissue specificity remains to be determined, although elucidation of HIF target genes in cells permissive to VHL-associated cancers (compared to that in non-permissive cells) will be key in future studies. Of note, the G1/S-phase cellcycle regulator cyclin D1 (CCND1) has been found to be a HIF-2 responsive gene, which is not regulated by HIF-1 and is unique to ccRCC cells [50]. Furthermore, CCND1 is required for ccRCC cell growth in mice [128]. This indicates that CCND1 and likely other tissue-specific HIF target genes may render certain cell types receptive to tumorigenesis upon VHL inactivation.

As well as being affected by somatic alterations and cell-type-specific features, the HIF pathway can also be modified by inherited genetic variants. Polymorphisms that predispose individuals to kidney cancer have been studied, and several of these have been shown to affect HIF target genes. Such variants have been identified by genome wide-association studies (GWAS), which compare the genome sequence of renal cancer patients with healthy control individuals [129–135]. Although these variants likely only account for about 5% of kidney cancer heritability [129], a disproportionately high number of these susceptibility loci overlap with cis-acting components of the HIF pathway [136]. This indicates that specific aspects of the HIF pathway are under genetic selection during the development of kidney cancer.

Many of these RCC-susceptibility loci lie in intergenic regions and so the functional target of these polymorphisms is not immediately apparent. However, several susceptibility loci overlap with, or lie adjacent to, HIF-binding sites [136]. In-depth analysis of chromatin looping and HIF-dependent gene regulation has identified a number of HIF target genes associated with these loci [127, 136–138]. At each locus, the renal cancer susceptibility polymorphism affects both HIF binding and expression of the HIF-target gene, either by generating a second HRE

#### **Figure 4.**

*Rebalancing the HIF pathway to favor tumorigenesis. HIF target genes include those that promote tumor growth (depicted in red), restrict tumor growth (depicted in green) and those that do not influence tumor growth (depicted in gray). Depending on the context (i.e. in a permissive or non-permissive context), activation of the HIF pathway may or may not be conducive to tumorigenesis. Features that could 'tip the balance' in a HIFactivated cell include genetic mutations, epigenetic features and the cell state (e.g. the underlying gene expression program).*

motif or by altering chromatin accessibility. Most notable are polymorphisms at the 11q13.3 locus, which affect HIF-2-dependent expression of cyclin D1 (CCND1) [137]; polymorphisms at the 8q24.21 locus, which affect HIF binding and expression of the oncogenic transcription factor MYC [127]; and polymorphisms at the 12p12.1 locus, which alter HIF-1 dependent expression of the basic helix–loop– helix transcription factor BHLHE41 (also known as DEC2) [138]. Furthermore, RCC-susceptibility polymorphisms have been identified at the 2p21 locus, lying in the first intron of the EPAS1 gene that encodes HIF-2α, although whether these affect HIF-2α expression remains unclear [130, 139]. Importantly, each renal cancer susceptibility locus affects a single component of the HIF pathway. This directly implicates these genes in the pathogenesis of kidney cancer. Furthermore, it helps distinguish them from HIF target genes with neutral effects on RCC susceptibility that might be simply co-activated as part of large pathway upregulation. Therefore, these analyses have highlighted specific 'driver' genes that may provide attractive targets for future therapeutic approaches or as biomarkers that might predict tumor behavior.
