**7. The HIF transcriptional response**

Once stabilized, both HIF-1α and HIF-2α, in complex with HIF-1β, are able to bind chromatin at either gene promoters or promoter-distant enhancers that contain one or more 5′-RCGTG-3′ recognition motifs, termed hypoxia response elements (HREs) [84, 85]. These short motifs are highly numerous across the genome and only a small proportion of accessible motifs are occupied by HIF, indicating that additional factors are involved in HIF DNA-binding [85]. HIF-binding sites may lie several hundreds of kilobases from the target promoter, interacting with it through chromatin looping, which can make it difficult to identify the transcriptional target of any given binding site. Therefore, much effort has been directed at determining both direct and indirect targets of the HIF transcriptional pathway in multiple settings, including in VHL-defective ccRCC cells, using both transcriptomic assays such as RNA-seq and assays of chromatin binding such as ChIP-seq [85–89].

These sequencing studies indicate that HIF acts as a gene activator rather than a repressor; causing the induction of hundreds to thousands of genes and triggering massive pathway activation [90–93]. These genes mediate diverse cellular functions including angiogenesis, erythropoiesis, glycolysis and the cell cycle [77, 94, 95]. This triggers a physiological response that enables cells to survive in low oxygen conditions. For example, HIF-dependent angiogenesis increases blood supply to oxygen-starved tissue; HIF-dependent erythropoiesis improves systemic oxygen delivery; HIF-dependent glycolysis allows cells to generate ATP in the absence of oxygen; and HIF-dependent cell cycle arrest can allow cells to conserve energy and reduce oxygen consumption.

Importantly, HIF-binding sites and HIF-regulated genes are highly cell-type specific. Thus, whilst HIF may regulate many hundreds of genes in any given cell type, only a small, core set of well-described genes are regulated in the majority of tissues [90, 93]. Furthermore, although both HIF-1α and HIF-2α share the same binding motif and their binding sites often overlap, HIF-1α tends to be more prevalent at gene promoters whereas HIF-2α is more prevalent at promoter-distant enhancers [90, 92]. In addition to this binding site specificity, post-DNA-binding mechanisms likely contribute to transcriptional selectivity between the two isoforms [96], such that specific genes may be regulated by either HIF-1α or HIF-2α only, even when both isoforms are bound [50, 97] For example, cyclin D1 (CCND1), transforming growth factor alpha (TGFA), vascular endothelial growth factor A (VEGFA), glucose uptake transporter 1 (SLC2A1/GLUT1), the MYC oncogene, and the stemness-related transcription factor OCT4/POU5F1 are specifically induced by HIF-2, whilst BCL2-interacting protein 3 (BNIP3) and carbonic anhydrase 9 (CA9) are positively regulated by HIF-1 [97–102].

Although primarily a physiological response, the HIF pathway is also relevant to the pathophysiology of cancer and many HIF target genes are central to the hallmarks of cancer described by Hanahan and Weinberg [103]. These include genes with prominent roles in angiogenesis, glycolysis, cell proliferation, cell invasion and immune evasion among other oncogenic processes (**Figure 3**). Indeed, HIF is activated in many types of solid tumor, largely as a result of intra-tumor hypoxia and is almost universally associated with a poor prognosis [104].

In particular, HIF promotes the metabolic switch from oxidative phosphorylation to anaerobic glycolysis by inducing a range of target genes, including those encoding transmembrane proteins that import glucose into the cell (SLC2A1/ GLUT-1 and SLC2A3/GLUT-3) as well as multiple catalytic enzymes in the glycolytic pathway [71]. Oxidative phosphorylation is oxygen-dependent, therefore switching to oxygen-independent glycolysis allows hypoxic cancer cells to generate energy. However, glycolysis causes accumulation of byproducts in the form of acidic metabolites, which can be toxic to cancer cells. Therefore, HIF also upregulates genes encoding transmembrane proteins that rebalance intracellular pH to promote cancer cell survival. For example, the HIF target genes CA9 and CA12, encoding carbonic anhydrases, generate alkaline sodium bicarbonate ions in the extracellular space [105]. Sodium bicarbonate can then be imported into cells by ion channels to counteract intracellular acidity. Furthermore, once a tumor outgrows its blood supply and becomes hypoxic, HIF induces genes encoding pro-angiogenic secreted factors, such as VEGFA and placental growth factor (PGF), that serve to transmit extracellular signals and stimulate blood vessel production [106]. This increases delivery of nutrients and oxygen to cancer cells, enabling the tumor to further expand. Furthermore, HIF has recently been found to upregulate genes that help cancer cells evade destruction by the immune system. One such example is CD274,

#### **Figure 3.**

*HIF target genes that promote or restrict tumorigenesis. HIF regulates hundreds to thousands of target genes, which mediate diverse and sometimes conflicting cellular processes. For example, such processes can either promote or restrict tumor growth. Those that are typically considered tumor-promoting processes are depicted in red, whereas those that are typically considered tumor-suppressive are depicted in green. Cellular processes that can be either tumor-promoting or -suppressive (depending on the context) are depicted in red and green. Exemplar HIF target genes involved in each process are listed. Note that whilst some HIF target genes appear to be consistent across cell types and conditions, others are context-dependent.*

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

encoding the transmembrane protein termed programed death ligand 1 (PD-L1), which is expressed in cancer cells [107]. PD-L1 interacts with its receptor termed programed cell death protein 1 (PD-1), which is expressed on the cell surface of T cells. The PD-L1/PD-1 interaction prevents T cell-mediated killing of cancer cells, therefore HIF may exacerbate this oncogenic mechanism.

However, since HIF evolved to mediate physiological responses to hypoxia, not all HIF target genes are advantageous in a cancer setting. Paradoxically, although HIF activates many pro-tumorigenic target genes, there are also anti-tumorigenic HIF targets (**Figure 3**). These may represent in-built tumor suppressor mechanisms that counterbalance oncogenic target genes when HIF is activated in response to physiological hypoxia. Tumor suppressive HIF target genes include BNIP3 and BNIP3L, which are pro-apoptotic proteins. BNIP3 and BNIP3L can promote either cell death or autophagy in response to hypoxia, depending on the context [108]. Furthermore, some HIF target genes may not influence cancer pathogenesis whatsoever and may represent genes that are only important in other contexts. This is epitomized by VHL loss in the earliest stages of ccRCC formation, which causes HIF activation in an inappropriate context (i.e. causing a cellular response to hypoxia when the cell is not hypoxic). In this setting, HIF causes a change in cell state that is unwarranted since the cell is exposed to normal oxygen levels. Therefore, many activated HIF target genes may confer no survival advantage or may even result in a "fitness penalty" to the cell in this context. Taken together, the overall consequences of massive HIF pathway activation in ccRCC will be a balance of many positive, neutral and negative effects [109]. The contribution of each effect may change during cancer pathogenesis as a result of subsequent somatic mutation, epigenetic events or changes in the tumor microenvironment allowing cancer cells to escape the long prodromal dormancy that occurs following VHL inactivation. Alternatively, the poise of the HIF transcriptional pathway may be partially pre-set prior to VHL inactivation due to cell-type specific differences in HIF target genes. In turn, this could render specific cell types particularly susceptible to VHL inactivation. Furthermore, genetic differences between individuals might alter specific HIF target genes, thus making that individual more or less susceptible to developing kidney cancer.

Activation of contrasting and aberrant pathways as part of large transcriptional programs is an emerging theme in cancer biology. For example, MYC, like HIF, has transcriptional targets with both oncogenic and tumor suppressive properties [110, 111]. Therefore, HIF activation in ccRCC serves as a model for studying large transcriptional cascades in cancer more generally.
