VHL and HIF in clear cell renal cell carcinoma: molecular abnormalities and potential clinical applications
Ronald M. Bukowski , Robert A. Figlin, Robert J. Motzer (Eds). Renal cell carcinoma. Molecular targets and clinical applications. 3 Ed. Springer Science+Business Media New York (2015)
Von Hippel-Lindau (VHL) disease (also known as VHL syndrome) is a hereditary, autosomal dominant, neoplastic disease caused by germline mutations in the VHL tumour suppressor gene . Patients inherit a single faulty copy of the gene, but the development of disease depends on spontaneous inactivation or loss of the second, wild-type VHL allele. VHL disease is associated with clear cell renal cell carcinomas (ccRCCs), central nervous system and retinal haemangioblastomas, phaeochromocytomas and pancreatic neuroendocrine tumours, in addition to pancreatic cysts, endolymphatic sac tumours and epididymal papillary cystadenomas.
Two key observations led groups to question whether mutations within the VHL gene are also responsible for the development of sporadic ccRCC. First, the leading cause of death in patients suffering from VHL disease is ccRCC . Second, the reintroduction of wild-type, but not mutant, VHL into RCC cell lines that lack the protein has no demonstrable effect on their growth in vitro but inhibits their ability to form tumours in nude mice [3–5]. Subsequently, it became clear that most sporadic ccRCCs do exhibit VHL alterations [6–9]. Consistent with the two-hit hypothesis of the tumour suppressor gene theory , biallelic inactivation of VHL occurs in the majority of sporadic ccRCCs due to a combination of somatic mutations, VHL promoter hypermethylation (which effectively turns off gene expression) and loss of heterozygosity (LOH) by allele deletion. In those ccRCCs which harbour VHL mutations, VHL is mutated ubiquitously in all areas of the tumour, supporting a key driver role for VHL mutations in the pathogenesis of most ccRCCs . This is in contrast to mutations in other proposed driver genes which are often not detectable across every region of the tumour.
The compelling correlation between VHL inactivation and the development of ccRCC has been the subject of extensive research for almost 20 years, resulting in a heightened appreciation of the intricate relationship between the tumour endothelial vascular network and ccRCC inception and progression. In turn, this has facilitated the development of a variety of targeted therapies for ccRCC which not only reduce tumour burden but also significantly improve the clinical outcome in patients with advanced disease.
In this chapter, we examine in detail the role of the VHL gene and protein in ccRCC, alongside that of its main downstream target, hypoxia-inducible factor (HIF).
VHL gene and protein
The VHL gene is located on 3p25, has been evolutionarily conserved and consists of 854 nucleotides in three exons  (Fig. 4.1). An alternative splice variant that lacks exon 2 has been described but is thought to lack tumour suppression activity . As a result of two alternative in-frame start codons, two pVHL isoforms exist in the cell: a 213-amino acid, 30 kDa form (pVHL30), and a 160-amino acid, 19 kDa form (pVHL19) [5, 13, 14]. pVHL19 lacks a 53-amino acid N-terminal pentameric acid repeat domain and seems to predominate in many tissues. Since both isoforms behave similarly in biochemical and functional assays and possess tumour suppressor activity in vivo , the term pVHL is used to describe both these proteins generically. Though pVHL shuttles between the nucleus and the cytoplasm, under steady-state conditions, most of the protein is located in the cytoplasm [4, 15–22]. Some pVHL is also found in mitochondria and associated with the endoplasmic reticulum [23, 24].
Fig. 4.1. VHL gene and protein structure. The VHL gene consists of 854 nucleotides in three exons. Two pVHL isoforms, pVHL19 and pVHL30, exist as a result of two alternative in-frame start codons. pVHL19 lacks an N-terminal (GXEEX) repeat domain, but both isoforms possess tumour suppressor activity in vivo
pVHL consists of two tightly coupled domains, a and β (Fig. 4.2). The β domain consists of a 7-stranded β-sandwich (amino acids 63–154) and an a-helix (residues 193–204) and has the properties of a substrate docking site . The a domain, residues 155–192, consists of three a-helices and binds elongin C; an a-helix of elongin C completes a folded leaf four-helix structure instigated by the three Pvhl a-domain helices . In turn, this nucleates a complex containing elongin B, cullin 2 (Cul2) and RING finger protein Rbx1 (VCB-CR complex) [17, 25–28]. The elongin B/elongin C complex thus acts as an adaptor that links a substrate-recognition subunit (pVHL β domain) to heterodimers of Cul2 and Rbx1. pVHL is directly stabilised by associating with elongins B and C, and in turn elongins B and C are stabilised through their interactions with each other and pVHL . The entire pVHL-elongin complex is thus resistant to proteasomal degradation. In contrast, VHL proteins harbouring mutations which disrupt elongin binding are unstable and rapidly degraded by the proteasome. Structurally, the VCB-CR complex resembles yeast Skp1-Cdc53-F-Box protein (SCF) ubiquitin ligases, and functionally, both the VCB-CR and SCF complexes have ubiquitin ligase activity and are capable of targeting proteins for proteasomal degradation [30, 31].
pVHL and hypoxia-inducible factors
The best-documented function of pVHL relates to its role as the substrate-recognition component of the VCB-CR E3 ubiquitin ligase complex. This complex is best known for its ability to target hypoxia-inducible factors (HIFs) for polyubiquitination and proteasomal degradation [32, 33] (Fig. 4.3). The alpha subunit of HIF interacts exclusively with the beta domain of pVHL, binding alongside the β-sandwich [34, 35] (Fig. 4.2). This binding is dependent on the hydroxylation of one of two conserved proline residues within HIFα by prolyl hydroxylases (PHDs) 1–3, which require oxygen as a co-substrate and are thus only active under normoxic conditions [35–39] (Fig. 4.4). Prolyl hydroxylation of HIFα enables its recognition and ubiquitination by the VCB-CR complex, and polyubiquitinated HIFs are recognised and degraded by the cellular proteasome (Fig. 4.3). Under hypoxic physiological conditions (or in the absence of functional pVHL), HIFα accumulates and forms heterodimers with HIF1Β. These heterodimers translocate to the nucleus where they bind to hypoxia response elements that contain the consensus sequence 5’RCGTG-3′ . Based on genome-wide chromatin immunoprecipitation combined with DNA sequencing or mRNA microarrays, the number of direct HIF target genes is currently greater than 800 [41, 42]; many of these genes promote adaptation to acute or chronic hypoxia . A list of selected HIF-induced genes with their functions is shown in Table 4.1. HIFs also indirectly regulate gene expression by transactivating genes encoding microRNAs  and chromatin-modifying enzymes [41, 43, 45].
Fig. 4.2. Ribbon diagrams illustrating the secondary structure of the VHL-elongin C-elongin B complex and interactions with HIFα. (a) Secondary structure of the VHL-elongin C-elongin B complex. pVHL (pink) consists of two tightly coupled domains, α and β. The β domain consists of a 7-stranded β-sandwich and an α-helix and has the properties of a substrate docking site. The α domain, residues 155–192, consists of three α-helices and binds elongin C (blue). The H4 helix of elongin C fits into an extended groove formed by the H1, H2 and H3 helices of the VHL a domain. The VHL-elongin C complex nucleates a complex containing elongin B (green), cullin 2 (Cul2) (not shown) and RING finger protein Rbx1 (not shown) (VCB-CR complex). (b) The HIFα hydroxyproline binding pocket of pVHL. A 15-amino acid portion of HIFα (yellow) adopts an extended beta strand-like conformation and interacts exclusively with the beta domain of pVHL (pink) binding alongside the beta sandwich. (b) Key interactions between pVHL and the HIFα hydroxyproline (HYP). The hydroxyproline of HIF binds in a pocket on pVHL lined by residues W88, Y98, S111, H115 and W117. The hydroxyl group of S111 and the H115 imidazole amino group serve as hydrogen-bonding partners to the HYP564 hydroxyl group. All of the residues that form the pocket are frequently mutated in ccRCC
Fig. 4.3. Oxygen-dependent HIF regulation. In normoxic conditions, HIFα is hydroxylated by prolyl hydroxylases (PHDs) 1–3. Prolyl hydroxylated HIFα is recognised by the VHL-elongin C-elongin B-cullin 2-Rbx1 (VCB-CR) E3 ubiquitin ligase complex and targeted for ubiquitination and proteasomal degradation. In hypoxic conditions, prolyl hydroxylases 1–3 are inactive. HIFα therefore accumulates and forms heterodimers with HIF1Β which translocate to the nucleus, bind to hypoxia response elements and induce transcription of genes involved in adaptations to hypoxia. Though HIF1α and HIF2α have significant overlap in function, they are not functionally redundant and activate different pathways to different extents
HIF thus plays a critical role in cellular adaptation to reduced oxygen tension; functional pVHL is necessary to switch off this adaptation under normoxic conditions. The loss of pVHL function, occurring, for example, secondary to biallelic inactivation of the VHL gene, impairs HIFα destabilisation. This promotes inappropriate activation of downstream target genes which would normally only be activated under hypoxic conditions and thereby contributes directly to tumorigenesis. This phenomenon provides an explanation for why pVHL-defective tumours including haemangioblastomas, phaeochromocytomas and ccRCCs are sometimes associated with paraneoplastic erythrocytosis . In keeping with the notion that the regulation of HIFα is the key tumour suppressor function of pVHL, a large proportion of disease-associated VHL mutations are predicted to and have been demonstrated to abolish the interaction between pVHL and HIF [34, 35, 47] (Table 4.4).
Other than HIFα, additional potential pVHL ubiquitination substrates, including atypical protein kinase C  and the large subunit of RNA polymerase II , have been described (reviewed within [32, 50–52]), though their significance in ccRCC tumorigenesis is uncertain.
Fig. 4.4. HIF transcription factors. HIF proteins are members of the basic helix-loop-helix (bHLH per-Arnt-SIM (PAS) family of DNA-binding transcription factors. The bHLH and PAS domains are involved in DNA binding and heterodimerisation; the oxygen-dependent degradation (ODD) domain is required for oxygen-dependent hydroxylation and degradation; and the N-terminal and C-terminal transactivation domains (NTAD and CTAD, respectively) are required for transcriptional activation. HIF1α and HIF2α both have two transcriptional activation domains. HIF1Β has just one transcriptional activation domain. The hydroxylation of conserved proline residues in the ODD of HIFα proteins by oxygen-dependent prolyl hydroxylase enzymes (PHDs) is required for pVHL to bind and degrade HIFα subunits under normoxic conditions. Hypoxia limits PHD activity. Hypoxia also inhibits hydroxylation of a conserved asparagine in the CTAD by factor-inhibiting HIF1 (FIH1); this blocks the interaction between HIFα and the transcriptional co-activators p300/CBp. FIH1 hydroxylates HIF2α at a lower efficiency (broken arrow) than HIF1α (unbroken arrow). HIF3α undergoes extensive mRNA splicing; many of the ensuing splice variants (e.g. IPAS) lack a transactivation domain and function as dominant-negative regulators of HIFs
The HIF transcription factors
Three HIFα family members (HIF1α, HIF2α, HIF3α) and two HIFβ family members (HIF1Β and HIF2β) exist (HIFβ is often referred to as ARNT [aryl hydrocarbon receptor nuclear translocator]) (reviewed in ). While HIF1α is ubiquitously expressed, the expression of HIF2α is restricted to endothelial, lung, renal and hepatic cells. HIF proteins are part of the basic helix-loop-helix PER-ARNT-SIM (PAS) family of DNA-binding transcription factors (Fig. 4.4). HIF1α and HIF2α both have two transcriptional activation domains: the N-terminal transactivation domain (NTAD) and the C-terminal transactivation domain (CTAD) [52, 53].
Table 4.1. Selected shared and unique target genes regulated by HIF1α and HIF2α in ccRCC
In contrast, HIF3α undergoes extensive mRNA splicing; many of the ensuing splice variants lack a transactivation domain and can competitively inhibit transcriptional activation by HIF1α and HIF2α [54–57], although little is yet known about the impact of HIF3α on hypoxic tumour progression.
HIF1α, but not HIF2α, can also be recognised by at least two hydroxylation-insensitive ubiquitin ligase complexes that do not contain pVHL [58, 59] (Fig. 4.5). Firstly, HIF-αssociated factor (HAF) binds and destabilises HIF1α under normoxic and hypoxic conditions in a pVHL-independent, proteasome-dependent manner, but has no effect on HIF2α levels. Instead, HAF binds HIF2α at a distinct C-terminal region and promotes HIF2α transcriptional activity, effectively switching cells from an HIF1α to an HIF2α transcriptional programme. Secondly, heat shock protein 70 (HSP70) and carboxyl terminus of Hsc70-interacting protein (CHIP), a recently identified E3 ubiquitin ligase, bind and degrade HIF1α (but not HIF2α) under conditions of prolonged hypoxia in cultured cells [58, 60]. In addition, the hydroxylation of HIFα can occur on a conserved asparaginyl residue within the CTAD by the asparaginyl hydroxylase factor-inhibiting HIF1 (FIH1). This hydroxylation prevents its interaction with the transcriptional co-activator p300 and thereby impairs CTAD activity [61–63]. Though the asparaginyl hydroxylation reaction also requires molecular oxygen, FIH1 remains active at intermediate levels of hypoxia which would render the prolyl hydroxylases inactive . On the whole, FIH1 seems to play a role in fine-tuning the hypoxic response [65, 66]. The HIF1α CTAD is more sensitive to FIH1 than the HIF2α CTAD [67, 68]. Consequently, different HIF target genes exhibit different sensitivities to FIH1 inhibition, presumably resulting from their relative dependency on HIF1α versus HIF2α, and/or on NTAD versus CTAD activity. Interestingly, some HIF target genes are induced by HIF in a broad variety of cells and tissues, while others are more constrained. For example, the expression of erythropoietin in adults is principally restricted to specialised cells in the kidney.
Fig. 4.5. Role of VHL, HIF1α and HIF2α in clear cell renal cell carcinoma. (a) In normoxic conditions, pVHL inhibits activity of both HIF1α and HIF2α by targeting them for ubiquitination and degradation. Evidence suggests that on balance, HIF2α is a renal oncoprotein which promotes growth of ccRCC, while HIF1α is a renal tumour suppressor which inhibits growth of ccRCC. Biallelic loss of VHL secondary to a combination of mutation, promoter methylation and/or LOH results in increased HIF1α and HIF2α levels. (b) Loss of HIF1α’s tumour-suppressive activity relative to HIF2α’s oncoprotein activity may result from the following: (1) Reduced HIF1α expression or activity secondary to 14q deletion, deletion of the HIF1α locus or loss-of-function mutations in HIF1α. (2) The greater sensitivity of HIF1α to FIH1 compared to HIF2α. HIF1α would therefore theoretically be silenced by FIH1 in VHL null cells unless those cells were profoundly hypoxic. (3) HIF-αssociated factor (HAF) binds and destabilises HIF1α but promotes HIF2α activity. (4) Heat shock protein 70 (HSP70) and carboxyl terminus of Hsc70-interacting protein (CHIP) bind and degrade HIF1α but not HIF2α. The gene sets regulated by HIF1α and HIF2α overlap but are not entirely congruent. HIF1α targets may be biased towards ccRCC tumour suppressors, while HIF2α targets might be biased towards ccRCC oncoproteins. (c) Alternatively, it is possible that the differences between HIF1α and HIF2α result from differential regulation of HIF target genes. For example, in some systems, while HIF1α suppresses c-Myc activity, HIF2α enhances c-Myc activity. Similarly, HIF1α enhances and HIF2α suppresses p53 function
HIF2α is more oncogenic than HIF1α
Both HIF1α and HIF2α are stabilised and activated by hypoxia and dimerise with HIF1β. Likewise, both isoforms activate transcription of target genes by binding to the same hypoxia response element. However, although significant overlap in their function exists, HIF1α and HIF2α are not functionally redundant. Array studies indicate that while HIF1α induces apoptotic pathways not targeted by HIF2α and preferentially drives the expression of genes involved in the glycolytic pathway, HIF2α preferentially promotes growth and angiogenesis [69–71]. Furthermore, the relative contributions of the two paralogs to the control of specific HIF target genes can differ in different cellular contexts. For example, VEGF is primarily regulated by HIF2α in pVHL-defective renal carcinoma cells but by HIF1α in breast cancer cells .
Moreover, in vitro and cell line xenograft studies suggest that although HIF2α is both necessary and sufficient for the growth of transformed RCC cell lines, HIF1α is not (reviewed in [72, 73]). There are four lines of evidence for this. First, all VHL null ccRCC cell lines examined to date express HIF2α, while not all express HIF1α [74, 75]. Second, the downregulation of HIF2α expression (using short hairpin RNAs delivered by a viral vector) in human VHL null RCC cells is sufficient to prevent tumour formation in nude mice [76, 77]. Conversely, the overproduction of HIF2α but not HIF1α can override pVHL’s tumour suppressor activity in such xenograft assays [70, 76, 78]. Third, in animal models, HIF2α variants that lack prolyl hydroxylation sites (and cannot therefore be targeted for ubiquitination by the E3m ubiquitin ligase complex) prevent tumour inhibition by pVHL, whereas analogous HIF1α variants do not [78, 79]. Finally, the levels of HIF2α are highest in cells engineered to produce variants of pVHL associated with type 1 and type 2B VHL disease (associated with a high risk of RCC), intermediate in cells with forms of pVHL linked with type 2A VHL disease (associated with a low risk of RCC) and essentially normal in cells with pVHL mutations seen in type 2C disease (which are not associated with ccRCC) [80, 81]. HIF2α seems to be both necessary and sufficient for much of the pathology that has been described in genetically engineered mouse models in which VHL has been inactivated in specific tissues [82–86]. Interestingly, HIF2α polymorphisms have recently been linked to the risk of developing kidney cancer in the general population .
HIF1α is a renal tumour suppressor gene
Four further lines of evidence suggest that HIF1α is not merely dispensable in the context of ccRCC but actually functions as a tumour suppressor gene. First, targeted exon sequencing of ccRCC has shown rare inactivating mutations in HIF1α , and copy-number analyses of RCC cell lines and primary tumours suggest that the HIF1α locus is frequently lost, either alone or along with the long arm of chromosome 14 (14q) where the HIF1α gene resides . The loss of chromosome 14q in this setting is associated with a poor prognosis [89, 90]. In other cases, alternative mRNA splicing around deleted HIF1α exonic sequences leads to the production of aberrant HIF1α isoforms . Second, though all VHL-defective ccRCCs overexpress HIF2α, approximately one third of these tumours also lack HIF1α expression . Third, functional in vitro and in vivo studies suggest that the overexpression of HIF1α in VHL wild-type cells restrains tumour growth, whereas the suppression of HIF1α in VHL-deficient cells enhances tumour growth [75, 91]. Finally, though uncommon, HIF1α mutations have been described in ccRCC. These include missense mutations which compromise HIF1α’s ability to suppress proliferation when reintroduced into ccRCC cell lines that lack endogenous, wild-type, HIF1α [75, 77, 88].
In keeping with these findings, many ccRCCs produce no, or very low, levels of HIF1α, and 14q-deleted tumours exhibit a transcriptional signature indicative of decreased HIF1α activity [74, 75, 92]. However, in contrast to ccRCC cell lines, ccRCC tumours often appear to retain a wild-type HIF1α allele , suggesting that HIF1α haploinsufficiency is sufficient to promote tumorigenesis in vivo. Since many ccRCC cell lines are established from metastatic lesions, it is possible that reduction to nullizygosity is a late event in renal carcinoma and thus underrepresented in primary tumours, particularly in patients with early disease who have undergone a nephrectomy with curative intent.
Collectively, these observations suggest that while HIF1α is a tumour suppressor gene in renal cancer development and is one of the relevant targets of the 14q deletions that are typical of ccRCC, HIF2α is the key driver of renal cancer progression.
Differences between HIF1α and HIF2α
Explanations for the difference in oncogenicity between HIF2α and HIF1α may relate to the relative resistance of the HIF2α CTAD to FIH1 compared with the HIF1α CTAD; HIF2α may be able to escape from proteins such as FIH1 that would otherwise limit HIFα activity in cells lacking VHL (Fig. 4.5). In contrast, the HIF1α CTAD would theoretically be silenced by FIH1 in VHL null ccRCC cells, unless the cells were severely hypoxic. A transcriptionally inactive HIF1α could, in principle, act as a dominant negative, by competitively displacing HIF2α, which is relatively insensitive to FIH1, from specific HIF target genes. In support of this theory, some HIF target genes are paradoxically increased when HIF1α is downregulated in VHL null RCC cells , and the HIF2α NTAD and CTAD cooperate to promote renal tumorigenesis in vivo . The differential sensitivities of HIF1α and HIF2α to HAF, HSP70 and CHIP may also play a role.
Alternatively, it is possible that some genes that are preferentially activated by HIF2α relative to HIF1α are particularly oncogenic. One which has attracted significant interest relates to the opposing roles of HIF1α and HIF2α in the regulation of c-Myc activity; while HIF1α suppresses c-Myc activity, HIF2α promotes the transactivation or transrepression of c-Myc-specific target genes [74, 93, 94]. In keeping with this notion, RCC tumours that exclusively express HIF2α have increased proliferation rates . Intriguingly, a subset of ccRCC tumours seem to have copynumber amplification of 8q24 where c-Myc resides .
Similarly, HIF target genes that are regulated primarily by HIF1α may suppress ccRCC growth. For example, HIF1α enhances and HIF2α suppresses p53 function [96, 97]. Other potential tumour suppressor genes that are regulated by HIF1α in VHL null ccRCCs include BNIP3, REDD1, TXNIP and ZAC1 . Interestingly, ZAC1 maps to chromosome 6q23, which is often deleted in VHL-associated renal cancers, haemangioblastomas and phaeochromocytomas. Other potential tumour suppressor genes that are regulated by HIF1α in VHL null ccRCCs include BNIP3, REDD1, TXNIP and ZAC1 . Interestingly, ZAC1 maps to chromosome 6q23, which is often deleted in VHL-associated renal cancers, haemangioblastomas and phaeochromocytomas [98, 99].
HIF-independent functions of pVHL (Table 4.2)
Despite pVHL’s well-characterised role in targeting HIFs for polyubiquitination and proteasomal degradation, evidence has accrued to indicate that pVHL also has functions independent of HIF1α and HIF2α that may be important for its tumour suppressor action. These include the assembly and regulation of the extracellular matrix, microtubule stabilisation and maintenance of the primary cilium, regulation of apoptosis, control of cell senescence and transcriptional regulation (Table 4.2).
Table 4.2. Mechanisms that involve pVHL
These roles are less thoroughly characterised than those that involve HIFs, and many have been discovered through biochemical interactions. Nonetheless, gene expression studies also support the notion that there are HIF-independent gene expression changes induced by VHL loss [100, 101], though to what extent the HIFindependent functions of pVHL cooperate with HIF dysregulation in ccRCC tumorigenesis currently remains unknown.
VHL and regulation of the extracellular matrix
One of the better characterised, though still incompletely understood, HIFαindependent roles of pVHL is its effect on the assembly of the extracellular matrix (ecm). The ecm is a complex structural component, composed of proteoglycans, hyaluronic acid and glycoproteins such as fibronectin and collagen . The disruption of its architecture has been associated with tumour growth, angiogenesis and metastasis, and pVHL plays an important role in its regulation. pVHL interacts directly with fibronectin and collagen IV, and all pVHL mutants tested to date fail to bind fibronectin and collagen IV and lose the ability to assemble an ecm [80, 81, 103–108]. The inability of VHL-deficient cells to bind ecm components is not mediated by HIF [103, 107, 109] and does not require binding to the other components of the pVHL complex such as elongins B and C and cullin 2.
The inactivation of the VHL-ecm assembly pathway results in highly vascularised tumours with a remodelled fibronectin and collagen IV matrix and increased invasive capacity , and it has been speculated that ecm remodelling may promote angiogenesis by providing a path for blood vessels to in?ltrate tumours, thereby supporting tumorigenicity. This is in contrast to inactivation of the VHLHIFα regulation pathway, which results in tumours with high VEGF levels but decreased angiogenesis, a tightly assembled fibronectin and collagen IV matrix and low invasive capacity.
The interaction of pVHL with fibronectin is mediated by pVHL neddylation  which acts as a molecular switch in conferring selectivity to fibronectin binding over CUL2 . The interaction of pVHL with collagen IV is dependent on endoplasmic reticulum hydroxylation  and is competed by hydroxylated, but not unmodified, HIF1α peptides, implying that this interaction requires the hydroxyproline binding pocket in the β domain of pVHL. pVHL does not affect fibronectin and collagen IV production or secretion and does not result in collagen IV proteasomal degradation [105, 106].
It is well recognised that tumour cell invasion is dependent on the adhesion and proteolytic remodelling of the ecm, and it is now known that pVHL influences both these processes. VHL promotes E-cadherin transcription through HIF-dependent mechanisms , and inactivation of pVHL results in downregulation of the adherens junction protein E-cadherin . VHL inactivation also leads to downregulation of the tight junction proteins occludin and claudin in an E-cadherin-independent manner , and pVHL has been reported to downregulate integrins in an HIFα-independent manner . As a consequence of these mechanisms, pVHL inactivation results in the disruption of both adherens and tight junctions. Cells lacking pVHL also fail to form β1 ?brillar adhesions, which may contribute to the increased cell motility and invasiveness seen in the absence of a functional pVHL .
VHL also regulates the matrix metalloproteinases, a family of matrix-degrading enzymes involved in ecm turnover. Firstly, HIF2α has been shown to influence RCC cell invasiveness by regulating membrane type 1 MMP expression [115, 116]. Secondly, the loss of VHL-ECM pathway regulation in RCC cells results in increased cell invasiveness and activation of MMP-2 . Compared to VHL WT ccRCC cell lines, VHL null ccRCC cells show increased invasiveness in growth factor-reduced Matrigel, overproduce MMP-2 and MMP-9 and display an extensive branching morphogenesis phenotype in response to hepatocyte growth factor/ scatter factor . MMP-induced proteolytic remodelling of the ecm has been shown to expose cryptic sites in collagen IV which are required for in vivo angiogenesis .
As yet, the precise mechanisms of the interplay between pVHL, the ecm and suppression of tumorigenesis, angiogenesis and invasiveness are not resolved. In particular, how an intracellular protein such as pVHL can modulate the assembly of the extracellular ecm components remains to be elucidated. pVHL may mediate fibronectin and collagen IV modification to allow their proper assembly into the ecm. Loss of these interactions resulting from loss of VHL would lead to an aberrant ecm, activation of MMPs, release of ecm-sequestered growth factors and stimulation of tumorigenesis, angiogenesis and invasion. The disruption of integrins and cell adhesion molecule regulation would further enhance the invasive RCC phenotype. In principle, a more complete understanding of the mechanisms of ecm regulation by pVHL could lead to novel therapies for patients with ccRCC, though extensive future work in this field is required.
Microtubule stabilisation and maintenance of the primary cilium
pVHL can also associate with microtubules . This association has been reported to result in microtubule stabilisation  and directional growth of microtubules towards the cell periphery  and appears to be independent of pVHL’s ability to downregulate HIF or its ubiquitin ligase function. In turn, this may explain the loss of primary cilia seen in renal cysts from VHL disease patients and ccRCC cell lines devoid of functional pVHL [120–122]. The primary cilium is a specialised structure on the cell surface that acts as an antenna of the cell and regulates the transduction of both chemical and mechanical signals . The ciliary axoneme is composed of microtubules that are arranged out from the basal body or mother centriole; thus, microtubule dynamics and formation and maintenance of the primary cilium are intimately linked.
An interaction between pVHL and the Par3-Par6-atypical protein kinase C polarity complex has been suggested as a mechanism for linking polarity pathways to microtubule capture and ciliogenesis . The phosphorylation of pVHL by glycogen synthase kinase 3β (GSK3β) has been reported to prevent pVHL from stabilising microtubules, without disrupting their interaction with pVHL . This phosphorylation occurs on Ser68, after a priming phosphorylation at Ser72 by an unidentified kinase. One hypothesis is that GSK3β maintains cilia independently of pVHL. However, when GSK3β is inactivated, pVHL is active and can regulate the microtubules and primary cilia independently of GSK3 . Interestingly, a pVHL variant with phosphomimetic substitutions at Ser68 and Ser72 was also impaired with respect to HIF polyubiquitination, suggesting GSK3β may regulate more than one pVHL function. In keeping with the notion that GSK3β and pVHL redundantly maintain primary cilia, it appears that the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney ; the combined loss of VHL and PTEN in a genetically engineered mouse model cooperate to promote renal and genital tract cysts. pVHL’s effects on microtubule dynamics appear to be HIF independent, though some studies suggest that HIF dysregulation may play at least a partial role in the loss of microtubule stability imparted by VHL inactivation [121, 122, 125].
Surprisingly, pVHL’s ability to stabilise microtubules is lost in VHL mutations that predispose to the development of haemangioblastomas and phaeochromocytomas, but not those associated with the development of ccRCC . This apparent paradox, whereby VHL mutants predisposing to RCC maintain the ability to regulate microtubule dynamics, is perplexing. It has been suggested that renal cysts which develop secondary to the loss of primary cilia on renal tubular cells lack significant malignant potential and that the majority of ccRCCs associated with VHL disease may arise without a preceding cystic phase . This speculation is not proven, though, to some extent, it is in keeping with the observation that patients with polycystic kidney disease, despite having large numbers of renal cysts, are not clearly at a significantly higher risk of developing ccRCC .
Regulation of apoptosis
In comparison to the majority of other tumour types, ccRCCs are insensitive to cytotoxic chemotherapies. Failure of cytotoxic chemotherapy is tightly associated with failure of p53-mediated apoptosis . However, p53 mutations or loss is rare in ccRCC [129–131]. Subsequently, groups have considered whether HIF or pVHL is able to influence p53 function or activate alternative anti-apoptotic pathways in ccRCC.
Indeed, pVHL loss has been demonstrated to result in p53 inactivation by both HIF-dependent and HIF-independent effects. Firstly, HIF can directly bind to and modulate p53 activity [132–134]. Secondly, pVHL has been shown to directly associate with and stabilise p53 by suppressing Mdm2-mediated ubiquitination and nuclear export of p53 and by subsequently recruiting p53-modifying enzymes, resulting in an increase in its transcriptional activity . VHL-deleted RCC cells show attenuated apoptosis and abnormal cell cycle arrest upon DNA damage, which normalises on restoration of pVHL .
Resistance to chemotherapy-induced apoptosis is also mediated through the nuclear factor kB (NF-kB) pathway. pVHL has been shown to facilitate TNFα-induced cytotoxicity in RCC cells, at least in part, through the downregulation of NF-kB activity and subsequent attenuation of anti-apoptotic proteins c-FLIP, survivin, c-IAP-1 and c-IAP-2 [137, 138]. pVHL’s effect on NF-kB is at least in part dependent on HIF signalling . In addition, pVHL can modulate NF-kB activity directly by serving as an adaptor that promotes the inhibitory phosphorylation of the NF-kB agonist CARD9 by casein kinase 2 .
Interestingly, 11 % of apparently sporadic phaeochromocytomas (defined by a lack of a family history or a spectrum of tumours suggestive of VHL disease) are actually due to occult germline mutations of VHL . However, somatic VHL mutations are uncommon in truly sporadic phaeochromocytomas. Furthermore, type 2C VHL disease mutations (which are associated only with phaeochromocytomas and not with other tumour types) retain their ability to downregulate HIFα [80, 81], suggesting that the development of VHL-associated phaeochromocytomas is related to an HIF-independent function of pVHL. Phaeochromocytomas derive from sympathetic neuronal precursor cells, many of which undergo c-Jun-dependent apoptosis during normal development as nerve growth factor (NGF) becomes limiting. Phaeochromocytoma-associated VHL mutations result in the HIF-independent accumulation of JUNB, which is known to blunt neuronal apoptosis during NGF withdrawal . Failure of developmental apoptosis may thus play a role in the development of phaeochromocytomas in patients inheriting phaeochromocytomaassociated VHL mutations.
Control of cell senescence
Cellular senescence is the phenomenon of irreversible growth arrest in response to DNA damage and is an important in vivo tumour suppressor mechanism . Studies have shown that the stabilisation of HIF occurring as a consequence of physiological oxygenation can extend the replicative lifespan of cells in culture [144, 145]. However, acute VHL inactivation, which would also result in the stabilisation of HIFα, has been shown to cause a senescent-like phenotype in vitro and in vivo . Interestingly, this phenotype was independent of p53 and HIF but dependent on the retinoblastoma protein (Rb) and the SWItch/Sucrose NonFermentable SWI2/SNF2 chromatin remodeller p400. This finding is somewhat surprising, since the induction of senescence would be expected to restrict the development of renal carcinoma in vivo. A subsequent study demonstrated that the induction of senescence secondary to VHL loss occurs under atmospheric conditions (21 % O2), but not under physiological oxygenation (2–5 % O2), suggesting that VHL inactivation sensitises cells to oxidative stress . The authors suggest that in vivo oxygenation may promote a tolerance of VHL loss in renal epithelia, which may allow cells to progress further towards a transformed state.
VHL has been shown to mediate the ubiquitination of the large subunit of RNA polymerase II, Rpb1, in response to oxidative stress, in a manner dependent on the hydroxylation of a specific proline [49, 148]. VHL has also been suggested to regulate transcription through a controlling influence on the RNA-binding protein HuR [149–151] and has been reported to bind the SP1 transcription factor [152–154].
It has long been known that the proper folding and functionality of pVHL requires its tight association with elongins B and C to form the VCB complex and that failure of correct folding and interaction with elongins B and C results in the proteolytic degradation of pVHL .
Following synthesis on ribosomes, nascent VHL is shuttled from the ribosomal machinery with the assistance of heat shock protein 70 (HSP70) . Formation of the VCB complex is then mediated by the chaperonin TCP-1 ring complex (TRiC; also called chaperonin-containing TCP-1 [CCT]) , a hetero-oligomeric complex which consists of two stacked rings with a central chamber in which unfolded polypeptides bind and fold . TRiC facilitates VHL folding, thereby enabling its association with elongins B and C to form the VCB complex which develops while VHL is bound to TRiC . Upon the formation of a mature VCB complex, pVHL is released from TRiC.
The binding of pVHL to TRiC occurs at amino acids 114–119 and 148–155 (called Box 1 and Box 2, respectively) . These two motifs are located in adjacent strands of the β domain, and both harbour tumour-associated mutations (e.g. W117A) that disrupt the association of pVHL with TRiC and lead to misfolding of newly translated pVHL and the absence of a mature VCB complex in the cell [158, 159]. Failure to generate a properly folded pVHL or a mature VCB results in the degradation of pVHL through the ubiquitin-proteasome system. pVHL degradation specifically requires another chaperone, Hsp90, which does not participate in pVHL folding . Since distinct chaperone pathways mediate the folding and quality control of pVHL, an enhanced understanding of the mechanisms by which destabilised pVHL mutants are targeted for proteasomal degradation may lead to strategies for refolding and stabilisation of pVHL, to allow its incorporation into the VCB complex and potential restoration of its tumour suppressor activity. Bortezomib and MG132 are both capable of increasing VHL levels, and a cell-based Prestwick compound screen has identified several compounds that upregulate VHL-W117A in VHL-W117A-infected cell lines . Further work is underway to analyse the functional consequence of pVHL upregulation using these compounds, as well as attempt to identify new compounds which rectify the interaction between point-mutated pVHL and the chaperones and chaperonins, and it is conceivable that such compounds may resuscitate the function of pVHL and thereby alter the disease phenotype and provide clinical benefit for patients with lesions possessing certain missense VHL mutations.
Genotype-phenotype correlations in von hippel-lindau disease
In VHL disease, there is clear evidence for strong genotype-phenotype correlations with specific classes of VHL mutations predisposing to different spectrums of morbidity and mortality (reviewed within , Table 4.3). While true null VHL alleles (i.e. large genomic deletions, frameshift mutations or nonsense mutations) are associated with a low risk of phaeochromocytoma (type 1 VHL disease), the majority of VHL mutations identified in families with an increased risk of phaeochromocytoma (type 2 VHL disease) are missense mutations. Type 2 VHL disease is further subdivided into type 2A (low risk of ccRCC), type 2B (high risk of ccRCC) and type 2C (phaeochromocytoma but no other manifestations of VHL disease). Subsequent analysis has suggested that surface amino acid substitutions confer a higher phaeochromocytoma risk than substitution of amino acids buried deep within the protein core .
Furthermore, the risk of developing ccRCC in VHL disease appears to be linked to the degree to which HIF activity is compromised ([47, 80, 81, 163, 164]. While type 1 and type 2B mutations (which are associated with a high risk of developing ccRCC) are grossly defective with respect to HIF regulation, type 2A mutations
Table 4.3. Genotype-phenotype correlations in VHL disease
(associated with a low risk of ccRCC) appear to be far less compromised with respect to HIFα regulation. Perhaps the clearest demonstration of this phenomenon is seen by undertaking a detailed biochemical analysis of the two most frequent type 2A mutations, Y98H and Y112H, in comparison to type 2B mutations in the same residues, Y98N and Y112N . While none of these mutations affected the assembly of the VCB complex, the type 2A mutant proteins exhibited higher stabilities at physiological temperature and higher binding affinities for HIF1α compared with the type 2B mutant proteins. Consistent with these results, the type 2A but not type 2B mutant VHL proteins retained significant ubiquitin ligase activity towards HIF1α in vitro .
Fascinatingly, individuals with homozygosity for the germline R200W VHL mutation develop Chuvash polycythaemia, a rare benign congenital erythrocytosis with no associated cancer risk [165, 166].
Therapeutic implications of pVHL in ccRCC
It has long been known that angiogenesis is a critical component for malignant tumour progression . This observation coupled with the hypervascular nature of ccRCCs led to intense interest in the molecular mechanisms of angiogenesis in ccRCCs. It is in this context that the discovery of the VHL gene and the identification of its critical role in regulating the HIF-mediated response to hypoxia have facilitated the dramatic recent shift in paradigm for the treatment of ccRCC. As described earlier, the inactivation of VHL triggers pro-angiogenic mechanisms through the activation of HIF with subsequent activation of transcription of many pro-angiogenic factors including the vascular endothelial growth factor (VEGF) family of proteins and TGFα. Numerous subtypes of VEGF exist (including VEGF-Agreed, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor-1) (reviewed within ). Most of these are regulated by pVHL and HIF and play key roles in angiogenesis by binding to cell membrane-associated tyrosine kinases, the VEGF receptors. The binding of VEGF ligand to its receptor initiates the activation of downstream signalling pathways which ultimately lead to endothelial cell activation, proliferation, migration and survival.
Drugs which modulate the VHL-HIF-VEGF pathway have proven benefit in treating ccRCC and are now the standard of care for patients with metastatic disease, with established superiority over cytokine therapies (reviewed within [72, 169–171]. Such drugs include sunitinib, sorafenib, pazopanib and axitinib (multiple tyrosine kinase inhibitors which inhibit the VEGFRs among others), inhibitors of the mTOR pathway such as temsirolimus and everolimus and the monoclonal anti-VEGF antibody bevacizumab. The mechanisms of action, benefits and toxicities of these drugs are detailed further in chapter x.
In general, these targeted therapies increase progression-free survival (PFS) compared with placebo or immunotherapy . However, very few trials have performed a direct head-to-head comparison of different targeted therapies or investigated which agents are most suitable for particular patient types, for example, patients at different levels of risk or different ages. The development of validated predictive and prognostic markers would be particularly valuable to optimise clinical management strategies and enhance clinical efficacy and cost-effectiveness. In ccRCC, the most widely used prognostic and predictive tools are based around the Motzer criteria or the Memorial Sloan Kettering Cancer Center risk status , which were developed in the immunotherapy era. In 2009, Heng et al.  conducted a large multi-centre study of 645 patients to better define the prognostic indicators for overall survival in mccRCC patients treated with VEGF-targeted therapy. They showed that six factors (performance status, haemoglobin levels, serum calcium concentration, time from diagnosis, neutrophil count and platelet count) could segregate patients into three prognostic categories: favourable, intermediate and poor prognosis groups. This model has since been externally validated and performs favourably compared with the MSKCC model . However, even this model performs only moderately well (concordance index for overall survival 0.71), begging the question as to whether it is possible to achieve better discrimination in outcomes.
On this note, it is highly likely that the addition of patient-specific or tumourspecific genetic biomarkers could help. These biomarkers can be broadly classified as prognostic markers (those mainly associated with the course or outcome of a disease) or predictive markers, which can be used to identify subpopulations of patients who are most likely to respond to a given therapy. Increasingly, treatments for other cancers are targeted to patients with alterations in specific molecular pathways known to be important in the pathogenesis of these tumours (reviewed within ). For example, a breast cancer with amplification of HER2 might be treated with the anti-HER2 monoclonal antibody trastuzumab or the HER2 tyrosine kinase inhibitor lapatinib. In contrast, there are currently no validated molecular markers used to guide therapeutic decisions in ccRCC. We now discuss whether VHL mutational status may be a clinically useful genetic biomarker in sporadic ccRCC.
Functional loss of VHL in sporadic ccRCC
The reported incidence of somatic VHL gene mutations in sporadic ccRCC varies from 18 % to 91 % in various studies [79, 176–191]. The reported frequency on COSMIC at the time of writing is 1,485/3,479 ccRCC samples (46.8 %), though this includes many older studies which used less sensitive sequencing methods . In addition, the methylation of VHL resulting in gene silencing occurs in between 5 and 30 % of sporadic ccRCC cases, and LOH occurs in up to 98 % of sporadic ccRCC cases [7, 188]. The wide variation in the reported prevalence of mutations may be explained by numerous confounding factors including the patient population examined, tumour histopathology, ratio of tumour to normal DNA in a sample and the method and depth of sequencing. As methods for detecting VHL gene alterations improve, the reported frequency of mutations in VHL is increasing.
Fig. 4.6. Spectrum of missense mutations in sporadic ccRCC. Distribution of coding mutations in VHL found in sporadic ccRCC samples as listed on COSMIC on 17 July 2013. The amino acid positions at which mutations were most commonly found are highlighted
Over 900 different mutations have been identified in sporadic ccRCC and VHL disease [129, 159]. More than half of these are frameshift and nonsense mutations which are likely to cause the loss of pVHL function. However, in total, nearly 200 different missense mutations have been described in sporadic ccRCC. Since both pVHL19 and pVHL30 possess tumour suppressor activity in vivo, it is unsurprising that mutations are rare in the first 53 amino acids . Otherwise, missense mutations are distributed fairly evenly across the three exons of VHL, with no dramatic hotspots for mutations (Fig. 4.6).
Dissecting the impact of missense mutations
The simplest assumption is that all missense mutations disable pVHL’s activity in equal amounts. However, numerous published studies which have examined the functional effects of VHL missense mutations in vitro and in cell culture systems demonstrate that this is not the case (Table 4.4). In fact, experimental data suggest that the impact of missense mutations on the function of pVHL is highly diverse, ranging from imperceptible to complete functional loss, particularly with respect to stability of the VCB complex and effects on HIFα ubiquitination and degradation. Interestingly, some mutations selectively influence HIF1α and HIF2α degradation .
To date, the preponderance of evidence can classify most missense mutations into four clear classes: (1) mutations which interfere with the binding of VHL to HIFα (e.g. Y98H, Y112H) [34, 35, 47], (2) mutations which inhibit the interaction between pVHL and elongins B and C (e.g. L158P, C162F and R167W) [17, 29, 80, 81, 193, 194], (3) mutations which inhibit the interaction with TRiC (e.g. G114R and A149P)  and (4) mutations which severely destabilise pVHL (e.g. G93D, W117R, L101P) . However, not all mutations fit into this classification system; indeed, there are a significant number of mutations described in sporadic ccRCC which appear to behave similarly to wild-type pVHL with respect to HIFα regulation and formation of the VCB complex  (Table 4.4). The most likely explanation is that these mutations are simply passenger mutations which don’t influence tumour growth, though the possibility remains that they may somehow interfere with HIFα-independent functions of pVHL.
Table 4.4. Functional effects of missense VHL mutations as determined experimentally
Mutations in non-coding regions of VHL
There is accumulating evidence that somatic mutations in non-coding regions of DNA may be pathogenic in other tumour types [195–208]. Furthermore, recent studies report that changes in the non-coding regions of VHL may have important physiological effects , suggesting that mutations in non-coding regions of VHL may be important physiologically. To date, there are 75 confirmed somatic variants (of which 47 are unique) in non-coding regions of VHL in tumour samples listed on COSMIC as of 13 June 2013 , and an additional study reports 49 somatic variants in non-coding regions of VHL in 37/128 sporadic ccRCC patients .
VHL as a biomarker in ccRCC
For several years, groups have been addressing the question as to whether VHL mutational status, namely, the presence or absence of mutation, the type of mutation/alteration or the effect of the mutation/alteration on the function of pVHL, is a useful biomarker in ccRCC. Are so-called “loss-of-function” mutations, such as nonsense or frameshift mutations, associated with a different prognosis to missense mutations, some of which appear to cause minimal changes in VHL function? Can VHL mutational status be used to predict response to treatment? Finally, do mutations in non-coding regions of VHL have important functional effects?
VHL alterations as potential prognostic markers in ccRCC
Numerous studies have investigated whether or not functional loss of VHL may influence prognosis in ccRCCs (Table 4.5). ccRCCs in VHL disease have an earlier age of onset than cases of sporadic ccRCC. However, they also appear to grow more slowly than cases of sporadic ccRCC and are associated overall with a better prognosis [210, 211]. However, while the results of some studies appear to support this hypothesis in principle [182, 187, 188, 212], others have found no association between the presence or absence of VHL alterations and prognosis or other adverse clinicopathological features ([79, 176, 178, 179, 181, 183–186 189–192, 213]), and one study reported that VHL mutation/hypermethylation was associated with advanced tumour stage  (Table 4.5). Overall, there is certainly no clear evidence that the presence or absence of VHL mutations per se influences the outcome in sporadic ccRCC.
Table 4.5. VHL alterations as potential prognostic markers in sporadic ccRCC
In part, the conflicting results may be explained by different methods used to assess the presence or absence of alterations, with not all studies assessing for the presence or absence of hypermethylation. Furthermore, different studies have included different patient populations with tumours of varying stages and grades. The majority of studies have used scanning techniques such as denaturing highperformance liquid chromatography or single-strand conformational polymorphism, followed by annotation of variants using Sanger sequencing. However, newer scanning techniques such as endonuclease scanning, and the incorporation of next-generation sequencing techniques with greater sensitivity and depth, may be more sensitive, and it is likely that the true prevalence of VHL alterations is higher than previously suspected . Including multiplex-ligation-dependent probe amplification and Southern blotting to detect copy-number changes in the VHL gene in the search for alterations may also increase the detected frequency of alterations, and it is possible that non-coding alterations in VHL may also have physiological significance.
Many groups have done further analyses on subgroups of VHL mutations to try and determine if the type or nature of mutation is associated with prognosis. For example, are mutations which would be predicted to result in complete loss of pVHL function, such as nonsense or frameshift mutations, associated with a worse prognosis than missense mutations, some of which may only mildly affect pVHL’s function? Alternatively, could nonsense mutations perhaps result in more aggressive tumours, perhaps due to retained “partial” activity of pVHL? Of course, categorising mutations in this way does not take into account the diverse effects of missense mutations on the function of pVHL (Table 4.4). While some missense mutations, even those distant from specific binding sites such as the HIF binding site, can have serious functional effects by globally destabilising protein folding, others seem to have little or no effect on pVHL’s function. The position of a missense mutation within the gene may therefore also be important prognostically. However, at present, there is no reliable way to easily discriminate between passenger mutations which do not influence tumour growth and driver mutations which do. The impact of individual missense mutations on either HIF regulation or other pro-tumorigenic activities thus remains largely uncertain, and the diverse spectrum of mutations presents a significant barrier to understanding the functional effects of missense mutations and other loss-of-function type events. In this respect, the development of validated in silico tools which can accurately predict the functional effects of missense mutations may be of benefit.
Consequently, it has proven difficult to develop a rational way of categorising mutations within VHL, and various groups have elected to use different classification systems. While some have divided mutations into “loss-of-function” mutations predicted to alter the open reading frame of VHL and “non-loss-of-function” mutations not predicted to alter the open reading frame , , others have classified mutations either according to their impact on the DNA sequence (frameshift/ missense/nonsense) or their location within the gene (e.g. exon number) or protein (e.g. deep/surface amino acids). To date, only one group has attempted to discriminate between those missense mutations with functional consequences and those without ; they subgrouped VHL missense mutations into predicted destabilising and neutral missense mutations using in silico prediction tools and in vitro assays but report no differing effects on patient outcome.
In VHL disease, there is an intimation that mean age of onset is earlier and agerelated risk of ccRCC is higher in patients with frameshift or nonsense mutations than in those with deletions or missense mutations that disrupt the structural integrity of pVHL . Another study of VHL disease patients reported an increased frequency of renal involvement in cases with a truncating mutation or large rearrangement than in those with missense mutations . Subsequently, some have speculated that truncating mutations may be associated with the expression of a truncated gene product that has retained a β-domain HIF binding site but not a functional a-domain elongin C binding site . Such a mutant protein might compete with wild-type pVHL to bind the HIFα subunits in normal renal cells, thereby protecting it from degradation. Further support for such a “dominant-negative” hypothesis comes from a study of sporadic ccRCC which reported a trend towards a worse prognosis for truncations resulting in late termination (termed as after codon 123) compared to truncations resulting in early termination (before codon 123) .
Mutations prior to codon 123 would be expected to disrupt the HIFα binding site, while later truncations may leave an intact HIFα binding site but disrupt the elongin C binding site and thus potentially protect HIFα from degradation.
In sporadic ccRCC, there are a handful of studies which suggest that nonsense mutations may be associated with worse prognosis and adverse pathological features [179, 181, 183]. Analysis of VHL mutations as part of a large kidney cancer case-control study suggested that nonsense mutations were significantly associated with increased grade and lymph node positivity and that these mutations were more prevalent among M1 than M0 cases . Another study of 113 ccRCCs subdivided VHL mutations into “loss-of-function” mutations predicted to alter the open reading frame of VHL and “DNA sequence variants of unknown biological consequence” (in-frame, missense, silent or intronic mutations) . Though “loss-of-function” mutations were not associated with tumour phenotype (grade, stage or metastasis), there was a significantly worse prognosis in tumours with “loss-of-function” mutations leading to truncated pVHL than in tumours with no mutations or mutations with unknown consequences for pVHL structure and function. However, on multivariate analysis, only histological grade and pT stage were independent predictors of adverse outcome, though this may simply reflect the small sample size. It should be noted that the prevalence of VHL mutations reported in this study was only 34 %, which is lower than that described in the majority of studies. Another small study of 56 patients found that loss-of-function mutations were associated with significantly decreased progression-free survival and overall survival .
It is not uncommon for a single tumour to have multiple VHL mutations with the reported prevalence of this up to 8 % in two studies [183, 215]. Multiple mutations of VHL within the same tumour have been described in up to 42 % of patients exposed to trichloroethylene . Though one study reported that late-stage metastatic lesions had more double mutations than M0 or Mx cases , other studies have not replicated these findings.
VHL alterations as potential predictive markers
As yet, only a few studies have examined a role for VHL as a potential predictive marker in ccRCC, largely because it is only recently that effective treatment options have come into widespread use (Table 4.6). One of the major hurdles relates to the collection of adequate quality tissue for DNA extraction and sequencing; the majority of clinical trials collect formalin-?xed paraffin-embedded (FFPE) tissue rather than fresh frozen tissue, and the quality of DNA extracted from FFPE tissue is generally inferior to that acquired from fresh frozen tissue. As such, the frequency of VHL mutations reported in many of these studies is lower than might be expected, implying a possible skewing of the results.
A recently reported study analysed 78 tumour tissue samples from a cohort of 225 metastatic clear cell RCC patients who received pazopanib, a standard first-line VEGF-targeted agent, as part of a clinical trial . The authors evaluated the association of several components of the VHL-HIF pathway (VHL gene inactivation [mutation and/or methylation], HIF1α and HIF2α immunohistochemistry staining and HIF1α transcriptional signature) with best overall response rate to pazopanib and progression-free survival. 70/78 (90 %) of patients had VHL mutations or methylation. Neither VHL gene status nor HIF1α or HIF2α protein expression or HIF1α gene expression signature was associated with clinical outcome to pazopanib.
The phase III Treatment Approaches in Renal Cancer Global Evaluation Trial (TARGET) randomised 903 patients with advanced ccRCC to sorafenib or placebo. VHL mutational status was available for 134 patients (though only 48 patients had all three coding exons of VHL successfully sequenced), and no correlation between VHL mutational status and sorafenib benefit was found .
One of the biggest earlier studies included 123 patients with metastatic ccRCC who had received treatment with sunitinib (51 %), sorafenib (23 %), axitinib (12 %) and bevacizumab (14 %) as part of a clinical trial . The incidence of VHL mutations in this group of patients was 49 %, with 78 % of these classified as “lossof-function” mutations (i.e. frameshift, nonsense, splice and in-frame deletions/ insertions). In addition, 10 % of patients had promoter methylation, though these patients were excluded from the analyses. Though there was no difference in response rate between patients with inactivated (mutated or methylated) VHL and those with wild-type VHL (41 % versus 31 %, p = 0.34), on subgroup analysis, patients with “loss-of-function” mutations had a significantly higher response rate than those with wild-type VHL (52 % versus 31 %, p = 0.04). This remained an independent predictor of response on multivariate analysis, even after adjusting for the specific anti-VEGF drug used. Further analyses showed that while patients who received sunitinib or axitinib had significant responses independent of VHL status, no responses (0/21) were seen in patients with wild-type VHL treated with bevacizumab or sorafenib. At present, the survival data are immature. A separate small study of ccRCC patients treated with first-line sunitinib found no association between VHL alterations and response to sunitinib .
Table 4.6. VHL alterations as potential predictive markers (all studies of patients with metastatic ccRCC)
These results at first appear to challenge those of previous studies, suggesting that “loss-of-function” mutations are associated with adverse outcomes. This may be because the advent of VEGF-targeted therapy means that patients who once had the worst outlook no longer do. The authors speculated that the differential effect of VHL mutations on therapeutic response could be explained either if sunitinib and axitinib have additional non-VHL-related inhibitory mechanisms on ccRCC or perhaps have superior inhibition of the VEGF receptor compared with bevacizumab and sorafenib. A subset of the data included in this analysis had previously been analysed and reported retrospectively in a study of 43 patients with metastatic ccRCC who received therapy with sunitinib, axitinib or interferon-a plus bevacizumab . In this report, there was no association between the presence of a VHL mutation or methylation and objective response rate or tumour shrinkage, and VHL alterations did not impact upon overall survival.
A very small study of 13 patients included in a phase II study testing the efficacy of axitinib found no correlation between the somatic mutational status of the VHL gene and the objective response to axitinib . However, this was a tiny study which did not analyse the methylation status of the VHL promoter, and among the 13 patients, only two showed the presence of sequence variants, suggesting the patient cohort within this study may not be entirely representative of the usual ccRCC population. There is very little work examining VHL mutational status as a predictive marker for response to mTOR-directed therapy, though a retrospective analysis on a subset of patients (n = 20) enrolled within a phase II trial of temsirolimus in advanced ccRCC found no correlation between VHL mutational status and response to temsirolimus . VHL alterations were not associated with response to immunotherapy in a retrospective analysis of 56 patients .
Categorising VHL mutations according to their functional effects
The preponderance of evidence suggests that not all VHL mutations are equal and that, in fact, a significant percentage of mutations described in sporadic ccRCC may simply be passenger mutations. Designing a system of categorising VHL mutations, perhaps by using in silico prediction tools to predict the effect of the mutation on the function of pVHL, may allow us to use this information practically to separate highand low-risk patient groups and select patients appropriately for targeted therapies.
Is VHL alone too simplistic?
As our understanding of the finer details of the molecular pathways downstream of VHL expands, various groups are investigating whether a combination of VHL mutational status and other molecular markers may prove more useful as prognostic markers than VHL alone. For example, one study reported that low CAIX expression and absence of VHL gene mutation are associated with aggressive clinicopathological features and poor survival of ccRCC , and another study by the same authors reported that the absence of VHL gene alteration, or altered VHL and high VEGF expression, is associated with poor survival .
An alternative way of classifying VHL-deficient tumours was described by Gordan et al.  who analysed VHL genotype, HIF1α, HIF2α and c-myc expression in 160 primary tumours. Based on immunohistochemical assays and mRNA profiling, they subdivided the tumours into three groups with distinct molecular characteristics: (1) tumours with wild-type VHL alleles and undetectable HIFα protein expression (designated VHL WT), (2) VHL-deficient tumours expressing detectable HIF1α and HIF2α proteins (designated H1H2) and (3) VHL-deficient tumours expressing HIF2α exclusively (designated H2). H2 tumours displayed enhanced c-Myc activity and higher rates of proliferation relative to H1H2 tumours regardless of stage and also displayed increased expression of genes involved in DNA repair, decreased levels of endogenous DNA damage and fewer genomic copy-number changes. In contrast, H1H2 and VHL WT tumours displayed increased activation of Akt/mTOR and ERK/MAPK1 growth factor signalling pathways and increased expression of glycolytic genes relative to H2 tumours. This study argues for the existence of two biologically distinct types of VHL-deficient ccRCCs: those that produce HIF1α and those that don’t.
Recent studies have used biologically driven clustering to define two robust subgroups of ccRCC, ccA and ccB, that are highly dichotomous by molecular phenotype and cancer-specific survival . ccA and ccB subtypes do not appear to be divided based on the expression of defined VHL or HIF profiles; however, a smaller, third subtype of tumours had a WT VHL signature and indications of variant histologies . Other studies have identified transcript patterns related to the expression of HIF1α and HIF2α [74, 95] and genetic sequence .
VHL and chromatin-remodelling genes
Studies of kidney cancers arising in VHL patients suggest that VHL inactivation in human kidneys leads to preneoplastic cysts but is not sufficient for malignant transformation [226, 227]. This explains why a mouse model of ccRCC does not exist today. Malignant transformation seems to require the accumulation of additional genetic and possibly epigenetic changes. Recently, genomic sequence analysis has identified several genes that are frequently mutated in ccRCC. These include polybromo 1 (PBRM1), SET domain containing 2 (SETD2), BRCA1-associated protein-1 (BAP1) (all of which lie on a relatively small, 43 Mb region of chromosome 3p and are therefore potentially deleted alongside VHL in tumours with 3p loss) and lysine (K)-specific demethylase 5C (JARID1c). It is likely that these genes function in pathways which would otherwise limit transformation driven by VHL loss. In this respect, acute VHL loss leads to senescence in many cell types [146, 147]. Interestingly, pVHL inactivation leads to the induction of JARID1c which then acts to block the proliferation in this setting .
It has been proposed that ccRCC development may evolve along two different paths . Following a VHL mutation and the loss of 3p which is frequently observed, mutations in the remaining PBRM1 or BAP1 allele may lead to tumours with different characteristics. Interestingly, in the mouse, Vhl is on a different chromosome to that of Pbrm1 and Bap1; thus, the loss of heterozygosity of the mouse Vhl region would not simultaneously inactivate one copy of Pbrm1 and Bap1. If this model is correct, simultaneous inactivation of Vhl and either Pbrm1 or Bap1 in the mouse should lead to the development of ccRCC.
It will be interesting to see whether subtypes of VHL alterations are linked in any way to mutations in chromatin-remodelling genes and whether these changes link to therapeutic response. As therapeutic strategies improve and specifically achieve more successful inhibition of VEGF activity and/or replacement of pVHL activity, closer correlations between drug responses and VHL alterations may be detected. A deeper understanding of VHL targets other than HIFα and particularly how VHL cooperates with BAP1, PBRM1 and other genes to cause ccRCC may ultimately lead to the identification of additional biomarkers and potentially novel therapeutic strategies.
HIFα as a biomarker in ccRCC
Many studies have investigated whether HIF1α or HIF2α is a useful prognostic marker in ccRCC (Table 4.7). Several earlier studies linked the expression of HIF1α to poor prognosis in ccRCC, though more recent studies suggest that the relative expression of HIF1α and HIF2α may be more important [74, 229]. A subset of ccRCC tumours seem to have copy-number amplification of 8q24, where c-Myc resides . Since HIF1α acts to suppress c-Myc activity while HIF2α promotes the transactivation or transrepression of c-Myc-specific target genes [74, 93, 94], it would be interesting for future studies to look at the expression of these genes in combination, particularly in conjunction with the mutational status of VHL, PBRM1 and BAP1.
Only a few studies have evaluated HIF expression as a predictor of response to targeted therapies (Table 4.7). Pretreatment HIF levels were associated with response to sunitinib in a cohort of 43 metastatic ccRCC patients , and positive HIF1α and HIF2α protein expression was reported to be an independent predictor of outcome for VEGFR tyrosine kinase inhibitor therapy in 71 patients with metastatic ccRCC . However, another study reported that the loss of chromosome14 or 14q(HIF1α locus) was not correlated with clinical response to pazopanib , and a very recent study found no association between HIF1α (65 samples) and HIF2α (66 samples) protein levels and overall response rate or progression-free survival to pazopanib .
Table 4.7. HIF1α and HIF2α as potential biomarkers in ccRCC
Since HIF2α seems to be an oncogene in ccRCC, targeting HIF2α would seem to be a sensible therapeutic strategy for ccRCC. However, with the exception of the steroid hormone receptors, targeting DNA-binding transcription factors with drug-like small organic molecules has historically been relatively unsuccessful. Despite this, several potential strategies to inhibit HIF2α have been identified. Proof-of-concept experiments intimate that, in principle, it may be possible to target HIF2α with DNA-binding polyamides that disrupt the HIFα-DNA interface, though at present the bioavailability of such agents is inadequate [233–235]. Acriflavine is a small molecule that inhibits the ability of HIF1α and HIF2α to dimerise with HIF1Β and has been shown to inhibit tumour growth and vascularisation . Alternatively, if reliable methods for systemic delivery of siRNA become available, siRNA targeting of HIF2α may become a future therapeutic option. Two groups have been screening for drugs that, at least indirectly, inhibit HIF2α in VHL null ccRCC cells, though the specificity of these compounds remains to be established [237–240].
Many other compounds are also known to indirectly inhibit HIFα, including mTOR inhibitors, HSP90 inhibitors and HDAC inhibitors . Unfortunately, the two currently used rapamycin-like mTOR inhibitors, everolimus and temsirolimus, primarily inhibit mTOR in the TORC1 complex and have less activity against mTOR present in the TORC2 complex . The inhibition of TORC1 inhibits HIF1α more than HIF2α  and can also paradoxically increase upstream receptor tyrosine kinase signalling due to a loss of TORC1-dependent negative feedback pathways [244, 245, 246].
Of course, many of the drugs currently used to treat ccRCC in the clinic indirectly inhibit HIF2α by inhibiting the action of one of its most tumorigenic downstream targets, VEGF. These include bevacizumab, sunitinib, sorafenib, pazopanib and axitinib.
pVHL and synthetic lethality
Two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death . Synthetic lethality thus provides a framework to discover drugs that might preferentially kill cancer cells harbouring a cancer-relevant gene yet leave normal cells unharmed. To date, the results from two synthetic lethality screens attempting to target VHL-deficient cells have been reported. Firstly, a cell-based small-molecule synthetic lethality screen identified a compound, STF-62247, that selectively induces autophagic cell death in VHLdeficient cells but not in those expressing wild-type VHL . From the same screen, a second compound, STF-31, was identified that inhibits glucose uptake by the Glut1 transporter and exhibits enhanced cytotoxicity against VHL-deficient ccRCC . Secondly, an shRNA screen targeting 88 kinases reported that silencing of CDK6, MET and MEK1 preferentially inhibited the growth of VHL null cells compared with their wild-type pVHL-reconstituted counterparts . Interestingly, in both screens, the selective killing of cells lacking VHL was HIF independent, suggesting that therapies targeting these pathways may cooperate with those targeting HIF. Another study showed that the lack of a functional VHL gene product sensitises renal cell carcinoma cells to the apoptotic effects of the protein synthesis inhibitor verrucarin A .
VHL, HIFα and metastasis
Since the main tumour-suppressive function of VHL is its role in mediating the degradation of HIF2α, and since at least one HIF2α target gene, chemokine (C-X-C motif) receptor 4 (CXCR4), is a direct mediator of metastatic colonisation [252, 253], it has previously been suggested that loss of VHL might directly lead to metastatic tumour phenotypes through HIF activation . However, even though CXCR4 expression correlates with metastasis in ccRCC [253–255], as described above, VHL mutation has not convincingly been shown to correlate with poor disease outcome and metastatic disease.
Therefore, Massaguй and colleagues examined whether the increased expression of CXCR4 and other potential metastatic genes downstream of the VHL-HIF axis occurs as a result of epigenetic changes. They selected highly metastatic subpopulations of the VHL-deficient ccRCC 786-0 cell line (which was originally established from a patient with metastatic disease) through tail vein injection into immunocompromised mice. Using genome-wide transcription profiling, they identified 155 genes associated with the metastatic phenotype of these cell variants. They then refined this gene set to a core set of 50 genes (termed the renal cancer metastasis signature 50 (RMS50)) that are also expressed in ccRCC gene expression profiles that form the GSE2109 data set in the Gene Expression Omnibus.
Additional gene expression profiling studies showed that a subset of these genes responded to VHL inactivation and were transcriptional targets of HIF2α. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, they showed that the loss of polycomb repressive complex 2 (PRC2)-dependent histone H3 Lys27 trimethylation (H3K27me3) activates HIF-driven chemokine (C-X-C motif) receptor 4 (CXCR4) expression in support of chemotactic cell invasion, whereas the loss of DNA methylation enables HIF-driven cytohesin 1-interacting protein (CYTIP) expression to protect cancer cells from death cytokine signals.
Previously, the pathways that drive metastasis have been considered to be separate from tumour-initiating functions . In contrast, this study suggests that metastasis in ccRCC is based on an epigenetically expanded output of the tumour-initiating pathway, namely, loss of VHL function.
HIF- and hypoxia-mediated epigenetic regulation
As discussed in high-throughput genetic studies of RCC have identified recurrent mutations in genes encoding several epigenetic regulators, including PBRM1, SETD2, JARID1c, KDM6A and MLL2 (reviewed in ). Interestingly, the hypoxia response pathway has been shown to have a direct effect on histone modification. Firstly, HIF has been shown to directly activate several chromatin demethylases, including KDM3A, KDM4B, KDM4C and KDM5B [41, 258–260]. Secondly, VHL inactivation has been shown to decrease H3K4Me3 levels through an HIF-dependent increase in the expression of JARID1c (an H3K4 Me3 demethylase) expression. The re-expression of pVHL in VHL-deficient cell lines reduces HIFα expression, resulting in decreased levels of JARID1c with a consequential increase in levels of H3K4me3 .
In contrast, hypoxia may also increase methylation through HIF-independent mechanisms; histone demethylases are members of the dioxygenase superfamily, which require oxygen for activity, and hypoxia suppresses JARID1A (KDM5A) activity, resulting in increased H3K4me3 levels . The loss of demethylases, and by implication, increased histone methylation, may thus be part of a hypoxia phenotype that is selected for in ccRCC. This hypoxia phenotype, which is mimicked by VHL loss, would also be mimicked by the loss of histone demethylase activity resulting from inactivating mutations.
Chromatin organisation also influences HIF function, and it seems that HIF is preferentially targeted to previously nucleosome-depleted chromatin regions . SWI/SNF has also been shown to regulate the cellular response to hypoxia by regulating HIF1α transcriptional activity .
As yet, the extent to which mutations of epigenetic regulators influence chromatin or HIF targeting is unknown. Since hypoxia directly influences demethylase activity, the relationship between epigenetic variation and HIF targeting might well differ depending on the conditions of hypoxia in primary cells and the context of specific epigenetic alterations in tumour cells.
VHL and HIF are undoubtedly key players in ccRCC pathogenesis. The loss of VHL with consequent deregulation of HIFα and its downstream targets is an early event for the majority of patients with ccRCC. In this disease, HIF2α acts as an oncoprotein, while HIF1α acts as a tumour suppressor. This knowledge underpins the rationale behind the development of drugs that inhibit HIF or selected HIF targets for the treatment of ccRCC. As our understanding of ccRCC biology continues to evolve rapidly, we must amalgamate the wealth of newly acquired information regarding chromatin-remodelling genes and ccRCC metabolics with the existing data regarding VHL and HIF in ccRCC. The challenge now is to establish a road map of tumour ontogeny for precursor lesions and early ccRCC and to clarify the mechanisms of tumour progression for more advanced disease. Studies of genomics, transcriptomics, epigenetic data and molecular biology must be co-ordinated and targeted. International collaboration and communication among experts is vital to ensure this information is used and interpreted as efficiently and effectively as possible.
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