Understanding the VHL pathway for RCC biomarker development

Kidney cancer. Principles and practice. Second edition. Primo N. Lara, Jr. Eric Jonasch (Editors). Springer International Publishing (2015)


Before embarking on an inventory of biomarkers for RCC, it is essential to understand the biology and molecular pathways which are known in this disease and from which the majority of biomarkers are derived (Fig. 4.1). A key event in the pathogenesis of clear cell RCC (ccRCC) appears to be the inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which is a biallelic event in over 90% of sporadic ccRCC [10]. The mechanisms that lead to the loss of VHL functionality include large-scale and small-scale deletions, missense mutations, early stop codons, truncations, and silencing of the locus by hypermethylation. The VHL gene is located on the short arm of chromosome 3. Large deletions of 3p are commonly identified in ccRCC. This causes a loss of heterozygosity in a majority of ccRCCs, leaving cells susceptible to loss of the remaining allele and full inactivation of VHL [11]. Overall, the disengagement of VHL in this unique tumor type is likely a critical and common event in ccRCC development.


The VHL protein (pVHL) performs a critical cellular function in regulating the cellular response to low oxygen. In the presence of sufficient oxygen, pVHL binds to a family of proteins called the hypoxia-inducible factor alpha (HIF-a) subunits, recruiting them to an E3 ubiquitin ligase complex which polyubiquitinates the HIF-a subunits, thus targeting them for proteasomemediated proteolysis [12]. The loss of pVHL activity, therefore, permits the constitutive stabilization of HIF-a factors, and high-level expression of HIF-a factors has been a widely recognized feature of ccRCC tumor biology. About 90% of all ccRCC display HIF-a stabilization apparently as a consequence of VHL loss or inactivation [13]. Recent evidence has accrued to indicate that pVHL has functions other than regulation of HIF-related pathways, such as regulation of apoptosis, control of cell senescence, and maintenance of the primary cilium [14].


HIF is a heterodimeric transcription factor complex consisting of an unstable alpha (a) subunit and a stable beta (β) subunit. Three HIF-a genes (HIF-1α, HIF-2α, and HIF-3a) have been identified in the human genome [15]. Both HIF-1α and HIF-2α function as classical transcription factors, although they can also cooperate with additional factors to maximize activity [16]. The role for HIF-3a, which does not clearly act as a transcriptional regulator and exists with many splicevariant isoforms, is poorly understood [17].

Despite many similarities, HIF-1α and HIF-2α are not fully redundant in function. The global gene expression changes induced by HIF-1α and HIF-2α show that they produce overlapping yet distinct gene expression profiles in both cells and in mice [18].

HIF plays a critical role in tumorigenesis. Indeed, there are several lines of evidence that implicate HIF-a and in particular HIF-2α as playing an active role in VHL-/renal cell carcinogenesis. First, RCC-associated pVHL mutants are at least partially defective with respect to HIF-2α polyubiquitination [19, 20]. Genetic manipulation of HIF expression in human tumor cell line xenografts has clearly demonstrated a growth advantage for cells expressing HIF-2α but not HIF-1α [12, 21]. Examination of human ccRCC tissues provided the ultimate demonstration of a dependence on HIF-2α stabilization, showing that all VHL-defective RCCs either stabilize dually both HIF-1α and HIF-2α or solely HIF-2α [13]. This observation provides an alternative way of classifying pVHL-deficient tumors based on this distinction of HIF expression. The VHL genotype and the protein expression of HIF-1α and HIF-2α proteins were analyzed in 160 primary tumors. The tumors were examined by immunohistochemistry (IHC) for HIF-1α and HIF-2α and messenger RNA profiling. VHLdeficient tumors that exclusively express HIF-2α (H2) tumors displayed greater c-Myc activity and higher rates of proliferation relative to those of VHL-deficient tumors expressing both HIF-1α and HIF-2α (H1H2), regardless of tumor stage. H2 tumors also demonstrated increased expression of genes involved in DNA repair, decreased levels of endogenous DNA damage, and fewer genomic copy-number changes. Moreover, those VHL-deficient H1H2 tumors and VHL wild-type tumors displayed increased activation of AKT/mTOR and ERK/MAPK1 growth factor signaling pathways and increased expression of glycolytic genes. Thus, there may be two biologically distinct types of VHL-deficient ccRCC: those that produce HIF-1α and those that do not. The relevance of this distinction as a biomarker remains to be demonstrated, although consistent with expectations, H2 tumors were of a higher T stage than their H1H2 counterparts.

HIF-responsive genes

HIF is a potent transcriptional activator of the cellular hypoxia response and more than 100 direct HIF-responsive genes have been described, with a number of these genes active in carcinogenesis [22]. Although some of these genes and their products are being studied in RCC, two deserve special attention: vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CAIX, CA9).


VHL-/ccRCCs are notoriously angiogenic and overproduce a variety of proangiogenic molecules including the HIF-responsive VEGF. VEGF stimulates endothelial cell proliferation, migration, maturation, and survival and is among the most potent endothelial mitogens. Furthermore, the VEGF receptor, kinase insert domaincontaining receptor (KDR), may be present on renal carcinoma cells, suggesting the possibility of an autocrine feedback loop, although receptor activity on tumor cells remains to be demonstrated [23, 24].

VEGF and VEGF receptors (VEGFR) have been thrust into the spotlight as a result of substantial activity of targeted therapies, which engage these factors. Bevacizumab is an antibody that binds circulating VEGF protein and has activity in metastatic RCC [25]. In addition, potent tyrosine kinase inhibitors, such as sunitinib, sorafenib, and pazopanib, target the intracellular signaling pathways of multiple members of the VEGF receptor family of proteins. Multiple phase III trials have demonstrated substantial clinical benefit from blocking VEGFRs with sunitinib, sorafenib, and pazopanib [26, 27, 28].

Below, we will discuss the potential utility of biomarkers of VEGF activity in the context of therapeutics that directly target this signaling pathway, either via tumor cells directly or via supporting cells of the endothelium.


CAIX is a transmembrane protein that may play a role in the regulation of cell proliferation, oncogenesis, and tumor progression. CAIX is a HIF-responsive, hypoxia-induced protein that accumulates in VHL-defective RCCs [29]. A study of CAIX expression in 317 primary and 42 metastatic renal neoplasms showed correlation between CAIX expression with ccRCC histology as well as histologic grade, suggesting that this HIF-dependent protein may provide an effective surrogate for HIF stabilization with the potential to independently serve as a biomarker [30].

AKT/mTOR/HIF pathway

A better understanding of the molecular biology underlying RCC will lead to the development of biomarkers reflecting aberrant signal transduction pathways within these tumors. Mammalian target of rapamycin (mTOR) is a kinase that activates substrates critical for protein synthesis. It directly phosphorylates the ribosomal subunit S6 kinase (S6K) as well as eukaryotic initiation factor 4E (eIF-4E), which is released from its inhibitory binding partner 4E-BP1 upon its phosphorylation by mTOR. Loss of function mutations of the PTEN tumor suppressor gene result in increased mTOR activity via AKTdependent inactivation of the tuberous sclerosis complex (TSC1 and TSC2), and key members of this pathway have been identified to have nonoverlapping mutations in a substantial percentage of tumors [31]. Inhibitors of mTOR decrease global translation of proteins including HIF, cyclin D1, and Myc [32]. There are now two FDA-approved mTOR inhibitors used in the clinic for advanced RCC: temsirolimus [33] and everolimus [34], which have led to both improved progression free survival (PFS) and overall survival. The detection of the effector molecules (phospho S6, phospho 4EBP1, and phospho AKT) has been linked with response to VEGFtargeted therapy [35] and is both prognostic for overall survival and predictive of response to mTOR therapy [36–38].

Other 3p genes involved in RCC

In addition to VHL, several other genes located on chromosome 3p have been recently shown via massively parallel sequencing to be commonly mutated in RCC. These genes are important in histone modification and chromatin remodeling. The most commonly mutated gene is PBRM1 (polybromo 1) [39]. Histone methyltransferase SETD2 (SET domain-containing protein) and the histone deubiquitinase BAP1 (BRCA1 associated protein-1) have also recently been described, in addition to several other less commonly mutated genes [40, 41]. There are both positive and negative genetic interactions among these genes, with PBRM1 mutations and SETD2 mutations commonly occurring in the same tumor and PBRM1 and BAP1 rarely occurring together [42, 43]. These genes, similar to VHL, are tumor suppressor genes in which one allele is typically inactivated by mutation or hypermethylation and the second is inactivated through a large deletion in chromosome 3p, resulting in loss of heterozygosity [44].

BAP1 is a nuclear deubiquitinase and tumor suppressor gene mutated in about 9–15% of ccRCCs [41, 45–48] (and commonly in other cancers, most notably metastatic uveal melanoma) [49]. It causes expression of a specific gene expression signature and is associated with increased mTOR activation. BAP1 mediates deubiquitination of histone H2A and binds to host cell factor-1 (HCF-1), a component of the chromatin-remodeling complex, and binding is required for suppression of cell proliferation. Therefore, loss or mutation of BAP1 is thought to result in loss of tumor suppression [41, 45, 50–52]. A missense variant of BAP1 has also been found as a germline mutation in familial RCC, although it rarely occurs [53].

SETD2 is a two-hit tumor suppressor gene located in the region of chromosome 3p that is deleted in a majority of ccRCCs. It is present in about 7–16% of ccRCCs [45, 46]. SETD2 functions as a histone modifier and methyltransferase and is responsible for trimethylation of lysine 36 of histone H3, causing decreased H3K36 levels in some tested ccRCC cell lines [54] and thereby possibly influencing gene expression and transcription activation.

PBRM1 encodes a chromatin/nucleosome remodeling complex protein BAF180 and is mutated in 30–45% of RCC [39, 46]. It is thought to work as a tumor suppressor gene through regulation of DNA accessibility and gene expression and therefore regulate cell proliferation, although the full mechanism of tumorigenesis due to loss of PBRM1 is not fully understood [42]. Nearly all PBRM1-mutated tumors exhibit a hypoxia signature, suggesting a loss of VHL even though not all cases are associated with a detectable VHL mutation [39]. Overall, the recently identified mutations of BAP1, PBRM1, and SETD2 represent novel genetic contributors to the pathogenesis of ccRCC, a finding that may reveal important prognostic classification groups and potentially inform therapeutic decisions in the future.

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