**1. Introduction**

Kidney cancer is the seventh most common malignancy in the Western world. In 2018, there were approximately 400,000 new kidney cancer cases and 180,000 kidney cancer-associated deaths worldwide [1]. The underlying causes of kidney cancer are complex and incompletely understood, although genetic factors (both inherited and somatic genetic mutations) are known to drive the disease. Additionally, certain lifestyle choices (such as smoking and a high protein diet) increase the risk of developing kidney cancer, consistent with its prevalence in the Western world. Unless surgically resectable, kidney cancer is largely incurable, and the 5-year survival rate for those with metastatic disease is only about 10% [2]. Systemic anti-cancer therapies, including those that inhibit the vascular response or enhance patients' immune response to the malignancy, have offered some hope [3]. However, these treatments confer limited efficacy and a considerable burden of toxicity. Therefore, there is a pressing need to better understand the drivers of kidney cancer in order to identify novel therapeutic strategies.

#### **2. Histological subtypes of renal cancers**

The most common form of kidney cancer is clear cell renal cell carcinoma (ccRCC), which arises from the adult renal tubular epithelium and accounts for approximately 75% of all kidney cancer cases. This subtype is termed as such due to the characteristic 'clear' cytoplasm of malignant cells observed histologically. This is caused by accumulation of excess glycogen and lipid in the cytoplasm (due to highly dysregulated metabolic pathways), which are dissolved by the tissue fixation process [4]. Other less common subtypes of adult renal cancers that also arise from the tubular epithelium include papillary RCC (types 1 and 2); chromophobe RCC; and oncocytoma. Each subtype is associated with different histological features, genetic drivers, and clinical behaviors. Rarely, cancers can arise from other cell types in the adult kidney, including transitional epithelial cells of the ureter and renal pelvis (giving rise to transitional cell carcinoma) and various mesenchymal cell types (e.g. interstitial cells, giving rise to renomedullary interstitial cell tumors). Although childhood kidney cancer is generally rare, the most common form is Wilms tumor, which originates in developing tubular cells during fetal development [5].

It should be noted that even within a specific renal cancer histological subtype there is evidence for substantial heterogeneity, which has initiated efforts to further refine subtype classification based on additional features. Recent studies have found that ccRCC can be further stratified based on architectural, cytological and microenvironmental features, and that these features can predict patient outcome and response to therapy [6]. The underlying cause of this variation remains to be determined but could be due to certain genetic or epigenetic differences between ccRCC tumors. Consolidation of histological and molecular heterogeneity in ccRCC will be important for disease subclassification, as well as better understanding ccRCC biology, going forward.

### **3. VHL syndrome**

Each kidney cancer subtype is associated with its own monogenic cancer syndrome [7]. Studying these rare family kindreds has provided unique insight into the genetic mechanisms underlying both inherited and sporadic cancers. In particular, clear cell renal cancer is associated with VHL syndrome, which is an autosomal dominant disorder, affecting 1 in 32,000 individuals, caused by heterozygous germline mutations of the VHL gene [8, 9]. As well as ccRCC, VHL syndrome is associated with a limited number of other tumors types, including hemangioblastomas of the retina and the central nervous system; pheochromocytomas; pancreatic lesions; endolymphatic sac tumors and epidydimal cysts [8, 9]. VHL syndrome can be further sub-divided according to which of these different tumor types develop in individuals within the kindred [10, 11]. Four distinct patterns have been identified: type 1 VHL disease, which is associated with hemangioblastoma and ccRCC; type 2A, which is associated with hemangioblastoma and pheochromocytoma; type 2B, which is associated with hemangioblastoma, pheochromocytoma and ccRCC; and type 2C, which is associated with pheochromocytoma alone. Each of these subtypes is linked to particular types of VHL mutation, which have been shown to have different downstream biological effects [12–17].

#### **4. The VHL gene**

The human VHL gene was first identified following classical linkage analysis of families with VHL syndrome and was cloned in 1993 [18]. In humans it is located on the short arm of chromosome 3 (3p25) and has three exons that encode a protein of 213 amino acids, with a molecular weight of around 30 kDa (termed p30). However, the gene also contains a second translation start site at codon 53, leading to the generation of a shorter protein of approximately 19 kDa (termed p19), which appears to retain canonical activity [19]. As a consequence, oncogenic mutations, most

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

typically single-nucleotide variants (SNVs) or short insertion/deletions (indels), are restricted to codons 53–213 in exons two and three.

VHL acts as a tumor suppressor gene [20, 21]. ccRCC and other cancer types are associated with inactivating mutations of VHL, which lead to loss-of-function of the gene product (termed pVHL). Although autosomal dominant at the level of the individual, both alleles of the VHL gene must be inactivated in a cell for cancer to develop, in line with Knudson's two-hit hypothesis [22, 23]. Since VHL syndrome is caused by germline VHL mutation, all cells of the affected individual harbor this mutation. The remaining wild-type (WT) allele is somatically inactivated in the tumor progenitor cell, which then multiplies to form the cancer [20, 21]. Typically, somatic inactivation of the WT allele occurs as a result of an arm-level loss of chromosome 3p (**Figure 1**), although promoter hypermethylation or a second SNV/ indel may also cause complete loss of functional VHL in the cell. Furthermore, since the cells of patients with VHL syndrome only require one somatic mutation to become functionally deficient in VHL, it is a relatively common event, accounting for the high tumor penetrance in these individuals. Indeed, over the course of their lifetime, these individuals often develop multiple tumors and close examination of their organs often reveals the presence of numerous synchronous tumors. However, VHL mutation is only associated with the very limited range of cancers outlined above, despite it being ubiquitously expressed. Therefore, VHL only appears to act as a tumor suppressor gene in very few tissues. Indeed, even within the kidney, ccRCCs appear to develop from a subset of proximal tubular cells [24]. It is assumed that somatic mutations in the wild-type copy of VHL do occur in other cell types, but it is not known whether these cells are eliminated by other tumor suppressor mechanisms, or simply fail to progress to overt cancer.

Importantly, VHL is also inactivated in the vast majority (approximately 90%) of sporadic ccRCC tumors, which occur in patients without a germline mutation in the VHL gene [25]. In order to develop cancer, these individuals require two somatic

#### **Figure 1.**

*VHL inactivation in ccRCC. Individuals with VHL syndrome are predisposed to ccRCC (termed hereditary ccRCC) as a result of a heterozygous germline VHL mutation. The second, wild-type allele is subsequently inactivated by somatic loss of chromosome 3p, resulting in biallelic VHL inactivation. On the other hand, in sporadic ccRCC, two somatic events are required for biallelic inactivation. Typically, one copy of chromosome 3p is lost followed by inactivation of the second VHL allele through mutation or promoter hypermethylation. Although the ordering is reversed, the same genetic aberrations are observed in both sporadic and hereditary ccRCC. However, because only one somatic event is required for biallelic VHL inactivation in patients with VHL syndrome, this is a much more likely event and occurs in multiple cells within the kidney, causing many pre-malignant lesions and multiple ccRCC tumors. chr= chromosome; CNAs= chromosomal copy number alterations; mut= mutation; WT= wild-type.*

events affecting both copies of the VHL gene in the same cell (**Figure 1**). As a result, this occurs much less frequently, accounting for the much lower overall prevalence of ccRCC in the general population of about 1%. However, in contrast to VHL syndrome, the order of events is typically reversed, with loss of chromosome 3p frequently occurring first and the remaining copy then being inactivated by singlenucleotide substitution (SNV) or small insertions or deletions (indels) [26].

Of note, although biallelic VHL inactivation is required for ccRCC (and other tumors) to develop, it does not appear to be sufficient on its own (**Figure 1**). Mitchell et al. have estimated that in sporadic ccRCC, VHL inactivation predates tumor formation by a number of years or even decades [26]. Consistent with this, examination of the kidneys from patients with VHL syndrome has identified multiple isolated VHL-defective cells, which may be present as single cells or small non-invasive cysts [27, 28]. Furthermore, *in vitro*, inactivation of VHL leads to cellular senescence rather than unrestricted proliferation [29, 30]. Therefore, it is thought that additional gene mutations are required for these early VHL-defective lesions to develop into mature ccRCC. Indeed, more recently, additional somatic mutations have been identified in ccRCC [25, 31–33]. Most notable among these are inactivating mutations in the PBRM1 (polybromo 1), SETD2 (SET domain-containing 2) and BAP1 (BRCA-associated protein 1) tumor suppressor genes, mutation of which typically follows loss of VHL. Importantly, these three genes also reside on the short arm of chromosome 3. As a result, the loss of chromosome 3p frequently observed in both familial and sporadic ccRCC can simultaneously result in copy loss of all 4 of these ccRCC-associated tumor suppressor genes; VHL, PBRM1, SETD2 and BAP1.
