SEARCHING FOR THE HEREDITARY CAUSES OF RENAL-CELL CARCINOMA

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Renal-cell carcinoma (RCC) affects approximately 150,000 people worldwide each year, causing close to 78,000 deaths annually, and its incidence seems to be ris-ing1,2. RCC is not a single entity, but rather comprises the class of tumours of renal epithelial origin. Extensive his-tological and molecular evaluation has resulted in the development of a consensus classification of different RCC subtypes (TABLE 1)3. Although most cases of RCC seem to occur sporadically, an inherited predisposition to renal cancer accounts for 1–4% of cases and could involve the same genes that cause sporadic renal cancer. Over the past two decades, studies of families with inher-ited RCC have laid the groundwork for the identification of seven hereditary renal cancer syndromes, and the pre-disposing genes for five of these have been identified (TABLE 2). The surprisingly diverse nature of these genes implicates various mechanisms and biological pathways in RCC tumorigenesis.

The year 2003 marked the tenth anniversary of the discovery of the von Hippel–Lindau (VHL) tumour-suppressor gene4, the first gene identified for hereditary RCC that is now known to be involved in the most cases of sporadic RCC. The VHLgene product is involved in the regulation of numerous pathways leading to extracel-lular-matrix assembly, cell-cycle regulation and, most importantly for tumorigenesis, oxygen sensing. Four years after the discovery ofVHL, interest in a gene

known for nearly two decades to have oncogenic poten-tial was rekindled when activating renal-cancer-causing mutations in patients with hereditary papillary renal car-cinoma(HPRC) were identified in the MET proto-oncogene5. Recently, the gene that encodes the KREBS CYCLE enzyme fumarate hydratase (FH) was found mutated in renal tumours from patients with a rare GENODERMATOSIS termed hereditary leiomyomatosis and renal-cell cancer

(HLRCC)6. Finally, new renal-cancer-predisposing genes were identified through linkage analysis in families

with another genodermatosis, the Birt–Hogg–Dubé

syndrome(BHD)7, and in families with

hyperparathy-roidism-jaw tumour syndrome (HPT-JT)8. These

predisposing genes — BHDand HRPT2,respectively —

are suspected to act as tumour suppressors, although their biological functions are as unknown.

Several renal-cancer-associated syndromes have been identified for which no predisposing gene has been found. Several families carry a balanced chromosome-3 translocation that predisposes family members to clear-cell RCC in the absence of germline VHLinactivation9–11. Other families have members affected with clear-cell renal carcinomas, but no detectable VHLinactivation or germline chromosome-3 translocations. Finally,familial papillary thyroid carcinoma (FPTC), which predisposes patients to thyroid cancer and nodular thyroid disease, can also predispose to papillary RCC and ONCOCYTOMA12.

SEARCHING FOR THE HEREDITARY

CAUSES OF RENAL-CELL

CARCINOMA

*Christian P. Pavlovich and

Laura S. Schmidt

Families with hereditary predispositions to cancer continue to provide a unique opportunity for the

identification and characterization of genes involved in carcinogenesis. A surprising number of

genetic syndromes predispose to the development of renal-cell carcinoma, and already genes

associated with five of these syndromes have been identified —

VHL, MET, FH, BHD

and

HRPT2

. These very different genes and the biochemical pathways in which they participate raise

interesting questions about the development of renal cancers and could lead to new therapeutic

approaches in the near future. So, what is known about hereditary renal cancer at present?

KREBS CYCLE

Also known as tricarboxylic-acid cycle. A series of enzymatic reactions that break down pyruvate to carbon dioxide and hydrogen atoms, which are in turn transferred to specific coenzymes for the oxidative generation of ATP in the mitochondria. GENODERMATOSIS An inherited syndrome involving a dermatological phenotype and possibly other phenotypes.

*Johns Hopkins Bayview Medical Center, Brady Urological Institute, A-345, 4940 Eastern Ave., Baltimore, Maryland 21224, USA.Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Bldg 560, Rm. 12-69, Frederick, Maryland 21702, USA. Correspondence to C.P.P or L.S.S. e-mails: cpavlov2@jhmi.edu; schmidt@ncifcrf.gov doi:10.1038/nrc1364

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ONCOCYTOMA

Large tumour cells with poorly defined borders, granular eosinophilic cytoplasm and large, basophilic nuclei. PENETRANCE The frequency with which individuals who carry a given gene mutation will show the manifestations associated with the gene syndrome. If penetrance of a disease allele is 100%, then all individuals carrying that allele will express the associated phenotype. LOSS OF HETEROZYGOSITY (LOH). In cells that carry a mutated allele of a tumour-suppressor gene, the gene becomes fully inactivated when the cell loses a large part of the chromosome carrying the wild-type allele. Regions with a high frequency of LOH are believed to harbour tumour-suppressor genes.

Hereditary syndromes with known genes VHL syndrome.VHL disease, named after the physi-cians who first described the hallmark retinal angiomas and central nervous system (CNS) haemangioblas-tomas, is an autosomal-dominant, inherited multisys-tem disorder that is characterized by solid vascular tumours of the kidney, CNS, retina, adrenal gland and endolymphatic sac, and by vascular/cystic lesions of the kidney, pancreas, epididymis and broad ligament16. VHL occurs at a prevalence of about 1/35,000 and VHL-associated tumours with relatively high PENETRANCE (80–90%) develop in the second to fourth decades of life. The renal tumours are almost exclusively clear-cell renal carcinomas or cystic variants that are often, but not always, multifocal and bilateral, and usually acquire metastatic potential when they reach more than 3–7 cm in diameter (TABLE 1). VHL disease is

caused by germline mutations in the VHL

tumour-suppressor gene4,17,18accompanied by inactivation of the wild-type copy of the VHLgene in a susceptible cell through LOSS OF HETEROZYGOSITY(LOH), promoter HYPERMETHYLATIONor somatic mutation, according to the KNUDSON TWO-HIT TUMOUR-SUPPRESSOR MODEL19–21. In fact, the two-hit event is documented very early in micro-scopic pre-neoplastic renal lesions and cysts22,23. Over half of all sporadic conventional renal carcinomas carry biallelic inactivation of both VHLalleles24–26.

Since the cloning of the VHLgene in 1993, an enor-mous number of experimental studies have been undertaken to unravel the pathway by which VHL inactivation leads to cancer (for reviews, see REFS 27–29). The frequent overexpression of vascular endothelial growth factor (VEGF) and erythropoietin (EPO) in VHL-associated tumours, both of which are encoded by hypoxia-inducible genes, offered clues to a potential role for VHL in regulating genes that are controlled by cellular oxygen tension (for reviews, see REFS 30,31). Subsequently, VHL was found to regulate expression of hypoxia-inducible factor (HIF)32, a heterodimeric protein made up ofα-subunits (HIF-1α,HIF-2αor

HIF-3α) and HIF-1β33. Under normoxic conditions, the α-subunit of HIF is hydroxylated at two proline This review will focus on the syndromes that are

associated with hereditary renal cancer, the attempts to understand the molecular biology of the genes already discovered, and on the search for new predisposing genes. Two forms of renal tumour that are associated with hereditary syndromes will not be covered — angiomyolipoma associated with tuberous sclerosis, and hereditary Wilms’ tumour— as both are not strictly of renal epithelial origin and both have heritable genetics that have recently been reviewed elsewhere13–15.

Summary

• A predisposition to renal cancer has been identified in several autosomal-dominant inherited cancer syndromes.

• von Hippel–Lindau (VHL) disease, associated with conventional (clear-cell) renal-cell carcinomas and multi-organ neoplasia, is caused by germline mutations in the VHL

tumour-suppressor gene and loss of the wild-type VHLallele.

• Patients with hereditary papillary renal carcinoma (HPRC) harbour germline-activating mutations in the METproto-oncogene, which can cause renal cancers with papillary type-1 histology.

• Papillary type-2 renal carcinomas and cutaneous and uterine smooth-muscle tumours are associated with the syndrome of hereditary leiomyomatosis and renal-cell cancer (HLRCC), which is caused by germline loss-of-function mutations in the fumarate-hydratase (FH) gene.

• The Birt–Hogg–Dubé syndrome (BHD) predisposes to cutaneous nodules (benign tumours of the hair follicle), spontaneous pneumothorax and an increased risk for renal cancers of various histological types, such as chromophobe renal-cell carcinoma and oncocytic hybrid renal tumour. BHD is caused by germline mutations in a newly discovered tumour-suppressor gene,BHD.

• Hyperparathyroidism-jaw tumour syndrome (HPT-JT) is associated with parathyroid adenomas, fibro-osseous tumours of the jaw, and unusual renal tumours containing a mixture of epithelial and stromal elements. This syndrome is caused by germline mutations in HRPT2.

• The identification of non-VHL families affected with clear-cell renal carcinomas, termed familial clear-cell renal carcinoma (FCRC), indicates that additional renal-cancer-predisposing genes remain to be identified.

• Diagnosis and appropriate treatment of these hereditary renal-cancer-associated syndromes relies on an understanding of their clinical spectrum, accurate histological evaluation of renal tumours from patients and on genetic testing for predisposing genes.

Table 1 | Classification schema for renal epithelial tumours

Histological type Cell of origin Behaviour Genes implicated* Chromosomal abnormalities

Conventional (clear-cell) Proximal renal tubule Malignant‡ VHL, BHD -3p, +5q, -Y, -8p, -9p, -14q;

renal-cell carcinoma t(3;5)(p;q)

Papillary renal-cell Proximal renal tubule Malignant‡ MET, FH, HRPT2 +7,+17, -Y, +12, +16, +20;

carcinoma t(X;1)(p11.2;q21.2),

t(X;17)(p11.2;q25.3)

Chromophobe renal Intercalated cell of Rarely BHD -1, -2, -6, -10, -13, -17, -21 carcinoma renal collecting duct malignant

Oncocytoma Intercalated cell of Benign BHD -1, -Y; t(5;11)(q35;q13), renal collecting duct t(9;11)(p23;q13)

Collecting-duct Renal collecting duct Aggressively FH -1q32, -6p, -8p, -21q

carcinoma malignant

*Genes potentially involved in sporadic neoplasms of each particular type, which have been identified by sequence abnormalities found in

cases of hereditary renal tumours of similar histology. ‡Rarely metastasize if less than at least 3 cm in diameter; if bigger than this, tumours

have an increase in metastatic potential. Tumours smaller than this are occasionally classified as tumours of ‘low malignant potential’ or as ‘adenomas’. BHD, Birt–Hogg–Dubé (encoding folliculin); FH, fumarate hydratase; HRPT2, hyperparathyroidism 2; VHL, von Hippel–Lindau.

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neovascularization of these proliferating renal tumours46–52. In addition, transforming growth factor-α (TGF-α), another HIF target gene, might function in the development of renal tumours, as TGF-αand its recep-tor, epidermal growth factor receprecep-tor, are commonly overexpressed in renal carcinoma53,54. Genes encoding enzymes that are involved in glucose uptake and metab-olism (glucose transporter 1 and phosphoglycerate kinase), pH regulation (carbonic anhydrase 9) and tis-sue-matrix metabolism (matrix metalloproteinases) are also transcriptionally activated by HIF.

Additional functions of VHL are rapidly emerging. VHL is clearly involved in assembly of the extracellular fibronectin matrix55and has been implicated as a regu-lator of epithelial-cell differentiation and cell-cycle exit56–59, perhaps related to its ability to downregulate

cyclin D1(REFS 59,60). VHL also downregulates the

chemokine receptor CXCR4, which is involved in

organ-specific metastasis and is overexpressed in VHL-deficient renal cancers from individuals with poor tumour-specific survival61. However, how these func-tions relate to the development of VHL-associated tumours remains to be established.

Interesting genotype–phenotype correlations are emerging for VHL disease that relate to the

develop-ment of RCC. A group ofVHLmutations termed type

1, comprising mostly deletions and premature-termina-tion mutapremature-termina-tions that cause total loss of VHL funcpremature-termina-tion, predispose to the entire spectrum of VHL-syndrome manifestations except PHEOCHROMOCYTOMAS. By contrast, type 2 mutations, which are mostly missense changes that reduce VHL activity, predispose to the entire VHL spectrum, including pheochromocytomas with or with-out RCC, called type 2B and type 2A, respectively62,63. Studies that dissect the molecular consequences of these mutation types on HIF binding64have revealed that type-1 and type-2B mutations, which predispose to RCC, show complete loss of HIF-1αubiquitylation and residues by an oxygen-dependent mechanism

involving one of several prolyl hydroxylases34.

Crystallographic studies have shown that VHL, through its α-domain, binds to elongin C and forms a complex with elongin B35and CUL2(REF. 36)to pro-duce the VHL SCF-like E3 ubiquitin ligase complex. This complex, through VHL, recognizes and binds to HIF-αafter prolyl hydroxylation37–40and polyubiqui-tylates HIF-α41, targeting it for destruction by the proteasome42. During hypoxic conditions, this oxygen-dependent prolyl hydroxylation can not take place, and HIF-αaccumulates, moves into the nucleus, dimerizes

with HIF-1βand activates expression of

hypoxia-inducible genes (FIG. 1). A second hypoxia-sensing region of HIF-α, the carboxy-terminal transactivation domain (CTAD), binds transcriptional co-activators

p300and CREB-binding protein (CBP) to activate

transcription of HIF-regulated genes under hypoxic conditions. Hydroxylation of a crucial asparaginyl residue in CTAD by factor inhibiting HIF-1 (FIH-1) during normoxia negatively regulates the function of

the HIF-α transactivation domain by preventing

recruitment of p300/CBP43,44.

Experiments in nude mice have confirmed that downregulation of HIF-αis required for tumour sup-pression by VHL45.VHL–/–RCC cell lines produce tumours in nude mice, but re-expression of wild-type VHL prevents tumorigenesis, an effect that is, in turn, reversed by expression of mutant HIF-2α46,47. Therefore, the absence of functional VHL — as occurs in renal cells with VHLinactivation that have also lost their wild-type

VHLallele — prevents degradation of HIF-αby the

proteasome and mimics the cellular response to hypoxia. The hypoxic response, as a result of dysregula-tion of HIF-αsubunits, results in transcriptional activa-tion of hypoxia-inducible genes (FIG. 1). These genes encode growth and angiogenic factors such as VEGF, EPO and platelet-derived growth factor-βthat enhance HYPERMETHYLATION

Methylation of a CpG island in a promoter of a gene usually prevents expression of the gene and can be used to regulate gene expression in a tissue-specific manner. Tumour-suppressor genes might be inactivated by mutation or hypermethylation of promoter regions. KNUDSON TWO-HIT HYPOTHESIS In 1971, Alfred Knudson proposed that two successive genetic ‘hits’ are required to turn a normal cell into a tumour cell and that, in familial cancers, one ‘hit’ was inherited.

PHEOCHROMOCYTOMA A neuroendocrine tumour that typically arises in the adrenal medulla. These tumours can be benign or malignant. Symptoms often relate to the ability of these tumours to secrete

catecholamines.

Table 2 | Heritable syndromes associated with renal-cell neoplasia

Syndrome Causative gene, Renal manifestations Other manifestations

location

Von Hippel–Lindau (VHL) VHL, 3p25 Clear-cell RCC: solid and/or cystic, Retinal and CNS haemangioblastomas; multiple and bilateral pheochromocytomas; pancreatic cysts and

neuroendocrine tumours; endolymphatic-sac tumours; epididymal and broad-ligament cystadenomas

Hereditary papillary renal MET, 7q31 Papillary RCC type 1: solid, multiple None carcinoma (HPRC) and bilateral

Hereditary leiomyomatosis FH, 1q42–43 Papillary RCC type 2, collecting-duct Uterine leiomyomas and leiomyosarcomas; renal-cell cancer (HLRCC) carcinoma: solitary, aggressive cutaneous nodules (leiomyomas)

Birt–Hogg–Dubé (BHD) BHD, 17p11.2 Hybrid oncocytic RCC, chromophobe Cutaneous papules (fibrofolliculomas); lung RCC, clear-cell RCC, oncocytoma: cysts, spontaneous pneumothoraces, multiple, bilateral possibly colon polyps

Hyperparathyroidism-jaw HRPT2, 1q25–32 Mixed epithelial and stromal tumours, Parathyroid tumours, fibro-osseous tumour (HP-JT) papillary RCC: cysts mandibular and maxillary tumours Constitutional chromosome-3 Unknown gene, Clear-cell RCC: multiple, bilateral None

translocation possibly VHL

Familial papillary thyroid cancer Unknown gene, Papillary RCC, oncocytoma Papillary thyroid cancer, nodular thyroid

(FPTC) 1q21 disease

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disrupting proper protein folding mediated by the chap-eronin TriC/CCT67. A recent study of the VHLlocus in 55 affected families has demonstrated that families with partial germline VHLdeletions are more often affected with RCC than families with complete VHLdeletion68.

The development of animal models in which the Vhl gene is deleted should facilitate our understanding of

the biochemical consequence ofVHLinactivation.

Homozygous inactivation ofVhlis embryonically lethal in mice69; however, two mouse models have recently been described in which the Vhlgene was conditionally inactivated using CRE/LOX RECOMBINATION. In the first model, Cre-mediated Vhl inactivation was targeted to the liver and resulted in hepatic haemangiomas70. In the second model,Vhlwas inactivated mosaically in several

organs by Cre recombination under a β-actin

pro-moter; this produced a highly vascular phenotype, including hepatic haemangiomas, abnormal blood ves-sels and angiogenesis in several organs, and defective spermatogenesis71. Interestingly, renal tumours failed to develop in either model, indicating that additional genetic effectors or modifier genes might be necessary for expression of the renal cancer phenotype. The avail-ability of a mouse model in which Vhlis inactivated in the proximal tubule of the kidney will be useful for understanding the renal phenotype of VHL disease.

HPRC.Families with HPRC, which is inherited in an autosomal-dominant pattern, develop multifocal, bilat-eral papillary RCC with a papillary type-1 histology (TABLE 2).An early report describing ten HPRC families noted a late age of onset and a male/female ratio of 2:1 among affected members72. Metastasis is less frequent, and age-dependent penetrance in mutation carriers seems to be reduced relative to penetrance in VHL syn-drome. A genome-wide scan in three families with HPRC localized the disease locus to chromosome 7q31, and activating germline mutations were subsequently identified in the tyrosine kinase domain of the MET proto-oncogene5,73–75.

The METproto-oncogene encodes a receptor

tyro-sine kinase that is activated by hepatocyte growth factor (HGF)76. MET–HGF signalling is important for the EPITHELIAL–MESENCHYMAL TRANSITION, cell proliferation, branching morphogenesis, differentiation and regulation of cell migration in many tissues, and integration of these pathways causes ‘invasive growth’. Phosphorylation of crucial tyrosines in the carboxyl terminus of MET generates a docking site for second messengers (FIG. 2), which activate several signalling pathways involving RAS, phosphatidylinositol 3-kinase, STATs and phospholipase Cγ (for reviews, see REFS 77–79).METis overexpressed in many cancers80, but had not previously been found to be mutated in human cancer. Most of the HPRC-associated germline mutations lie within the MET activation loop or in the ATP-binding pocket, and cause ligand-indepen-dent MET activation81. Several of the HPRC-associated

METmutations lie in codons that are homologous to

sites of disease-causing mutations in other receptor tyro-sine kinases, indicating that these residues are required for the function of receptor tyrosine kinases74,82–84. regulation, whereas type-2A mutations result in an

incomplete defect in HIF regulation. However, type-2A mutations have been shown to disrupt binding of VHL to microtubules and abrogate the associated micro-tubule-stabilizing function of VHL, implicating defective cytoskeleton organization in this VHL phenotype65. An interesting third VHL-syndrome subclass (type 2C) pre-disposes almost exclusively to pheochromocytomas. Type-2C mutations produce VHL that regulates HIF but is defective in fibronectin assembly, indicating a possible link between fibronectin-matrix assembly and

pheochromocytoma development66. Another class

of VHL point mutations inactivates VHL function by CRE/LOX RECOMBINATION

A method in which the Cre recombinase enzyme catalyses recombination between loxP sequences. If the loxPsequences are arranged as a direct repeat, recombination will delete the DNA between the sites.

HIF-1α HIF-1α HIF-1α Hypoxia OH OH HIF-1α OH Prolyl and asparaginyl hydroxylases RBX1 VHL HIF-1α VHL VHL inactivation Ubiquitylation 26S Proteasome Degradation p300/CBP p300/CBP Transcription of HIF target genes

Nucleus Cytoplasm Elongin B Elongin C CUL2 RBX1 Elongin B Elongin C CUL2 E3 ubiquitin ligase complex N P N HRE HIF-1α HIF-1β Fe2+ 02

Figure 1 |Dysregulation of HIF-1αby VHL inactivation leads to clear-cell renal tumours in patients with von Hippel–Lindau disease.Under normoxic conditions, hypoxia-inducible factor-1α(HIF-1α) is hydroxylated (-OH) on two conserved proline residues (for simplicity, only one is shown) by a family of prolyl hydroxylases at its oxygen-dependent degradation domain. This hydroxylation provides a substrate-recognition site for the von Hippel–Lindau (VHL) E3 ubiquitin ligase complex, which contains elongins C and B, cullin-2 (CUL2) and RBX1. Polyubiquitylation of HIF1-α by the VHL complex leads to its proteasomal degradation by the 26S proteasome. HIF1-α is also hydroxylated at an asparagine residue in its carboxy-terminal transactivation domain by FIH-1, an asparaginyl hydroxylase. This blocks binding of the transcriptional coactivators CREB-binding protein (CBP) and p300 to HIF-1α, thereby inhibiting transcription of HIF target genes. Hypoxic conditions block both types of hydroxylation, allowing HIF-1αsubunits to accumulate and activate transcription of hypoxia-responsive genes. VHLinactivation — as occurs in renal cells from patients with a germline VHLmutation and loss of the wild-type allele — mimics the hypoxic response by preventing degradation of HIF-1αsubunits. Loss of VHL function causes accumulation of HIF-1α subunits in the cytoplasm and their translocation to the nucleus. HIF-1αdimerizes with HIF-1βand is coactivated by CBP/p300. HIFα/βbinds to hypoxia response elements (HRE) in gene promotors, thereby activating transcription of genes upregulated in clear-cell renal tumours, including vascular endothelial growth factor, erythropoietin and platelet-derived growth factor-β. (HIF-1αis shown, but HIF-2αis also recognized as a VHL substrate.)

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been described in association with HLRCC99,100. Lack of multifocal disease in the kidney and the presence of seg-mental distribution of skin lesions indicate the possibility ofMOSAICISMin some affected individuals99. Phenotypic features vary among families and among members within families who carry the same germline mutation. In addition, some families develop the cutaneous and uter-ine features and only rarely renal cancer, a syndrome called multiple cutaneous and uterine leiomyomatosis101.

METmutations were not identified in papillary RCC from patients with HLRCC; linkage analysis performed by two independent groups mapped the predisposing gene to chromosome 1q42–44 (REFS 95,101). Subsequently and in collaboration, these groups identified germline HLRCC-associated mutations in FH, a mitochondrial Krebs-cycle enzyme that converts fumarate to malate6 (FIG. 3). The spectrum of mutations includes missense, insertion/deletion and NONSENSE MUTATIONSthat are pre-dicted to truncate the protein, or substitute or delete highly conserved amino acids, along with several whole-gene deletions. FH activity evaluated in lymphoblastoid cell lines from patients with HLRCC shows a 20–50% reduction compared with unaffected individuals, and when FH levels were measured in CUTANEOUS LEIOMYOMAS, no activity was observed relative to normal skin

sam-ples. So, the consequence of mutations in FHis a

severe reduction in enzyme activity. LOH on chromo-some 1q42 as well as several acquired somatic muta-tions have been observed in papillary RCC, and LOH was detected in UTERINE LEIOMYOMASfrom patients with HLRCC. These experimental observations clearly

sup-port a role for FHas a tumour-suppressor gene.

However, unlike VHL, few FHmutations (<2%) have been identified in sporadic-tumours including RCCs, uterine and cutaneous leiomyomas, and sporadic prostate and breast tumours102–104.

FHmutations are found throughout the entire gene, with no particular genotype–phenotype correlations for HLRCC being identified so far. Nearly 40 different mutations have been identified in FHthat predispose to cutaneous and uterine leiomyomas and renal can-cer6,99,100. Several of the mutations occur in many fami-lies, which could reflect a founder effect; notably, the Arg190His mutation, which is the most frequent muta-tion (33%) in a North American family study, and the Arg58X and Asn64Thr mutations in studies by the European-based Multiple Leiomyoma Consortium. Arg190His and Lys187Arg mutations were detected in both North American and European families with HLRCC. Types of mutations in patients with HLRCC are similar to those seen in patients with recessive FUMARASE DEFICIENCY, except that the latter tend to be located in the 3′ end of the gene. More truncating and whole gene deletion mutations are seen in HLRCC than in fumarase deficiency105.

The overall risk for renal tumour development is

unclear and the mechanism ofFH-mutation-driven

tumorigenesis is unknown at present. One can speculate that defects in the Krebs cycle might lead to blockage and feedback effects on oxidative metabolism and, therefore, the cell cycle. Energy-independent apoptosis is mediated To understand the mechanism of MET activation by

these mutations, molecular modelling studies of the MET kinase domain were performed using insulin receptor kinase as a model74,85. In its inactive form, the MET kinase domain assumes a bi-lobal configuration, with a self-inhibitory activation loop blocking access of ATP to its binding pocket. It is predicted that all of the missense mutations found in the germline of HPRC patients either destabilize the self-inhibited inactive form of MET by dis-placing the activation loop from the ATP-binding pocket, or stabilize the active form, thereby allowing ATP to access the binding pocket and activate the kinase. However, addition of HGF to cells expressing mutant MET further activated the kinase86, indicating that the need for HGF stimulation for full activation of MET was lowered, but not completely eliminated, by these missense mutations (FIG. 2). Although two sequential tyrosine phosphorylation events in the catalytic domain are required for wild-type MET activation, HPRC-associated MET mutants only required phosphorylation of one of the two tyrosines. So, the threshold that is nec-essary for activation of mutant MET is lowered as the second phosphorylation event is no longer required87. Duplication of the chromosome 7 which carries the mutant METallele within the patient’s renal cells (FIG. 2) provides the second activating event and primes the cell to develop a papillary renal tumour88,89. It is possible that the additional cytogenetic changes documented in many of these tumours are required for progression from a small papillary adenoma to papillary renal-cell carcinoma (TABLE 1).

Met-knockout mice develop an embryonic-lethal phenotype presenting liver, placenta, muscle and nerve

defects90–92. A mutant METmouse model has been

described in which one of the HPRC-associated MET mutations was introduced as a transgene; however, these mice develop mammary carcinoma, not renal cancer93, a phenotype that is also observed in transgenic animals

expressing a constitutively activated form of MET

known as TPR-MET94. Targeted overexpression of

HPRC-associated METmutations in the kidney will

possibly produce a mouse model of papillary RCC.

HLRCC.Individuals with the autosomal-dominant syndrome HLRCC have an increased risk for papillary RCCs of type-2 morphology, which is an aggressive can-cer less commonly encountered than papillary RCC type 1 (REFS 95,96;TABLE 2). Papillary RCC type 1 is char-acterized by cells with scant cytoplasm that are arranged in single layers on papillary cores and often contain foamy macrophages, whereas papillary RCC type 2 is characterized by larger cells with eosinophilic cytoplasm and pseudostratified nuclei97. Type-2 papillary RCC also differs cytogenetically from type-1 RCC and confers a worse prognosis, as shown in a multivariate analysis that included tumour grade and stage98,99. The renal tumours in patients with HLRCC are usually found as solitary and unilateral tumours; this is in contrast to those in VHL syndrome, HPRC and BHD, which are often mul-tiple and bilateral. In addition, at least two cases of aggressive renal collecting-duct carcinoma have also EPITHELIAL–MESENCHYMAL

TRANSITION

Conversion from an epithelial to a mesenchymal phenotype, which is a normal component of embryonic development. In carcinomas, this transformation results in altered cell morphology, the expression of mesenchymal proteins and increased invasiveness. MOSAICISM

Postzygotic mutations resulting in some, but not all, of a patient’s tissues being affected by gene mutation. Patients with mosaicism represent de novo cases of a disease within the family. The patient might be asymptomatic or have less severe disease than offspring and might test negative for a germline mutation, making them difficult to diagnose.

NONSENSE MUTATION A sequence alteration in the DNA that changes a codon specific for one amino acid to a chain termination codon, that is, TAA, TAG or TGA. Termination codons produce premature truncated proteins that are likely to abolish protein function. CUTANEOUS LEIOMYOMAS Benign smooth-muscle tumours of the skin presenting as firm, skin-coloured papules and nodules.

UTERINE LEIOMYOMAS Also known as a ‘fibroids’, benign smooth-muscle tumours of the uterus, the most common gynaecological tumours in women of reproductive age. Fibroids can interfere with child-bearing.

FUMARASE DEFICIENCY An autosomal-recessive disorder in which biallelic FHmutations cause gross developmental delay and death in the first decade.

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GAB1 β α β α MET MET MET MET β α β α HGF HGF P P P P P P P P P P P P Y1234 Y1235 Y1234 Y1235 β α β α HGF Transphosphorylation of catalytic domain GRB2 Docking-site autophosphorylation and second- messenger binding • Cell polarity • Actin cytoskeleton • Motility Auto-inhibited MET • Proliferation • Cell-cycle progression • Cell-junction formation • Migration • Invasion Survival • Cell polarity • Actin cytoskeleton • Motility • Proliferation • Cell-cycle progression • Cell-junction formation • Migration • Invasion Survival β α β α HGF β α β α HGF P Lower activation threshold Transphosphorylation Y1235 only Docking-site autophosphorylation and second- messenger binding Kinase-activated MET

a Normal renal cell

b HPRC cell with MET mutation

Additional trisomies of Chr 16, Chr 17 and Chr 20 Nucleus Chromosomes MET MET Y1349 Y1356 GAB1 P P P P P P β α β α HGF GRB2 MET MET Y1349 Y1356

MET MET MET

P Y1235 MET Y1235 Dimerization Increased activation Duplication of Chr 7 carrying mutant MET

Figure 2 |Activating missense mutations in METlead to papillary renal carcinoma. a| In normal cells, hepatocyte growth factor (HGF) binds the MET receptor to induce MET dimerization and release auto-inhibition by the MET carboxyl terminus. This permits transphosphorylation of catalytic tyrosine (Tyr)1234 and Tyr1235. Subsequent phosphorylation of the multisubstrate docking sites Tyr1349 and Tyr1356 promotes binding of second-messenger molecules, such as GRB2, GAB1, phosphatidylinositol 3-kinase (not shown), and downstream signalling leading to morphogenic, motogenic and mitogenic programmes. b| Renal cells from patients with hereditary papillary renal carcinoma (HPRC) can harbour germline mutations (star) in the tyrosine kinase domain of MET. These mutations are predicted to release the auto-inhibition by the MET carboxyl terminus, allowing the receptor to transition to the active kinase form in the absence of ligand stimulation85. However, addition of HGF fully activates mutant MET kinase by stimulation of transphosphorylation of Tyr1235 only87. Signals for proliferation, invasion and survival occur after docking-site phosphorylation and second-messenger binding. Additional steps such as duplication of mutant MET-bearing chromosome 7 and trisomy of chromosomes 16, 17 and 20 might be necessary for the development of these late-onset papillary renal carcinomas.

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thought to be hypermutable because of slippage of the DNA polymerase during replication117.

Several animal models of BHD have been reported. The Nihon rat RCC model develops hereditary renal can-cer, which is inherited in an autosomal-dominant man-ner with complete penetrance by 6 months of age. The gene locus was mapped by linkage analysis to

chromo-some 10p, which is SYNTENICwith human chromosome

17p11.2 (REFS 118–119), and a disease-associated insertion mutation in the first coding exon of the rat Bhd ortho-logue was identified120. Most renal tumours displayed LOH at the Bhdlocus and the homozygous mutant con-dition resulted in embryonic lethality. Canine hereditary multifocal renal cystadenocarcinoma and nodular der-matofibrosis (RCND) is a naturally occurring inherited cancer syndrome in German-Shepherd dogs that is char-acterized by bilateral, multifocal renal tumours and numerous firm skin nodules made up of dense collagen fibres121. The RCND locus maps to canine chromosome 5, which corresponds to human chromosome 17p11.2 (REF. 122). A missense mutation that changes a highly con-served histidine to arginine was found in the canine Bhd orthologue, which co-segregated with disease in the canine families with RCND and was not found in 264 control dogs123. Homozygosity of the mutation is embry-onically lethal in dogs with RCND, as in the Nihon rat. by oxygen free radicals, and impairment of

mitochondr-ial function leads to severe energy deficits and the gener-ation of large amounts of oxygen free radicals, which cause hypoxia (FIG. 3). Subsequent upregulation of HIF-1αand transcriptional activation of hypoxia-inducible genes, reminiscent ofVHLinactivation, could explain the development of papillary type-2 RCC in response to FHinactivation106. Recently, germline mutations in suc-cinate dehydrogenase B, another enzyme in the Krebs cycle, were found in patients with hereditary paragan-glioma syndrome who developed early onset renal can-cer107, further supporting the idea that impairment of mitochondrial function creates a hypoxic environment that can promote RCC development.

BHD.Birt–Hogg–Dubé (BHD) syndrome, named

after the three Canadian physicians who first described the dermatological features108, is an autosomal-domi-nant genodermatosis that predisposes individuals to FIBROFOLLICULOMASon the face and neck,SPONTANEOUS PNEUMOTHORAXand/or lung cysts, and an increased risk of renal neoplasia (TABLE 2). Phenotypic manifestations vary within and between families. A strong connection between the BHD skin lesions and renal manifesta-tions was made when members of several families with inherited renal oncocytomas were identified with the classic BHD hallmark, fibrofolliculoma109,110. In a large risk-assessment study of family members affected with BHD skin lesions, a 7-fold increased risk for development of renal tumours and a 50-fold increased risk for sponta-neous pneumothorax were found111. Although some have reported a co-association of BHD with colon polyps or

colon cancerin other families112, no increased risk for colon manifestations was identified in this study111.

Renal tumours found in individuals with BHD can be multifocal and bilateral, or unilateral and single, and display various histological features, including CHROMOPHOBE RCC, oncocytoma, clear-cell RCC and even, rarely, papillary RCC (TABLE 2). However, the most frequent renal tumours are chromophobe RCCs and ONCOCYTIC HYBRID TUMOURScomprising elements of

both oncocytomas and chromophobe RCCs113,114.

Microscopic oncocytosis found in the renal parenchyma indicated that these lesions might be precursors of hybrid oncocytic tumours, chromophobe renal carcinomas and perhaps clear-cell renal carcinomas in individuals with BHD syndrome. As individuals with large renal tumours have died of metastatic RCC, the renal manifestations of the syndrome can not be considered benign113.

Using fibrofolliculomas as an indicator of affected

status, the BHDlocus was identified on chromosome

17p11.2 by linkage115,116, and mutations that co-segre-gated in BHD families were identified in a new gene7. BHDencodes folliculin — named for the fibrofollicu-lomas seen in BHD patients. Folliculin has no signifi-cant homology to any known human proteins although it is highly conserved across species. Almost all BHD mutations are insertions, deletions or non-sense mutations, predicted to truncate or prematurely terminate folliculin7,112. Interestingly, nearly half of the mutations occur in a mononucleotide tract of cytosines FIBROFOLLICULOMAS

Benign hamartomas of the hair follicle, characterized by anastomozing (branching) strands of proliferating epithelial cells extending from a central hair follicle encapsulated by loose, mucin-rich connective-tissue stroma.

SPONTANEOUS PNEUMOTHORAX

A sudden rupture of lung tissue, resulting in air escaping from the lung into the pleural cavity. CHROMOPHOBE RCC Tumour with well-defined cell borders, fluffy eosinophillic cytoplasm, pyknotic nuclei and perinuclear halos.

ONCOCYTIC HYBRID TUMOUR Hybrid tumours contain zones classic for more than one type of tumour histology. Oncocytic hybrid tumours contain areas with cells histologically consistent with oncocytoma, areas consistent with chromophobe RCC and mixed areas.

SYNTENY

In this context, this term is used to refer to gene loci in different organisms that are located on a chromosomal region of common evolutionary ancestry.

Mitochondrion HIF-1α HIF-1β p300/CBP p300/CBP HRE Transcription of

HIF target genes Hypoxia Nucleus HIF-1α stabilization HIF-1α HIF-1β Fumarate FH Malate ↑O2ATP

Figure 3 |Fumarate-hydratase-inactivating mutations in hereditary leiomyomatosis renal-cell carcinoma lead to papillary type-2 renal-cell carcinoma.It is likely that impaired mitochondrial function due to fumarate hydratase (FH)-inactivating mutations (star) which block the conversion of fumarate to malate by the Krebs cycle lead to severe energy deficits (depletion of ATP) and the formation of oxygen free radicals (O2). This is sensed by the mitochondria as hypoxia,

which leads to stabilization of HIF-1αsubunits (see Fig. 1 for details) and transcriptional upregulation of hypoxia-inducible genes such as vascular endothelial growth factor, erythropoietin, platelet-derived growth factor-βand transforming growth factor-β106. These proteins promote cell proliferation that could activate tumour growth.

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tumorigenicity in FHIT-negative cells indicates a tumour-suppressor role138; however, how FHITcontributes to tumour development remains to be established. The other breakpoint-spanning gene in this translocation family is TRC8, which is located on chromosome 8 and is similar to the Drosophilasegment-polarity gene patched (ptc)139. PTCH, the human homologue ofptc, acts as a tumour-suppressor gene and is involved in basal-cell carcinoma and medulloblastoma.DrosophilaTrc8 protein was found to interact with the DrosophilaVhl protein, presenting a

possible common pathway for TRC8 and VHL in

humans140. Disruption of three novel genes,DIRC1 (2q33)141,DIRC2(3q21)142, and DIRC3(2q35)143, has been identified in two other families that have translocations with breakpoints at t(2;3)(q33;q21) and t(2;3)(q35;q21). DIRC3was shown to form fusion transcripts with HSP-BAP1, a JmjC-Hsp27 domain gene, and might affect chro-matin remodelling or stress-response signals leading to renal-cell carcinoma.LSAMP(1q32), an IgLON family

member, and NORE1(3q13), which is homologous to

a family of RAS-binding proteins, were identified as breakpoint-spanning genes in a Japanese family with t(1;3)(q32.1;q13.3), and hypermethylation of promoter regions of both genes was reported in renal tumours of patients144. However, causative roles for each of these translocation breakpoint genes in the development of RCC remain to be determined.

Given the role ofVHLin the development of clear-cell renal tumours, several renal tumours from affected members of these families with the chromosome-3 translocation were tested for 3p loss and VHL muta-tions. Nearly all cases showed loss of the derivative

chromosome 3, and VHLmutations were confirmed in

12 of 22 renal tumours135,145. These molecular and cytogenetic results indicate a three-step model for development of renal carcinoma in these families with chromosome-3 translocations11,145: inheritance of a germline chromosome-3 balanced translocation; non-disjunctional loss of the derivative chromosome that carries the short arm of chromosome 3; and somatic mutation or hypermethylation of the remaining tumour-suppressor gene on 3p, such as VHL. It is possible that other tumour-suppressor genes on chro-mosome 3p, such as RASSF1A, which is commonly hypermethylated in clear-cell RCC, are involved in the development of the renal carcinomas that are seen in these families with chromosome-3 translocations.

Papillary thyroid carcinoma with associated papillary renal neoplasia.Papillary thyroid carcinoma is usually sporadic, but evidence for a genetic predisposition occurs in about 5% of cases. FPTC is characterized by autosomal-dominant inheritance with age-dependent partial penetrance and an association with benign nodular thyroid disease. Linkage to several loci has been reported. An unusually large three-generation family with FPTC was recently described in which two affected members had multifocal papillary renal neoplasms and one affected member developed renal oncocytoma12. METmutation analysis and analysis of genetic linkage to chromosome 1q21 was negative. This ruled out In contrast to VHL,BHDis infrequently mutated in

sporadic renal tumours in humans, as several studies show a mutation rate of less than 10%124,125. Although 17%–36% of sporadic renal tumours show LOH at the

BHDlocus and 11–33% show hypermethylation of the

BHDpromoter, depending on their histology124,126, only

one case with both somatic BHDmutation and LOH

has been reported125indicating that BHDprobably has only a minor role in the development of sporadic RCC. One study also reported LOH at the BHDlocus in 81% of colorectal carcinomas and another detected a low fre-quency ofBHDmutations in a set of colon carcinoma samples with MICROSATELLITE INSTABILITY125,127,128. Further clarification of the role ofBHDin colorectal carcinoma will require additional family studies.

The function ofBHDin normal cells and how pro-tein-truncating mutations in folliculin lead to renal tumours, fibrofolliculomas and spontaneous pneumo-thorax remain to be established. Functional studies of the BHD protein have not been reported and therefore its role in the cell is unclear. Studies ofBHDorthologues indicate a tumour-suppressor role for BHD,further sup-ported by the high frequency of inactivating mutations found in the germline of individuals with BHD. Identification of ‘second-hit’ mutations in BHD-associ-ated renal tumours would confirm a tumour-suppressor function for folliculin.

HPT-JT.HPT-JT is a rare autosomal dominantly inher-ited syndrome, which predisposes individuals to develop multiple parathyroid adenomas and multiple fibro-osseous tumours of the jaw129,130. Affected individuals from some families present with renal disease, including cystic kidney disease,HAMARTOMAS, mesoblastic nephro-mas, late-onset Wilms’ tumours and, in one patient, papillary renal-cell carcinoma131,132. Recently, germline mutations were identified in a new gene — HRPT2— on chromosome 1q, which are predicted to cause deficient or impaired protein function8. Associated LOH of the wild-type chromosome in the region of 1q21–q32, which is found in renal hamartomas131, and frequent biallelic inac-tivation ofHRPT2in sporadic parathyroid tumours133,134 indicate that HRPT2acts as a tumour suppressor. The function of the HRPT2 protein — parafibromin — is now under investigation.

Hereditary syndromes with unknown genes Breakpoint genes.At least seven families that carry a constitutional chromosome-3 translocation have been described135. These individuals have an increased risk for developing bilateral multifocal RCC, most often with conventional (clear-cell) histology, and genes at or near the breakpoints of the two chromosomes involved might be involved in RCC development. The fragile his-tidine triad (FHIT) gene spans the chromosome-3 breakpoint that was first identified in the first transloca-tion family reported by Cohen8,136.This family harbours a t(3;8)(p14;q24) balanced translocation.FHIT colocal-izes with the most common fragile site in the human

genome — FRA3B— and its expression is reduced or

lost in various neoplasms137.FHITsuppression of MICROSATELLITE INSTABILITY

Describes diploid tumours in which genetic instability is due to a high mutation rate, primarily in short nucleotide repeats. Cancers with the microsatellite instability phenotype are associated with defects in DNA mismatch-repair genes.

HAMARTOMAS

Tumours comprising cells of more than one histological type.

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If an RCC syndrome is not clearly evident after considering family history, physical examination and abdominal imaging, an effective way to tailor subsequent diagnostic studies is to obtain a pathological diagnosis of renal tumour type. Histological type varies between hereditary tumour syndromes; for example, in VHL and HPRC a characteristic histology is noted in all cases (TABLE 2). So, histological differences between multiple renal tumours from the same individual virtually rule out VHL and HPRC, but suggest BHD. Careful pathological re-review of all tumours resected, as well as tumours resected from first-degree relative(s) can help focus sub-sequent testing for specific syndromes. For example, if a genodermatosis (HLRCC or BHD) is suspected, skin biopsies and a high-resolution chest computed-tomogra-phy scan to look for lung cysts or evidence of previous pneumothoraces are recommended. If VHL is suspected, standard practice includes central nervous system imag-ing and biochemical testimag-ing for pheochromocytoma.

The cloning of causative genes for VHL, HPRC, HLRCC, BHD and HPT-JT has made definitive genetic testing a reality for most syndromes that predispose to RCC. Germline genetic testing requires evaluation and referral by a certified genetic counsellor. At present,VHL screening and karyotyping are performed at numerous diagnostic laboratories in the United States, and tests for germline MET,FH and HRPT2mutations are now avail-able (for further information, see the GeneTests web site

in the online links box). The following recommendations can tentatively be made at this time.

If the diagnostic criteria for VHL are met (for a review, see REF. 151), then germline VHLanalysis is

rec-ommended. Genetic analysis of the VHL locus has

demonstrated VHLinactivation in 100% of patients

presenting with VHL by clinical criteria152. Families with a documented predisposition to clear-cell RCC should be screened for germline VHLabnormalities and kary-otyped to rule out chromosome-3 translocations. If papillary RCCs have been detected in a familial setting, then the sub-type — papillary type 1 or type 2 — must be determined histologically so that appropriate genetic testing can be considered: mutational screening of exons 16–19 ofMETfor HPRC and mutational analyses ofFH for HLRCC (enzymatic-activity assays at present, but mutational analyses should be available soon). Genetic screening for BHDshould be considered once this test becomes available. Any patient with hybrid oncocytic neoplasms, multifocal chromophobe RCC, and/or oncocytomas should ultimately be tested for mutations in BHD, as should families with discordant renal tumour histologies within or between family members.

Genetic screening of ‘at risk’ relatives, that is, first-degree relatives of patients suspected to have hereditary renal neoplasia, should only occur once the proband has been found to have a heritable genetic anomaly causative of renal cancer. In cases where germline genetic testing is not available (for example, in BHD, FCRC and FPTC), an updated search for such tests, followed by a history and physical examination of first-degree relatives are required if suspicion is high for a syndrome. These individuals should also be offered abdominal imaging at this time. HPRC and indicated that a new renal-cancer-associated

gene could be responsible for this familial syndrome.

FCRC and future renal cancer gene discovery.Eleven small families, each with 2–5 members affected with familial clear-cell renal carcinoma (FCRC) without VHLmutations have also been reported146,147. Linkage to chromosome 3p was excluded in all eleven families, nine of which were screened and found to be negative for mutations in MET andCUL2, indicating that additional renal carcinoma genes must exist. A large population-based study was undertaken in Iceland to assess the role of heredity in the development of RCC in this isolated country148. An extensive genealogical database ascer-tained the relatedness of all individuals diagnosed with RCC over a period of 45 years; first-degree relatives of RCC patients (siblings and parents) were found to have a 2–3-fold higher risk of developing RCC compared with the general population. Interestingly, a similar 2.5-fold ODDS RATIOfor those with first-degree relatives affected by RCC was noted in a population from the western United States149, strongly supporting a genetic component in common non-VHL RCC. An extensive recruitment effort is now underway in the United States for families with two or more individuals affected with non-VHL RCC for the purpose of mapping cancer-susceptibility genes for familial RCC (W.M. Linehan and B. Zbar, personal communication).

Diagnostic considerations

There are no generally accepted screening guidelines for hereditary RCC syndromes; however, some recommen-dations can be made. A hereditary predisposition to renal cancer should be suspected whenever an individual who is diagnosed with renal cancer has a close relative also diagnosed with the disease, and/or when an individual presents with multifocal renal tumours or a history of previous renal tumour. Family history should be obtained and a pedigree created, paying specific attention to relatives with a known history of cancer. The relatively well-characterized syndromes VHL, HPRC, HLRCC, BHD and HPT-JT are all inherited in an autosomal-dominant fashion. VHL, HLRCC, BHD, HPT-JT and FPTC have non-renal manifestations, whereas other syn-dromes, such as HPRC, FCRC and chromosome-3 translocation, generally do not. Therefore, a thorough physical examination, focusing on skin, ophthalmologi-cal, neurologiophthalmologi-cal, parathyroid and thyroid abnormalities should be carried out. HLRCC and BHD are associated with dermatological lesions that can be observed by astute clinicians. Abdomino-pelvic computed tomogra-phy or magnetic resonance imaging with and without intravenous contrast medium are recommended for all individuals with suspected primary renal tumour, as ultrasound is insensitive for renal masses, particularly HPRC150. Multifocal and bilateral renal tumours are com-monly found in RCC associated with hereditary syn-dromes (with the exception of HLRCC), and extra-renal manifestations of disease — such as uterine tumours in HLRCC, pancreatic cysts or tumours, or adrenal pheochromocytomas in VHL — might also be noted. ODDS RATIO

The odds ratio is a way of comparing whether the probability of a certain event is the same for two groups, and is calculated using a 2 × 2 table. An odds ratio of one implies that an event is equally likely in both groups. An odds ratio greater than one implies that an event is more likely in the first group. An odds ratio less than one implies that the event is less likely in the first group.

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sporadic neoplasia. This is well demonstrated for colon cancer, where hereditary cancer syndromes have led to the discovery of genes involved in sporadic colon can-cer, and the sequential inactivation of different genes and genetic instability result in the malignant degener-ation of precancerous lesions157. For renal cancer, the paradigm that the germline abnormality in hereditary syndromes is commonly found in sporadic tumours is true for VHLin most, and for MET in some, cases of RCC. Further research is needed to determine whether the other syndrome-related genes discussed in this review contribute to sporadic RCC. This line of investi-gation would determine whether the progression of RCC depends on sequential alterations in distinct genes or whether RCC truly comprises distinct tumour types each with a classic mutation or set of genetic alterations, as might be predicted from most histological, molecular and cytogenetic analyses. So far, only a handful of renal tumours have been described where inactivation of more than one renal cancer tumour-suppressor gene

has been found (BHD and VHL), indicating that

tumours possibly progress from a more benign to a more malignant histology113.

A key goal in clinical oncology is the development of medical therapies specific to pathways that are altered in cancer. Understanding the biological pathways involving

VHL, MET, FH, BHD andHRPT2will provide new

therapeutic approaches for kidney cancer that were unimaginable before the identification of these target genes. Although the oncogenic pathways resulting from

HRPT2and BHDmutation remain unknown, and

those indicated by FH mutation remain speculative106, much information is known about the pathways down-stream of VHL and MET. Several groups have shown that VHL and MET are part of a common signalling pathway that is deregulated in RCC. Hypoxia activates METtranscription and amplifies HGF signalling to pro-mote MET-dependent invasive growth158. Expression of wild-type VHL in renal carcinoma cell lines inhibits HGF-induced invasion and branching morphogenesis by increasing levels of tissue inhibitors of metallopro-teinases159. Tumour invasion and metastasis requires dysregulation and degradation of the tissue matrix in cancer cells, and this complex process has been associ-ated with HGF/MET signalling. The specific targeting of such pathways in the hope of preventing RCC in high-risk individuals or medically treating existing RCC is the main goal of kidney oncology.

Specific therapeutic options for VHL-related neopla-sia involve restoring VHL protein function, inhibiting HIF activity, and/or targeting the downstream pathways that are activated by HIF (FIG. 4). Therapeutic gene replacement is a future possibility for VHL syndrome and for any syndrome that involves an inactivated tumour suppressor, possibly FH, BHD and HRPT2. As the hypoxia pathway is activated in clear-cell RCC, sev-eral trials have already targeted angiogenesis in individ-uals with metastatic RCC. These include attempted

therapy with thalidomide160and the VEGF-receptor

inhibitor SU5416 (REF. 161), and a successful trial using an anti-VEGF antibody162. Current trials of imatinib Follow-up imaging scans should be obtained at

reg-ular intervals for carriers of germline predispositions to renal cancer. These studies are generally obtained every 6–12 months depending on the growth rate of a patient’s previous or existing tumours.

Implications for the treatment of RCC

Individuals diagnosed with hereditary RCC should be considered for nephron-sparing treatment regimens, as these individuals are at risk of recurrent renal tumours throughout their lifetime. Such regimens include regular observation for most hereditary RCC tumours less than 3 cm in diameter153, percutaneous ablation of renal tumours by radiofrequency154(heat) or cryotherapy155 (cold), and/or nephron-sparing surgical approaches such as partial nephrectomy156. Regular observation of patients with hereditary forms of RCC, followed by the removal of all lesions in the same kidney once a single lesion reaches greater than 3 cm, has been instituted by the National Cancer Institute in an attempt to minimize the number of operations an affected individual must have during his/her lifetime. Observation should include bi-yearly to yearly imaging, depending on the tumour growth rates. Very few deaths from metastatic RCC in families with VHL, HPRC and BHD have been noted using this strat-egy, which has led to fewer surgeries in individuals affected by recurrent hereditary renal tumours153. HLRCC is an exception, as RCC in these patients is often large and already metastatic at presentation. Surgical ther-apy is therefore recommended when HLRCC tumours are detected at any size before metastasis has occurred.

Future directions

Hereditary cancer syndromes often represent uncom-mon germline genetic abnormalities, which are similar to more common somatic abnormalities that cause HIF-1α HIF-1α VHL p300/CBP Replace VHL gene, protein or function Inhibit HIF-1 activity

Inhibit one or more HIF-induced gene products

Transcription of HIF target genes

Nucleus 26S proteosome degradation HIF-1α HIF-1β HRE

Figure 4 |Possible targets for VHL-related therapeutics.Targeting the von Hippel–Lindau (VHL) pathway for therapeutic intervention can theoretically occur at many sites. VHL protein function could be replaced, restoring binding to hypoxia-inducible factor-1α(HIF-1α) and allowing its proteasomal degradation. The activity of HIF-1 could be a target for inhibition. Finally, molecules upregulated by HIF-1 also provide specific targets for potential downstream inhibition of the VHL pathway.

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become commonplace. Gene-expression profiling of renal tumours and of affected individuals should help to identify those individuals that benefit most from mole-cule-specific cancer therapies, as has been elegantly shown for haematopoietic malignancies and melanoma165,166. Such work in renal neoplasia has already generated expression profiles for the common renal can-cer histologies167and expression profiles with prognostic relevance for conventional (clear-cell) RCC168,169. Current immunotherapeutic approaches to systemic treatment of advanced RCC are likely to benefit from the wealth of new molecular information obtained from such studies until more effective therapies are developed.

Taken together, the advent of better diagnostic and discriminatory tools for both the hereditary and spo-radic forms of renal cancer will no doubt improve the prognosis of diseases that, at present, are almost always lethal once metastatic. The increase in availability of genetic testing and counselling for high-risk families should prove both helpful and cost-effective, as geneti-cally unaffected family members are reassured regarding their health status and removed from lifelong follow-up screening programmes.

(Glivec), an inhibitor of the BCR–ABLfusion gene

product that also inhibits HIF-upregulated platelet-derived growth factor, and of EGF receptor inhibitors are also underway for clear-cell RCC.

MET inhibitors are also being studied. SU11274 is a small-molecule inhibitor of MET that competitively binds its ATP pocket and has had encouraging in vitro results; SU11274 adversely affects cellular growth in a dose-dependent fashion. Autophosphorylation of MET was reduced and the inhibitor blocked phosphorylation of AKT, glycogen synthase kinase-3βand the pro-apop-totic transcription factor FKHR163. The drug gel-danamycin has also shown in vitroeffects against MET expression and its downstream effects164. The develop-ment of agents that inactivate MET tyrosine kinase activity could provide future targeted therapies for oncogene-related renal tumour syndromes like HPRC.

Ultimately, the study of families with increased rates of cancer will continue to yield more insight into the factors that increase cancer risk. Genetic predispo-sitions in the form of mutations and polymorphisms will increasingly be catalogued and DNA-level genetic profiling of high-risk families and individuals will

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Crystal structure of VHL protein in complex with elongins B and C that identified the frequently mutated α- and β-domains.

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40. Jaakkola P. et al. Targeting of HIF-αto the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science292, 468–472 (2001).

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