**2. Genes associated with renal tumors**

While kidney cancer ranked 9th in 2002 in the European Union and the United States (Baldewijns et al., 2008), its mortality rate was not high in Japan (12th in 2002 and 2007: Matsuda et al., 2012). Although this difference could be attributable to risk factors such as smoking, hypertension and long-term dialysis, there might be a contribution of genes associated with the cancer. In spite that RCC shows a poor survival rate (less than 19%) for patients with metastasis, molecular pathological tests, such as those dividing good and poor prognosis groups, have not been established (Stewart et al., 2011). A lack of such effective tests may be one of the reasons why the mortality rate in Japan has been gradually increasing from 1.8% (2002) to 2.0% (2007).

A large majority of RCC cases are sporadic and only 2-4% are hereditary. There are cas‐ es where gene expression profiling cannot distinguish between them (Beroukhim et al., 2009), suggesting common genetic factors between them. Several genes are known to be associated with RCC, such as *VHL*, *TSC1* and *TSC2*, which play different roles in the mechanism of cancer and so have different advantages in diagnostics/therapeutics. The information about genes can be categorized by the levels of genomics, transcriptomics, proteomics and others including metabolomics, and used to understand the mechanism of cancer, to support diagnostic or therapeutic processes. In this section, we focus on the roles and merits of these genes.

### **2.1. Genes associated with tumorigenesis**

Since a majority of sporadic cancers originate from a recessive mutation that causes a loss of function of a particular type of gene, loss of heterozygosity (LOH) is an important step in the disabling of a functional gene (or a wild-type allele) to give a mutated and cancer phenotype. Such genes are termed tumor suppressor genes, and so far, more than 100 have been reported (Fearon, 2002; Polinsky, 2007). Among them, twenty well-characterized genes showed both familial and sporadic phenotypes (Sherr, 2004). Since a cancer phenotype can be revealed by morphological changes, growth stimulation, gaining immortality and/or others, there are quite a few functions associated with tumor suppressor genes. Thus, it is easier to examine tumori‐ genesis in association with genomic status, mutations and/or epigenetic modifications, by analyzing the loci specific to RCC.

### *2.1.1. VHL gene*

The gene best known to be associated with RCC is the von Hippel-Lindau (*VHL*) gene, whose inactivation accounts for nearly 100% of hereditary cases and sporadic clear cell RCC cases (Baldewijns et al., 2008). This gene was found by positional cloning from the locus associated with the VHL disease, a familial syndrome accompanying cancer in the eye, brain, spinal cord, kidney, pancreas and adrenal glands. The *VHL* gene encodes the 30-kDa protein VHL, 213 amino acid residues long, and is implicated in the regulation of hypoxia-inducible factors (HIFs) (Maher et al., 2011). The VHL protein forms a complex with elongin B, elongin C and cullin-2, and the complex has ubiquitin ligase E3 activity and is involved in the ubiquitination and degradation of HIFα, the α subunits of transcription factors HIF-1 and HIF-2, which form a dimer with HIFβ and regulate the transcription of hypoxia-inducible genes such as those for VEGF (vascular endothelial growth factor), PDGF (platelet derived growth factor) and TGFα (transforming growth factor α) (Kondo and Kaelin, 2001; Kaelin, 2009; Fig. 1). However, the cancer found in VHL disease is sporadic and the lifetime risk of RCC in VHL disease patients is about 70% (Maher et al., 2011). So, it is reasonable to assume that additional genes are involved in RCC and the mutations in *VHL* are not the definitive cause of RCC, which is one of the reasons to explore new genes and genetic loci (see below). Meanwhile, the status of the *VHL* gene is important for the treatment of VHL disease and kidney cancer patients. HIFresponsive gene products, such as VEGF and PDGF, activate the angiogenesis of tumors and therefore are good therapeutic targets. Inhibitors of VEGF and PDGF, sunitinib and sorafenib, have been approved by the US Food and Drug Administration (Kaelin, 2009).

also discuss a methodology for collecting information on multiple gene functions with a simple

While kidney cancer ranked 9th in 2002 in the European Union and the United States (Baldewijns et al., 2008), its mortality rate was not high in Japan (12th in 2002 and 2007: Matsuda et al., 2012). Although this difference could be attributable to risk factors such as smoking, hypertension and long-term dialysis, there might be a contribution of genes associated with the cancer. In spite that RCC shows a poor survival rate (less than 19%) for patients with metastasis, molecular pathological tests, such as those dividing good and poor prognosis groups, have not been established (Stewart et al., 2011). A lack of such effective tests may be one of the reasons why the mortality rate in Japan has been gradually increasing from 1.8%

A large majority of RCC cases are sporadic and only 2-4% are hereditary. There are cas‐ es where gene expression profiling cannot distinguish between them (Beroukhim et al., 2009), suggesting common genetic factors between them. Several genes are known to be associated with RCC, such as *VHL*, *TSC1* and *TSC2*, which play different roles in the mechanism of cancer and so have different advantages in diagnostics/therapeutics. The information about genes can be categorized by the levels of genomics, transcriptomics, proteomics and others including metabolomics, and used to understand the mechanism of cancer, to support diagnostic or therapeutic processes. In this section, we focus on the

Since a majority of sporadic cancers originate from a recessive mutation that causes a loss of function of a particular type of gene, loss of heterozygosity (LOH) is an important step in the disabling of a functional gene (or a wild-type allele) to give a mutated and cancer phenotype. Such genes are termed tumor suppressor genes, and so far, more than 100 have been reported (Fearon, 2002; Polinsky, 2007). Among them, twenty well-characterized genes showed both familial and sporadic phenotypes (Sherr, 2004). Since a cancer phenotype can be revealed by morphological changes, growth stimulation, gaining immortality and/or others, there are quite a few functions associated with tumor suppressor genes. Thus, it is easier to examine tumori‐ genesis in association with genomic status, mutations and/or epigenetic modifications, by

The gene best known to be associated with RCC is the von Hippel-Lindau (*VHL*) gene, whose inactivation accounts for nearly 100% of hereditary cases and sporadic clear cell RCC cases (Baldewijns et al., 2008). This gene was found by positional cloning from the locus associated with the VHL disease, a familial syndrome accompanying cancer in the eye, brain, spinal cord,

pathological system (Section 4).

4 Renal Tumor

(2002) to 2.0% (2007).

roles and merits of these genes.

analyzing the loci specific to RCC.

*2.1.1. VHL gene*

**2.1. Genes associated with tumorigenesis**

**2. Genes associated with renal tumors**

**Figure 1.** Summary of signal transduction pathways associated with RCC.

### *2.1.2. MET gene*

The *MET* protooncogene was found in hereditary papillary RCC without mutations in the *VHL* gene (Schmidt et al., 1997). *MET* encodes a membrane receptor (MET) for hepatocyte growth factor (HGF). MET has tyrosine kinase activity, and HGF activates this kinase activity and initiates signaling for mitogenesis and migration (Fig. 1). While aberrantly active MET triggers tumor growth, angiogenesis and metastasis, such cases are relatively rare (~5%) among sporadic papillary RCC, suggesting other genes to play a major role in the tumorigenesis (Baldewijns et al., 2008).

From the analysis of their mutations, this gene is considered as a tumor suppressor gene (Sudarshan et al., 2007). Although the mechanism that leads *FH* alterations to cancer is not clearly understood, there is a link between fumarate dysregulation and impaired HIF hydrox‐

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 7

*FLCN*, on the other hand, is the gene responsible for Birt-Hogg-Dubé (BHD) syndrome, which is a rare autosomal dominant disease including kidney tumors, predominantly chromophobe RCC. Mutations in this gene were found in approximately 80% of BHD kindreds and loss of expression of this gene were frequently found in kidney tumors from BHD patients, suggesting

Several genes were recently implicated in association with RCC, including *BAP1*, *SETD2* and *NF2*, by means of advanced technologies such as the next-generation sequencing, a microarraybased analysis and a mouse transgene analysis. *BAP1* plays a role of a tumor suppressor and encodes a nuclear deubiquitinase, which is inactivated in 15% of clear cell RCC cases (Peña-Llopis et al., 2012). Mutations in *BAP1* anticorrelates with those in another tumor suppressor gene, *PBRM1*, and these mutations comprise a subtype of clear cell RCC (70% of all clear cell RCC cases). The BAP1 protein may work with host cell factor-1 (HCF-1), a scaffold protein, to

*SETD2* was found by the analysis of accumulated transcripts containing premature termina‐ tion codons and encodes a histone methyltransferase, which is responsible for trimethylation of the lysine residue at position 36 of histone H3 and may play a role in suppressing tumor

*NF2* was identified as a tumor suppressor gene by the analysis of knock-out mice (Mor‐ ris and McClatchey, 2009). The mice developed kidney tumors in 6-10 months with char‐ acteristics of hyperactive epidermal growth factor receptor (EGFR) signaling. Merlin, the *NF2* gene product, was implicated in suppressing tumorigenesis by inhibiting hyperacti‐

The recurrence of RCC is 20 to 40%, depending on the stage and grade of tumor (Chin et al., 2006). So, it is important to understand the genes (and their products) associated with pro‐ gression/metastasis to predict the outcome of cancer. The classification of RCC subtypes is apparently not possible by a single marker, but could be done using combinations of markers such as vimentin, epithelial cell adhesion molecule (EpCAM), glutathione S-transferase α (GSTα), carbonic anhydrase II (CA II), cytokeratin 7 (CK7) and cluster of differentiation 10

Important prognostic markers for RCC represent specific cellular signaling pathways, such as the VHL and mTOR pathways. The VHL pathway gives several well-studied markers, such as VHL, HIFs, VEGF and carbonic anhydrase 9 (CAIX), although their ap‐

this gene to be a tumor suppressor gene (Baldewijns et al., 2008).

regulate transcription factors and suppress cell proliferation.

**2.2. Genes implicated in diagnostic markers and therapeutic targets**

ylation (Isaacs et al., 2005).

development (Duns et al., 2012).

vated EGFR signaling.

(CD10) (Stewart et al., 2011).

*2.1.6. Other genes*

### *2.1.3. TSC1/TSC2 genes*

Two tumor suppressor genes, *TSC1* and *TSC2*, were found in a study of tuberous sclerosis complex (TSC), which is known to develop into various types of RCCs, including clear cell RCC, papillary RCC and chromophobe RCC (Borkowska et al., 2011). The TSC1 and TSC2 proteins form a heterodimer and inhibit the mammalian target of rapamycin (mTOR; a key signaling mediator for cell growth), by inactivating a small GTPase Rheb (an activator of mTOR) as a GTPase-activating protein (GAP) (Linehan et al., 2010; (Fig. 1). However, muta‐ tions are not frequently found in sporadic RCC (Parry et al., 2001) and therefore its role is not completely clear yet.

### *2.1.4. PBRM1 gene*

Several genes, *UTX* (or *KDM6A*), *JARID1C* (or *KDM5C*) and *SETD2*, were found in close association with clear cell RCC by a recent technology of the next-generation sequencing (Dalgliesh et al., 2010). As these genes are related with the methylation status of lysine residues of hitone H3, further mutation studies were conducted to identify a SWI/SNF chromatin remodeling complex gene, *PBRM1*, to be frequently (over 40%) mutated in clear cell RCC (Varela et al., 2011). *PBRM1* is mapped to chromosome 3p21 and encodes the BAF180 protein, a chromatin targeting subunit of a SWI/SNF chromatin remodeling complex, which regulates replication, DNA repair and cell proliferation/differentiation. Knock-down of this gene enhanced colony formation and migration of cancer cells, suggesting this gene to be a tumor suppressor gene. Further studies are needed to reveal a mechanism of cancer involving *PBRM1* and to find its clinical application.

### *2.1.5. Genes related to hereditary renal cancer syndromes*

Approximately 2-4% of RCC cases are hereditary and some genes have been identified as the genes responsible for hereditary renal cancer (HRC) syndromes (Verine et al., 2010). Apart from the genes already mentioned above (*VHL*, *MET*, *TSC1* and *TSC2*), several more genes have been described in association with HRC syndromes, including *FH* and *FLCN* genes. *FH* is the gene responsible for a HRC syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC), in which affected individuals often develop cutaneous and uterine leiomyoma and an aggressive form of papillary RCC (Linehan et al., 2004). The *FH* gene encodes an enzyme (FH) catalyzing the conversion of fumarate to malate in the tricarboxylic acid (Krebs) cycle. From the analysis of their mutations, this gene is considered as a tumor suppressor gene (Sudarshan et al., 2007). Although the mechanism that leads *FH* alterations to cancer is not clearly understood, there is a link between fumarate dysregulation and impaired HIF hydrox‐ ylation (Isaacs et al., 2005).

*FLCN*, on the other hand, is the gene responsible for Birt-Hogg-Dubé (BHD) syndrome, which is a rare autosomal dominant disease including kidney tumors, predominantly chromophobe RCC. Mutations in this gene were found in approximately 80% of BHD kindreds and loss of expression of this gene were frequently found in kidney tumors from BHD patients, suggesting this gene to be a tumor suppressor gene (Baldewijns et al., 2008).

### *2.1.6. Other genes*

*2.1.2. MET gene*

6 Renal Tumor

(Baldewijns et al., 2008).

*2.1.3. TSC1/TSC2 genes*

completely clear yet.

and to find its clinical application.

*2.1.5. Genes related to hereditary renal cancer syndromes*

*2.1.4. PBRM1 gene*

The *MET* protooncogene was found in hereditary papillary RCC without mutations in the *VHL* gene (Schmidt et al., 1997). *MET* encodes a membrane receptor (MET) for hepatocyte growth factor (HGF). MET has tyrosine kinase activity, and HGF activates this kinase activity and initiates signaling for mitogenesis and migration (Fig. 1). While aberrantly active MET triggers tumor growth, angiogenesis and metastasis, such cases are relatively rare (~5%) among sporadic papillary RCC, suggesting other genes to play a major role in the tumorigenesis

Two tumor suppressor genes, *TSC1* and *TSC2*, were found in a study of tuberous sclerosis complex (TSC), which is known to develop into various types of RCCs, including clear cell RCC, papillary RCC and chromophobe RCC (Borkowska et al., 2011). The TSC1 and TSC2 proteins form a heterodimer and inhibit the mammalian target of rapamycin (mTOR; a key signaling mediator for cell growth), by inactivating a small GTPase Rheb (an activator of mTOR) as a GTPase-activating protein (GAP) (Linehan et al., 2010; (Fig. 1). However, muta‐ tions are not frequently found in sporadic RCC (Parry et al., 2001) and therefore its role is not

Several genes, *UTX* (or *KDM6A*), *JARID1C* (or *KDM5C*) and *SETD2*, were found in close association with clear cell RCC by a recent technology of the next-generation sequencing (Dalgliesh et al., 2010). As these genes are related with the methylation status of lysine residues of hitone H3, further mutation studies were conducted to identify a SWI/SNF chromatin remodeling complex gene, *PBRM1*, to be frequently (over 40%) mutated in clear cell RCC (Varela et al., 2011). *PBRM1* is mapped to chromosome 3p21 and encodes the BAF180 protein, a chromatin targeting subunit of a SWI/SNF chromatin remodeling complex, which regulates replication, DNA repair and cell proliferation/differentiation. Knock-down of this gene enhanced colony formation and migration of cancer cells, suggesting this gene to be a tumor suppressor gene. Further studies are needed to reveal a mechanism of cancer involving *PBRM1*

Approximately 2-4% of RCC cases are hereditary and some genes have been identified as the genes responsible for hereditary renal cancer (HRC) syndromes (Verine et al., 2010). Apart from the genes already mentioned above (*VHL*, *MET*, *TSC1* and *TSC2*), several more genes have been described in association with HRC syndromes, including *FH* and *FLCN* genes. *FH* is the gene responsible for a HRC syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC), in which affected individuals often develop cutaneous and uterine leiomyoma and an aggressive form of papillary RCC (Linehan et al., 2004). The *FH* gene encodes an enzyme (FH) catalyzing the conversion of fumarate to malate in the tricarboxylic acid (Krebs) cycle.

Several genes were recently implicated in association with RCC, including *BAP1*, *SETD2* and *NF2*, by means of advanced technologies such as the next-generation sequencing, a microarraybased analysis and a mouse transgene analysis. *BAP1* plays a role of a tumor suppressor and encodes a nuclear deubiquitinase, which is inactivated in 15% of clear cell RCC cases (Peña-Llopis et al., 2012). Mutations in *BAP1* anticorrelates with those in another tumor suppressor gene, *PBRM1*, and these mutations comprise a subtype of clear cell RCC (70% of all clear cell RCC cases). The BAP1 protein may work with host cell factor-1 (HCF-1), a scaffold protein, to regulate transcription factors and suppress cell proliferation.

*SETD2* was found by the analysis of accumulated transcripts containing premature termina‐ tion codons and encodes a histone methyltransferase, which is responsible for trimethylation of the lysine residue at position 36 of histone H3 and may play a role in suppressing tumor development (Duns et al., 2012).

*NF2* was identified as a tumor suppressor gene by the analysis of knock-out mice (Mor‐ ris and McClatchey, 2009). The mice developed kidney tumors in 6-10 months with char‐ acteristics of hyperactive epidermal growth factor receptor (EGFR) signaling. Merlin, the *NF2* gene product, was implicated in suppressing tumorigenesis by inhibiting hyperacti‐ vated EGFR signaling.

### **2.2. Genes implicated in diagnostic markers and therapeutic targets**

The recurrence of RCC is 20 to 40%, depending on the stage and grade of tumor (Chin et al., 2006). So, it is important to understand the genes (and their products) associated with pro‐ gression/metastasis to predict the outcome of cancer. The classification of RCC subtypes is apparently not possible by a single marker, but could be done using combinations of markers such as vimentin, epithelial cell adhesion molecule (EpCAM), glutathione S-transferase α (GSTα), carbonic anhydrase II (CA II), cytokeratin 7 (CK7) and cluster of differentiation 10 (CD10) (Stewart et al., 2011).

Important prognostic markers for RCC represent specific cellular signaling pathways, such as the VHL and mTOR pathways. The VHL pathway gives several well-studied markers, such as VHL, HIFs, VEGF and carbonic anhydrase 9 (CAIX), although their ap‐ plicability is sometimes questionable (Stewart et al., 2011). HIF-responsive gene products are potential markers representing angiogenesis (VEGF, PDGF, SDF, CXCR4, TGFβ and CTGF), glucose uptake and metabolism (HK2 and PDK4), pH control (CAIX and CAXII), invasion/metastasis (MMP1, SDF, CXCR4 and c-Met), and proliferation and survival (TGFα) (Smaldone and Maranchie, 2009).

gene on the X chromosome and either of *ASPL* (17q25), *PRCC* (1q21), *PSF* (1q34), *NonO* (Xq12) and *CLTC* (17q23) (Kuroda et al., 2012). All of the gene fusions result in overexpression of the TFE3 protein, a transcription factor. Among them, the translocation of t(X;17)(p11.2q25), which fuses the *ASPL* and *TFE3* genes, is most frequently observed. Meanwhile, there are some unclassified cases, such as those where trisomy 7/17 in areas typical of papillary RCC and both trisomy 7/17 and 3p loss in areas with clear cell RCC were observed (Ross et al., 2012).

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 9

Even though a number of genetic markers have been reported, they are not currently used for the diagnosis of RCC. As discussed in Section 1, this is because understanding a single gene or a few genes is not enough for a diagnosis of sufficient reliability. For diagnosing more complex and more specific states of diseases or disease phenotypes, groups of markers that are able to more accurately distinguish the phenotypes are needed. Such markers should be derived from the direct process of the disease and therefore would represent the signal transduction that occurs within the cell. There are several new technologies which might open the door to a more comprehensive understanding of RCC especially at the level of cellular signaling: array-based genome-wide association studies, microRNA (miRNA) studies and

**Figure 2.** Significant LOH regions in RCC. LOH regions of less than ~10 Mb were identified as the minimum overlap‐ ping regions of LOH by subtraction cloning of mutated regions followed by the quantitative allelic analysis of over 60

RCC cases using microsatellite markers (see Hatano et al., 2001; Sarkar et al., 2002).

**2.5. Exploration of new genetic markers**

next-generation sequencing-based expression profiling.

Another pathway for potential makers is the mTOR pathway (Fig. 1). The main cascade of this pathway is PI3K/AKT/mTOR, which mediates signals by activating phosphoinositide 3-kinase (PI3K) through kinases such as receptor tyrosine kinases to generate phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which further activates AKT via phosphorylation and phospho-AKT activates mTOR complex 1 (mTORC1) through inhibition of the TSC1/TSC2 complex (Allory et al., 2011). Then, mTORC1 phosphorylates proteins such as P70-S6 kinase and activates protein synthesis and cell proliferation. Importantly, HIF-1α expression is dependent on mTORC1 signaling (Toschi et al., 2008). Potential markers in this pathway include P70-S6 kinase, PTEN (a phosphatase that decreases PIP3) and phospho-AKT.

### **2.3. Mutation sites and LOH loci**

A comprehensive analysis of RCC genomes has been done through genomic (Hatano et al., 2001; Cifola et al., 2008), transcriptomic (Takahashi et al., 2001; Takahashi et al., 2003; Cifola et al., 2008) and proteomic/metabolic (Perroud et al., 2006; Raimondo et al., 2012) approaches. We used a genome-subtraction technique, or the in-gel competitive reassociation method (Kiyama et al., 1995; Rodley et al., 2003), for cloning the sites of LOH that occurred in a RCC genome by subtracting normal DNA from cancer DNA of the same patient (Hatano et al., 2001). The minimum size of LOH (caused by hemizygous deletions) detected by this method was roughly 50 kb. This resolution was made possible by *Mse*I, which recognizes TTAA, a sequence appearing frequently in human genomic DNA, and completely digests genomic DNA to sizes mostly below 1 kb. Such a high resolution has not been used even in recent genome-wide association studies (see Jacobs et al., 2012, for example). A total of 187 clones were mapped on the chromosomes and a total of 44 candidate regions, where at least two clones were mapped within 5 Mb, were selected and analyzed for mapping the sites of LOH in 61 cancer cases (Table 1). Among them, we found interesting LOH sites at 5q32-q34, 6q21 q22, 8p12 and 9p24, whose frequencies are relatively high among RCC and whose lengths are less than ~10 Mb (Hatano et al., 2001; Sarkar et al., 2002; Fig. 2). A tumor suppressor gene, *Kank1*, was found at 9p24 after extensive analysis of the LOH site by examining the loss of function upon its mutation; the loss of expression of the gene at mRNA and protein levels in RCC, and the loss of suppression of tumor growth in renal tumor cells (Sarkar et al., 2002).

### **2.4. Chromosomal abberations in RCC**

Chromosomal abberations are often observed in RCC (Ross et al., 2012). Deletions of chromo‐ some 3p, where the *VHL* gene resides, are found in most sporadic and familial clear cell RCCs. Distinctive abnormalities were reported for papillary RCC, where, in contrast to clear cell RCC, most of the tumors are characterized by trisomy of chromosomes 7 and 17 along with loss of Y, while the 3p arm is intact. In Xp11.2 RCC, the gene fusion was observed between the *TFE3* gene on the X chromosome and either of *ASPL* (17q25), *PRCC* (1q21), *PSF* (1q34), *NonO* (Xq12) and *CLTC* (17q23) (Kuroda et al., 2012). All of the gene fusions result in overexpression of the TFE3 protein, a transcription factor. Among them, the translocation of t(X;17)(p11.2q25), which fuses the *ASPL* and *TFE3* genes, is most frequently observed. Meanwhile, there are some unclassified cases, such as those where trisomy 7/17 in areas typical of papillary RCC and both trisomy 7/17 and 3p loss in areas with clear cell RCC were observed (Ross et al., 2012).

### **2.5. Exploration of new genetic markers**

plicability is sometimes questionable (Stewart et al., 2011). HIF-responsive gene products are potential markers representing angiogenesis (VEGF, PDGF, SDF, CXCR4, TGFβ and CTGF), glucose uptake and metabolism (HK2 and PDK4), pH control (CAIX and CAXII), invasion/metastasis (MMP1, SDF, CXCR4 and c-Met), and proliferation and survival

Another pathway for potential makers is the mTOR pathway (Fig. 1). The main cascade of this pathway is PI3K/AKT/mTOR, which mediates signals by activating phosphoinositide 3-kinase (PI3K) through kinases such as receptor tyrosine kinases to generate phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which further activates AKT via phosphorylation and phospho-AKT activates mTOR complex 1 (mTORC1) through inhibition of the TSC1/TSC2 complex (Allory et al., 2011). Then, mTORC1 phosphorylates proteins such as P70-S6 kinase and activates protein synthesis and cell proliferation. Importantly, HIF-1α expression is dependent on mTORC1 signaling (Toschi et al., 2008). Potential markers in this pathway include P70-S6

A comprehensive analysis of RCC genomes has been done through genomic (Hatano et al., 2001; Cifola et al., 2008), transcriptomic (Takahashi et al., 2001; Takahashi et al., 2003; Cifola et al., 2008) and proteomic/metabolic (Perroud et al., 2006; Raimondo et al., 2012) approaches. We used a genome-subtraction technique, or the in-gel competitive reassociation method (Kiyama et al., 1995; Rodley et al., 2003), for cloning the sites of LOH that occurred in a RCC genome by subtracting normal DNA from cancer DNA of the same patient (Hatano et al., 2001). The minimum size of LOH (caused by hemizygous deletions) detected by this method was roughly 50 kb. This resolution was made possible by *Mse*I, which recognizes TTAA, a sequence appearing frequently in human genomic DNA, and completely digests genomic DNA to sizes mostly below 1 kb. Such a high resolution has not been used even in recent genome-wide association studies (see Jacobs et al., 2012, for example). A total of 187 clones were mapped on the chromosomes and a total of 44 candidate regions, where at least two clones were mapped within 5 Mb, were selected and analyzed for mapping the sites of LOH in 61 cancer cases (Table 1). Among them, we found interesting LOH sites at 5q32-q34, 6q21 q22, 8p12 and 9p24, whose frequencies are relatively high among RCC and whose lengths are less than ~10 Mb (Hatano et al., 2001; Sarkar et al., 2002; Fig. 2). A tumor suppressor gene, *Kank1*, was found at 9p24 after extensive analysis of the LOH site by examining the loss of function upon its mutation; the loss of expression of the gene at mRNA and protein levels in RCC, and the loss of suppression of tumor growth in renal tumor cells (Sarkar et al., 2002).

Chromosomal abberations are often observed in RCC (Ross et al., 2012). Deletions of chromo‐ some 3p, where the *VHL* gene resides, are found in most sporadic and familial clear cell RCCs. Distinctive abnormalities were reported for papillary RCC, where, in contrast to clear cell RCC, most of the tumors are characterized by trisomy of chromosomes 7 and 17 along with loss of Y, while the 3p arm is intact. In Xp11.2 RCC, the gene fusion was observed between the *TFE3*

kinase, PTEN (a phosphatase that decreases PIP3) and phospho-AKT.

(TGFα) (Smaldone and Maranchie, 2009).

8 Renal Tumor

**2.3. Mutation sites and LOH loci**

**2.4. Chromosomal abberations in RCC**

Even though a number of genetic markers have been reported, they are not currently used for the diagnosis of RCC. As discussed in Section 1, this is because understanding a single gene or a few genes is not enough for a diagnosis of sufficient reliability. For diagnosing more complex and more specific states of diseases or disease phenotypes, groups of markers that are able to more accurately distinguish the phenotypes are needed. Such markers should be derived from the direct process of the disease and therefore would represent the signal transduction that occurs within the cell. There are several new technologies which might open the door to a more comprehensive understanding of RCC especially at the level of cellular signaling: array-based genome-wide association studies, microRNA (miRNA) studies and next-generation sequencing-based expression profiling.

**Figure 2.** Significant LOH regions in RCC. LOH regions of less than ~10 Mb were identified as the minimum overlap‐ ping regions of LOH by subtraction cloning of mutated regions followed by the quantitative allelic analysis of over 60 RCC cases using microsatellite markers (see Hatano et al., 2001; Sarkar et al., 2002).


based on more than 5,000 RCC cases revealed two loci, 2p21 and 11q13.3, to be associated with RCC susceptibility (Purdue et al., 2011). Although the authors claimed these sites to be previously unidentified, both of the loci were actually identified in 2001 (Hatano et al., 2001; Table 1). While the association is statistically significant, the frequencies among RCC cases are not very high (less than 20%), and therefore, it is doubtful that these sites alone can be used for diagnosis. The candidate genes in these loci which contribute to the association are *EPAS1* encoding hypoxia-inducible factor-2α (HIF2α) and *SCARB1* encoding a scavenger receptor. While HIF2α was known to be associated with RCC though it has not yet been used clinically, SCARB1 is new and its association with RCC may indicate a new signaling pathway. The arraybased genome-wide association technique was also applied to the study of copy-number

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 11

The study of miRNA is rapidly providing as new information about disease phenotypes. MiRNA, a group of short non-coding RNA with lengths of 19-22 nucleotides, differs from mRNA in that it has a role in gene function, and, while information about mutations is important for mRNA, quantity is mostly emphasized for miRNA. So, while there are cases where mRNA bearing a mutation without a change in its quantity contributes to a disease phenotype, there would be few such cases for miRNA. Naturally, the linkage of a disease to a genomic location reveals in most cases a mutation in a gene. This may indicate that miRNA contributes to quantitative change as a group as a result of changes in transcriptional efficiency caused by alterations to the transcriptional machinery or genomic location/status, or by epigenetic modifications. In contrast to mRNA, however, the quantity of miRNA can be controlled rapidly and specifically, and thus, miRNA could be more advantageous for the rapid control of the amount of specific proteins, which is important in signal transduction. Such cases were reported for TGFβ, WNT, Notch and EGF signaling in association with homeostasis, cancer, metastasis, fibrosis and stem cell biology (Inui et al., 2010), and VHLsignaling and VEGF-signaling in association with RCC (Fendler et al., 2011). Several miRNAs were reported to be induced or repressed by VHL-induced hypoxia in RCC and regulate the PI3K/AKT/mTOR pathway and Wnt signaling/β-catenin pathway to control cell proliferation,

Next-generation sequencing technology was applied to genome-wide expression profiling of miRNA related to clear cell RCC (Osanto et al., 2012). By analyzing 22 RCCs, 100 miRNA differentially expressed between clear cell RCC and matched normal tissues were found. While the biological relevance of these novel miRNAs is unknown, they may be potential diagnostic

The human *Kank1* gene was found as a candidate tumor suppressor gene for renal tumors at 9p24, and encodes a protein containing ankyrin-repeats at the C-terminus and coiled-coil motifs near the N-terminus (Sakar et al., 2002). Based on domain and phylogenetic analyses,

tumorigenesis and other cellular functions (Redova et al., 2011).

variations (Krill-Burger et al., 2012).

markers or targets for therapeutics.

**3.1. Structure of** *Kank***-family genes**

**3. Kank family genes and renal tumors**

**Table 1.** Summary of LOH at significant locations among 44 sites. For details, see Hatano et al. (2001) and Sarkar et al. (2002). Only the loci in which more than 10 % of RCC patients had LOH are shown. The LOH analysis is applicable to only female patients for the cluster at Xq26-q28. There were a total of 44 clusters containing more than two of 187 clones analyzed within 5 Mb. The locations of the clusters other than those shown above are as follows: 1p31.1, 1p13.3-p22.3, 1p13.3-q12, 1q12-p21.1, 2p21-p22, 2p12-q11.2, 4p14, 4p13.3-p21.1, 4q22, 4q32, 10p14-p15.1, 10p12.1-p12.2, 12q13.3-q15, 13q13-q14.1, 13q14.2-q14.3, 16q12.1-q12.2, and 20p11.2-p12.

Recent advances in high-resolution genomic arrays have enabled us to analyze 1,000 or more disease cases efficiently, and thus to give statistically significant loci associated with the diseases. Such an approach was applied to the study of RCC. A genome-wide association study based on more than 5,000 RCC cases revealed two loci, 2p21 and 11q13.3, to be associated with RCC susceptibility (Purdue et al., 2011). Although the authors claimed these sites to be previously unidentified, both of the loci were actually identified in 2001 (Hatano et al., 2001; Table 1). While the association is statistically significant, the frequencies among RCC cases are not very high (less than 20%), and therefore, it is doubtful that these sites alone can be used for diagnosis. The candidate genes in these loci which contribute to the association are *EPAS1* encoding hypoxia-inducible factor-2α (HIF2α) and *SCARB1* encoding a scavenger receptor. While HIF2α was known to be associated with RCC though it has not yet been used clinically, SCARB1 is new and its association with RCC may indicate a new signaling pathway. The arraybased genome-wide association technique was also applied to the study of copy-number variations (Krill-Burger et al., 2012).

The study of miRNA is rapidly providing as new information about disease phenotypes. MiRNA, a group of short non-coding RNA with lengths of 19-22 nucleotides, differs from mRNA in that it has a role in gene function, and, while information about mutations is important for mRNA, quantity is mostly emphasized for miRNA. So, while there are cases where mRNA bearing a mutation without a change in its quantity contributes to a disease phenotype, there would be few such cases for miRNA. Naturally, the linkage of a disease to a genomic location reveals in most cases a mutation in a gene. This may indicate that miRNA contributes to quantitative change as a group as a result of changes in transcriptional efficiency caused by alterations to the transcriptional machinery or genomic location/status, or by epigenetic modifications. In contrast to mRNA, however, the quantity of miRNA can be controlled rapidly and specifically, and thus, miRNA could be more advantageous for the rapid control of the amount of specific proteins, which is important in signal transduction. Such cases were reported for TGFβ, WNT, Notch and EGF signaling in association with homeostasis, cancer, metastasis, fibrosis and stem cell biology (Inui et al., 2010), and VHLsignaling and VEGF-signaling in association with RCC (Fendler et al., 2011). Several miRNAs were reported to be induced or repressed by VHL-induced hypoxia in RCC and regulate the PI3K/AKT/mTOR pathway and Wnt signaling/β-catenin pathway to control cell proliferation, tumorigenesis and other cellular functions (Redova et al., 2011).

Next-generation sequencing technology was applied to genome-wide expression profiling of miRNA related to clear cell RCC (Osanto et al., 2012). By analyzing 22 RCCs, 100 miRNA differentially expressed between clear cell RCC and matched normal tissues were found. While the biological relevance of these novel miRNAs is unknown, they may be potential diagnostic markers or targets for therapeutics.
