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

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

**Location LOH (%)** 3p22-p23 15/16 (93.8) 3p21.2-p21.3 21/23 (91.3) 3q13.3-q21 13/31 (41.9) 5q12-q13.1 10/31 (32.3) 5q13.1-q14 7/28 (25.0) 5q23.3 10/27 (37.0) 5q31.3-q32 7/28 (25.0) 5q32-q34 14/30 (46.7) 6q22.3 9/34 (26.5) 7p12-p14 5/32 (15.6) 7q11.23 7/33 (21.2) 7q22.1-q31.2 5/38 (13.2) 8p12 5/29 (17.2) 8q13 6/34 (17.6) 9p21-p22 5/20 (25.0) 9p12-q11 6/33 (18.2) 9q31 8/41 (19.5) 11q13.3-q14.1 5/28 (17.9) 11q22.3 2/17 (11.8) 14q11.2-q12 9/26 (34.6) 14q13-q21 10/34 (29.4) 14q24.1-q31 12/36 (33.3) 14q32.1-q32.3 7/22 (31.8) 15q23 5/28 (17.9) 18p11.1-q11.2 8/35 (22.9) 18q22 7/39 (17.9) Xq26-q28 3/4 (75.0)

10 Renal Tumor

**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,

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

10p12.1-p12.2, 12q13.3-q15, 13q13-q14.1, 13q14.2-q14.3, 16q12.1-q12.2, and 20p11.2-p12.

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, *Kank2*, *Kank3* and *Kank4* were found to form a family with *Kank1* (Zhu et al., 2008). Five repeats of the ankyrin-repeat motif comprise the basic structure of all Kank proteins (Fig. 3A). In addition, each Kank protein contains different combinations of four types of coiled-coil motifs. They also have a conserved region close to the N-terminus, named the KN-motif (Zhu et al., 2008; Fig. 3A), which contains a leucine-rich region and an arginine-rich region.

motif, serine at position 167, located between the first and second coiled-coil motifs. Kif21a is a unique protein found to interact with the ankyrin-repeat domain of Kank1 (Kakinuma et al., 2008 & 2009; Roy et al., 2009; Suzuki et al., unpublished data). Although the function of the KN-motif is not clear, it contains several potential motifs for a nuclear localization signal (NLS) and nuclear export signal (NES). These signals may contribute to nucleo-cytoplasmic shuttling of Kank1, and further affect the subcellular distribution of β-catenin (Wang et al., 2006; Previdi

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 13

Some studies have demonstrated that *Kank*-family genes are related to various cell func‐ tions. VAB-19, an ortholog of the Kank1 protein in *C. elegans*, was reported to occur with components of an epidermal attachment structure. It plays an antagonistic role in the regulation of actin cytoskeleton and halts basement membrane opening associated with cell invasion and tissue remodeling (Ding et al., 2003; Ihara et al., 2011). The deletion of *Kank1* is associated with parent-of-origin-dependent inheritance of familial cerebral palsy (Lerer et al., 2005). There have also been reports that *Kank1* was fused with the gene for platelet-derived growth factor receptor β (PDGFRβ) and that the fusion protein was a vi‐ tal regulator of hematopoietic cell proliferation (Medves et al., 2010 & 2011). Meanwhile, *Kank1* expression was down-regulated in patients with polycythemia vera, suggesting this gene to be related to myeloproliferative disorders (Kralovics et al., 2005). Some stud‐ ies have described about the functions of other *Kank*-family members. Kank2, found as a novel podocyte-associated protein, may contribute to the regulation of actin dynamics in podocyte foot processes in the renal filter physiology and diseases (Xu et al., 2011). In addition, NBP, an ortholog of Kank3 in zebrafish, interacts with Numb, an adaptor pro‐ tein implicated in various basic cellular processes, through the PTB domain, which is well conserved among vertebrate *Kank* genes. In embryogenesis, NBP accumulates at the cell periphery during gastrulation and, later in the development, is concentrated at the basal poles of differentiated cells. These findings suggest a role for NBP in regulating

*Kank1* may contribute to several regulatory activities, such as regulation of the actin cytoske‐ leton, cell migration and the cell cycle through interactions with the proteins described above (Sakar et al., 2002; Kakinuma et al., 2008 & 2009; Roy et al., 2009; Suzuki et al., unpublished data; summarized in Fig. 3B). Kank1 regulates the Rac1-dependent formation of lamellipodia and the activity of RhoA, resulting in the inhibition of cell migration. This function is mediated through two binding partners of Kank1, 14-3-3 and IRSp53. Kank1 binds to the Akt-phos‐ phrylation motif of 14-3-3θ, 14-3-3γ, 14-3-3η and 14-3-3ε. Interaction between these two proteins is enhanced by growth factors such as insulin and epidermal growth factor (EGF) (Kakinuma et al., 2008). This interaction regulates the activation of RhoA through the PI3K/Akt signaling pathway. When a 14-3-3 binding motif is phosphorylated by Akt, 14-3-3 is separated from an activation complex for RhoA, and binds to Kank1 resulting in the inhibition of RhoA activities, and thereby decreases the formation of actin stress fibers and inhibition of cell migration (Kakinuma et al., 2008). The coiled-coil domain of IRSp53, which

et al., 2010).

**3.2. Functions of** *Kank***-family genes**

cell adhesion and tissue integrity (Boggetti et al., 2012).

**Figure 3.** (A) Schematic structure of human Kank family proteins. Black boxes indicate the Kank N-terminal (KN) motif. Gray boxes indicate coiled-coil motifs. White boxes indicate the ankyrin-repeat (ANK) motifs. (B) A hypothetical model of Kank1 functions. Kank1 is transported to areas of membrane ruffling, such as lamellipodia, through association with Kif21a. Kank1 regulates RhoA and Rac1 activities through interaction with 14-3-3 in PI3K/Akt signaling and IRSp53 in Rac1 signaling, respectively. These interactions negatively regulate the formation of actin stress fibers and lamellipodia, and finally decrease cell migration. Kank1 and BIG1 may exist in a multimolecular complex that affects Golgi/MTOC orientation and regulates cell polarity during directed migration. Kank1 may inhibit Rho activation by binding to Rho-regulating proteins, like Daam1, which may result in negative regulation of cytokinesis.

Yeast-two hybrid or mass-spectrometrical studies have shown that Kank1 can directly bind to several proteins, such as 14-3-3 proteins, insulin receptor substrate (IRS) p53, Kif21a and Disheveled-associated activator of morphogenesis 1 (Daam1). Kank1 binds to IRSp53 and Daam1 at its coiled-coil domain (Kakinuma et al., 2011). In addition, there is a 14-3-3-binding motif, serine at position 167, located between the first and second coiled-coil motifs. Kif21a is a unique protein found to interact with the ankyrin-repeat domain of Kank1 (Kakinuma et al., 2008 & 2009; Roy et al., 2009; Suzuki et al., unpublished data). Although the function of the KN-motif is not clear, it contains several potential motifs for a nuclear localization signal (NLS) and nuclear export signal (NES). These signals may contribute to nucleo-cytoplasmic shuttling of Kank1, and further affect the subcellular distribution of β-catenin (Wang et al., 2006; Previdi et al., 2010).

### **3.2. Functions of** *Kank***-family genes**

*Kank2*, *Kank3* and *Kank4* were found to form a family with *Kank1* (Zhu et al., 2008). Five repeats of the ankyrin-repeat motif comprise the basic structure of all Kank proteins (Fig. 3A). In addition, each Kank protein contains different combinations of four types of coiled-coil motifs. They also have a conserved region close to the N-terminus, named the KN-motif (Zhu et al.,

**Figure 3.** (A) Schematic structure of human Kank family proteins. Black boxes indicate the Kank N-terminal (KN) motif. Gray boxes indicate coiled-coil motifs. White boxes indicate the ankyrin-repeat (ANK) motifs. (B) A hypothetical model of Kank1 functions. Kank1 is transported to areas of membrane ruffling, such as lamellipodia, through association with Kif21a. Kank1 regulates RhoA and Rac1 activities through interaction with 14-3-3 in PI3K/Akt signaling and IRSp53 in Rac1 signaling, respectively. These interactions negatively regulate the formation of actin stress fibers and lamellipodia, and finally decrease cell migration. Kank1 and BIG1 may exist in a multimolecular complex that affects Golgi/MTOC orientation and regulates cell polarity during directed migration. Kank1 may inhibit Rho activation by

Yeast-two hybrid or mass-spectrometrical studies have shown that Kank1 can directly bind to several proteins, such as 14-3-3 proteins, insulin receptor substrate (IRS) p53, Kif21a and Disheveled-associated activator of morphogenesis 1 (Daam1). Kank1 binds to IRSp53 and Daam1 at its coiled-coil domain (Kakinuma et al., 2011). In addition, there is a 14-3-3-binding

binding to Rho-regulating proteins, like Daam1, which may result in negative regulation of cytokinesis.

2008; Fig. 3A), which contains a leucine-rich region and an arginine-rich region.

12 Renal Tumor

Some studies have demonstrated that *Kank*-family genes are related to various cell func‐ tions. VAB-19, an ortholog of the Kank1 protein in *C. elegans*, was reported to occur with components of an epidermal attachment structure. It plays an antagonistic role in the regulation of actin cytoskeleton and halts basement membrane opening associated with cell invasion and tissue remodeling (Ding et al., 2003; Ihara et al., 2011). The deletion of *Kank1* is associated with parent-of-origin-dependent inheritance of familial cerebral palsy (Lerer et al., 2005). There have also been reports that *Kank1* was fused with the gene for platelet-derived growth factor receptor β (PDGFRβ) and that the fusion protein was a vi‐ tal regulator of hematopoietic cell proliferation (Medves et al., 2010 & 2011). Meanwhile, *Kank1* expression was down-regulated in patients with polycythemia vera, suggesting this gene to be related to myeloproliferative disorders (Kralovics et al., 2005). Some stud‐ ies have described about the functions of other *Kank*-family members. Kank2, found as a novel podocyte-associated protein, may contribute to the regulation of actin dynamics in podocyte foot processes in the renal filter physiology and diseases (Xu et al., 2011). In addition, NBP, an ortholog of Kank3 in zebrafish, interacts with Numb, an adaptor pro‐ tein implicated in various basic cellular processes, through the PTB domain, which is well conserved among vertebrate *Kank* genes. In embryogenesis, NBP accumulates at the cell periphery during gastrulation and, later in the development, is concentrated at the basal poles of differentiated cells. These findings suggest a role for NBP in regulating cell adhesion and tissue integrity (Boggetti et al., 2012).

*Kank1* may contribute to several regulatory activities, such as regulation of the actin cytoske‐ leton, cell migration and the cell cycle through interactions with the proteins described above (Sakar et al., 2002; Kakinuma et al., 2008 & 2009; Roy et al., 2009; Suzuki et al., unpublished data; summarized in Fig. 3B). Kank1 regulates the Rac1-dependent formation of lamellipodia and the activity of RhoA, resulting in the inhibition of cell migration. This function is mediated through two binding partners of Kank1, 14-3-3 and IRSp53. Kank1 binds to the Akt-phos‐ phrylation motif of 14-3-3θ, 14-3-3γ, 14-3-3η and 14-3-3ε. Interaction between these two proteins is enhanced by growth factors such as insulin and epidermal growth factor (EGF) (Kakinuma et al., 2008). This interaction regulates the activation of RhoA through the PI3K/Akt signaling pathway. When a 14-3-3 binding motif is phosphorylated by Akt, 14-3-3 is separated from an activation complex for RhoA, and binds to Kank1 resulting in the inhibition of RhoA activities, and thereby decreases the formation of actin stress fibers and inhibition of cell migration (Kakinuma et al., 2008). The coiled-coil domain of IRSp53, which is the site for the interaction with active Rac1, binds to Kank1. Endogenous Kank1 and IRSp53 are co-localized at the site of membrane protrusions such as lamellipodia, which are needed for cell migration. Overexpression of Kank1 inhibits the formation of lamellipodia induced by active Rac1 in NIH3T3 cells, and knockdown of Kank1 enhances the formation. Therefore, Kank1 negatively regulates membrane protrusions at the leading edge of cells, by inhibiting the association between active Rac1 and IRSp53 (Roy et al., 2009). Taken together, Kank1 regulates cell migration through inhibition of IRSp53 in Rac1 signaling and inactivation of RhoA activity through PI3K/Akt signaling (Fig. 3B). As the Kank1 locus shows loss of heter‐ ozygosity in RCC and the expression of the Kank1 gene is suppressed in RCC, Kank1 may contribute to the malignant transformation of cells such as metastasis.

**3.3.** *Kank***-family genes and renal tumors**

and function as tumor suppressors.

in tumorigenesis.

**3.4. Clinical study of** *Kank1* **gene in renal cancer patients**

*3.4.1. Genetic and clinical characteristics of renal tumors*

The *Kank1* gene was found at 9p24 by a comprehensive analysis of human chromosomes for loss of heterozygosity (LOH) in RCC (Sakar et al., 2002). Kank1 family proteins localize at the area of cytoplasma in renal tubular cells and glandular cells of some digestive and endocrine organs (Roy et al., 2005). Kank family genes show different expression patterns at the mRNA and protein levels in normal and tumor kidney tissues and some kidney tumor cell lines (Zhu et al., 2008; Wang et al., 2005). Loss of expression of *Kank1* in RCC was confirmed by Western blotting, RT-PCR and immunohistochemical analyses (Sakar et al., 2002, Roy et al., 2005). In addition, immunostaining in RCC showed decreased expression of Kank1 in high grade tumors (Zhu et al., 2011). Therefore, the *Kank* family genes may be related to renal carcinoma,

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 15

A growth inhibitory effect of Kank1 has been reported. Overexpression of *Kank1* in HEK293 cells resulted in cell cycle arrest at G0/G1. On the other hand, growth suppres‐ sion of tumor cells was caused by *Kank1* gene expression using nude mice abdominally injected with HEK293 cells stably expressing *Kank1* (Sakar et al., 2002). These findings demonstrated that Kank1 can regulate the growth of cells and can also regulate the ab‐ normal growth of cancer cells. Kank1 may exert its growth inhibitory effect by regulating Rho activity mediated via its association with Daam1, resulting in abnormal nuclear divi‐ sion, and thus blocking the cytokinesis of cancer cells (Suzuki et al., unpublished data). According to recent studies, Kank1 can negatively regulate the formation of actin stress fibers and cell migration (Kakinuma et al., 2008; Roy et al., 2009). When cells need to con‐ trol migration, Kank1 could be transferred to the leading edge of the moving cells' mem‐ branes, mediated by Kif21a, and co-localized with IRSp53. Kank1 may bind to IRSp53 competing with active Rac1, and thus inhibits integrin-induced cell spreading and the formation of lamellipodia. Simultaneously, Kank1 may inactivate RhoA, which is control‐ led by binding with 14-3-3, inhibit the formation of actin stress fibers and ultimately in‐ hibit cell migration. Loss of expression of Kank-family proteins may enhance cell migration in renal cell carcinoma. Since enhancement of cell migration is related to meta‐ stasis, Kank-family proteins might be related to the malignancy of renal cell carcinoma. According to studies to date, the Kank1 protein may act as a tumor suppressor through inhibition of cell migration and cell cycle. These functions are facilitated by several proteins interacting with Kank1, including 14-3-3, IRSp53, Kif21a and Daam1. Further studies of the interactions of these proteins will help us to understand clearly the role of Kank family proteins

Kidney cancer accounts for about 4% of adult cancers, with an estimated 64,770 new cases annually in the US (Siegel et al., 2012). Of kidney cancers, 92% are pathologically diagnosed as RCC. This "RCC" has interesting and unique characteristics when investigated from a clinical view point. Although 95% of patients with T1-T2 RCC survived 5 to 10 years, among

Kank1 regulates cell migration by inhibiting Rac1 signaling and RhoA activity as descri‐ bed above. To fulfill this function, Kank1 needs to be located at the leading edge of cells and affect the neighboring membrane. Because Kank1 has no membrane-targeting motif or membrane protein to associate with, some proteins may help transport Kank1 to the site of membrane ruffling. Kank1 interacts with the third and fourth coiled-coil domains of KIF21a, a member of the Kif4-class superfamily of kinesin motors that acts as a plusend kinesin motor (Marszalek et al., 1999; Kakinuma et al., 2009), at its ankyrin-repeat domain. Overexpression of Kif21a or one of the Kif21a mutants (R954W) enhances the translocation of Kank1 to the membrane. In contrast, knockdown of Kif21a decreases the amount of Kank1 at the membrane (Yamada et al., 2005; Kakinuma et al., 2009). Al‐ though the mechanisms involved need further study, translocation of Kank1 mediated by Kif21a may affect cell migration (Fig. 3B). Kank1 is also functionally associated with a protein, brefeldin A-inhibited guanine nucleotide-exchange 1 (BIG1), a binding partner of Kif21a. Although there is no direct interaction between these two proteins, they may ex‐ ist in a multimolecular complex that maintains the orientation of the Golgi/microtubuleorganizing center (MTOC) and regulates cell polarity during directed migration. Furthermore, a protein complex containing BIG1, Kif21a and Kank1 may contribute to di‐ rected transport along microtubules (Li et al., 2011).

Overexpression of Kank1 suppresses the cell cycle and cell growth (Sakar et al., 2002). We observed that the overexpression of Kank1 blocked cytokinesis and generated bi‐ nucleated cells. We also found co-localization of endogenous Kank1 with Rho, a key molecule required in cytokinesis for regulating the constriction of the contractile ring, at the contractile ring during cytokinesis of NIH3T3 cells (Kamijo et al., 2006; Li et al., 2010; Kakinuma et al., 2011; Suzuki et al., unpublished data). The coiled-coil domain of Kank1 binds to another protein, Daam1 (Suzuki et al., unpublished data). Daam1 belongs to a novel protein family containing formin homology domains and has been implicated in the regulation of cell polarity associated with the Wnt/Frizzled/Rho signaling pathway (Jantsch-Plunger V et al., 2000; Kosako H et al., 2000). Although the mechanism is still not clear, Kank1 may block cytokinesis by regulating Rho activity through the interac‐ tion with Daam1 (Fig. 3B). Therefore, it may reveal a new mechanism of regulation of cytokinesis and tumor suppression.

### **3.3.** *Kank***-family genes and renal tumors**

is the site for the interaction with active Rac1, binds to Kank1. Endogenous Kank1 and IRSp53 are co-localized at the site of membrane protrusions such as lamellipodia, which are needed for cell migration. Overexpression of Kank1 inhibits the formation of lamellipodia induced by active Rac1 in NIH3T3 cells, and knockdown of Kank1 enhances the formation. Therefore, Kank1 negatively regulates membrane protrusions at the leading edge of cells, by inhibiting the association between active Rac1 and IRSp53 (Roy et al., 2009). Taken together, Kank1 regulates cell migration through inhibition of IRSp53 in Rac1 signaling and inactivation of RhoA activity through PI3K/Akt signaling (Fig. 3B). As the Kank1 locus shows loss of heter‐ ozygosity in RCC and the expression of the Kank1 gene is suppressed in RCC, Kank1 may

Kank1 regulates cell migration by inhibiting Rac1 signaling and RhoA activity as descri‐ bed above. To fulfill this function, Kank1 needs to be located at the leading edge of cells and affect the neighboring membrane. Because Kank1 has no membrane-targeting motif or membrane protein to associate with, some proteins may help transport Kank1 to the site of membrane ruffling. Kank1 interacts with the third and fourth coiled-coil domains of KIF21a, a member of the Kif4-class superfamily of kinesin motors that acts as a plusend kinesin motor (Marszalek et al., 1999; Kakinuma et al., 2009), at its ankyrin-repeat domain. Overexpression of Kif21a or one of the Kif21a mutants (R954W) enhances the translocation of Kank1 to the membrane. In contrast, knockdown of Kif21a decreases the amount of Kank1 at the membrane (Yamada et al., 2005; Kakinuma et al., 2009). Al‐ though the mechanisms involved need further study, translocation of Kank1 mediated by Kif21a may affect cell migration (Fig. 3B). Kank1 is also functionally associated with a protein, brefeldin A-inhibited guanine nucleotide-exchange 1 (BIG1), a binding partner of Kif21a. Although there is no direct interaction between these two proteins, they may ex‐ ist in a multimolecular complex that maintains the orientation of the Golgi/microtubuleorganizing center (MTOC) and regulates cell polarity during directed migration. Furthermore, a protein complex containing BIG1, Kif21a and Kank1 may contribute to di‐

Overexpression of Kank1 suppresses the cell cycle and cell growth (Sakar et al., 2002). We observed that the overexpression of Kank1 blocked cytokinesis and generated bi‐ nucleated cells. We also found co-localization of endogenous Kank1 with Rho, a key molecule required in cytokinesis for regulating the constriction of the contractile ring, at the contractile ring during cytokinesis of NIH3T3 cells (Kamijo et al., 2006; Li et al., 2010; Kakinuma et al., 2011; Suzuki et al., unpublished data). The coiled-coil domain of Kank1 binds to another protein, Daam1 (Suzuki et al., unpublished data). Daam1 belongs to a novel protein family containing formin homology domains and has been implicated in the regulation of cell polarity associated with the Wnt/Frizzled/Rho signaling pathway (Jantsch-Plunger V et al., 2000; Kosako H et al., 2000). Although the mechanism is still not clear, Kank1 may block cytokinesis by regulating Rho activity through the interac‐ tion with Daam1 (Fig. 3B). Therefore, it may reveal a new mechanism of regulation of

contribute to the malignant transformation of cells such as metastasis.

14 Renal Tumor

rected transport along microtubules (Li et al., 2011).

cytokinesis and tumor suppression.

The *Kank1* gene was found at 9p24 by a comprehensive analysis of human chromosomes for loss of heterozygosity (LOH) in RCC (Sakar et al., 2002). Kank1 family proteins localize at the area of cytoplasma in renal tubular cells and glandular cells of some digestive and endocrine organs (Roy et al., 2005). Kank family genes show different expression patterns at the mRNA and protein levels in normal and tumor kidney tissues and some kidney tumor cell lines (Zhu et al., 2008; Wang et al., 2005). Loss of expression of *Kank1* in RCC was confirmed by Western blotting, RT-PCR and immunohistochemical analyses (Sakar et al., 2002, Roy et al., 2005). In addition, immunostaining in RCC showed decreased expression of Kank1 in high grade tumors (Zhu et al., 2011). Therefore, the *Kank* family genes may be related to renal carcinoma, and function as tumor suppressors.

A growth inhibitory effect of Kank1 has been reported. Overexpression of *Kank1* in HEK293 cells resulted in cell cycle arrest at G0/G1. On the other hand, growth suppres‐ sion of tumor cells was caused by *Kank1* gene expression using nude mice abdominally injected with HEK293 cells stably expressing *Kank1* (Sakar et al., 2002). These findings demonstrated that Kank1 can regulate the growth of cells and can also regulate the ab‐ normal growth of cancer cells. Kank1 may exert its growth inhibitory effect by regulating Rho activity mediated via its association with Daam1, resulting in abnormal nuclear divi‐ sion, and thus blocking the cytokinesis of cancer cells (Suzuki et al., unpublished data). According to recent studies, Kank1 can negatively regulate the formation of actin stress fibers and cell migration (Kakinuma et al., 2008; Roy et al., 2009). When cells need to con‐ trol migration, Kank1 could be transferred to the leading edge of the moving cells' mem‐ branes, mediated by Kif21a, and co-localized with IRSp53. Kank1 may bind to IRSp53 competing with active Rac1, and thus inhibits integrin-induced cell spreading and the formation of lamellipodia. Simultaneously, Kank1 may inactivate RhoA, which is control‐ led by binding with 14-3-3, inhibit the formation of actin stress fibers and ultimately in‐ hibit cell migration. Loss of expression of Kank-family proteins may enhance cell migration in renal cell carcinoma. Since enhancement of cell migration is related to meta‐ stasis, Kank-family proteins might be related to the malignancy of renal cell carcinoma.

According to studies to date, the Kank1 protein may act as a tumor suppressor through inhibition of cell migration and cell cycle. These functions are facilitated by several proteins interacting with Kank1, including 14-3-3, IRSp53, Kif21a and Daam1. Further studies of the interactions of these proteins will help us to understand clearly the role of Kank family proteins in tumorigenesis.

### **3.4. Clinical study of** *Kank1* **gene in renal cancer patients**

### *3.4.1. Genetic and clinical characteristics of renal tumors*

Kidney cancer accounts for about 4% of adult cancers, with an estimated 64,770 new cases annually in the US (Siegel et al., 2012). Of kidney cancers, 92% are pathologically diagnosed as RCC. This "RCC" has interesting and unique characteristics when investigated from a clinical view point. Although 95% of patients with T1-T2 RCC survived 5 to 10 years, among those with metastatic disease the 5 year survival rate was 26% (DeCastro and McKiernan, 2008). Renal cancer is resistant to conventional chemotherapeutic agents and also to radiation therapy. Many cancer-related genes have been found in renal cancer, including a multi-drug resistance gene (Walsh et al., 2009), anti-apoptotic genes (Bilim et al., 2009), and radiation resistant components (Kransny et al., 2010). The most characteristic genomic structure in renal cancer is the *VHL*-related hypoxia-inducible factor gene and its cascades shown in hereditary RCC and sporadic RCC cases (Linehan et al., 2011). The down-regulation in expression of *Kank1*, our main theme, was also found from the study of renal cancer and normal renal tubular cells (Sarkar et al., 2002), as we mentioned in other sections. The current WHO classification of RCC in 2004 (Deng and Melamed, 2012) follows the earlier Heidelberg and Rochester classifications, recognizing the heterogeneity of RCC, and describes distinct types of RCC with unique morphologic and genetic characteristics. The most popular histological type, clear cell RCC, accounts for 80 % of all RCC cases. Compared with clear cell RCC, papillary RCC (10%) and chromophobe RCC (5%) are more benign. Collecting duct (bellini) (1%) type or other rare sarcomatous types of RCCs are more aggressive (Deng and Melamed, 2012). However, once metastasis occurs, papillary and choromophobe RCCs are more resistant to immunological and new molecular targeting agents than clear cell RCC (Chowdhury et al., 2011). These clinical features characterize the complexity of the clinical categorization of RCC.

reported method (Roy et al., 2005). In brief, amino acids 406 to 580 of the Kank1 protein were fused in-frame with the glutathione S-transferase gene in the vector pGEX. After induction of the fusion protein in *E. coli*, it was purified and used to immunize mice. A mouse hybridoma

The histological subtypes of RCC analyzed here were as follows; 92 clear cell RCCs, 11 papillary RCCs, 5 chromophobe RCCs and 7 other histological types. We compared all histological subtypes with clear cell RCC. The evaluation of positivity of staining was done by two independent examiners, who decided that the sample was positive when more than 30 % of cells were stained with the antibody, weakly positive (±) when 5 to 30 % cells were stained, and negative when less than 5 % cells were stained. The 2004 WHO histological classification (Eble et al., 2004), 2002 TNM classification (Edge et al., 2010) and Fuhrman nuclear grade (Fuhrman et al., 1982) were used in this study. Kaplan-Meyer cause-specific survival was determined and statistical difference in positivity was evaluated by the Kluskal-Wallis test

Representative examples of positive and negative staining for the Kank1 protein in clear cell RCC and positive staining in normal renal tubular cells are indicated in Fig. 4. Normal renal tubules usually expressed Kank1. Of 92 clear cell RCCs, Kank1 was positive in 47 cases (52%). Kank1 was weakly positive (less than 30% of cells) in 14 cases (15%). Kank1 was negative in 29 cases (33%). The results grouped by clinical outcome (clear cell RCC) and histology are summarized in Table 2. There was no relation or special tendency between the staining results and clinical results on Kank1 expression. Kank1 was expressed in 87.5% of other histological

Clear cell Alive without cancer 29 11 18

Others 16 alive, 7 dead 21 2 1

**Table 2.** Immunohistological staining of Kank1 antibody classified by clinical outcome (clear cell RCC) and histological subtypes. Sums of the numbers of patients do not match all the evaluated numbers due to inavailabilty of follow-up

There were no differences in the survival curves for clear cell RCC among the groups (Fig. 5). However, when the positivity rate was evaluated among the groups divided by the Furman nuclear grade, a highly malignant grade of clear cell RCC showed high Kank1 positivity (*p* < 0.05), while the others did not (Table 3). In clear cell RCC, 42% of grade 1 tumors were Kank1 negative, while 80% of grade 3 tumors were Kank1 positive. In other histological types, there was no apparent difference among nuclear grades (most of them showed Kank1). When subdivided by pathological T stages, higher T stages of clear cell RCC showed a tendency to

Alive with cancer 7 1 4 Dead 11 2 7

**Kank1 (+) (±) (-)**

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 17

cell producing an anti-Kank1 antibody was selected and amplified for further use.

using Stat View™ software following the instructions.

RCC subtypes.

to judge the clinical outcome.

*Kank1* was found by a genome subtraction method among the genes at 9p24 susceptible to RCC (Sarkar et al., 2002). A devoted study revealed that *Kank1* belongs to a fourmember family, has splice variants, and plays a role in cell migration, intracellular trans‐ port and cell division, suggesting that *Kank1* has a kind of tumor suppressor function (Kakinuma et al., 2009). In this section, the expression of the Kank1 protein in renal can‐ cer specimens resected from RCC patients is indicated using immunohistochemical meth‐ ods, and the relationship between the expression and tumor pathology, patient status, and clinical outcomes is examined.

### *3.4.2. Expression of Kank1 protein in renal cancer and autologous normal kidney*

**1.** Expression of *Kank1* in RCC

We tried to find a RCC-related gene at 9p24, which lead to the discovery of *Kank1*. Of nine ESTs analyzed in the 9p24 region, only three (WI-17492, WI-12779 and WI-19184) were expressed in the kidney. The *Kank1* gene was associated with WI-12779. This *Kank1*-associated EST lost its expression in six out of eight cancer cases. Kank1 expression was examined in 5 matched normal kidney and cancer pairs by Western blotting using an anti-Kank1 antibody, which was obtained as mentioned below. Reduced or loss of Kank1 expression in cancer was observed in all 5 cases.

**2.** Immunohistochemical study of *Kank1* expression in RCC and the relationship between its expression and clinical-pathological outcomes

One hundred and five formalin-fixed paraffin-embedded slides including normal renal tubular cells and RCC were subjected to immunohistological staining for Kank1 with a monoclonal antibody. An anti-Kank1 (total Kank1) antibody was generated by a previously reported method (Roy et al., 2005). In brief, amino acids 406 to 580 of the Kank1 protein were fused in-frame with the glutathione S-transferase gene in the vector pGEX. After induction of the fusion protein in *E. coli*, it was purified and used to immunize mice. A mouse hybridoma cell producing an anti-Kank1 antibody was selected and amplified for further use.

those with metastatic disease the 5 year survival rate was 26% (DeCastro and McKiernan, 2008). Renal cancer is resistant to conventional chemotherapeutic agents and also to radiation therapy. Many cancer-related genes have been found in renal cancer, including a multi-drug resistance gene (Walsh et al., 2009), anti-apoptotic genes (Bilim et al., 2009), and radiation resistant components (Kransny et al., 2010). The most characteristic genomic structure in renal cancer is the *VHL*-related hypoxia-inducible factor gene and its cascades shown in hereditary RCC and sporadic RCC cases (Linehan et al., 2011). The down-regulation in expression of *Kank1*, our main theme, was also found from the study of renal cancer and normal renal tubular cells (Sarkar et al., 2002), as we mentioned in other sections. The current WHO classification of RCC in 2004 (Deng and Melamed, 2012) follows the earlier Heidelberg and Rochester classifications, recognizing the heterogeneity of RCC, and describes distinct types of RCC with unique morphologic and genetic characteristics. The most popular histological type, clear cell RCC, accounts for 80 % of all RCC cases. Compared with clear cell RCC, papillary RCC (10%) and chromophobe RCC (5%) are more benign. Collecting duct (bellini) (1%) type or other rare sarcomatous types of RCCs are more aggressive (Deng and Melamed, 2012). However, once metastasis occurs, papillary and choromophobe RCCs are more resistant to immunological and new molecular targeting agents than clear cell RCC (Chowdhury et al., 2011). These clinical

features characterize the complexity of the clinical categorization of RCC.

*3.4.2. Expression of Kank1 protein in renal cancer and autologous normal kidney*

expression and clinical-pathological outcomes

and clinical outcomes is examined.

16 Renal Tumor

**1.** Expression of *Kank1* in RCC

observed in all 5 cases.

*Kank1* was found by a genome subtraction method among the genes at 9p24 susceptible to RCC (Sarkar et al., 2002). A devoted study revealed that *Kank1* belongs to a fourmember family, has splice variants, and plays a role in cell migration, intracellular trans‐ port and cell division, suggesting that *Kank1* has a kind of tumor suppressor function (Kakinuma et al., 2009). In this section, the expression of the Kank1 protein in renal can‐ cer specimens resected from RCC patients is indicated using immunohistochemical meth‐ ods, and the relationship between the expression and tumor pathology, patient status,

We tried to find a RCC-related gene at 9p24, which lead to the discovery of *Kank1*. Of nine ESTs analyzed in the 9p24 region, only three (WI-17492, WI-12779 and WI-19184) were expressed in the kidney. The *Kank1* gene was associated with WI-12779. This *Kank1*-associated EST lost its expression in six out of eight cancer cases. Kank1 expression was examined in 5 matched normal kidney and cancer pairs by Western blotting using an anti-Kank1 antibody, which was obtained as mentioned below. Reduced or loss of Kank1 expression in cancer was

**2.** Immunohistochemical study of *Kank1* expression in RCC and the relationship between its

One hundred and five formalin-fixed paraffin-embedded slides including normal renal tubular cells and RCC were subjected to immunohistological staining for Kank1 with a monoclonal antibody. An anti-Kank1 (total Kank1) antibody was generated by a previously The histological subtypes of RCC analyzed here were as follows; 92 clear cell RCCs, 11 papillary RCCs, 5 chromophobe RCCs and 7 other histological types. We compared all histological subtypes with clear cell RCC. The evaluation of positivity of staining was done by two independent examiners, who decided that the sample was positive when more than 30 % of cells were stained with the antibody, weakly positive (±) when 5 to 30 % cells were stained, and negative when less than 5 % cells were stained. The 2004 WHO histological classification (Eble et al., 2004), 2002 TNM classification (Edge et al., 2010) and Fuhrman nuclear grade (Fuhrman et al., 1982) were used in this study. Kaplan-Meyer cause-specific survival was determined and statistical difference in positivity was evaluated by the Kluskal-Wallis test using Stat View™ software following the instructions.

Representative examples of positive and negative staining for the Kank1 protein in clear cell RCC and positive staining in normal renal tubular cells are indicated in Fig. 4. Normal renal tubules usually expressed Kank1. Of 92 clear cell RCCs, Kank1 was positive in 47 cases (52%). Kank1 was weakly positive (less than 30% of cells) in 14 cases (15%). Kank1 was negative in 29 cases (33%). The results grouped by clinical outcome (clear cell RCC) and histology are summarized in Table 2. There was no relation or special tendency between the staining results and clinical results on Kank1 expression. Kank1 was expressed in 87.5% of other histological RCC subtypes.


**Table 2.** Immunohistological staining of Kank1 antibody classified by clinical outcome (clear cell RCC) and histological subtypes. Sums of the numbers of patients do not match all the evaluated numbers due to inavailabilty of follow-up to judge the clinical outcome.

There were no differences in the survival curves for clear cell RCC among the groups (Fig. 5). However, when the positivity rate was evaluated among the groups divided by the Furman nuclear grade, a highly malignant grade of clear cell RCC showed high Kank1 positivity (*p* < 0.05), while the others did not (Table 3). In clear cell RCC, 42% of grade 1 tumors were Kank1 negative, while 80% of grade 3 tumors were Kank1 positive. In other histological types, there was no apparent difference among nuclear grades (most of them showed Kank1). When subdivided by pathological T stages, higher T stages of clear cell RCC showed a tendency to express Kank1 (*p* = 0.07) (Table 4). Other factors such as patient's age, gender and the size of the tumor (largest diameter) had no relation to the expression of Kank1 in clear cell and other RCCs (data not shown).

**Kank1 (+) (±) ( - )**

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 19

**Kank1 (+) (±) ( - )**

Clear cell RCC grade 1 9 6 11

Others grade 1 5 0 0

Clear cell RCC pT1 27 10 17

Others pT1 11 1 1

Many RCC cells showed inactivation of the *Kank1* gene as shown here. This inactivation presumably occurs at the early stage of carcinogenesis in normal renal tubular cells. Because hemizygous methylation of *Kank1* was observed in many cancer cells (Sarkar et al., 2002), inactivation of *Kank1* could be caused in both alleles by an epigenetic modification such as

Concerning the genetic abnormality of RCC, mutations in the *VHL* gene are most prevalent especially in clear cell RCC (Arai and Kanai, 2011). While *VHL* mutations can be found quite often in sporadic clear cell RCC, they are not significant in other RCC histological subtypes or benign oncocytoma. *VHL* mutations affect the activation of hypoxia-inducible factors, and

**Table 3.** Results of Kank1 staining classified by histological grade.

**Table 4.** Results of Kank1 staining classified by pathological stage.

*3.4.3. Meaning of Kank1 expression and clinical outcome*

methylation, rather than by mutations.

grade 2 31 6 19 grade 3 8 2 0

grade 2 12 1 1 grade 3 5 1 0

pT2 5 2 8 pT3 14 2 4 pT4 2 0 0

pT2 5 0 0 pT3 5 0 0 pT4 1 1 0

**Figure 4.** Immunohistochemical analysis of Kank1 protein in clear cell RCC. There was a case of positive staining of Kank1 protein in both normal renal tubular cells (upper left) and clear cell RCC (lower left), while another case indi‐ cates negative staining of Kank1 in clear cell RCC (lower right) while it was positive in the normal renal cells (upper right) (reduced from 40× images).

**Figure 5.** Kaplan-meyer's overall survival curve of RCC patients classified by Kank1 positivity (○ psitive; △ weakly posi‐ tive; □ negative). None of these survival curves showed statistical differences.


**Table 3.** Results of Kank1 staining classified by histological grade.

express Kank1 (*p* = 0.07) (Table 4). Other factors such as patient's age, gender and the size of the tumor (largest diameter) had no relation to the expression of Kank1 in clear cell and other

**Figure 4.** Immunohistochemical analysis of Kank1 protein in clear cell RCC. There was a case of positive staining of Kank1 protein in both normal renal tubular cells (upper left) and clear cell RCC (lower left), while another case indi‐ cates negative staining of Kank1 in clear cell RCC (lower right) while it was positive in the normal renal cells (upper

**Figure 5.** Kaplan-meyer's overall survival curve of RCC patients classified by Kank1 positivity (○ psitive; △ weakly posi‐

tive; □ negative). None of these survival curves showed statistical differences.

RCCs (data not shown).

18 Renal Tumor

right) (reduced from 40× images).


**Table 4.** Results of Kank1 staining classified by pathological stage.

#### *3.4.3. Meaning of Kank1 expression and clinical outcome*

Many RCC cells showed inactivation of the *Kank1* gene as shown here. This inactivation presumably occurs at the early stage of carcinogenesis in normal renal tubular cells. Because hemizygous methylation of *Kank1* was observed in many cancer cells (Sarkar et al., 2002), inactivation of *Kank1* could be caused in both alleles by an epigenetic modification such as methylation, rather than by mutations.

Concerning the genetic abnormality of RCC, mutations in the *VHL* gene are most prevalent especially in clear cell RCC (Arai and Kanai, 2011). While *VHL* mutations can be found quite often in sporadic clear cell RCC, they are not significant in other RCC histological subtypes or benign oncocytoma. *VHL* mutations affect the activation of hypoxia-inducible factors, and investigation of this pathway will contribute to a new molecular targeting therapy for RCC (Suwaki et al., 2011). The difference in *VHL* mutations among the RCC histological subtypes suggests a difference in carcinogenesis for each histological subtype, though the origin of the cancer is always a renal tubular cell.

sampling and processing. In immunohistochemisty, protein cross-linking at the preparation steps disturbs antibody binding. Sampling of homogenously expressed proteins is crucial for the stability of assays, but would not be possible for most sampling cases as the tissue itself is not homogenous. However, diagnosis even for such cases could be possible with markers sufficiently distinguishing heterogenously expressed proteins in different parts of the diseased tissue. In all cases, a statistical significance analysis should be included as a standard evaluation step for quality control of multi-marker systems such as DNA microarrays (Shi et al., 2010).

Genetics of Renal Tumors http://dx.doi.org/10.5772/54588 21

Biologically relevant markers will be made available in the future based on the analysis of signal transduction, because, as shown in Fig. 1 (Section 2), there are a number of markers available even within a single signaling pathway and there are sufficient numbers of different pathways affected by the disease, which will contribute to the stability of assays. As discussed, the VHL and mTOR pathways have drawn much attentions to prognosis/diagnosis and therapeutic targets for RCC, but there are more pathways such as the Myc and FLCN pathways and pathways related to VEGF, PDGF and TGFα, and some are specific to subtypes of RCC

Meanwhile, pathologically relevant markers will also be made available in the future, although the situation is different from other technologies due to the technical limit in the number of

One obstacle to improving immunohistochemistry is the availability of markers. Immu‐ nostaining is a relatively simple technique and thus can be used in unequipped laborato‐ ries and hospitals, because the preparation, storage and handling of samples are relatively simple. However, ordinary immunostaining is based on single-dye (or singlemarker) colorimetric techniques such as the alkaline phosphatase-based method. This is because of a lack of multi-dye (or multi-marker) colorimetric techniques due to expen‐ sive devices and, especially, inavailability of stable fluorescent dyes. Fluorescent dyes have been used in many technologies although this has not happened yet in immunohis‐ tochemistry because of the lack of their sufficient stability. Stable fluorescent dyes are

We reported applications of a new fluorescent dye, Fluolid, for DNA microarray assays and immunohistochemistry (Zhu et al., 2011). Fluolid dyes, including Fluolid-Orange, show stability against heat and excess light compared with other dyes (Fig. 6) and thus can be stored for more than a year without losing fluorescence (data not shown). So, multi-color immuno‐ histochemistry with stable fluorescent dyes will change the pathological diagnostics in several ways: long-term storage of stained sections, simultaneous multi-marker detection and handling of fluorescently stained sections. Heat and light stable fluorescent dyes will enable us to store fluorescently stained sections at room temperature for a long time, which will be

(Linehan et al., 2010; Allory et al., 2011).

markers to examine simultaneously.

**4.2. A new fluorescence-based immunohistochemical technique**

thus needed for progress in immunohistochemistry.

important for follow-up studies by microdissection of specific regions.

Given that the alteration of *Kank1* expression occurred at the early stage of carcinogenesis, our findings that *Kank1* expression differed among the histological subtypes of RCC might reflect a difference in cancer development (Kim et al., 2005). In clear cell RCC, the loss of *Kank1* expression occurred at a high rate in the lower grade tumors, and the expression was reoc‐ curred as the malignant grade increased. Although the reason for this is not clear, it is presumed that epigenetic modifications such as methylation might have been removed when the malignant grade increased, and consequently, the expression reoccurred (Kisseljova and Kisseljov, 2005). There was no difference in *Kank1* expression between the samples obtained from the groups of patients who survived or not (Table 2). This may reflect the fact that histological grade does not necessarily contribute to clinical outcome, but clinical stage (i.e. the presence of metastasis) is more crucial to obtaining a good prognosis (RCC patients diagnosed at the early stage have more than a 90% five year survival rate) (Lane and Kattan, 2008). The discordance of T stage (tumor size) and the malignant grade on *Kank1* expression could also be supposed for the same reason. A similar result was found for the expression of *CDKN2A* encoding a growth suppressor protein, which is located at 9p21 and close to *Kank1* (9p24) (unpublished data). Although the loss of *Kank1* expression resulted in increased proliferation and poor differentiation in *in vitro* study (Sarkar et al., 2002), our results about the *in vivo* expression of *Kank1* in clinical cases proved that reduced expression does not necessarily reflect a high grade malignancy or poor clinical outcome. These contradictory experimental and clinical results are very interesting, because they suggest that malignant transformation of a normal renal tubular cell has many genetic alterations and clinical outcome is contributed to by many factors in RCC.
