A Novel Gene "Niban" Upregulated in Renal Carcinogenesis: Cloning by the cDNA-amplified Fragment Length Polymorphism Approach

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named "Niban" ("second" in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, "Niban" is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This "Niban" gene is a candidate as a marker for renal tumor, especially early-stage renal carcinogenesis.     

Niban gene is commonly expressed in the renal tumors: a new candidate marker for renal carcinogenesis

Functional inactivation of tuberous sclerosis 2 gene (Tsc2) leads to renal carcinogenesis in the hereditary renal carcinoma Eker rat models. Recent studies revealed a role of tuberin, a TSC2 product, in suppressing the p70 S6 kinase (p70S6K) activity via inhibition of mammalian target of rapamycin (mTOR). Phosphorylated S6 protein, a substrate of p70S6K, was expressed in the early lesions in Eker rats, and this expression was suppressed by the treatment of rapamycin, an inhibitor of mTOR. We previously isolated the novel gene Niban expressed in renal carcinogenesis of Eker rats. In this study, we demonstrated that the expression of Niban was detected from early preneoplastic lesions in Eker rats. Interestingly, in contrast to the phosphorylated S6 protein, the expression of Niban was unchanged and early lesions still remained even after treatment with rapamycin. These results might suggest the existence of another pathway independent of mTOR-S6K pathway in Tsc2 mutant renal carcinogenesis. In addition, Niban was also expressed in other renal carcinoma models, including Tsc1 and Tsc2 knockout mice, and various types of human renal cell carcinomas. Thus, Niban was commonly expressed in renal carcinomas and might be a new marker for renal carcinogenesis.     

Renal carcinogenesis: genotype,phenotype and dramatype

Cancer is a heritable disorder of somatic cells. Environment and heredity are both important in the carcinogenic process. The Eker rat model of hereditary renal carcinoma (RC) is an example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. Forty years after the discovery of the Eker rat in Oslo, we and Knudson's group independently identified a germline retrotransposon insertion in the rat homologue of the human tuberous sclerosis ( TSC2 ) gene. To our knowledge, this was the first isolation of a Mendelian dominantly predisposing cancer gene in a naturally occurring animal model. Recently, we discovered a new hereditary renal carcinoma in the rat. This rat was named the "Ninon'rat and its predisposing ( Nihon ) gene could be a novel renal tumor suppressor gene. This article will review the utility of these unique models for the study of problems in carcinogenesis; e.g., species-specific differences in tumorigenesis, cell stage and tissue/cell-type specific tumorigene-sis, multistep carcinogenesis, modifier gene(s) in renal carcinogenesis, cancer prevention and the development of therapeutic treatments which can be translated to human patients, as well as how environmental factors interact with cancer susceptibility gene(s). (Cancer Sci 2003; 94: 142 – 147)     

A Novel Renal Cell Carcinoma Susceptibility Gene Maps on Chromosome 10 in the Eker Rat

Hereditary renal cell carcinoma (RC) in the rat, originally reported by Eker in 1954, is an example of Mendelian dominant predisposition to a specific cancer in an experimental animal. We previously reported that this predisposing inherited gene is a tumor suppressor gene fitting Knudson's "two-hit" model. This study was designed to map the RC susceptibility gene in the Eker rat using backcross animals. Our present data clearly show that the RC gene is genetically linked to the protamine-1 gene (Lod score = 11.65) and the interleukin-3 gene (Lod score=4.13), both of which are located on the proximal part of rat chromosome 10. Rat chromosome 10 is currently believed to have no syntenic relationship to human chromosome 3p, the presumed site of the putative tumor suppressor gene for human RC and the locus of von Hippel Lindau disease (affected patients develop multiple RCs). Thus, the Eker rat might have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.     

Presence of a modifier gene(s) affecting early renal carcinogenesis in the Tsc2 mutant (Eker) rat model

The Eker (Tsc2 mutant) rat model of renal carcinoma is an example of Mendelian dominantly inherited predisposition to a specific cancer. Effects of genetic background on renal carcinogenesis in the Eker rat model (Eker/Eker > Eker/BN strain) indicate the presence in the BN rat genome of a modifier gene(s) that suppresses tumorigenesis. The identification of such a modifier gene(s) might help clarify the diversity of tuberous sclerosis in humans. i) We found that preneoplastic lesions in 8-week-old F1 rats [(Eker x LE) and (Eker x BN)] were more numerous in the LE strain than in the BN strain although the difference was not large. ii) We next administered N-ethyl-N-nitrosourea (ENU; single injection, i.p.) at the age of 4 weeks to amplify the strain difference in tumorigenesis, as we had done in an earlier study to identify the predisposing gene. iii) This experiment was also done in BN congenic Eker rats to confirm the strain difference in tumorigenesis. Preneoplastic lesions were fewer in BN congenic rats than in Eker rats by a factor of 100. We used this ENU system to perform a backcross experiment [F1(Eker x BN) x Eker] and finally succeeded in mapping a new modifier locus on rat chromosome 5 (the LOD score of the D5Rat12 was 3.13).     

Establishment of the enzyme-linked immunosorbent assay system to detect the amino terminal secretory form of rat Erc/Mesothelin

By representational difference analysis, we previously identified the rat Erc (Expressed in renal carcinoma) gene that was more abundantly expressed in the renal carcinoma tissues of Eker rats than in the rat normal kidney. In this study, we raised antibodies against the amino-terminal portion of the rat Erc, and demonstrated the existence of a approximately 30-kDa secretory form in the supernatant of cultured cells derived from rat renal carcinoma. The enzyme-linked immunosorbent assay (ELISA) system using these antibodies detected high concentrations of this form in the sera of Eker rats bearing renal carcinomas, and in the sera of rats transplanted with mesothelioma cells. Mesothelin, a human homolog of the rat Erc, was recently reported to be a serum marker of malignant mesothelioma. The prognosis of mesothelioma is poor and there is no effective treatment at present. There are several rat model systems of mesothelioma that may be promising tools in the development of an antimesothelioma treatment. We hope our ELISA to detect the soluble form of rat Erc/Mesothelin is useful in the rat model system to exploit the antimesothelioma therapy to be used in human cases.     

A novel renal carcinoma predisposing gene of the Nihon rat maps on chromosome 10.

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley (SD) strain in Japan, and named the "Nihon" rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross animals. Our present data clearly show that the Nihon gene is genetically linked to interleukin-3 (IL3) gene (chi(2) = 93.6, Lod score = 25.16), lethal (2) giant larvae (LLGL1) locus (chi(2) = 109.0, Lod score = 31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE) gene (chi(2) = 90.6, Lod score = 23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)--21.3 cM--IL3 gene (human 5q23-31)--4.4 cM--Nihon gene--0.9 cM--LLGL1 locus (human 17p11.2)--4.4 cM--MYHSE gene (human 17p13.1). We also detected loss of the wild-type allele at the MYHSE locus, fitting Knudson's "two hit" model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.     

A Novel Renal Carcinoma Predisposing Gene of the Nihon Rat Maps on Chromosome 10

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Spra-gue-Dawley (SD) strain in Japan, and named the "Nihon" rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross annuals. Our present data clearly show that the Nihon gene is genetically linked to interleukin-3 (IL3 ) gene (χ²=93.6, Lod score=25.16), lethal (2) giant larvae (LLGL1 ) locus (χ²=109.0, Lod score=31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE ) gene (χ²=90.6, Lod score=23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2 ) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)–21.3 cM– IL3 gene (human 5q23-31)–4.4 cM– Nihon gene–0.9 cM–LLGL1 locus (human 17p11.2)-4.4 cM– MYHSE gene (human 17pl3.1). We also detected loss of the wild-type allele at the MYHSE locus, fitting Knudson's "two hit" model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.     

Cloning of the Rat Homologue of the von Hippel-Lindau Tumor Suppressor Gene and Its Non-somatic Mutation in Rat Renal Cell Carcinomas

Recently, von Hippel-Lindau ( VHL ) gene mutations were detected in non-inherited, sporadic human renal cell carcinomas (RCs) at a high frequency. In order to determine whether or not the VHL gene is also a critical gene in rat RCs, we cloned and sequenced the rat homologue of human VHL gene and searched for mutations of the VHL gene in rat RCs. Mutations in the VHL gene were not detected in spontaneous RCs of the Eker rat model or in ferric nitrilotriacetate-induced rat RCs using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. These data indicate that mutation of the VHL tumor suppressor gene is not an event in rat renal carcinogenesis, at least in our present systems.     

Regulation of PI 3-K, PTEN, p53, and mTOR in Malignant and Benign Tumors Deficient in Tuberin

The tuberous sclerosis complex (TSC) is caused by mutation in either of 2 tumor suppressor genes, TSC-1 (encodes hamartin) and TSC-2 (encodes tuberin). In humans, deficiency in TSC1/2 is associated with benign tumors in many organs, including renal angiomyolipoma (AML) but rarely renal cell carcinoma (RCC). In contrast, deficiency of TSC function in the Eker rat is associated with RCC. Here, we have investigated the activity of PI 3-K and the expression of PTEN, p53, tuberin, p-mTOR, and p-p70S6K in both Eker rat RCC and human renal AML. Compared to normal tissue, increased PI 3-K activity was detected in RCC of Eker rats but not in human AML tissue. In contrast, PTEN was highly expressed in AML but significantly reduced in the renal tumors of Eker rats. Phosphorylation on Ser(2448) of mTOR and Thr(389) of p70S6K were significantly increased in both RCC and AML compared to matching control tissue. Total tuberin was significantly decreased in AML while completely lost in RCC of Eker rats. Our data also show that while p53 protein expression is lost in rat RCC, it was highly elevated in AML. These novel data provide evidence that loss of TSC-2, PTEN, and p53 as well as activation of PI 3-K and mTOR is associated with kidney cancer in the Eker rat, while sustained expression of TSC-2, PTEN, and p53 may prevent progression of kidney cancer in TSC patients.     

Carcinogen-specific gene expression profiles in short-term treated Eker and wild-type rats indicative of pathways involved in renal tumorigenesis

Eker rats heterozygous for a dominant germline mutation in the tuberous sclerosis 2 (Tsc2) tumor suppressor gene were used as a model to study renal carcinogenesis. Eker and corresponding wild-type rats were exposed to genotoxic aristolochic acid (AA) or non-genotoxic ochratoxin A (OTA) to elucidate early carcinogen-specific gene expression changes and to test whether Eker rats are more sensitive to carcinogen-induced changes in gene expression. Male Eker and wild-type rats were gavaged daily with AA (10 mg/kg body weight) or OTA (210 microg/kg body weight). After 1, 3, 7, and 14 days of exposure, renal histopathology, tubular cell proliferation, and Affymetrix gene expression profiles from renal cortex/outer medulla were analyzed. AA-treated Eker and wild-type rats were qualitatively comparable in all variables assessed, suggesting a Tsc2-independent mechanism of action. OTA treatment resulted in slightly increased cortical pathology and significantly elevated cell proliferation in both strains, although Eker rats were more sensitive. Deregulated genes involved in the phosphatidylinositol 3-kinase-AKT-Tsc2-mammalian target of rapamycin signaling, among other important genes prominent in tumorigenesis, in conjunction with the enhanced cell proliferation and presence of preneoplastic lesions suggested involvement of Tsc2 in OTA-mediated toxicity and carcinogenicity, especially as deregulation of genes involved in this pathway was more prominent in the Tsc2 mutant Eker rat.     

Generation of Metastatic Variants of Eker Renal Carcinoma Cell Lines for Experimental Investigation of Renal Cancer Metastasis

We and others have demonstrated that a mutation in Tsc2 is the rate-limiting step for renal carcinogenesis in the Eker rat model. Although inactivation of Tsc2 results in development of renal tumors, it is not sufficient for metastatic renal cell carcinomas (RCs) in the Eker rat. To investigate the additional genetic event(s) necessary for cancer metastasis, we have established highly metastatic S-Lk9d-SLM cell lines from a non-metastatic RC cell line (Lk9dL) by co-implantation with a foreign body (gelatin sponge). Since these cell lines were remarkably different in metastatic performance (all and none, respectively) despite having the same genetic background, they should be useful experimental tools to investigate metastasis-promoting events in renal carcinogenesis.     

p53 status in spontaneous and dimethylnitrosamine—induced renal cell tumors from rats

Rats carrying the Eker tumor–susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT → CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non–p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer. © 1995 Wiley-Liss Inc.     

A novel "Nihon" rat model of a Mendelian dominantly inherited renal cell carcinoma.

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan, and named the rising "Nihon" rat. In this strain, RCs develop from early preneoplastic lesions, which begin to appear at 4 weeks of age, forming adenomas by the age of 16 weeks. The RCs are predominantly of clear cell type. Southern blot, northern blot and SSCP analyses revealed no change in the Tsc1, Tsc2, VHL, and c-Met genes. Thus, the Nihon rat should be a valuable experimental model for understanding renal carcinogenesis, especially clear cell type, which is common among human RCs.     

Suppression of cytochrome P450 1A1 and 4A1 gene expression in renal carcinomas of TSC2 gene mutant (Eker) rats

In the kidney, cytochrome P450 (CYP) is involved in arachidonic acid metabolism and the maintenance of homeostasis, but only scarce information is available as to how CYP expression is altered in rodent renal carcinomas (RCs). TSC2 gene mutant (Eker) rat RCs are an example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. In the present study, the expression of CYP in Eker RCs was studied. In the normal kidney, CYP 1A1 and 4A1 mRNAs were expressed, but this expression was suppressed in spontaneously-induced Eker RCs and in cell line Lk9dL and Lk9dR. In Lk9dL and Lk9dR, Ah receptor nuclear translocator and haemoxygenase-1 mRNAs were expressed, but this expression was inconsistent in spontaneously-induced Eker RCs. The present results showed the suppression of CYP 1A1 and 4A1 mRNA expression in spontaneously-induced Eker RCs and this suppression indicates altered metabolic conditions in Eker RCs.     

Allelic Loss at the Tuberous Sclerosis (Tsc2) Gene Locus in Spontaneous Uterine Leiomyosarcomas and Pituitary Adenomas in the Eker Rat Model

Hereditary renal carcinomas (RCs) develop in virtually all Eker rats by the age of one year. Investigation of extra-renal primary tumors co-occurring in Eker rats late in life (at 2 years) additionally revealed enhanced development of hemangiosarcomas of the spleen, uterine leiomyosarcomas and pituitary adenomas, although the demonstrated predilection for these extra-renal tumors was not as complete as with RCs. We identified the germline mutated tuberous sclerosis ( Tsc2 ) gene as the predisposing Eker gene and revealed the tumor suppressor nature of Tsc2 gene function in renal carcinogenesis. In the present study, we examined allelic loss at the Tsc2 gene locus in uterine leiomyosarcomas and pituitary adenomas developing in hybrid F1 rats carrying the Eker mutation as well as in pituitary adenomas from non-carrier rats. We detected loss of heterozygosity in 4 of 11 uterine leiomyosarcomas (36%) and 11 of 31 pituitary adenomas (35%) from Eker rats but in none of 9 pituitary adenomas from non-carrier rats ( P <0.05), suggesting that inactivation of the Tsc2 gene is also a critical event in the pathogenesis of these extra-renal tumors. Our present data indicate that there might be different pathways for tumorigenesis of pituitary adenomas between Eker and non-carrier rats.     

Estrogen treatment enhances hereditary renal tumor development in Eker rats

Hormonal influences are known to affect the development of renal cell carcinoma in man and laboratory animal models. We tested the hypothesis that estrogen treatment or ovariectomy of rats modulates renal tumor development using tuberous sclerosis 2 (Tsc2) heterozygous mutant (Eker) rats in which a germline mutation predisposes the animals to renal cell tumor development. Two-month-old female wild-type and Eker rats were ovariectomized or sham-operated and treated with placebo or 5 mg 17beta-estradiol in s.c. pellets for 6 or 10 months. Rats were examined at 8 or 12 months of age, at which time the numbers of renal tumors and preneoplastic foci were quantitated and the severity of nephropathy was assessed. In contrast to what may have been expected, prolonged estrogen treatment enhanced the development of hereditary renal cell tumors, with a 2-fold greater number of preneoplastic and neoplastic renal lesions compared with untreated Eker rats. Ovariectomized Eker rats had 33% fewer renal lesions than the unmanipulated control group. No tumors or preneoplastic lesions were present in wild-type rats at either time point. Estrogen treatment increased the severity of nephropathy in both wild-type and Eker rats, whereas ovariectomy was protective against nephropathic changes. Although estrogen is not a rat renal carcinogen, it enhanced the development of hereditary renal cell tumors when administered to Eker rats. Eker rats heterozygous for a mutation in the Tsc2 locus provide a good model in which to study how genetic and hormonal factors contribute to the development of renal cell tumors and to understand the influence genetic susceptibility has on the development of renal cell carcinoma.