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.     

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.     

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.      

Isolation and characterization of a rat homologue of the human tuberous sclerosis 1 gene (Tsc1) and analysis of its mutations in rat renal carcinomas

In the Eker rat, a germ-line mutation in the homologue of the human tuberous sclerosis gene (Tsc2) causes renal cell carcinomas (RCs) with a complete penetrance in all heterozygotes. Tsc2 mutations have also been found in a subset of chemically induced non-Eker rat RCs. Because tuberous sclerosis patients with alteration of either of the two predisposing genes (TSC1 and TSC2) show identical symptoms, the products of these two genes are thought to be involved in a common biological pathway. In this study, to analyze the possible overlap between the functions of Tsc2 and Tscl gene products, we isolated and characterized a rat homologue of the TSC1 gene (Tsc1). The rat Tsc1 gene, which has an identical exon-intron structure to that of human TSC1 and is localized on rat chromosome 3, has been shown to encode a protein (hamartin) that is highly homologous to the human counterpart with an approximately 86% amino acid sequence identity. Using PCR-single-strand conformational polymorphism analysis, we identified two splicing donor site mutations in one chemically induced rat RC (1 of 15). This suggests that alterations of the Tsc1 gene may be involved in the development of a subset of rat RCs.     

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.     

Interstitial chromosomal deletion of the tuberous sclerosis complex 2 locus is a signature for radiation‐associated renal tumors in Eker rats

Ionizing radiation can damage DNA and, therefore, is a risk factor for cancer. Eker rats, which carry a heterozygous germline mutation in the tumor‐suppressor gene tuberous sclerosis complex 2 (Tsc2), are susceptible to radiation‐induced renal carcinogenesis. However, the molecular mechanisms involved in Tsc2 inactivation are unclear. We subjected Fischer 344 × Eker (Long Evans Tsc2^+/?) F1 hybrid rats to gamma‐irradiation (2 Gy) at gestational day 19 (GD19) or postnatal day 5 (PND5) and investigated the patterns of genomic alterations in the Tsc2 allele of renal tumors that developed at 1 year after irradiation (N = 24 tumors for GD19, N = 10 for PND5), in comparison with spontaneously developed tumors (N = 8 tumors). Gamma‐irradiation significantly increased the multiplicity of renal tumors. The frequency of LOH at the chromosome 10q12 region, including the Tsc2 locus, was 38%, 29% and 60% in renal carcinomas developed from the nonirradiated, GD19 and PND5 groups, respectively. Array comparative genomic hybridization analysis revealed that the LOH patterns on chromosome 10 in renal carcinomas were classified into chromosomal missegregation, mitotic recombination and chromosomal deletion types. LOH of the interstitial chromosomal deletion type was observed only in radiation‐associated carcinomas. Sequence analysis for the wild‐type Tsc2 allele in the LOH‐negative carcinomas identified deletions (nonirradiated: 26%; GD19: 21%) and base‐substitution mutations (GD19: 4%). Reduced expression of Tsc2 was also observed in the majority of the LOH‐negative carcinomas. Our results suggest that interstitial chromosomal deletion is a characteristic mutagenic event caused by ionizing radiation, and it may contribute to the assessment of radiation‐induced cancer risk.     

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.     

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.     

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 "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.     

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.     

Aberrant expression of HMGA2 in uterine leiomyoma associated with loss of TSC2 tumor suppressor gene function

Unregulated proliferation of mesenchymal cells in leiomyomas, lipomas, hamartomas,and other diseases has been linked to the high mobility group (HMGA) family of DNA architectural proteins. HMGA genes are primarily expressed during embryonal development and silenced in adult tissues but can become reactivated in neoplasia as a result of chromosomal rearrangements. Although the genetic data suggesting a role for HMGA proteins in tumorigenesis are compelling, the biological role of these proteins in mesenchymal proliferation and differentiation is incompletely defined. Uterine myometria and spontaneous leiomyomas from the Eker rat, which carries a germ-line mutation in the tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene, were analyzed for genetic defects in and expression of the Tsc2 and HMGA proteins. Eker leiomyomas exhibited a 50% incidence of loss of the wild-type Tsc2 allele and an almost uniform loss of protein expression, implicating loss of function of the Tsc2 gene in these tumors. Concomitantly, HMGA2 protein, which was completely absent in normal myometria, was expressed in 16 of 19 Eker leiomyomas. HMGA1 was expressed in both leiomyoma and normal myometria. No structural alterations were observed at the HMGA2 locus in either primary rat leiomyomas or leiomyoma-derived cell lines that expressed HMGA2. These data support a role for HMGA2 in the development of smooth muscle neoplasms and suggest HMGA2 expression is a point of convergence between the human disease and the Eker rat model. Furthermore, these data indicate that aberrant HMGA2 expression can result from dysfunction of the Tsc2 tumor suppressor gene, in the absence of structural alterations involving the HMGA2 locus.     

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).     

Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice

Germ-line mutations of the human TSC2 tumor suppressor gene cause tuberous sclerosis (TSC), a disease characterized by the development of hamartomas in various organs. In the Eker rat, however, a germ-line Tsc2 mutation gives rise to renal cell carcinomas with a complete penetrance. The molecular mechanism for this phenotypic difference between man and rat is currently unknown, and the physiological function of the TSC2/Tsc2 product (tuberin) is not fully understood. To investigate these unsolved problems, we have generated a Tsc2 mutant mouse. Tsc2 heterozygous mutant (Tsc2+/-) mice developed renal carcinomas with a complete penetrance, as seen in the Eker rat, but not the angiomyolipomas characteristic of human TSC, confirming the existence of a species-specific mechanism of tumorigenesis caused by tuberin deficiency. Unexpectedly, approximately 80% of Tsc2+/- mice also developed hepatic hemangiomas that are not observed in either TSC or the Eker rat. Tsc2 homozygous (Tsc2-/-) mutants died around embryonic day 10.5, indicating an essential function for tuberin in mouse embryonic development. Some Tsc2-/- embryos exhibited an unclosed neural tube and/or thickened myocardium. The latter is associated with increased cell density that may be a reflection of loss of a growth-suppressive function of tuberin. The mouse strain described here should provide a valuable experimental model to analyze the function of tuberin and its association with tumorigenesis.     

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.     

Development of High-grade Renal Cell Carcinomas in Rats Independently of Somatic Mutations in the Tsc2 and VHL Tumor Suppressor Genes

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular damage that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. The RCCs are characterized by 1) high incidence of pulmonary metastasis and peritoneal invasion, 2) high incidence of tumor-associated mortality and 3) possible involvement of reactive oxygen species in carcinogenesis. The present study investigated the possible role of Tsc2 and VHL tumor suppressor genes in this model. Thirty-four Fe-NTA-induced primary RCCs and 20 other primary or metastatic tumors of rats were searched for genetic alteration in all the coding exons of both genes by polymerase chain reaction-single-strand-conformation polymorphism analysis and sequencing in conjunction with morphological evaluation. In the Fe-NTA-induced RCCs, frequency of metastasis or invasion was proportionally associated with the nuclear grade of the tumor (grades 1–3). Only one Fe-NTA-induced RCC of grade 1 revealed missense mutations with loss of heterozygosity in exon 10 of the Tsc2 gene (codons 334, GTG (Val) to GCG (Ala), and 336, TAT (Tyr) to CAT (His)). No mutation was found in the VHL gene. The results suggest that 1) high-grade RCCs can develop in the absence of mutations in the Tsc2 and VHL genes in rats, and that 2) Tsc2 gene somatic mutation can nonetheless be one of the causes of non-Eker rat RCCs.     

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.