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.     

Heterozygosity for the tuberous sclerosis complex (TSC) gene products results in increased astrocyte numbers and decreased p27-Kip1 expression in TSC2+/- cells

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor predisposition syndrome characterized by benign proliferations (hamartomas). In the brain, individuals with TSC develop autism, mental retardation and seizures associated with focal cortical dysplasias, subependymal nodules, and subependymal giant cell astrocytomas (SEGAs). We hypothesize that dysregulated astrocyte function due to mutations in the tumor suppressor genes, TSC1 and TSC2, may contribute to the pathogenesis of these brain abnormalities. In this report, we demonstrate that mice heterozygous for a targeted defect in either the Tsc1 or Tsc2 genes(Tsc1+/- and Tsc2+/- mice) exhibit a 1.5-fold increase in the number of astrocytes in vivo. Whereas increased astrocyte numbers in vivo were suggestive of a proliferative advantage, Tsc2+/- primary astrocyte cultures did not show a cell-autonomous growth advantage, anchorage-independent growth, increased saturation density, or increased fluid-phase endocytosis compared to wild type astrocytes. Tsc2 null mouse embryonic fibroblasts (MEFs) however, did exhibit increased saturation density compared to Tsc2 wild type controls. In both Tsc2+/- astrocytes and Tsc2 null mouse embryonic fibroblasts, p27-Kip1 expression was decreased compared to wild type cells, and was reversed by tuberin re-expression in Tsc2-/- MEFs. In contrast, no change in endocytosis was observed upon tuberin re-expression in Tsc2-/- MEFs. Collectively, these results suggest Tsc heterozygosity may provide a non-cell-autonomous growth advantage for astrocytes that may involve p27-Kip1 expression.     

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.     

Mourning Dr. Alfred G. Knudson: the two‐hit hypothesis,tumor suppressor genes,and the tuberous sclerosis complex

On July 10, 2016, Alfred G. Knudson, Jr., MD, PhD, a leader in cancer research, died at the age of 93 years. We deeply mourn his loss. Knudson's two‐hit hypothesis, published in 1971, has been fundamental for understanding tumor suppressor genes and familial tumor‐predisposing syndromes. To understand the molecular mechanism of two‐hit‐initiated tumorigenesis, Knudson used an animal model of a dominantly inherited tumor, the Eker rat. From the molecular identification of Tsc2 germline mutations, the Eker rat became a model for tuberous sclerosis complex (TSC), a familial tumor‐predisposing syndrome. Animal models, including the fly, have greatly contributed to TSC research. Because the product of the TSC2/Tsc2 gene (tuberin) together with hamartin, the product of another TSC gene (TSC1/Tsc1), suppresses mammalian/mechanistic target of rapamycin complex 1 (mTORC1), rapalogs have been used as therapeutic drugs for TSC. Although significant activity of these drugs has been reported, there are still problems such as recurrence of residual tumors and adverse effects. Recent studies indicate that there are mTORC1‐independent signaling pathways downstream of hamartin/tuberin, which may represent new therapeutic targets. The establishment of cellular models, such as pluripotent stem cells with TSC2/Tsc2 gene mutations, will facilitate the understanding of new aspects of TSC pathogenesis and the development of novel treatment options. In this review, we look back at the history of Knudson and animal models of TSC and introduce recent progress in TSC research.     

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.     

Tsc1 haploinsufficiency without mammalian target of rapamycin activation is sufficient for renal cyst formation in Tsc1+/- mice

Tuberous sclerosis complex (TSC) is caused by mutations in either the TSC1 or TSC2 gene. Both genes are generally considered to act as tumor suppressors that fulfill Knudson's "two-hit hypothesis" and that function within the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin (mTOR) pathway. We previously generated Tsc1(+/-) mice that are predisposed to renal cysts, which develop into cystadenomas and renal cell carcinomas. Here, we identified somatic Tsc1 mutations (second hits) in approximately 80% of cystadenomas and renal cell carcinomas, but only 31.6% of cysts from Tsc1(+/-) mice (P < 0.0003), raising the possibility that haploinsufficiency for Tsc1 plays a role in cyst formation. Consistent with this proposal, many cysts showed little or no staining for phosphorylated mTOR (53%) and phosphorylated S6 ribosomal protein (37%), whereas >90% of cystadenomas and renal cell carcinomas showed strong staining for both markers (P < 0.0005). We also sought somatic mutations in renal lesions from Tsc1(+/-) Blm(-/-) mice that have a high frequency of somatic loss of heterozygosity, thereby facilitating the detection of second hits. We also found significantly less somatic mutations in cysts as compared with cystadenomas and renal cell carcinomas from these mice (P = 0.017). Our data indicate that although activation of the mTOR pathway is an important step in Tsc-associated renal tumorigenesis, it may not be the key initiating event in this process.     

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.     

TSC2 loss in lymphangioleiomyomatosis cells correlated with expression of CD44v6, a molecular determinant of metastasis

Lymphangioleiomyomatosis (LAM), a rare multisystem disease found primarily in women of childbearing age, is characterized by the proliferation of abnormal smooth muscle-like cells, LAM cells, that form nodules in the pulmonary interstitium. Proliferation of LAM cells results, in part, from dysfunction in tuberous sclerosis complex (TSC) genes TSC1 (hamartin) and/or TSC2 (tuberin). Identification of LAM cells in donor lungs, their isolation from blood, and their presence in urine, chylous ascites, and pleural effusions are consistent with their ability to metastasize. Here, we investigated the presence on LAM cells of the hyaluronic acid receptor CD44 and its splice variants associated with metastasis. The heterogeneous populations of cells grown from lungs of 12 LAM patients contain cells expressing mRNA for the variant CD44v6. Histologically, CD44v6 was present in LAM lung nodules, but not in normal vascular smooth muscle cells. CD44v6-positive sorted cells showed loss of heterozygosity at the TSC2 locus; binding of CD44v6 antibody resulted in loss of cell viability. Levels of CD44 were higher in cultured Eker rat (Tsc2-/-) cells than in Tsc2+/+ cells, but unlike human LAM cells, the Tsc2-/- Eker rat cells did not contain CD44v6 splice variant mRNA. CD44 splicing and signaling is regulated by osteopontin. Plasma from LAM patients contained higher concentrations of osteopontin than plasma of healthy, age-, and sex-matched volunteers (P = 0.00003) and may be a biomarker for LAM. The cell surface receptor CD44 and its splice variant CD44v6 may contribute to the metastatic potential of LAM cells.     

Induction of renal tumorigenesis with elevated levels of somatic loss of heterozygosity in Tsc1+/- mice on a Blm-deficient background

A Bloom's deficient mouse model (Blm(m3/m3)) has been shown to induce colorectal tumorigenesis when crossed with Apc+/Min mice. Here, we investigated whether the Blm(m3/m3) genotype could induce tumorigenesis in extracolonic tissues in tuberous sclerosis 1-deficient (Tsc1+/-) mice that are predisposed to renal cystadenomas and carcinomas. Genotyping of offspring from Tsc1+/- Blm+/m3 intercrosses showed that a approximately 24% excess of Tsc1+/- over Tsc1+/+ mice died before weaning (P = 0.016), although Blm deficiency had no cumulative effect on Tsc1+/- survival. Tsc1+/- Blm(m3/m3) mice had significantly more macroscopic and microscopic renal lesions at 3 to 6 months compared with Tsc1+/- Blm+/m3 mice (P =0.0003 and 0.0203, respectively), and their tumors showed significantly increased levels of somatic loss of heterozygosity (LOH) of the wild-type Tsc1 (Tsc1wt) allele compared with those from Tsc1+/- Blm+/+ mice (P < 0.0001). Tsc1+/- Blm+/m3 mice did not show significantly more renal lesions compared with Tsc1+/- Blm+/+ animals; however, their lesions still showed significantly increased levels of somatic LOH of the Tsc1wt allele (P = 0.03). Ninety-five percent (19 of 20) of lesions from Tsc1+/- Blm+/m3 mice retained the wild-type Blm (Blm(wt)) allele, indicating that the increased somatic LOH at Tsc1 was mediated by Blm haploinsufficiency. Renal lesions from a Blm-deficient background stained positively with anti-phospho-S6 ribosomal protein (Ser240/244), suggesting that these lesions develop through the normal pathway of Tsc-associated tumorigenesis. This work shows the use of the Blm(m3/m3) mice for inducing renal tumorigenesis, and the high levels (approximately 87%) of LOH in the resultant tumors will help facilitate mapping of loci involved in tumor progression.     

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.     

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).     

The G1556S-type tuberin variant suppresses tumor formation in tuberous sclerosis 2 mutant (Eker) rats despite its deficiency in mTOR inhibition

Tuberin, a tumor-suppressor protein produced by the tuberous sclerosis gene TSC2, downregulates the Rheb-mTOR-S6K pathway (mTOR axis). Comparison of the effects of human tuberin mutations, such as G1556S, suggests that pathways other than the mTOR axis might also be involved in the pathogenesis of tuberous sclerosis. Here we test this possibility using the rat G1556S-type mutation (GSM) and a transgenic Eker (Tsc2 mutant) rat system. Cells expressing GSM-tuberin failed to downregulate the mTOR axis. GSM-tuberin had an altered localization, which underlie its reduced ability to form a complex with hamartin, and a site-specific alteration in phosphorylation status indicating diverse regulation by Akt. GSM-transgenic (GSM-Tg) rats exhibited suppression of macroscopic renal tumors following N-ethyl-N-nitrosourea treatment. Intriguingly, rats with weaker GSM-Tg expression showed microscopic cystic and pre-tumorous lesions that were restricted in size and expansion, although they had hyper-phosphorylation of ribosomal protein S6. These results highlight a novel pathway involving tuberin that regulates tumor suppression independently of the mTOR inhibitory function. Identification of such a novel pathway will provide clear implications for generation of new therapeutic targets in the treatment of these tumors.     

Selective inhibition of growth of tuberous sclerosis complex 2 null cells by atorvastatin is associated with impaired Rheb and Rho GTPase function and reduced mTOR/S6 kinase activity

Inactivating mutations in the tuberous sclerosis complex 2 (TSC2) gene, which encodes tuberin, result in the development of TSC and lymphangioleiomyomatosis (LAM). The tumor suppressor effect of tuberin lies in its GTPase-activating protein activity toward Ras homologue enriched in brain (Rheb), a Ras GTPase superfamily member. The statins, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have pleiotropic effects which may involve interference with the isoprenylation of Ras and Rho GTPases. We show that atorvastatin selectively inhibits the proliferation of Tsc2-/- mouse embryo fibroblasts and ELT-3 smooth muscle cells in response to serum and estrogen, and under serum-free conditions. The isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) significantly reverse atorvastatin-induced inhibition of Tsc2-/- cell growth, suggesting that atorvastatin dually targets a farnesylated protein, such as Rheb, and a geranylgeranylated protein, such as Rho, both of which have elevated activity in Tsc2-/- cells. Atorvastatin reduced Rheb isoprenylation, GTP loading, and membrane localization. Atorvastatin also inhibited the constitutive phosphorylation of mammalian target of rapamycin, S6 kinase, and S6 found in Tsc2-/- cells in an FPP-reversible manner and attenuated the high levels of phosphorylated S6 in Tsc2-heterozygous mice. Atorvastatin, but not rapamycin, attenuated the increased levels of activated RhoA in Tsc2-/- cells, and this was reversed by GGPP. These results suggest that atorvastatin may inhibit both rapamycin-sensitive and rapamycin-insensitive mechanisms of tuberin-null cell growth, likely via Rheb and Rho inhibition, respectively. Atorvastatin may have potential therapeutic benefit in TSC syndromes, including LAM.     

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.     

Tuberin haploinsufficiency is associated with the loss of OGG1 in rat kidney tumors

Background  

Tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors. Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) and in RCC in the Eker rat. Tuberin downregulates the DNA repair enzyme 8-oxoguanine DNA-glycosylase (OGG1) with important functional consequences, compromising the ability of cells to repair damaged DNA resulting in the accumulation of the mutagenic oxidized DNA, 8-oxo-dG. Loss of function mutations of OGG1 also occurs in human kidney clear cell carcinoma and may contribute to tumorgenesis. We investigated the distribution of protein expression and the activity of OGG1 and 8-oxo-dG and correlated it with the expression of tuberin in kidneys of wild type and Eker rats and tumor from Eker rat.     

Estrogen enhances whereas tamoxifen retards development of Tsc mouse liver hemangioma: a tumor related to renal angiomyolipoma and pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis and abdominal angiomyolipoma are related lesions for which there is no authentic animal model. Both of these proliferative lesions occur in sporadic patients, and at much higher frequency in patients with tuberous sclerosis, which is due to mutations in the TSC1 and TSC2 genes. Tsc1+/- and Tsc2+/- mice frequently develop liver hemangioma. We found that the Tsc mouse liver hemangioma are composed predominantly of endothelial cells but with a smooth muscle component, and express HMB45 antigen, estrogen receptor, and progesterone receptor, similar to lymphangioleiomyomatosis and angiomyolipoma. Estrogen treatment significantly accelerated the development of liver hemangioma in Tsc1+/- female mice, with 91% having liver hemangioma and 55% having severe lesions by 7 months of age. Similarly, an increased frequency and severity of liver hemangiomas was seen in Tsc1+/- males treated with estrogen. In contrast, tamoxifen treatment for 9 months significantly reduced the frequency and severity of hemangiomas in Tsc1+/- female mice. In addition, estrogen treatment significantly increased serum vascular endothelial growth factor levels in Tsc1+/- mice, whereas tamoxifen reduced vascular endothelial growth factor levels. These mouse model observations indicate the importance of estrogen signaling in vivo for the growth of tuberous sclerosis lesions, suggesting the possible benefits of tamoxifen therapy for the treatment of angiomyolipoma and lymphangioleiomyomatosis.     

Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors

Disruption of the TSC1 or TSC2 gene leads to the development of tumors in multiple organs, most commonly affecting the kidney, brain, lung, and heart. Recent genetic and biochemical studies have identified a role for the tuberous sclerosis gene products in phosphoinositide 3-kinase signaling. On growth factor stimulation, tuberin, the TSC2 protein, is phosphorylated by Akt, thereby releasing its inhibitory effects on p70S6K. Here we demonstrate that primary tumors from tuberous sclerosis complex (TSC) patients and the Eker rat model of TSC expressed elevated levels of phosphorylated mammalian target of rapamycin (mTOR) and its effectors: p70S6K, S6 ribosomal protein, 4E-BP1, and eIF4G. In the Eker rat, short-term inhibition of mTOR by rapamycin was associated with a significant tumor response, including induction of apoptosis and reduction in cell proliferation. Surprisingly, these changes were not accompanied by significant alteration in cyclin D1 and p27 levels. Our data provide in vivo evidence that the mTOR pathway is aberrantly activated in TSC renal pathology and that treatment with rapamycin appears effective in the preclinical setting.     

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.      

siRNA-mediated Erc gene silencing suppresses tumor growth in Tsc2 mutant renal carcinoma model

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery of siRNAs for stable treatment except short hairpin RNAs (shRNAs). On the other hand, there are many reports of systemic delivery of siRNAs for transient treatment using liposome carriers and others. With regard to shRNAs, a report showed fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Therefore, we decided to use original siRNA microspheres instead of shRNA for stable treatment of disease. In this study, we designed rat-specific siRNA sequences for Erc/mesothelin, which is a tumor-specific gene expressed in the Eker (Tsc2 mutant) rat model of hereditary renal cancer and confirmed the efficacy of gene silencing in vitro. Then, by using siRNA microspheres, we found that the suppression of Erc/mesothelin caused growth inhibition of Tsc2 mutant renal carcinoma cells in tumor implantation experiments in mice.     

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.