# Patent

## Title
Methods for detecting, diagnosing and treating human renal cell carcinoma

## Abstract

Gene expression profiling and hierarchical clustering analysis readily identify differential gene expressions in normal renal epithelial cells and renal cell carcinomas. Genes identified by this analysis would be useful for diagnosis, prognosis and development of targeted therapy for the prevention and treatment of conventional renal cell carcinoma.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This is a continuation application under 35 U.S.C. §120 of nonprovisional application U.S. Ser. No. 10/938,973, filed Sep. 10, 2004, now abandoned, which claims benefit of provisional application U.S. Ser. No. 60/539,838, filed Jan. 28, 2004, now abandoned, and of provisional application U.S. Ser. No. 60/502,038, filed Sep. 10, 2003, now abandoned, the entirety of all of which are hereby incorporated by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of cancer research. More specifically, the present invention relates to gene expression profiling for human renal cell carcinoma.
2. Description of the Related Art
Renal cell carcinoma (RCC) represents a major health issue. The American Cancer Society predicts 31,900 new cases will be diagnosed in the United States alone in the year 2003, with 11,900 people dying of the disease. When clinically localized or even locally advanced, renal cell carcinoma can be surgically resected for cure using a variety of approaches. With metastatic progression, however, renal cell carcinoma is incurable, and existing systemic therapies are largely ineffective in impacting disease response or patient survival. The lack of effective systemic therapy for metastatic renal cell carcinoma is, in part, due to a fundamental lack of understanding of the molecular events that result in cellular transformation, carcinogenesis, and progression in human kidney.
The advent of gene array technology has allowed classification of disease states at molecular level by examining changes in all mRNAs expressed in cells or tissues. Gene expression fingerprints representing large numbers of genes may allow precise and accurate grouping of renal cell carcinoma. Moreover, large scale gene expression analysis have the potential of identifying a number of differentially expressed genes in renal cell carcinoma compare to normal renal epithelial cells. These genes or markers may further be tested for clinical utility in the diagnosis and treatment of renal cell carcinoma.
Thus, the identification of novel renal cell carcinoma markers to be used for detection, diagnosis and development of effective therapy against the disease remains a high priority. The prior art is deficient in understanding the molecular differences between renal cell carcinoma and normal renal epithelium. The present invention fulfills this need in the art by providing gene expression profiling for these two types of tissues.
SUMMARY OF THE INVENTION
The present invention identifies genes with a differential pattern of expression between different subtypes of renal cell carcinomas (RCC) and normal renal epithelium. These genes and their products can be used to develop novel diagnostic and therapeutic markers for the treatment of renal cell carcinomas.
Genomic profiling of conventional renal cell carcinoma tissues and patient-matched normal kidney tissue samples was carried out using stringent statistical analyses (ANOVA with full Bonferroni corrections). Subtypes of renal cell carcinoma include stage I, II, III, and IV (reflecting differences in tumor size, lymph node and organ metastasis), stage I papillary renal cell carcinoma, and benign oncocytoma. Hierarchical clustering of the expression data readily distinguished normal tissue from renal cell carcinomas. The identified genes were verified by real-time FCR and immunohistochemical analyses.
Different subtypes of conventional renal cell carcinomas can be diagnosed either by drawing blood and identifying secreted gene products specific to renal cell carcinoma or by doing a biopsy of the tissue and then identify specific genes that are altered when renal cell carcinoma is present. An example of when this may be especially important is in distinguishing the deadly conventional renal cell carcinomas (account for 85% of all renal cell carcinomas) from renal oncocytoma (benign renal cell carcinoma) as well as identifying the histologic subtypes of papillary and sarcomatoid renal cell carcinoma. Identification of specific genes will also help in determining which patients will have a good prognosis verses that of a poor prognosis. In addition, subsets of genes identified in the present invention can be developed as targets for therapies that could cure, prevent, or stabilize the disease. Thus, results from the present invention could be used for diagnosis, prognosis, and development of therapies to treat or prevent renal cell carcinoma.
In one embodiment, there are provided methods of detecting conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein. In another embodiment, conventional or clear cell renal cell carcinoma is detected based on decreased expression of type III TGF-β receptor.
In yet another embodiment, there are provided methods of detecting stage I conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
The present invention also provides methods of detecting stage II conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
The present invention also provides methods of detecting papillary renal cell carcinoma or benign oncocytoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
In another embodiment, there is provided a method of targeting conventional or clear cell renal cell carcinoma cells based on generating antibodies or small molecules directed against a cell surface molecule over-expressed in conventional renal cell carcinoma cells.
In yet another embodiment, there is provided a method of treating conventional or clear cell renal cell carcinoma by replacing down-regulated tumor suppressor gene in conventional renal cell carcinoma.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows hierarchical clustering of genes expressed in normal renal cortex (12 patient tissue samples) verse stage I conventional renal cell carcinoma (6 patient tissue samples). Red indicates that a gene is highly expressed and green is indicative of low expression. Four hundred eighty eight genes were depicted in FIG. 1A. FIG. 1B shows hierarchical clustering of genes expressed in normal renal cortex (12 patient tissue samples) verse stage II conventional renal cell carcinoma (6 patient tissue samples). Red indicates that a gene is highly expressed and green is indicative of low expression. Six hundred twenty eight genes were depicted in FIG. 1B. FIG. 1C shows hierarchical clustering of genes selected from a Venn analysis in which the chosen genes were expressed in common in both stage I and II at a 99% confidence level. One hundred eighty eight genes were depicted in FIG. 1C. C, cancer cells; N, normal cells; S1, stage 1; S2, stage 2.
FIG. 2 shows TGF-β1 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR TGF-β1 mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
FIG. 3 shows TGF-α mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF-α mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
FIG. 4 shows adrenomedulin mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. Adrenomedulin mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
FIG. 5 shows TGF-β2 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF-β2 mRNA levels were not altered between normal and tumor matched samples.
FIG. 6 shows TGF-β3 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF-β3 mRNA levels were not altered between normal and tumor matched samples.
FIG. 7 shows tumor suppressor gene Wilms Tumor 1 (WT1) mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. WT1 mRNA levels were down-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
FIG. 8 shows von Hippel Lindau mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. A small percentage of tumor tissues demonstrated attenuated von Hippel Lindau mRNA levels when compared to matched normal tissue
FIG. 9 shows calbindin mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. Calbindin mRNA was completely lost in all stage I renal cell carcinoma. p<0.05 compared to matched control. *Stage I tumor: 0±0; stage III tumor: 0.0009±0.0004; stage IV tumor: 0.003±0.0004/
FIG. 10 shows MUC1 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. MUC1 mRNA levels were down-regulated in all tumor tissues as early as stage I. *p<0.05 compared to matched control.
FIGS. 11A-11B show stepwise loss of type III αreceptor (TBR3) and type II TGF-β receptor (TBR2) mRNA expression during renal cell carcinogenesis and progression in patient tissue samples. FIG. 11A shows gene array data from 10 patients—five diagnosed with localized renal cell carcinoma and five with metastatic disease. ‘+’ (P<0.05) indicates statistical difference for TBR3 mRNA levels as compared to normal tissue and ‘*’ (P<0.28) indicates statistical difference for TBR2 mRNA levels as compared to normal controls. Data are expressed as mean±s.e. FIG. 11B shows real-time RT-PCR verification of TBR1, TBR2, and TBR3 mRNA levels of tissue samples described in FIG. 11A. Data are expressed as mean±s.d.
FIG. 12 shows immunohistochemistry of patient tissue demonstrating loss of type III αreceptor (TBR3) expression (top row) in all tumors, loss of type II αreceptor (TBR2) expression (middle row) in patients diagnosed with metastatic tumors, and no change in type I αreceptor (TBR1) protein expression (bottom row).
FIG. 13 demonstrates down-regulation of TGF-β-regulated genes in human tumor tissues by real-time PCR. Genes known to be up-regulated by α are suppressed in tumor tissues. Down-regulation of collagen IV type 6, fibulin 5, and connective tissue growth factor (CTGF) mRNA in tumor tissues were compared to matched normal tissue controls. Values were normalized to 18 s mRNA. Each matching tumor value was compared to its respective normal control. The mean±s.d. was calculated for each sample group with n values of 10-15 matched samples.
FIGS. 14A-14B show tumor cell lines that lose type III αreceptor (TBR3) and type I TGF-β receptor (TBR2) expression. FIG. 14A shows semi-quantitative RT-PCR measurements of mRNA levels of TBR1, TBR2, and TBR3 for UMRC3, UMRC6 and normal renal epithelial (NRE) cells. FIG. 14B shows immunohistochemistry of protein expression for TBR1, TBR2, and TBR3 (×40 magnification).
FIGS. 15A-15B show loss of type III TGF-β receptor (TBR3) and type II αreceptor (TBR2) expression in renal tumor cell lines correlate with loss of TGF-β-regulated growth inhibitory and transcriptional responses. FIG. 15A shows cell proliferation was inhibited as assessed by DNA content 3 days after αtreatment. Percent of each respective untreated control was used for comparisons. Transient transfection using 3TP/Ix along with a renilla luciferase control demonstrates loss of responsiveness to 2 ng/ml TGF-β1 with loss of TGF-β receptor expression (FIG. 15B). Firefly luciferase activity was normalized using the ratio of firefly luciferase/renilla luciferase. Data are expressed as mean±s.d.
FIG. 16A demonstrates RT-PCR derived mRNA expression of type III αreceptor (TBR3), type II αreceptor (TBR2), and type I αreceptor (TBR1) in UMRC3 cells and cells stably transfected with TBR2 and TBR3. FIG. 16B shows UMRC3 cells stably transfected with type II TGF-β receptor (UMRC3+TBR2) or type II and type III TGF-β receptor (UMRC3+TBR2+TBR3) demonstrated attenuated cell proliferation following the administration of exogenous TGF-β1 as compared to that of UMRC3 cells. FIG. 16C shows UMRC3 cells, UMRC3+TBR2 cells, and UMRC3+TBR2+TBR3 stable cell lines transfected with 3TP/lux were treated with or without TGF-β and examined for luciferase activity. FIG. 16D shows real-time PCR measuring mRNA levels for collagen IV type 6 in UMRC3, UMRC3+TBR2 cells, and UMRC3+TBR2+TBR3 cells in the presence of 2 ng/ml TGF-β1 for 24 h. FIG. 16E shows colony formation assay demonstrates that UMRC3+TBR2+TBR3 cells have completely lost anchorage-independent growth, while attenuated growth in UMRC3+TBR2 cells occurs as compared to that of UMRC3 cells. The number of colonies were stained and counted after 45 days of growth. Data are expressed as mean±s.d.
FIG. 17A shows growth inhibition after re-expressing human type III TGF-β receptor (TBR3) in UMRC3 cells. UMRC3 cells were stably transfected with TBR3 or infected using an adenoviral vector expressing TBR3. Cells were plated in culture dishes at 20,000 cells/well. Cell number was determined at the indicated times using a Coulter cell counter. FIG. 17B shows RT-PCR data demonstrating the mRNA expression levels of type I, II, or III TGF-β receptors (TBR1, TBR2, TBR3) in UMRC3 cells in the presence or absence of the adenoviral vector expressing TBR3. Unmodified UMRC3 cells only express TBR1.
FIG. 18 shows re-expression of human type II or III TGF-β receptors (TBR2 or TBR3) inhibits tumor growth in nude mice. One million UMRC3 cells stably transfected with human type II or type III TGF-β receptors were implanted into nude mice ectopically and tumor growth was measured weekly. Tumor volume (mm3) was calculated by width×length×height×0.5236.
FIG. 19 shows hierarchical clustering of genes expressed in normal renal cortex verse stage I papillary renal cell carcinoma. Red indicates that a gene is highly expressed and green is indicative of low expression.
FIG. 20 shows hierarchical clustering of genes expressed in normal renal cortex verse benign oncocytoma. Red indicates that a gene is highly expressed and green is indicative of low expression.
FIG. 21 shows venn analysis of gene distribution among stage I renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.
FIG. 22 shows venn analysis of gene distribution among stage II renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.



DETAILED DESCRIPTION OF THE INVENTION
High-throughput technologies for assaying gene expression, such as high-density oligonucleotide and cDNA microarrays, offer the potential to identify clinically relevant genes differentially expressed between normal and tumor cells. The present invention discloses a genome-wide examination of differential gene expression between renal cell carcinomas (RCC) and normal renal epithelial cells.
Currently, there are no proven molecular markers useful clinically for the diagnosis, staging, or prognosis of sporadic renal cell carcinoma. The present invention detects genes that have differential expression between renal cell carcinomas and normal renal epithelial cells. The known function of some of these genes may provide insight into the biology of renal cell carcinomas while others may prove to be useful as diagnostic or therapeutic markers against renal cell carcinomas. Subtypes of renal cell carcinomas disclosed herein include stage I, II, III, and IV renal cell carcinomas (reflecting differences in tumor size, lymph node and organ metastasis), stage I papillary renal cell carcinoma, and benign oncocytoma.
The present invention provides methods of detecting conventional renal cell carcinoma based on determining the expression level of a number of genes that are found to have 2-fold or higher differential expression levels between tumor and normal tissue. In general, biological samples (e.g. tissue samples, serum samples, urine samples, saliva samples, blood samples or biopsy samples) are obtained from the individual to be tested and gene expression at RNA or protein level is compared to that in normal tissue. The normal tissue samples can be obtained from the same individual who is to be tested for renal cell carcinoma. It will be obvious to one of ordinary skill in the art that gene expression can be determined by DNA microarray and hierarchical cluster analysis, real-time PCR, RT-PCR, or northern analysis, whereas secreted gene products can be measured in blood samples by standard procedures.
In one embodiment, there is provided a method of detecting conventional or clear cell renal cell carcinoma based on differential expression of one or more of the following genes or proteins: TGF-β1, TGF-α, adrenomedulin, fibroblast growth factor 2 (FGF2), vascular epidermal growth factor (VEGF), osteonectin, follistatin like-3, inhibin beta A, spondin 2, chemokine X cytokine receptor 4 (CXCR4), fibronectin, neuropilin 1, frizzled homolog 1, insulin-like growth factor binding protein 3, laminin alpha 3, integrin beta 2, semaphorins 6A, semaphorins 5B, semaphorins 3B, caspase 1, sprouty 1, CDH16, PCDH9, compliment component 1-beta, compliment component 1-alpha, compliment component 1-gamma, CD53, CDW52, CD163, CD14, CD3Z, CD24, RAP1, angiopoietin 2, cytokine knot secreted protein, MAPKKKK4, 4-hydroxyphenylpyruvate dioxygenase, pyruvate carboxyknase 2, 11-beta-hydroxysteroid dehydrogenase 2, GAS1, CDKN1, nucleolar protein 3, interferon induced protein 44, NR3C1, vitamin D receptor, hypothetical protein FLJ14957 (Affy#225817_at), metallothionein 2A, metallothionein-If gene, metallothionein 1H, secreted frizzled related protein 1, connective tissue growth factor, and epidermal growth factor.
In another embodiment, there is provided a method of detecting conventional renal cell carcinoma by examining the expression level of type III TGF-β receptor, wherein decreased expression of type III TGF-b receptor indicates the presence of renal cell carcinoma. In general, the expression level of type III TGF-β receptor can be determined at the mRNA or protein level.
The present invention also provides methods of detecting stage I conventional renal cell carcinoma, stage II conventional renal cell carcinoma, stage I papillary renal cell carcinoma, or benign oncocytoma based on over-expression or down-regulation of a number of genes identified in the present invention. The present invention discloses a number of genes that are up- or down-regulated specifically in these subtypes of renal cell carcinoma. Determining the expression levels of these genes would provide specific diagnosis for these different subtypes of renal cell carcinoma.
For example, stage I conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 1, (ii) down-regulation of one or more genes listed in Table 2, or (iii) over-expression of one or more genes listed in Table 1 and down-regulation of one or more genes listed in Table 2. Similarly, stage II conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 3, (ii) down-regulation of one or more genes listed in Table 4, or (iii) over-expression of one or more genes listed in Table 3 and down-regulation of one or more genes listed in Table 4.
The present invention also discloses a number of genes that are up- or down-regulated in both stage I and stage II conventional renal cell carcinoma (Tables 5 and 6 respectively). These genes would also provide diagnosis for stage I or stage II conventional renal cell carcinoma. Hence, stage I or stage II conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 5, or (ii) down-regulation of one or more genes listed in Table 6.
In another embodiment, stage I papillary renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 8, (ii) down-regulation of one or more genes listed in Table 9, or (iii) over-expression of one or more genes listed in Table 8 and down-regulation of one or more genes listed in Table 9.
In yet another embodiment, benign oncocytoma can be detected based on (i) over-expression of one or more genes listed in Table 10, (ii) down-regulation of one or more genes listed in Table 11, or (iii) over-expression of one or more genes listed in Table 10 and down-regulation of one or more genes listed in Table 11.
In still yet another embodiment, there are provided methods of utilizing genes over-expressed on the cell surface of renal carcinoma tissue to develop antibodies or other small molecules for the purpose of specifically targeting the renal tumor cells. The present invention discloses a number of genes that are up-regulated in stage I renal cell carcinoma (RCC), stage II RCC tumor, stage I papillary RCC, and benign oncocytoma. Antibodies or small molecules directed against proteins encoded by these genes can be linked with a therapeutic drug to deliver drug to the tumor tissue, or be linked with dye, nanoparticle or other imaging agents for cancer imaging. Some of the novel genes identified herein for the first time include, but are not limited to, the following genes: calcitonin receptor-like (206331_at; 210815_s_at); receptor (calcitonin) activity modifying protein 2 (RAMP2; 205779_at); endothelin receptor type B (206701_x_at); beta 2 integrin (202803_s_at); alpha 5 integrin (201389_at); chemokine X cytokine receptor 4 (CXCR4); fibronectin; neuropilin 1 (212298_at; 210510_s_at); CD24; CD14; Cd163; CD53; Compliment Componenet 1-beta, 1-alpha, and 1-gamma; CDH4; integrin beta2; ADAM28; FK506 binding protein; collagen Valpha2; tumor necrosis factor receptor superfamily, member 6; tumor necrosis factor receptor superfamily, member 5; tumor necrosis factor (ligand) superfamily, member 13b; tumor necrosis factor receptor superfamily, member 12A; and the FGF receptor.
In another embodiment, there is provided a method of treating conventional or clear cell renal cell carcinoma. The method involves replacing tumor suppressor genes (e.g., via gene therapy) whose expression is down-regulated in tumor tissues or introducing a molecule that induces the down-regulated gene to be re-expressed in the tumor. The present invention discloses a number of genes that are down-regulated in stage I renal cell carcinoma (RCC), stage II RCC tumor, stage I papillary RCC, and benign oncocytoma. Some examples of down-regulated genes identified in stage I and/or II RCC tumors include, but are not limited to, CDKN1, secreted frizzled related protein 1, semaphoring 6D, semaphoring 3B, CDH16, TNF alpha, calbindin D28, defensin beta1, beta-catenin interacting protein 1, GAS1, vitamin D receptor, Kruppel-like factor 15. This method of treatment can be combined with other therapies to provide combinatorial therapy.
The genes that are found to have altered expression in stage I and stage II renal cell carcinoma would also be useful for determining patient prognosis. These genes or gene products (i.e., proteins) would have the unique characteristic of being altered in tumor verses normal samples in a subset of patients. For example, basic transcription element binding protein 1 is down-regulated in 7 out of 12 renal cell carcinoma tumors. Other examples include CD164, decreased 5/12; Map kinase kinase kinase 7, increased 6/12; Endoglin, increased 7/12; SERPIN A1, increased 6/12; Metalloprotease 11 (MMP11), increased 7/12; Integrin 3 alpha, increased 4/12; carbonic anhydrase II, decreased 7/12; protein tyrosine kinase 2, increased 4/12; fibroblast growth factor 11, increased 6/12; fibroblast growth factor 2, increased 7/12; VEGF B, increased 5/12.
Moreover, the levels of change may be a useful determinant of patient outcome and/or rationale for strategy of treatment course. An example of this is found for chemokine (C—X—C motif) ligand 14 (CXCL14, 222484_s_at). Six patients with stage I and six patients with stage II renal cell carcinoma were analyzed by genomic profiling. A patient with stage I renal cell carcinoma has CXCL14 mRNA expression levels of 19862 and 24.49 in his normal tissue and tumor tissue respectively. This patient would be predicted to have a poor prognosis or poor response to therapy based upon this result along with other gene predictors. On the other hand, a patient with stage II RCC has CXCL14 mRNA expression levels of 20435 and 18557 in his normal tissue and tumor tissue respectively. This patient would be predicted to have a good prognosis and good response to chemotherapy.
The following examples are given for illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
EXAMPLE 1
Tissue Banking
Renal tissue (normal and tumor) was transported to a sterile hood on ice and under sterile conditions. Tissue was dissected under the direction of a pathologist. The tissue was frozen in liquid nitrogen for isolation of RNA, DNA, and protein or processed to establish primary cell cultures. The tissue was fixed in formalin for immunohistochemistry and in situ hybridization and RNAlater (Ambion) for RNA isolation. Primary normal renal epithelial (NRE) cell cultures were established using standard collagenase/Dnase techniques to digest tissue and isolate single cells. NREs were easily isolated and grew well in culture for up to 10 passages. These cells were further analyzed for homogeneity with regard to epithelial population using appropriate immunohistochemical markers such as vimentin, cytokeratin, and megalin.
EXAMPLE 2
Genomic Gene Array and Microarray Data Analysis
Gene expression profiling was performed using Affymetrix HU95A oligonucleotide gene arrays (>12,600 genes) or HG-U133 A&B GeneChip® oligonucleotide microarrays (33,000+ probe sets). Total RNA (Trizol®, Ambion) was extracted from patient-matched normal renal cortex and tumor tissue from patients diagnosed with local disease confined to the kidney. Alternatively, the investigators analyzed metastatic disease defined by lesions in lymph nodes, adrenal, or other organs. Data were analyzed by a combination of two-dimensional ANOVA, Affymetrix MAS5.0®, and hierarchical cluster analysis using Spotfire®. Procedure that were used to identify altered expression of large sets of genes, as well as other issues concerning microarray analyses can be found in a recent review article by Copland et al. (2003).
EXAMPLE 3
Real-Time PCR
Applied Biosystems' assays-by-design or assays-on-demand 20× assay mix of primers and TaqMan® MGB probes (FAM® dye-labeled) for all target genes and predeveloped 18S rRNA (VIC® dye-labeled probe) TaqMan® assay reagent for internal control were used for real-time PCR measurements. These assays were designed to span exon-exon junctions so as not to detect genomic DNA and all primers and probes sequences were searched against the Celera database to confirm specificity. Validation experiments were performed to test the efficiency of the target amplification and the efficiency of the reference amplification. All absolute values of the slope of log input amount versus DCT is less than 0.1.
Separate tubes (singleplex) for one-step RT-PCR was performed with 50 ng RNA for both target genes and endogenous controls using TaqMan® one-step RT-PCR master mix reagent kit (Applied Biosystems). The cycling parameters for one-step RT-PCR were: reverse transcription 48° C. for 30 min, AmpliTaq® activation 95° C. for 10 min, denaturation 95° C. for 15 s, and annealing/extension 60° C. for 1 min (repeat 40 times) on ABI7000®. Duplicate CT values were analyzed with Microsoft Excel® using the comparative CT(DDCT) method as described by the manufacturer (Applied Biosystems). The amount of target (2−DDCT) was obtained by normalizing to an endogenous reference (18smRNA) and relative to a calibrator (normal tissue).
EXAMPLE 4
Immunohistochemical Analyses of Protein Expression
For immunohistochemical analyses of type I TGF-β receptor (TBR1), type II TGF-β receptor (TBR2), and type Ill TGF-β receptor (TBR3) expression, patient-matched normal renal and tumor tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks. Consecutive sections were cut 5 um thick, deparaffinized, hydrated, and immunostained using antibodies recognizing human TBR1, TBR2, and TBR3 (1:100; Santa Cruz Biotechnology). Biotinylated secondary antibody (1:600; Santa Cruz Biotechnology) was detected using avidin-biotin-peroxidase detection according to the manufacturer's instructions (Vectastatin Elite ABC kit; Vector Lab). All slides were lightly counterstained with hematoxylin before dehydration and mounting.
For cell lines, cells were plated on glass coverslips in wells. Prior to the detection of TGF-β receptor expression as described above, cells were fixed onto the coverslips with 3% formalin.
EXAMPLE 5
Gene Expression Profiling of Renal Cell Carcinoma
Gene expression profiling was performed using Affymetrix oligonucleotide gene arrays. RNA was extracted from patient-matched normal renal cortical and tumor tissues from patients diagnosed with localized and metastatic renal cell carcinoma. Data were analyzed by a combination of two-dimensional ANOVA, Affymetrix MAS5.0®, and hierarchical cluster analysis using Spotfire® (reviewed in Copland et al., 2003).
A primary goal of microarray analysis is to discover hidden patterns of differential expression within a large data field. Normal renal cortical and primary tumor tissue with no metastasis were collected from patients diagnosed with local disease. Normal tissue, primary tumor, and metastatic tissue were also collected from patients diagnosed with metastatic disease. Comparison of patient-matched normal and tumor tissue allowed for the discovery of changes in mRNA levels between normal and tumor tissue, as well as local and metastatic disease.
Heatmaps with two-way dendograms depicting genes specifically altered in tumor tissue as compared to normal renal cortex are shown in FIG. 1. FIG. 1A shows hierarchical clustering of genes expressed in normal renal cortex verses stage I conventional renal cell carcinoma. FIG. 1B shows hierarchical clustering of genes expressed in normal renal cortex verses stage II renal cell carcinoma. FIG. 1C shows hierarchical clustering of genes selected from a Venn analysis in which the chosen genes were expressed in common in both stage I and II at a 99% confidence level.
TGF-β1, TGF-α and adrenomedulin mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts (FIGS. 2-4), whereas TGF-β2 and TGF-β3 mRNA levels were not altered between normal and tumor matched samples (FIGS. 5-6).
Tumor suppressor gene Wilms Tumor 1 (WT1) was down-regulated in all stages of renal cell carcinoma (FIG. 7). A small percentage of tumor tissues demonstrated attenuated von Hippel Lindau mRNA levels when compared to matched normal tissue (FIG. 8). Calbindin mRNA was completely lost (FIG. 9) while MUC1 was greatly attenuated in stage I renal cell carcinoma (FIG. 10).
The present analysis identifies 278 genes that were up-regulated in stage I renal cell carcinoma, whereas 380 genes were up-regulated in stage II renal cell carcinoma. Among these genes, 82 were up-regulated in both stages I and II renal cell carcinoma. One hundred fifty nine genes were down-regulated in stage I renal cell carcinoma, whereas 195 genes were down-regulated in stage II RCC. Among these genes, 82 were down-regulated in both stage I and II renal cell carcinoma.
Genes over-expressed and down-regulated in stage I renal cell carcinoma are listed in Table 1 and Table 2 respectively. Genes over-expressed and down-regulated in stage I renal cell carcinoma are listed in Table 3 and Table 4 respectively. Genes over-expressed in both stage I and II renal cell carcinoma are listed in Table 5. Genes down-regulated in both stage I and II renal cell carcinoma are listed in Table 6.







TABLE 1







Genes With Up-Regulated Expression


In stage I Renal Cell Carcinoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





NM004356.1
CD81
NM004079.1
CTSS


NM002293.2
LAMC1
NM001784.1
CD97


NM000980.1
RPL18A
AF151853.1
PREI3


AK002091.1
MGEA5
NM000491.2
C1QB


NM005721.2
ACTR3
BC000125.1
TGFB1


NM002668.1
PLP2
NM004520.1
KIF2


NM021038.1
MBNL
NM000321.1
RB1


AF070656.1
YME1L1
NM012262.2
HS2ST1


NM021029.1
RPL36A
NM000560.1
CD53


NM002945.1
RPA1
NM005502.1
ABCA1


NM002480.1
PPP1R12A
AF285167.1
ABCA1


NM001349.1
DARS
BG170541
MET


NM005496.1
SMC4L1
NM021642.1
FCGR2A


AW163148
MARCKS
BE967532
KIAA0220


NM002356.4
MARCKS
NM006526.1
ZNF217


M68956.1
MARCKS
NM000570.1
FCGR3B


AI589086
LAPTM5
N26005
PPP1R3C


NM006762.1
LAPTM5
NM006153.1
NCK1


NM014267.1
SMAP
NM001549.1
IFIT4


NM000235.1
LIPA
NM003141.1
SSA1


NM000176.1
NR3C1
NM014705.1
KIAA0716


NM005737.2
ARL7
NM005197.1
CHES1


NM005737.2
ARL7
NM002907.1
RECQL


BC001051.1
ARL7
U43328.1
CRTL1


NM006169.1
NNMT
NM017925.1
FLJ20686


NM005862.1
STAG1
NM006773.2
DDX18


AI356412
LYN
U20350.1
CX3CR1


NM002350.1
LYN
NM005761.1
PLXNC1


BG107456
TRIP-Br2
NM004834.1
MAP4K4


NM021913.1
AXL
NM021644.1
HNRPH3


NM002194.2
INPP1
NM006640.1
MSF


NM019058.1
RTP801
NM004180.1
TANK


NM002110.1
HCK
AW148801
NAP1L1


NM030755.1
TXNDC
AB011118.1
KIAA0546


NM030984.1
TBXAS1
AU145005
SP3


NM014350.1
GG2-1
N80918
CG018


BC001312.1
P5
BF439472
ATP11A


U14990.1
RPS3
BE968801
RPL35A


D83043.1
HLA-B
AI985751
NAP1L1


AI888672
NAP1L1
AI735692
LST1


BC002387.1
NAP1L1
AA995910
ALOX5


M60334.1
HLA-DRA
M12679.1
HUMMHCW1A


AF161522.1
C3orf4
AL133053.1
FLJ23861


BG256677
IFI16
X03348.1
NR3C1


M26880.1
UBC
AC005339
N/A


U17496.1
PSMB8
AK024836.1
HLA-C


AF141347.1
TUBA3
AC003999
SCAP2


L01639.1
CXCR4
AJ224869
CXCR4


NM005445.1
CSPG6
AL022067
PRDM1


AB030655.1
EFEMP2
AL110158.1
KIAA1078


AF165520.1
APOBEC3C
S81916.1
N/A


AF009670.1
ABCC3
M80469
N/A


AF020314.1
CMRF-35H
NM002860.1
PYCS


BC001606.1
NCF2
NM020198.1
GK001


BC005352.1
GG2-1
NM016304.1
C15orf15


AF281030.1
HRIHFB2122
AA102574
BAZ1A


BC001052.1
RECQL
NM024844.1
PCNT1


L32610.1
HNRPH3
NM015938.1
CGI-07


M23612.1
RASA1
NM018200.1
HMG20A


AF109683.1
LAIR1
NM025235.1
TNKS2


BC002841.1
HSA9761
NM015991.1
C1QA


D29640.1
IQGAP1
NM016090.1
RBM7


L25259.1
CD86
NM024554.1
PGBD5


M60333.1
HLA-DRA
NM017718.1
FLJ20220


U13698.1
CASP1
NM017923.1
FLJ20668


U90940.1
FCGR2C
NM030921.1
DC42


M90685.1
HLA-G
BC004470.1
ASC


M90684.1
HLA-G
AK021413.1
LARS


M90686.1
HLA-G
BF444916
FAD104


L22453.1
RPL3
BC004819.1
PLDN


U01351.1
NR3C1
AF247167.1
AD031


U62824.1
HLA-C
U39402.1
N/A


L07950.1
HLA-B
BC006112.1
DKFZP434B195


AF348491.1
CXCR4
BG388615
N/A


NM003079.1
SMARCE1
AB033007.1
KIAA1181


BE646386
EXO70
BG250721
N/A


AI972475
N/A
AK024221.1
C40


AA195999
MAPK1
BF477658
N/A


AL049397.1
N/A
BG251556
KIAA1949


BE895685
KIAA0853
AB033091.1
KIAA1265


M82882.1
ELF1
AK024350.1
AMOTL1


AB020633.1
KIAA0826
NM018440.1
PAG


AL031781
N/A
AW500180
N/A


BF209337
MGC4677
AW026543
N/A


AI709406
N/A
AI092770
N/A


AI806905
N/A
NM020679.1
AD023


AI392933
FLJ36090
AK024855.1
CTSS


AH42096
N/A
AK000119.1
N/A


AL137430.1
N/A
AW977527
PRDM1


AV724266
FLJ20093
BE671060
N/A


BF589359
N/A
AL037450
N/A


AW084125
CAPZA1
AI401535
N/A


N20927
RAP2B
AV683852
N/A


AI627666
LOC115548
BF055144
N/A


AV726322
N/A
AA352113
N/A


AI697657
LANPL
BF056209
N/A


BF002625
N/A
X60592
TMFRSF5


BF439533
N/A
















TABLE 2







Genes With Down-Regulated Expression In


stage I Renal Cell Carcinoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





L38487
ESRRA
AK024386.1
GRHPR


NM004415.1
DSP
AL109716.2
N/A


NM005327.1
HADHSC
AK026411.1
ALDOB


NM003321.1
TUFM
M10943
N/A


NM002084.2
GPX3
AW088547
N/A


AI983043
N/A
NM018049.1
GNRPX


NM006066.1
AKR1A1
NM017900.1
AKIP


NM006384.2
CIB1
NM006548.1
IMP-2


NM001685.1
ATP5J
NM025135.1
KIAA1695


NM014652.1
IMP13
NM016458.2
LOC51236


NM013410.1
AK3
NM022128.1
RBSK


NM016725.1
FOLR1
NM015974.1
CRYL1


NM021151.1
CROT
NM013333.1
EPN1


NM005951.1
MT1H
AA133341
C14orf87


NM005952.1
MT1X
AF226732.1
NPD007


AL080102.1
N/A
AF265439.1
MRPS15


BC000931.2
ATP5C1
AI743534
DKFZP564B1162


BC005398.1
DKFZP566D193
AB042647.1
B29


D87292.1
TST
AL522667
ORF1-FL49


AU151428
IDH2
BG255416
KIAA0114


BC000109.1
ILVBL
AF308301.1
MRPS26


AF333388.1
N/A
BE408081
N/A


NM005953.1
MT2A
AL521634
FLJ32452


BF217861
N/A
BF203664
MGC14288


AA594937
COBL
BE645551
MGC39329


AW052179
COL4A5
AW193698
TGFBR3


AI884867
LOC155066
BF540829
N/A


BF246115
N/A
W72455
FLJ25476


AW028110
KIAA0500
AI457453
N/A


AW242315
N/A
BF056892
N/A


AW080549
FUT3
AK024386.1
GRHPR


AW149846
GPX3
AL109716.2
N/A


AI038402
N/A
AA442776
N/A


AI051046
MGC4614
AI913600
N/A


AI659456
N/A
AW771908
N/A


AW664964
N/A
AI807887
N/A


AI631895
SGK2
AW102941
N/A


AI263078
FLJ31168
AW024656
N/A


BF057634
HOXD8
AB002342
PRKWNK1


AA746038
GPR110
















TABLE 3







Genes With Up-Regulated Expression In


stage II Renal Cell Carcinoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





NM006096.1
NDRG1
NM002053.1
GBP1


NM006098.1
GNB2L1
NM000089.1
COL1A2


NM001780.1
CD63
NM021105.1
PLSCR1


NM003118.1
SPARC
NM002467.1
MYC


NM000291.1
PGK1
NM001284.1
AP3S1


NM003870.1
IQGAP1
AI825926
PLSCR1


AB032261.1
SCD
NM014736.1
KIAA0101


NM002629.1
PGAM1
AF161461.1
LEPROTL1


NM003564.1
TAGLN2
NM014873.1
KIAA0205


NM000310.1
PPT1
AI005043
N/A


NM003405.1
YWHAH
NM000416.1
IFNGR1


U82164.1
MIC2
NM004172.1
SLC1A3


NM002305.2
LGALS1
NM004207.1
SLC16A3


NM001096.1
ACLY
AI761561
HK2


NM002121.1
HLA-DPB1
Y09216.1
N/A


NM021038.1
MBNL
NM002922.1
RGS1


NM003651.1
CSDA
NM005990.1
STK10


AV685920
CAPZA2
NM014863.1
GALNAC4S-6ST


NM002654.1
PKM2
NM014737.1
RASSF2


NM001175.1
ARHGDIB
NM000418.1
IL4R


BC000182.1
ANXA4
BC000658.1
STC2


NM001153.2
ANXA4
NM003751.1
EIF3S9


NM001975.1
ENO2
NM002339.1
LSP1


NM006435.1
IFTTM2
NM004604.1
STX4A


NM001387.1
DPYSL3
NM006404.1
PROCR


BG398414
RPA1
AF275945.1
EVA1


NM004039.1
ANXA2
NM004221.1
NK4


NM005534.1
IFNGR2
NM004556.1
NFKBIE


AL136877.1
SMC4L1
NM004688.1
NMI


NM014876.1
KIAA0063
NM003332.1
TYROBP


NM024830.1
FLJ12443
NM015136.1
STAB1


NM005505.1
SCARB1
NM006019.1
TCIRG1


NM003025.1
SH3GL1
NM004877.1
GMFG


NM013285.1
HUMAUANTIG
NM002317.1
LOX


NM005720.1
ARPC1B
NM025201.1
PP1628


AW157070
EGFR
NM014800.1
ELMO1


NM002835.1
PTPN12
L41944.1
IFNAR2


NM004428.1
EFNA1
NM007268.1
Z39IG


AW006290
SUDD
NM006994.2
BTN3A3


NM014791.1
MELK
AF091352.1
VEGF


NM014882.1
KIAA0053
AB035482.1
ICB-1


NM003864.1
SAP30
Z24727.1
TPM1


NM001558.1
IL10RA
M19267.1
TPM1


NM003264.1
TLR2
U13700.1
CASP1


NM014221.1
MTCP1
M27281.1
VEGF


AV756141
CSF2RB
BC005838.1
N/A


AI123251
LCP2
BC005858.1
FN1


NM006433.2
GNLY
BC005926.1
EVI2B


NM000861.2
HRH1
BE513104
YARS


NM001870.1
CPA3
AU147399
CAV1


NM003586.1
DOC2A
AK023154.1
HN1L


NM004271.1
MD-1
AK021757.1
KIAA0648


NM014932.1
NLGN1
H95344
VEGF


NM014947.1
KIAA1041
AB023231.1
FNBP4


NM000647.2
CCR2
AL523076
N/A


NM002562.1
P2RX7
NM030666.1
SERPINB1


NM006058.1
TNIP1
AB018289.1
KIAA0746


NM013447.1
EMR2
AW043713
SULF1


NM013416.1
NCF4
BE880591
EP400


NM001776.1
ENTPD1
AU158495
NOTCH2


NM020037.1
ABCC3
BE965029
N/A


NM006135.1
CAPZA1
AL564683
CEBPB


NM007036.2
ESM1
AA349595
RAB6IP1


AF034607.1
CLIC1
AI809341
PTPRC


BC000915.1
PDLIM1
AW205215
KIAA0286


AL162068.1
NAP1L1
BE349017
HA-1


NM006947.1
SRP72
AF070592.1
HSKM-B


L12387.1
SRI
AI769685
CARS


AF141349.1
N/A
AI935123
LOC113146


AF263293.1
SH3GLB1
BG255188
N/A


BC000389.1
TM4SF7
AI088622
PRKCDBP


AF007162.1
CRYAB
BE222709
N/A


D38616.1
PHKA2
AW007573
DKFZP586L151


AV717590
ENTPD1
BG332462
N/A


U87967.1
ENTPD1
AI862658
FEM1C


H23979
MOX2
AI934469
KIAA0779


AF063591.1
MOX2
AB018345.1
KIAA0802


BC005254.1
CLECSF2
W87466
LOC92689


BC000893.1
H2BFT
BE908217
ANXA2


L22431.1
VLDLR
NM005615.1
RNASE6


AI741056
SELPLG
BE300252
K-ALPHA-1


AF084462.1
RIT1
BF740152
MYO1F


U62027.1
C3AR1
AV711904
LYZ


M87507.1
CASP1
AW072388
N/A


J04132.1
CD3Z
AW190316
SHMT2


M31159.1
IGFBP3
NM005412.1
SHMT2


AF257318.1
SH3GLB1
NM006417.1
IFI44


BC001388.1
ANXA2
AL008730
C6orf4


AF130095.1
FN1
L16895
LOC114990


AF022375.1
VEGF
Z21533.1
HHEX


AA807529
MCM5
AK022955.1
DKFZp762L0311


AK026737.1
FN1
BF001267
N/A


X14355.1
N/A
AL558987
N/A


AK025608.1
KIAA0930
AA577672
LOC151636


AF183421.1
RAB31
BE620734
ZAK


NM002695.1
POLR2E
AI937446
N/A


AF288391.1
C1orf24
H99792
N/A


NM003730.2
RNASE6PL
BE966748
N/A


NM016359.1
ANKT
AI659418
MGC21854


NM014164.2
FXYD5
AI990891
DKFZp761K2222


NM022736.1
FLJ14153
AA827892
N/A


NM021158.1
C20orf97
AL135264
N/A


NM017792.1
FLJ20373
AI375753
N/A


NM020142.1
LOC56901
AA573502
TAP2


NM016448.1
RAMP
BG387557
CASP2


NM005767.1
P2Y5
AA554833
MAP1B


NM020169.1
LXN
AK026764.1
N/A


NM022834.1
FLJ22215
AU146532
PDK1


NM018460.1
BM046
BE348597
N/A


NM024629.1
FLJ23468
AL577758
LOC133957


NM018641.1
C4S-2
AI133452
FGG


NM018295.1
FLJ11000
AU157224
N/A


NM024576.1
FLJ21079
AI742057
N/A


NM016582.1
PHT2
BE500942
N/A


NM003116.1
SPAG4
N25631
RFXANK


NM018454.1
ANKT
AU145366
N/A


NM018099.1
FLJ10462
AW270037
KIAA0779


NM007072.1
HHLA2
BF526978
N/A


NM022445.1
TPK1
AW182575
N/A


AW173623
TDE1
BF339831
MGC13114


AB044088.1
BHLHB3
AI056992
N/A


AF043244.1
NOL3
BE222668
N/A


AF133207.1
H11
BG165011
N/A


AF313468.1
CLECSF12
AI188445
MGC14289


AA191576
NPM1
BE551416
HAK


AI765383
KIAA1466
AI972498
a1/3GTP


BC003654.1
SLC27A3
AW662189
N/A


W60806
N/A
AA142842
N/A


AI335263
NETO2
BF939473
N/A


AI378406
EGLN3
AI681260
N/A


BC005400.1
FKSG14
AA551090
AP1S2


AI761520
CENTA2
AA045175
MS4A6A


BC000771.1
TPM3
W05495
N/A


BC000190.1
HSPC216
AI093231
N/A


BC002776.1
SEMA5B
AI565054
N/A


AF132203.1
SCD
AL553774
N/A


BC006107.1
ARHGAP9
AK023470.1
MGC15875


AK024263.1
N/A
AL157377
ENPP3


AK024846.1
SET7
AL139109
TEX11


BE878463
N/A
AK025631.1
POLH


AW304786
PTR4
AI873425
N/A


AI769269
N/A
BF541967
N/A


AI935334
N/A
AI686890
N/A


BF437747
SAMHD1
AI936034
ITGA4


AW300953
N/A
U88964
ISG20


H37811
N/A
AJ243797
TREX1


AA603344
SAMHD1
D29642
KIAA0053


AA742310
N/A
D87433
STAB1


AI248208
FLJ25804
AI129310
FLJ21562


AI962367
ECGF1
















TABLE 4







Genes With Down-Regulated Expression In


stage II Renal Cell Carcinoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





NM012248.1
SPS2
AB019695.1
TXNRD2


NM002300.1
LDHB
M61900.1
PTGDS


BC000306.1
HADHSC
BF967998
N/A


NM001640.2
APEH
BF967998
N/A


NM005875.1
GC20
AL526243
KIAA0446


NM003365.1
UQCRC1
NM000532.1
PCCB


BF031714
HYA22
BE042354
LDHB


NM005808.1
HYA22
AI587323
ATP5A1


AF113129.1
ATP6V1A1
AW195882
ATPW


NM002402.1
MEST
H71135
ADH6


NM006844.1
ILVBL
AV659180
ALDOB


NM004636.1
SEMA3B
AK027006.1
TNRC9


NM002496.1
NDUFS8
AV693216
PLXNB1


NM006556.1
PMVK
BG398937
N/A


NM004255.1
COX5A
NM002489.1
NDUFA4


NM002225.2
IVD
NM003849.1
SUCLG1


NM004524.1
LLGL2
NM014019.1
HSPC009


AI950380
BCL7A
NM024952.1
FLJ20950


AB020707.1
WASF3
NM014185.1
MOG1


NM000481.1
AMT
NM018013.1
FLJ10159


NM012317.1
LDOC1
NM018373.1
SYNJ2BP


NM006456.1
STHM
NM014067.2
LRP16


NM006614.1
CHL1
NM013261.1
PPARGC1


NM015393.1
DKFZP564O0823
NM021963.1
NAP1L2


AV729634
DNAJC6
NM018658.1
KCNJ16


NM002628.1
PFN2
NM014553.1
LBP-9


NM003500.1
ACOX2
AF112204.1
ATP6V1H


NM002655.1
PLAG1
AU145941
CDC14B


NM004393.1
DAG1
AF061264.1
MGC4825


NM003026.1
SH3GL2
BF941492
FLJ10496


NM002010.1
FGF9
AI984229
HSPC121


NM014033.1
DKFZP586A0522
N71923
FLRT3


NM004868.1
GPSN2
BC005050.1
NICN1


BC000649.1
UQCRFS1
AF172327.1
N/A


S69189.1
ACOX1
AF356515.1
HINT2


AF153330.1
SLC19A2
BE620739
RHOBTB3


AF094518.1
ESRRG
BF435123
N/A


M55575.1
BCKDHB
AW149498
BTBD6


BE044480
MGC32124
AW024437
LOC118491


BF382393
N/A
AW195353
N/A


AV751731
PNKP
BE044193
N/A


U55984
N/A
AI493303
FLJ31709


BF059512
DNER
AI636080
N/A


AK025934.1
Evi1
BF509031
ATP6V1G3


AL036088
SEMA6D
AW242920
N/A


BE964222
FLJ38482
BF002046
ANGPTL1


AW290940
N/A
BF130943
N/A


AL545998
N/A
AW452631
N/A


AW274874
N/A
AI792937
N/A


AI709389
N/A
AI810572
N/A


BF224092
MGC15854
BG165743
LOC112817


AU145805
N/A
AW466989
N/A


AW079843
MGC33338
R48991
N/A


AW138815
N/A
BF029215
MSI2


AW242286
N/A
D21851
LARS2


AW025023
N/A
Z83838
ARHGAP8


BE672659
N/A
















TABLE 5







Genes With Up-Regulated Expression In both


stage I & stage II Renal Cell Carcinoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





NM005566.1
LDHA
NM014812.1
KIAA0470


NM000291.1
PGK1
AF208043.1
IFI16


NM001219.2
CALU
BC002654.1
TUBB-5


NM002966.1
S100A10
BC006379.1
K-ALPHA-1


NM000034.1
ALDOA
BC006481.1
K-ALPHA-1


NM002627.1
PFKP
AF000426.1
LST1


NM006082.1
K-ALPHA-1
AF000424.1
LST1


AI922599
VIM
BG500301
ITGB1


NM020474.2
GALNT1
AL516350
ARPC5


NM006406.1
PRDX4
M27487.1
HLA-DPA1


NM015344.1
LEPROTL1
M27487.1
HLA-DPA1


NM014755.1
TRIP-Br2
AW517686
ATP2B4


AI796269
NBS1
AL581768
K-ALPHA-1


NM005783.1
APACD
AA524505
TSGA


BF197655
N/A
Z78330
ACTR3


NM001233.1
CAV2
Z78330
ACTR3


NM002845.1
PTPRM
BG532690
ITGA4


NM014302.1
SEC61G
AW005535
RAP2B


U47924
CD4
NM007161.1
LST1


NM004106.1
FCER1G
AK026577.1
ALDOA


NM015474.1
SAMHD1
AI091079
SHC1


NM004915.2
ABCG1
AV713720
LST1


NM002432.1
MNDA
NM021103.1
TMSB10


NM005565.2
LCP2
NM016337.1
RNB6


NM005531.1
IFI16
NM013260.1
HCNGP


NM005849.1
IGSF6
NM021199.1
SQRDL


NM002189.1
IL15RA
NM018149.1
FLJ10587


NM004353.1
SERPINH1
NM016951.2
CKLF1


NM017760.1
FLJ20311
AB033038.1
FLJ10392


NM022349.1
MS4A6A
AI184968
C1QG


NM023003.1
TM6SF1
AL161725
FLJ00026


NM016184.1
CLECSF6
NM018440.1
PAG


NM031284.1
DKFZP434B195
AL553942
FLJ31951


BC002342.1
CORO1C
AI394438
N/A


AA775177
PTPRE
T64884
N/A


AL162070.1
CORO1C
T64884
N/A


AF253977.1
MS4A6A
AW511319
N/A


AF237908.1
MS4A6A
AI640834
RA-GEF-2


W03103
DDEF1
AI655467
N/A


AK022888.1
FENS-1
AL161725
FLJ00026


AI141784
N/A
T92908
N/A
















TABLE 6







Genes With Down-Regulated Expression In Both


stage I And stage II Renal Cell Carcinoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





NM004092.2
ECHS1
BC002449.1
FLJ13612


NM000270.1
NP
J02639.1
SERPINA5


NM002354.1
TACSTD1
BC002571.1
DKFZP564O243


AF017987.1
SFRP1
U03884.1
KCNJ1


NM003012.2
SFRP1
AF173154.1
HYAL1


NM000666.1
ACY1
AF130103.1
PBP


NM000191.1
HMGCL
AL117618.1
PDHB


NM015254.1
KIF13B
AF063606.1
N/A


NM000140.1
FECH
BC005314.1
N/A


U75667.1
ARG2
BF686267
PBP


NM000196.1
HSD11B2
AI742553
PRKWNK1


NM014636.1
RALGPS1A
D83782.1
SCAP


NM001441.1
FAAH
AB029031.1
TBC1D1


NM005978.2
S100A2
AK025432.1
KIAA0564


NM001678.1
ATP1B2
AL117643.1
N/A


NM001099.2
ACPP
AW772192
N/A


NM014731.1
ProSAPiP1
NM003944.1
SELENBP1


BF343007
N/A
AL049977.1
CLDN8


NM000035.1
ALDOB
AK023937.1
THEA


NM005950.1
MT1G
AK025084.1
TNRC9


NM002371.2
MAL
X03363.1
ERBB2


NM006984.1
CLDN10
AK026411.1
ALDOB


NM002567.1
PBP
NM016026.1
RDH11


NM000019.1
ACAT1
NM016286.1
DCXR


NM001692.1
ATP6V1B1
NM019027.1
FLJ20273


X77737.1
N/A
BG338251
RAB7L1


NM006226.1
PLCL1
NM006113.2
VAV3


NM000893.1
KNG
NM018075.1
FLJ10375


NM000412.2
HRG
NM013271.1
PCSK1N


NM001963.2
EGF
NM017586.1
C9orf7


NM003361.1
UMOD
NM016321.1
RHCG


NM000050.1
ASS
NM025247.1
MGC5601


NM001438.1
ESRRG
BC002449.1
FLJ13612


NM020632.1
ATP6V0A4
AI379517
N/A


AI632015
SLC12A1
AA058832
MGC33926


NM000701.1
ATP1A1
AW274034
N/A


NM031305.1
DKFZP564B1162
AI580268
NUDT6


AF130089.1
ALDH6A1
AI761947
DKFZP564B1162


AK025651.1
N/A
AI793201
N/A


W45551
MMP24
AK025898.1
N/A


W67995
FXC1
AB046810.1
C20orf23


AL136566.1
IBA2
AK024204.1
N/A


AF105366.1
SLC12A6
BF594722
N/A


AF284225.1
DMRT2
R88990
N/A


AA191708
N/A
N73742
N/A


AL355708.1
N/A
AI697028
FLJ90165


BE783949
FLJ10101
BF590528
N/A


AL529672
N/A
AI733359
N/A


AL568674
MYBBP1A
H20179
N/A


AU147564
CLMN
AA991551
MGC14839


AK000208.1
N/A
AI758950
SLC26A7


AB051536.1
FLJ14957
AA911561
N/A


AI569747
TFDP2
AI769774
N/A


AK025562.1
N/A
AA669135
N/A


AI660243
TMPRSS2
AW136060
SLC13A2


N50413
N/A
AI733593
N/A


AI347918
N/A
BF739841
N/A


AL536553
GRP58
AA600175
N/A


BC000282.1
LOC89894
BF477980
N/A


BF106962
FAM3B
AI934557
N/A


AI051248
FLJ32115
BE326951
KNG


AI928242
N/A
AI632567
N/A


BG236006
N/A
BE300882
N/A


AI653107
N/A
BE855713
N/A


AI824037
FLJ25461
AA485440
DBP


R61322
N/A
AA915989
FLJ10743


AW071744
KCNJ10
AA085764
SIGIRR


BF059276
N/A









EXAMPLE 6
Loss of TGF-β Receptor Expression Demonstrated by Gene Array and Real-Time PCR in Renal Cell Carcinoma
Expression of type I TGF-β receptor (TBR1), type II TGF-β receptor (TBR2), and type III TGF-β receptor (TBR3) mRNA were compared in normal renal tissue, primary renal cell carcinoma without metastasis, primary lesions of metastatic renal cell carcinoma, and metastatic lesions. A summary of gene array analysis was presented as average signal intensities in FIG. 11A (mean±standard error). The signal intensity for TBR1 (cross-hatched bars) was relatively low, although TBR1 was scored as ‘Present’ in all samples. No significant changes in TBR1 expression were observed. TBR2 (gray bars) was abundantly expressed in normal epithelium and in primary lesions of nonmetastatic renal cell carcinoma. TBR2 was significantly reduced in primary lesions with metastatic disease (P<0.028 by ANOVA). TBR2 was even more reduced in metastatic lesions. TBR3 expression was high in normal epithelium, but was significantly reduced in each of the five primary tumors with nonmetastatic disease (black bars). TBR3 expression was also reduced in primary tumors with metastatic lesions and in metastatic lesions themselves.
These expression patterns were confirmed by real-time PCR (Tagman®) in the 10 patients used for gene array analysis. Means and standard errors for individual samples are shown in FIG. 11B. All data were normalized to 18S rRNA and calibrated to target abundance in the paired normal tissues. TBR1 mRNA abundance did not change (cross-hatched bars), consistent with the gene chip data. TBR2 (gray bars) was not reduced in primary tumors without metastases, whereas TBR2 was significantly reduced in primary tumors with metastatic disease and in metastatic lesions. TBR3 was reduced in all tumors (black bars).
The investigators have subsequently completed real-time PCR analysis of TBR1, TBR2, and TBR3 expression in 16 primary tumors without metastases (plus paired normal epithelium) and nine samples of primary tumors with metastatic disease, paired metastatic lesions, and paired normal tissue. The data were consistent with those shown for the samples analyzed in FIG. 11A. TBR3 expression was significantly reduced in all tumors; whereas TBR2 expression was reduced in only 1/16 primary tumors without metastatic lesions, but was reduced in primary tumors with metastatic lesions (8/9). These data show that loss of TBR3 is an early event in renal cell carcinoma, strongly suggesting that TBR3 plays a critical role in renal cell carcinoma carcinogenesis.
The loss of TBR3 mRNA expression was also correlated with TNM scores (T, histological score; N, lymph node number; M, number of organ metastases) from patient samples (data not shown). TBR3 mRNA expression was suppressed in the earliest stage, stage I, and was found to be suppressed in all tumor stages (I-IV). In addition, loss of TBR2 in the primary tumor is significantly associated with acquisition of the metastatic phenotype and clinically manifests as metastatic progression.
EXAMPLE 7
Attenuation of TGF-β-Mediated Signal Transduction in Human Renal Cell Carcinoma
Decreased type III TGF-β receptor (TBR3) mRNA expression in all tumors was associated with failure to detect TBR3 protein by immunohistochemistry (FIG. 12). Type I TGF-β receptor (TBR2) protein was detected in localized tumor (primary, no mets), but was not detectable in primary tumors with metastatic disease or in corresponding metastatic lesions. Type I TGF-β receptor (TBR1) protein was detected in normal tissue and in all tumor samples.
The investigators hypothesized that these losses seen in TGF-β receptor expression would manifest as an attenuation of TGF-β mediated signal transduction, and would significantly alter the expression of TGF-β regulated genes. From the gene array data disclosed above, 13 known TGF-β/Smad-regulated genes were down-regulated in renal cell carcinoma (Table 7). Using mRNA from 35 patient-matched samples, the investigators verified loss of expression of three of these genes by comparing matched normal and tumor tissue. Real-time PCR was used to measure the expression of Collagen IV type 6, fibulin-5, and connective-tissue growth factor (CTGF). Collagen IV type 6 (gray bars) is an extracellular matrix protein that plays a critical role in the regulation of membrane integrity and cell signaling. Fibulin-5 is a recently discovered TGF-β-regulated gene, which has tumor suppressor activity. Fibulin-5 is an extracellular matrix protein that is believed to signal through interaction with integrins. CTGF is a secreted protein involved in angiogenesis, skeletogenesis, and wound healing. CTGF enhances TGF-β1 binding to TBR2, and CTGF and TGF-β collaborate to regulate the expression of extracellular matrix proteins during renal fibrosis. As summarized graphically in FIG. 13, all the evaluated TGF-β-regulated genes were down-regulated in early tumor stages, suggesting that renal cell carcinoma undergoes loss of TGF-β responsiveness at an early stage. These data indicate that this loss of TGF-β sensitivity is due, primarily, to loss of type III TGF-β receptor (TBR3) in early tumor development and further loss of sensitivity in metastatic disease is mediated through subsequent loss of type II TGF-β receptor (TBR2).







TABLE 7







Known TGF-β-Regulated Genes Found To Be Down-Regulated


In Localized Tumors By Gene Array Analysis











Fold


GenBank No.
Gene Name
Attenuation












S81439
TGFβ-induced early growth factor (TIEG)
2.5


AF093118
Fibulin 5
4.0


U42408
Ladinin 1
15.4


U01244
Fibulin 1
4.8


J05257
Dipeptidase 1
7.7


D21337
Collagen, type IV, a6
3.6


X80031
Collagen, type IV, a3
2.4


M64108
Collagen, type XIV, a1
3.2


M98399
Collagen, type I receptor
4.2


L23808
Matrix metallo-proteinase 12
3.7


M35999
Integrin, b3
2.5


AI304854
p27Kip1
2.1


J05581
Mucin 1
6.5









Data were analysed by a combination of two-dimensional ANOVA, Affymetrix MAS5.0, and hierarchical cluster analysis using Spotfire to identify genes that are down-regulated in local tumors versus that of normal renal cortex tissue.

EXAMPLE 8
TGF-β Receptor Expression in Renal Cell Carcinoma Cell Lines
Human renal cell carcinoma cell lines were identified that recapitulate the clinical observations of TGF-β receptor biology described above. UMRC6 cells were derived from a clinically localized human renal cell carcinoma (Grossman et al., 1985). As shown in FIG. 14A, UMRC6 cells express type II TGF-β receptor (TBR2) mRNA, but not type III TGF-β receptor (TBR3). Immunohistochemical analysis (FIG. 14B) confirms the presence of TBR2 protein and the absence of TBR3 expression. UMRC3 cells were derived from the primary tumor of a patient with metastatic renal cell carcinoma. This highly aggressive cell line lacks detectable TBR2 and TBR3 mRNA (FIG. 14A) and protein (FIG. 14B).
In addition to these relevant laboratory models, normal renal epithelial (NRE) tissue was harvested from nephrectomy specimens and established as primary cultures (Trifillis, 1999). As shown in FIGS. 14A and 14B, these primary cultures of NRE expressed TBR3, TBR2, and TBR1 mRNA and protein in vitro. NRE cells can be grown in culture for 10 passages and were easily isolated and characterized. NRE cells were characterized for cytokeratin expression and tubule-specific gene expression, for example, megalin (data not shown). Thus, there are relevant cell models in which TBR2 and TBR3 expression can be manipulated to examine the impact of TGF-β receptor biology on the carcinogenesis and progression of human renal cell carcinoma in vitro.
EXAMPLE 9
TGF-β Activity in Renal Cell Carcinoma Cell Lines
It is well known that TGF-β1 inhibits cell proliferation in epithelial cells. The present example demonstrates the effects of TGF-β on renal tumor cell proliferation.
DNA content of cells was used as a measure of cell proliferation. Cells were plated at 20,000 cells/well in 12-well plates. Cells were grown in 10% FBS:DMEM:penicillin:streptomycin. The following day, media were exchanged with appropriate treatment added to the media. On day 3 of treatment, cells were analyzed for DNA content using Hoechst reagent. DNA standard was used to correlate DNA content per well.
As shown in FIG. 15A (squares), TGF-β1 inhibited the proliferation of normal renal epithelial cells in culture. URMC3 cells expressed neither type II or type III TGF-β receptors and, not surprisingly, were resistant to the inhibitory effects of TGF-β on cell proliferation (triangles, FIG. 15A). UMRC6 cells expressed type II but not type III TGF-β receptors, and were partially resistant to TGF-β1 (circles, FIG. 15A).
TGF-β transcriptional activity was also measured in the above cell models using transient transfection of the 3TP/lux reporter, which contains an AP-1/Smad3 response element from the PAI-1 promoter. This luciferase reporter construct demonstrates increased transcriptional activity in response to exogenous TGF-β-mediated signal transduction. 3TP/lux was transiently transfected along with SV/renilla luciferase (Promega) into cells using fugene (Roche) as the transfection agent. Cells were treated with or without TGF-β1 24 h after transfection and luciferase activity (Promega Luciferase Assay system and Lumat luminometer) was determined 24 h after TGF-β treatment. Firefly luciferase activity was normalized using the ratio of firefly luciferase/renilla luciferase. As shown in FIG. 15B, normal renal epithelial cells were highly responsive to 2 ng/ml (80 pM) of TGF-β1. UMRC6 cells demonstrated significantly less luciferase activity in response to TGF-β1, and UMRC3 cells were entirely unresponsive.
EXAMPLE 10
Recapitulation of TGF-β Signaling Through Reintroduction of TGF-b Receptor Expression into Renal Cell Carcinoma
To test whether reintroduction of TGF-β receptor expression would result in re-establishment of TGF-β signal transduction and reacquisition of TGF-β cellular sensitivity, UMRC3 cells were engineered to express stably either type II TGF-β receptor (+TBR2) alone or type II plus type III TGF-β receptor (+TBR2+TBR3).
Plasmid construction and transfection were described as follows. The complete coding sequences for human type II TGF-β receptor (TBR2) was cloned into the EcoRI/XbaI site of pcDNA3/FLAG. The expression vector was stably transfected into UMRC3 cells using fugene as DNA carrier and genticin as selection antibiotic (Sigma, 1 mg/ml). Ten clones (UMRC3/TBR2) were selected and verified for TBR 2 mRNA and protein expression such as Western analysis using the FLAG antibody (data not shown). From these cell clones, one was to be selected that had equivalent protein expression of TBR2 to that of normal renal epithelial (NRE) and UMRC6 cells.
The type III TGF-β receptor (TBR3) coding sequence was PCR amplified from a plasmid expressing wild-type TBR3 in pSV7d (a gift from Dr C-H Heldin). TBR3 was then cloned into the EcoRI site of pcDNA4/TO/myc-His® (InVitrogen) in the sense and antisense (negative control) orientation. The orientation and sequence of TBR3 was verified. The antisense TBR3 (As TBR3) vector was used as a control. TBR3/pcDNA4/TO/myc-His and As TBR3/pcDNA4/TO/myc-His vectors were stably transfected into UMRC3/TBR2 cells. A clone was selected that demonstrated an equivalent expression of TBR3 mRNA to that of normal renal epithelial cells. As a control for UMRC3+TBR2 and UMRC3+TBR2+TBR3, wild-type UMRC3 were stably transfected with both pcDNA/FLAG and pcDNA4/TO/myc-His vectors.
As shown in FIGS. 16A-16B, stable transfection of type II TGF-β receptor (TBR2) alone or type II plus type III TGF-β receptor (TBR2+TBR3) resulted in detectable levels of mRNA for each receptor on RT-PCR analysis. On examining the in vitro growth kinetics of these re-engineered cells, it was noted that reintroduction of TBR2 resulted in a twofold reduction in cell proliferation and reintroduction of both TBR2 and TBR3 resulted in a fourfold reduction in cell proliferation with the addition of exogenous TGF-β.
The investigators then examined TGF-β-mediated transcriptional activity as a consequence of TGF-β receptor re-expression. As shown in FIG. 16C, reintroduction of TBR2 partially restored transcriptional responsiveness, as evidenced by a 5.6-fold increase in 3TP/lux activity after addition of TGF-β1. Reintroduction of both TBR2 and TBR3 into UMRC3 cells resulted in 17.5-fold increase in 3TP/lux activity after addition of TGF-β1.
To demonstrate reestablishment of TGF-β-regulated gene expression, collagen IV type 6 mRNA expression was examined by real-time PCR in these re-engineered cell lines in the presence of TGF-β1. As shown in FIG. 16D, reexpression of TBR2 in UMRC3 cells results in a sevenfold increase in collagen IV type 6 mRNA levels over that of UMRC3 controls, while reintroduction of both TBR2 and TBR3 enhanced collagen IV type 6 mRNA expression 11-fold. These data are consistent with a number of published reports that indicate expression of TBR3 is essential for full TGF-β responsiveness.
UMRC3 cells have been shown to be tumorigenic in athymic nude mice (Grossman et al., 1985). Anchorage independent growth in soft agar is a well-established in vitro correlate of in vivo tumorigenicity. Colonies formation in soft agar was determined as follows. UMRC3 (pcDNA/FLAG and pcDNA4/T0/myc-His empty vectors), UMRC3+TBR2, or UMRC3+TBR2+TBR3 cells were plated at 1000 cells/60 mm dish in an agarose/FBS/media sandwich in the presence of 2 ng/ml TGF-β. No selection antibodies were added to the agarose media mixture. The cells were incubated for 45 days to insure that no colony formation would occur. Cells were then stained with 0.005% Crystal Violet, photographed, and assessed for number and size of colonies.
As shown in FIG. 16E, UMRC3 cells demonstrated anchorage independent growth in soft agar. Reintroduction of TBR2 into UMRC3 cells significantly decreased the number and size of colonies that formed in soft agar. Reintroduction of both TBR2 and TBR3 completely abrogated the ability of UMRC3 cells to form colonies in soft agar, even after 45 days in culture. These data demonstrate that reintroduction of TBR2 resensitizes UMRC3 cells to the effects of exogenous TGF-β through reacquisition of TGF-β signal transduction. More interestingly, however, reintroduction of TBR3 in the presence of TBR2 into UMRC3 cells significantly enhanced TGF-β-regulated gene transcription, growth inhibition, and loss of anchorage-independent growth over that seen with reintroduction of TBR2 alone. These data clearly show that renal cell carcinoma cells are TGF-β resistant. Loss of TBR3 expression occurs early and appears to be associated with a relatively less aggressive state that is partially TGF-β responsive. Loss of TBR2 results in frank TGF-β resistance and is associated with acquisition of a more aggressive phenotype.
FIGS. 17-18 demonstrate that re-expression of type II or type Ill TGF-β receptor in the highly metastatic human renal cell carcinoma cell line UMRC3 inhibited cell proliferation in cell culture and tumor growth in a nude mouse model. The TGF-β receptors were either re-expressed in a stable vector system or as an adenoviral vector. For clinical purposes, it would be envisioned to treat patients with an adenovirus expressing one or both of the TGF-β receptors to block tumor growth or cause tumor regression.
EXAMPLE 11
Stepwise Sequential Loss of Type III and Type II TGF-β Receptor Expression in Renal Cell Carcinoma
With genomic profiling in human renal cell carcinoma, the data presented above demonstrated a stepwise sequential loss of type III and type II TGF-β receptor expression in association with renal cell carcinogenesis and progression. These findings were confirmed by both immunohistochemistry and real-time PCR in patient-matched tissue samples. This clinical observation was brought to the laboratory to identify relevant in vitro models. Using these models, it was demonstrated that loss of type III TGF-β receptor expression resulted in incremental desensitization to TGF-β and attenuation of TGF-β signaling. Subsequent loss of type II TGF-β receptor resulted in complete loss of TGF-β sensitivity. With in vitro modulation of TGF-β receptor expression, it was demonstrated that reconstitution of the TGF-β signaling pathway resulted in significant growth inhibition and loss of the aggressive phenotype.
These experiments are unique in that clinically relevant observations, which are derived from the evaluation of gene expression in normal renal cortical tissue, localized renal cell carcinoma and metastatic renal cell carcinoma, were brought to the laboratory for validation and experimental manipulation in relevant in vitro models. Other investigators have examined human renal cell carcinoma cell lines and identified alterations in the expression of TGF-β signaling pathway intermediaries, but those observations have not been validated in the clinical biology of renal cell carcinoma. To the investigators' knowledge, few studies have methodically examined the expression of all three TGF-βreceptors in patient samples at the protein and mRNA level in an effort to correlate TGF-β receptor expression to disease-specific states of renal cell carcinoma (i.e. localized versus metastatic tumor). A major strength of the present study is that the investigators recognized distinct disease states in renal cell carcinoma, associated them with specific alterations in the TGF-β signaling pathway, and then validated and manipulated the clinical observations in the laboratory.
Although the mechanisms are not well understood, it is clear that TGF-β regulates a large number of diverse biological functions, including cell proliferation, differentiation, cell adhesion, apoptosis, extracellular matrix production, immune regulation, neuroprotection, and early embryonic development. In epithelial cells, the effect of TGF-β is generally to inhibit proliferation, promote cellular differentiation, and regulate interactions with the extracellular matrix. As a direct consequence, aberrations in TGF-β signaling can have a dramatic impact on cellular processes that are critically associated with neoplastic and malignant transformation. Given the well-documented observation that the end result of TGF-β signaling is largely growth inhibitory, it makes intuitive sense that cancer cell would develop mechanisms to escape TGF-β sensitivity. To date, these mechanisms have not been elucidated in human renal cell carcinoma.
Based on the data presented above, the investigators hypothesize that this escape from the growth-inhibitory effects of TGF-β is mediated through the stepwise sequential loss of type III and type II TGF-β receptor expression. To the investigators' knowledge, no one has linked sequential loss of these two types of receptors to carcinogenesis and metastatic progression in oncology. This is the first time that stepwise loss of a single transduction pathway has been associated with important biologic sequelae in a human cancer.
Results presented in the present invention demonstrate that loss of type III TGF-β receptor expression is an early event in renal cell carcinoma biology and that this loss has important sequelae with regard to renal cell carcinoma carcinogenesis and progression. All clinical samples of localized renal cell carcinoma demonstrated loss of type III TGF-β receptor, but had normal expression of type I and type II TGF-β receptors. Replication of this clinical observation in in vitro models demonstrated significant loss of TGF-β sensitivity, manifest as a significant reduction in the growth inhibitory effects of TGF-β1 and significantly reduced TGF-β-mediated transcription. Interestingly, cell lines derived from localized RCC retained type II TGF-β receptor expression and therefore, still demonstrated sensitivity, albeit reduced, to TGF-β. Only with metastatic progression and loss of type II TGF-β receptor expression does the cell become completely resistant to the effects of TGF-β. The investigators hypothesize that this retained, but attenuated, TGF-β signaling seen in local tumors must convey some as yet unrecognized biologic benefit for local tumors that is no longer required, and therefore discarded, with metastatic progression. In fact, this loss of type II TGF-β receptor expression may be an absolute integral component in the cascade of intracellular events that lead to the development of metastatic potential. In keeping with this hypothesis, it has been shown that loss of type I TGF-b receptor expression was one of 40 integral alterations of gene expression to predict for poor prognosis of patients diagnosed with renal cell carcinoma.
In summary, the above results demonstrate a clear link between loss of type III TGF-β receptor expression to a human disease state. Reduced type III TGF-β receptor (TBR3) expression has been reported in human breast tumor cell lines, suggesting that loss of TBR3 expression may be a more ubiquitous phenomena in carcinogenesis, rather than an isolated finding in human RCC biology. The fact that the investigators found down-regulation of TBR3 in every renal cell carcinoma specimen studied to date (35 patients) and that re-expression of TBR3 (in the presence of re-expressed TBR2) completely abolish growth on soft agar suggests an important role for TBR3 in normal renal epithelial homeostasis that must be abrogated for renal cell carcinogenesis and progression to occur. Little attention has been given to TBR3 in normal cell biology or the changes in expression that occur with carcinogenesis and progression. Observations from the present invention would suggest that TBR3 plays an important functional role in signaling and that loss of expression is an important event in the acquisition of the tumorigenic and metastatic phenotype
EXAMPLE 12
Genomic Profiling for Stage I Papillary Renal Cell Carcinoma and Benign Oncocytoma
FIG. 19 shows hierarchical clustering of genes over-expressed or down-regulated (with at least 2 fold differences) in stage I papillary renal cell carcinoma verses normal renal cortex. Genes over-expressed and down-regulated in stage I papillary renal cell carcinoma are listed in Table 8 and Table 9 respectively. FIG. 20 shows hierarchical clustering of genes over-expressed or down-regulated (with at least 2 fold differences) in benign oncocytoma verses normal renal cortex. Genes over-expressed and down-regulated in benign oncocytoma are listed in Table 10 and Table 11 respectively. FIG. 21 shows venn analysis of gene distribution among stage I renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma. Genes with at least 2-fold differences in expression were filtered at 95% confidence level (CL) in the following 3 t-tests: stage I RCC vs. normal; oncocytoma vs. normal; and stage I papillary renal cell carcinoma vs. normal. Six hundred twenty five genes were present only in stage I RCC (95% CL), 136 genes were present only in oncocytoma (95% CL), 344 genes were present only in stage I papillary renal cell carcinoma (95% CL), and 60 genes were common to stage I RCC, oncocytoma and stage I papillary renal cell carcinoma. FIG. 22 shows venn analysis of gene distribution among stage II renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma. Genes with at least 2-fold differences in expression were filtered at 95% confidence level (CL) in the following 3 t-tests: stage II RCC vs. normal; oncocytoma vs. normal; and stage I papillary renal cell carcinoma vs. normal. One thousand and five genes were present only in stage II RCC (95% CL), 152 genes were present only in oncocytoma (95% CL), 334 genes were present only in stage I papillary renal cell carcinoma (95% CL), and 43 genes were common to stage II RCC, oncocytoma and stage I papillary renal cell carcinoma.







TABLE 8







Genes With Up-Regulated Expression In


stage I Papillary Renal Cell Carcinoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





NM_003505
FZD1
AC004382
DKFZP434K046


AL035683
B4GALT5
NM_000248
MITF


R56118
N/A
NM_022154
SLC39A8


NM_014575
SCHIP1
AI436813
N/A


AI694320
ZNF533
AF007162
CRYAB


BC031322
N/A
NM_015392
NPDC1


BF346665
N/A
AL136585
DKFZp761A132


BC004283
LOC283639
AB040120
SLC39A8


AF302786
GNPTAG
NM_138473
SP1


AU121975
PAICS
AU144387
182-FIP


NM_016315
GULP1
NM_022763
FAD104


AL541302
SERPINE2
AI093231
APBB1IP


BG391217
C9orf80
NM_000235
LIPA


NM_000700
ANXA1
AI817079
EXOC7


N30188
N/A
NM_004385
CSPG2


NM_003651
CSDA
NM_024801
TARSH


AI830227
FLII
BF218922
CSPG2


U20350
CX3CR1
BF590263
CSPG2


NM_005692
ABCF2
NM_001233
CAV2


U34074
AKAP1
AB020690
PNMA2


AB056106
TARSH
AW188198
TNFAIP6


AU151483
CDH6
NM_007115
TNFAIP6


BC026260
TTC3
AI742838
DOCK11


AL133001
SULF2
AW117264
N/A


NM_003358
UGCG
AF016266
TNFRSF10B


NM_001282
AP2B1
NM_013952
PAX8


AF322067
RAB34
AA771779
ZFP90


NM_001540
HSPB1
W72333
FLJ21657


N58363
STATIP1
H23979
MOX2


AF072872
FZD1
BG542521
PPM2C


BF247552
SLC38A1
AF063591
MOX2


X69397
CD24
BF247383
BMPR2


BC000251
GSK3B
NM_005114
HS3ST1


BF691447
B4GALT5
BE466145
N/A


AB046817
SYTL2
BC005352
TNFAIP8


AF255647
DKFZP566N034
AC002045
LOC339047


BF344237
N/A
BC040558
D2LIC


AW242720
LOC143381
U13699
CASP1


AA115485
MGC3222
NM_002718
PPP2R3A


NM_006588
SULT1C2
BF476502
MPPE1


NM_000546
TP53
BC034275
LOC253982


N92494
JWA
AF279145
ANTXR1


W74580
MGC3222
AV724216
NDRG4


AF131749
PSK-1
BG165613
N/A


AW026491
CCND2
NM_018205
LRRC20


NM_012410
PSK-1
NM_022083
C1orf24


NM_002800
PSMB9
NM_006169
NNMT


BF512748
JAK3
AF141347
TUBA3


AA404269
PRICKLE1
NM_000064
C3


M33376
AKR1C1
AV710838
BCDO2


AF035321
DNM1
AI417917
EHD2


NM_002862
PYGB
AI681260
LILRB1


AF132000
DKFZP564K1964
NM_000389
CDKN1A


L07950
HLA-C
AF288391
C1orf24


AF114011
TNFSF13
NM_002627
PFKP


BF674052
VMP1
NM_001975
ENO2


AI922599
VIM
NM_030786
SYNCOILIN


AF044773
BANF1
NM_006169
NNMT


NM_015925
LISCH7
AI417917
EHD2


NM_001684
ATP2B4
NM_006868
RAB31


AI123348
CHST11
L03203
PMP22


NM_001304
CPD
AF199015
VIL2


NM_006762
LAPTM5
AI873273
SLC16A6


NM_000211
ITGB2
NM_017821
RHBDL2


AA995910
ALOX5
BF740152
MYO1F


NM_018965
TREM2
AA954994
N/A


AL353715
STMN3
AI458735
MGC26717


BC019612
C20orf75
NM_003254
TIMP1


AF086074
N/A
AI688631
N/A


NM_005045
RELN
AK026037
N/A


AI935123
C14orf78
BG327863
CD24


AL550875
C7orf28B
NM_016008
D2LIC


L27624
TFPI2
AI394438
LOC253981


AL574096
TFPI2
AA947051
D2LIC


AA005141
MET
AI819043
N/A


D86983
D2S448
AI378044
UGCG


AW439242
C6orf68
NM_024576
OGFRL1


AB000221
CCL18
M76477
GM2A


NM_002121
HLA-DPB1
NM_002214
ITGB8


U17496
PSMB8
AI879381
ADCK2


U05598
AKR1C1
NM_000152
GAA


BF342851
D2S448
H15129
MEIS4


BF311866
PTGFRN
L42024
HLA-C


NM_001449
FHL1
NM_002178
IGFBP6


AA954994
N/A
AI761561
HK2


Y13710
CCL18
AA722799
DCBLD2


BG170541
MET
NM_003255
TIMP2


AB037813
DKFZp762K222
NM_000107
DDB2


D28124
NBL1
AV699565
CTSC


NM_021103
TMSB10
NM_000861
HRH1


AI949772
N/A
















TABLE 9







Genes With Down-Regulated Expression In


stage I Papillary Renal Cell Carcinoma








Genbank ID
Gene Symbol













AF232217
N/A
NM_003877
SOCS2


AI823572
MGC45438
AI768894
CGN


AU154994
SLC13A3
AW772192
N/A


AW979271
N/A
AF094518
ESRRG


AF064103
CDC14A
T40942
ANGPTL3


AI524125
PCDH9
NM_001146
ANGPT1


AI733474
GPR155
AI242023
N/A


AI767756
HS6ST2
BF970431
N/A


NM_000412
HRG
NM_005670
EPM2A


NM_021614
KCNN2
AW071744
KCNJ10


M13149
HRG
AI928242
TFCP2L1


H17038
N/A
AI769774
LOC155006


NM_002010
FGF9
AW274034
USP2


AI635774
EMCN
NM_004633
IL1R2


AW007532
IGFBP5
NM_003289
TPM2


NM_004070
CLCNKA
BF512388
C10orf58


NM_014621
HOXD4
BC005830
ANXA9


AI733593
N/A
NM_000362
TIMP3


NM_020632
ATP6V0A4
NM_001438
ESRRG


AI697028
FLJ90165
AU146204
ENPP6


AA897516
PTGER4
AA775681
FLJ23091


NM_024307
MGC4171
AI393205
ACY-3


J02639
SERPINA5
AF017987
SFRP1


NM_000085
CLCNKB
NM_005951
MT1H


AA058832
MGC33926
NM_005950
MT1G


BF059276
N/A
NM_021805
SIGIRR


BC043647
LOC284578
AA557324
CYP4X1


AL161958
THY1
BF528646
DKFZP564I1171


AL121845
KIAA1847
AW340112
LOC401022


AY079172
ATP6V0D2
R73554
IGFBP5


AA928708
CYP8B1
AI826437
N/A


H71135
ADH6
AV720650
KIAA0888


NM_000102
CYP17A1
AA780067
HS3ST3B1


Z92546
SUSD2
NM_000640
IL13RA2


AL558479
THY1
AI806338
TBX3


BC005314
ALDOB
NM_003155
STC1


NM_173591
FLJ90579
AA931562
N/A


BF510426
N/A
AI694325
N/A


AF331844
SOST
AF205940
EMCN


X77737
SLC4A1
NM_001290
LDB2


NM_004392
DACH1
NM_016242
EMCN


BC001077
LOC87769
AW014927
CALB1


AA218868
THY1
AI758950
SLC26A7


BF478120
RECQL5
AK024256
KIAA1573


BC041158
CYP4A11
BF726212
ANK2


AI623321
MTP
AI985987
SCNN1G


AI796189
PAH
AW242408
UPP2


NM_021161
KCNK10
NM_000860
HPGD


NM_000163
GHR
BF447963
KIAA0962


AL136880
ESPN
BF941499
GPR116


NM_024426
WT1
AW242409
N/A


M61900
PTGDS
BF509031
ATP6V1G3


AW963951
SIAT7C
NM_000934
SERPINF2


AW340588
MAN1C1
BF248364
AF15Q14


AI263078
SLC23A3
AL534095
FLJ23091


BF130943
PPAPDC1
NM_004929
CALB1


AI732596
N/A
AI222435
N/A


AA603467
ZNF503
NM_005397
PODXL


R41565
N/A
AI090268
N/A


AI951185
NR2F1
AI300520
STC1


NM_002609
PDGFRB
BC006236
MGC11324


NM_006984
CLDN10
NM_024609
NES


BG413612
N/A
NM_002591
PCK1


D64137
CDKN1C
NM_005410
SEPP1


AK026344
PEPP2
AB020630
PPP1R16B


AI670852
PTPRB
AF022375
VEGF


AI693153
GABRB3
NM_016246
DHRS10


NM_001393
ECM2
AA873542
SLC6A19


N93191
PR1
U95090
PRODH2


BC005090
AGMAT
D26054
FBP1


NM_000717
CA4
AI732994
MGC13034


D38300
PTGER3
NM_000151
G6PC


AI650260
N/A
AK025651
PNAS-4


BC024226
IFRG15
AF161441
N/A


BC006294
DHRS10
AF161454
APOM


NM_003039
SLC2A5
NM_022129
MAWBP


AI675836
SORCS1
AI733515
MGC52019


NM_005276
GPD1
NM_001443
FABP1


NM_014298
QPRT
AI433463
MME


M10943
MT2A
AL049313
N/A


NM_005952
MT1X
BF195998
ALDOB


NM_002450
MT1X
NM_022829
SLC13A3


NM_002910
RENBP
NM_000035
ALDOB


BF246115
MT1F
NM_007287
MME


AF078844
MT1F
NM_003399
XPNPEP2


AF170911
SLC23A1
NM_000196
HSD11B2


AF333388
MT1H
BF431313
N/A


NM_003500
ACOX2
NM_004844
SH3BP5


AA995925
N/A
NM_003206
TCF21


NM_001218
CA12
AI311917
DPYS


BF432333
FLJ31196
AA843963
PRLR


NM_001385
DPYS
NM_017753
PRG-3


NM_003052
SLC34A1
NM_006633
IQGAP2


NM_000778
CYP4A11
NM_001133
AFM


AL136551
SESN2
T90064
N/A


NM_000792
DIO1
BF696216
N/A


NM_016725
FOLR1
NM_004413
DPEP1


NM_019101
APOM
Z98443
FLJ38736


NM_014270
SLC7A9
NM_018456
EAF2


AF124373
SLC22A6
AW771563
N/A


NM_016327
UPB1
NM_014495
ANGPTL3


NM_024734
CLMN
AI074145
KMO


NM_016527
HAO2
NM_000896
CYP4F3


NM_003645
SLC27A2
NM_001072
UGT1A6


AB051536
FLJ14957
AI631993
N/A


NM_025149
FLJ20920
NM_000277
PAH


BC005939
PTGDS
M74220
PLG


AL574184
HPGD
AI935789
UMOD


NM_000161
GCH1
NM_002472
MYH8


H57166
N/A
BC020873
CLCNKA


NM_000597
IGFBP2
NM_000550
TYRP1


NM_000790
DDC
AA806965
BTNL9


NM_004668
MGAM
NM_020163
LOC56920


NM_021027
UGT1A6
NM_004490
GRB14


AF348078
GPR91
AA788946
COL12A1


NM_016347
NAT8
AW242315
N/A


AF338650
PDZK3
AI735586
LOC152573


BE221817
CNTN3
R88990
N/A


NM_004476
FOLH1
NM_003278
TNA


NM_004615
TM4SF2
NM_007180
TREH


NM_023940
RASL11B
AW173045
TBX2


AI742872
SLC2A12
U28049
TBX2


BC001196
HS6ST1
NM_001395
DUSP9


AW195353
TFCP2L1
NM_000336
SCNN1B


NM_003122
SPINK1
U43604
N/A


NM_144707
PROM2
BC029135
N/A


AI653981
L1CAM
NM_005414
SKIL


AI796169
GATA3
BQ894022
PDE1A


M96789
GJA4
NM_013335
GMPPA


N74607
AQP3
NM_003221
TFAP2B


NM_014059
RGC32
BF057634
HOXD8


AI572079
SNAI2
AA523172
N/A


AI056877
N/A
AF319520
ARG99


NM_006206
PDGFRA
NM_002885
RAP1GA1


AW771314
MGC35434
NM_003361
UMOD


NM_016955
SLA/LP
NM_000142
FGFR3


AI569804
LOC375295
NM_000893
KNG1


NM_001584
C11orf8
BC029135
N/A


BG261252
EVI1
NM_147174
HS6ST2


NM_006226
PLCL1
NM_000218
KCNQ1


NM_001172
ARG2
U03884
KCNJ1


AL050264
TU3A
X83858
PTGER3


BC003070
GATA3
BF439270
N/A


AL120332
MGC20785
AA911235
MST1


NM_000459
TEK
NM_000955
PTGER1


AW242836
LOC120224
NM_022844
MYH11


AI926697
Gup1
BC042069
N/A


NM_000486
AQP2
NM_005518
HMGCS2


AI870306
IRX1
NM_001963
EGF


AW264204
CLDN11
AI632015
SLC12A1


BF431989
THRB
AF339805
N/A


AI459140
N/A
BF106962
FAM3B


NM_001864
COX7A1
NM_005019
PDE1A


AI471866
SLC7A13
AU146305
PDE1A


AI653107
NRK
NM_000663
ABAT


NM_004466
GPC5
AU119437
LOC144997


BF195936
KRT18L1
BC036095
DRP2


NM_022454
SOX17
R49295
N/A


AW299531
HOXD10
AI623202
PRDM16


AL137716
AQP6
AW452355
N/A


AI332407
SFRP1
AA563621
HSPB6


AL565812
PTN
X15217
SKIL


AI452457
LOC199920
AK095719
N/A


AI281593
DCN
AI056187
N/A


M21692
ADH1B
AI668598
N/A


AI660243
TMPRSS2
AI700882
SLC13A3


AI754423
FLJ38507
NM_000963
PTGS2


AA759244
FXYD4
AW051712
N/A


U75667
ARG2
AL832099
MGC33190


NM_000930
PLAT
AK057337
LOC145820


AF083105
SOX13
AW300204
SLC30A8


NM_013231
FLRT2
NM_005856
RAMP3


BI825302
PR1
AI458003
CYYR1


NM_003012
SFRP1
AK026877
N/A


AF138300
DCN
AI632567
N/A


AU155612
N/A
U91903
FRZB


BG435302
EBF
AF352728
FLJ12541


NM_005978
S100A2
BM128432
IGFBP5


NM_000900
MGP
NM_003102
SOD3


AK026748
DKFZP761M1511
BE676272
TACC1


J03208
DBT
AI692180
PPFIBP2


NM_002345
LUM
AL544576
LOC92162


NM_006623
PHGDH
NM_017688
BSPRY


AF063606
my048
AU146310
N/A


NM_001647
APOD
AI912976
RASGRF2


AI935541
N/A
U83508
ANGPT1


NM_005558
LAD1
L47125
GPC3


AW138125
PRODH2
NM_000663
ABAT
















TABLE 10







Genes With Up-Regulated Expression In Benign Oncocytoma












Genbank ID
Gene Symbol
Genbank ID
Gene Symbol







NM_005114
HS3ST1
AF178532
BACE2



AA650558
GNAS
AI521166
LOC283104



BF062244
LIN7A
AA005023
NOD27



NM_030674
SLC38A1
AV725364
GPRC5B



NM_014766
SCRN1
AW195581
GPSM2



BC002471
CPLX1
BG503479
B4GALT6



AF183421
RAB31
BF031829
DSG2



AK022100
KIAA0256
AW975728
SLC16A7



BF508244
AKR1C2
NM_022495
C14orf135



BG772511
N/A
AA703159
N/A



AB037848
SYT13
BF247552
SLC38A1



AK055769
N/A
NM_001673
ASNS



T58048
N/A
NM_024622
FLJ21901



NM_012105
BACE2
AI565054
N/A



AA992805
LEF1
AW058459
LOC134285



AK026420
DMN
NM_001233
CAV2



NM_024812
BAALC
BC036550
N/A



AI057226
N/A
BE464483
N/A



AW138767
ELOVL7
NM_002512
NME2



NM_013233
STK39
AF178532
BACE2

















TABLE 11







Genes With Down-Regulated Expression In Benign Oncocytoma










Genbank ID
Gene Symbol
Genbank ID
Gene Symbol





BF593625
SYK
AW274034
USP2


AI310001
FLJ22789
NM_147174
HS6ST2


NM_006206
PDGFRA
AA074145
PRODH


NM_003740
KCNK5
AL049176
CHRDL1


AW138125
PRODH2
NM_020353
PLSCR4


NM_000336
SCNN1B
NM_024803
TUBAL3


BC005314
ALDOB
D16931
ALB


AI796189
PAH
NM_019076
UGT1A10


NM_013363
PCOLCE2
AF138303
DCN


NM_004466
GPC5
D13705
CYP4A11


AI627531
N/A
NM_000587
C7


U28055
MSTP9
R49295
N/A


NM_152759
MGC35140
NM_000385
AQP1


AW052159
N/A
AI669229
RARRES1


NM_017712
PGPEP1
U36189
C5orf13


AI961231
TOX
AL110135
FLJ14753


AI767962
BNC2
AW271605
N/A


AF350881
TRPM6
BF358386
N/A


AU146418
N/A
NM_016270
KLF2


BE875072
N/A
AA905508
LOC128153


AI653981
L1CAM
NM_021630
PDLIM2


AI634662
SLC13A3
AA915989
TBC1D13


NM_000486
AQP2
AL565812
PTN


AW206292
AQP2
AI990790
N/A


AI572079
SNAI2
BC041158
CYP4A11


AI694118
N/A
NM_138474
N/A


NM_000142
FGFR3
NM_002899
RBP1


U78168
RAPGEF3
AK024256
KIAA1573


AI913600
UNQ846
AW779672
SLC17A1


W93847
MUC15
NM_021161
KCNK10


NM_004616
TM4SF3
BF196891
TPMT


AI935789
UMOD
AY028896
CARD10


NM_007180
TREH
NM_018456
EAF2


AL110152
CD109
NM_017806
FLJ20406


AW051599
N/A
X59065
FGF1


AI796169
GATA3
AI650353
DACH1


AF017987
SFRP1
AW771563
N/A


BE550027
DKFZp761N1114
BF431313
N/A


AA535065
KIAA1847
NM_000896
CYP4F3


NM_003361
UMOD
BC005090
AGMAT


AI263078
SLC23A3
U24267
ALDH4A1


M13149
HRG
AI090268
N/A


AF278532
NTN4
AW014927
CALB1


AI632015
SLC12A1
AL023553
PIPPIN


NM_000412
HRG
AL049313
N/A


NM_000893
KNG1
AK021539
NCAG1


BG398937
KNG
AI220117
MGST1


AL049977
CLDN8
NM_020300
MGST1


N74607
AQP3
NM_022568
ALDH8A1


AW071744
KCNJ10
BE874872
FAM20C


AW015506
AQP2
NM_004668
MGAM


AI927000
SOSTDC1
BF033242
CES2


AI471866
SLC7A13
BC004542
PLXNB2


NM_001099
ACPP
NM_000204
F


NM_005074
SLC17A1
NM_004525
LRP2


AA995925
N/A
AA442149
MAF


AF352728
FLJ12541
NM_000049
ASPA


BF343007
TFAP2A
AI830469
TFEC


NM_016929
CLIC5
NM_003759
SLC4A4


AA911235
MST1
AF169017
FTCD


AA639753
N/A
AF170911
SLC23A1


NM_004887
CXCL14
AA865601
LOC123876


AW771565
AIM1
AA863031
MGC32871


AI264671
N/A
AW136060
SLC13A2


BF510426
N/A
NM_003041
SLC5A2


AV728958
TLN2
NM_021924
MUCDHL


T90064
N/A
AW299568
N/A


AA218868
THY1
AI927941
N/A


NM_003104
SORD
AI433463
MME


AJ292204
AGXT2
AL365347
SLC7A8


AI056359
MAPT
AA502331
PRAP1


AL568422
DZIP1
NM_024709
FLJ14146


AF339805
N/A
AF289024
FTCD


NM_000163
GHR
NM_017614
BHMT2


AI042017
NPL
NM_016347
NAT8


AW340457
N/A
NM_000277
PAH


BF431199
DEHAL1
NM_000316
PTHR1


BF432254
MGC15937
NM_001091
ABP1


AI368018
GPD1
NM_000790
DDC


AF144103
CXCL14
BF217861
MT1E


NM_016725
FOLR1
BF447963
KIAA0962


NM_000050
ASS
NM_001081
CUBN


AA693817
N/A
NM_018484
SLC22A11


NM_004929
CALB1
AW192692
N/A


NM_000592
C4A
BF000045
TINAG


AL574184
HPGD
BC005830
ANXA9


AA676742
DMGDH
NM_025257
C6orf29


AI631993
N/A
NM_020973
GBA3


AI566130
AK3
NM_001977
ENPEP


AW024233
GLYAT
AI632692
N/A


AA873542
SLC6A19
BI825302
PR1


AK026966
AK3
L12468
ENPEP


NM_022829
SLC13A3
AL571375
SCD4


NM_005950
MT1G
AL136858
ZMYND12


AV700405
MGC52019
NM_024027
COLEC11


AI733515
MGC52019
NM_014934
DZIP1


NM_000860
HPGD
BG496631
FBI4


U95090
PRODH2
NM_018265
FLJ10901


NM_001385
DPYS
AI770035
UPB1


BG401568
SLC16A9
AF177272
UGT2B28


NM_000846
GSTA1
NM_004392
DACH1


BF195998
ALDOB
N95363
CDKN1C


NM_004413
DPEP1
AF261715
FOLH1


NM_000151
G6PC
NM_000042
APOH


NM_006744
RBP4
NM_001393
ECM2


NM_013410
AK3
R88990
N/A


NM_000035
ALDOB
AA557324
CYP4X1


AK026411
ALDOB
AF116645
ALB


AL135960
CYP4A11
BC015993
MGC27169


M74220
PLG
AL558479
THY1


NM_001713
BHMT
NM_000785
CYP27B1


AW614558
SLC39A5
AW051926
AMN


Z92546
SUSD2
AA928708
CYP8B1


NM_000778
CYP4A11
BE407830
KIFC3


NM_000792
DIO1
AI431643
RRAS2


AI222435
N/A
AF001434
EHD1


D26054
FBP1
BC005894
FMO2


AW025165
SLC22A8
NM_006798
UGT2A1


NM_007287
MME
BF217861
MT1E









The following references were cited herein:

    Copland et al., Recent Prog. Horm. Res. 58:25-53 (2003).
    Copland et al., Oncogene 22:8053-62 (2003).
    Grossman et al., J. Surg. Oncol. 28:237-244 (1985).
    Trifillis, Exp. Nephrol. 7:353-359 (1999).

## Claims

1. A method of detecting a renal cell cancer comprising the steps of:
obtaining one or more biological samples comprising renal tissue or renal cells from an individual;
determining an RNA gene expression level of secreted frizzled related protein 1; and
performing statistical analysis on the expression level of said gene as compared to that expressed in normal biological samples comprising renal tissue or renal cells, wherein statistically down-regulated gene expression levels would indicate said individual has papillary or clear cell renal cell cancer.

2. The method of claim 1, wherein statistically down-regulated secreted frizzled related protein 1 gene expression levels would indicate said individual has papillary renal cell cancer.

3. The method of claim 1, wherein statistically down-regulated secreted frizzled related protein 1 gene expression levels would indicate said individual has clear cell renal cell cancer.