CROSS-REFERENCE TO RELATED APPLICATION This is a continuation application under 35 U.S.C. §120 of pending nonprovisional application U.S. Ser. No. 10/938,973, filed Sep. 10, 2004, 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-tβ2 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF-β32 mRNA levels were not altered between normal and tumor matched samples.
FIG. 6 shows TGF-133 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 α receptor (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 α receptor (TBR3) expression (top row) in all tumors, loss of type II α receptor (TBR2) expression (middle row) in patients diagnosed with metastatic tumors, and no change in type I α receptor (TBR1) protein expression (bottom row).
FIG. 13 demonstrates down-regulation of TGF-3-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 α receptor (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 α receptor (TBR2) expression in renal tumor cell lines correlate with loss of TGF-3-regulated growth inhibitory and transcriptional responses. FIG. 15A shows cell proliferation was inhibited as assessed by DNA content 3 days after α treatment. Percent of each respective untreated control was used for comparisons. Transient transfection using 3TP/lx 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 α receptor (TBR3), type II α receptor (TBR2), and type I α receptor (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-β3 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 WIC® 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 III 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 TUBAS 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 IQGAPI 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 SMARCEI AB033007.1 KIAA1181
BE646386 EXO70 BG250721 N/A
A1972475 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 AMOTLI
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
AI142096 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 TNFRSF5
BF439533 N/A
TABLE 2
Genes With Down-Regulated Expression
In stage I Renal Cell Carcinoma
Genbank ID Gene Symbol
L38487 ESRRA
NM004415.1 DSP
NM005327.1 HADHSC
NM003321.1 TUFM
NM002084.2 GPX3
AI983043 N/A
NM006066.1 AKR1A1
NM006384.2 CIB1
NM001685.1 ATP5J
NM014652.1 IMP13
NM013410.1 AK3
NM016725.1 FOLR1
NM021151.1 CROT
NM005951.1 MT1H
NM005952.1 MT1X
AL080102.1 N/A
BC000931.2 ATP5C1
BC005398.1 DKFZP566D193
D87292.1 TST
AU151428 IDH2
BC000109.1 ILVBL
AF333388.1 N/A
NM005953.1 MT2A
BF217861 N/A
AA594937 COBL
AW052179 COL4A5
AI884867 LOC155066
BF246115 N/A
AW028110 KIAA0500
AW242315 N/A
AW080549 FUT3
AW149846 GPX3
AI038402 N/A
AI051046 MGC4614
AI659456 N/A
AW664964 N/A
AI631895 SGK2
AI263078 FLJ31168
BF057634 HOXD8
AA746038 GPR110
AK024386.1 GRHPR
AL109716.2 N/A
AK026411.1 ALDOB
M10943 N/A
AW088547 N/A
NM018049.1 GNRPX
NM017900.1 AKIP
NM006548.1 IMP-2
NM025135.1 KIAA1695
NM016458.2 LOC51236
NM022128.1 RBSK
NM015974.1 CRYL1
NM013333.1 EPN1
AA133341 C14orf87
AF226732.1 NPD007
AF265439.1 MRPS15
AI743534 DKFZP564B1162
AB042647.1 B29
AL522667 ORF1-FL49
BG255416 KIAA0114
AF308301.1 MRPS26
BE408081 N/A
AL521634 FLJ32452
BF203664 MGC14288
BE645551 MGC39329
AW193698 TGFBR3
BF540829 N/A
W72455 FLJ25476
AI457453 N/A
BF056892 N/A
AK024386.1 GRHPR
AL109716.2 N/A
AA442776 N/A
AI913600 N/A
AW771908 N/A
AI807887 N/A
AW102941 N/A
AW024656 N/A
AB002342 PRKWNK1
TABLE 3
Genes With Up-Regulated Expression
In stage II Renal Cell Carcinoma
Genbank ID Gene Symbol
NM006096.1 NDRG1
NM006098.1 GNB2L1
NM001780.1 CD63
NM003118.1 SPARC
NM000291.1 PGK1
NM003870.1 IQGAP1
AB032261.1 SCD
NM002629.1 PGAM1
NM003564.1 TAGLN2
NM000310.1 PPT1
NM003405.1 YWHAH
U82164.1 MIC2
NM002305.2 LGALS1
NM001096.1 ACLY
NM002121.1 HLA-DPB1
NM021038.1 MBNL
NM003651.1 CSDA
AV685920 CAPZA2
NM002654.1 PKM2
NM001175.1 ARHGDIB
BC000182.1 ANXA4
NM001153.2 ANXA4
NM001975.1 ENO2
NM006435.1 IFITM2
NM001387.1 DPYSL3
BG398414 RPA1
NM004039.1 ANXA2
NM005534.1 IFNGR2
AL136877.1 SMC4L1
NM014876.1 KIAA0063
NM024830.1 FLJ12443
NM005505.1 SCARB1
NM003025.1 SH3GL1
NM013285.1 HUMAUANTIG
NM005720.1 ARPC1B
AW157070 EGFR
NM002835.1 PTPN12
NM004428.1 EFNA1
AW006290 SUDD
NM014791.1 MELK
NM014882.1 KIAA0053
NM003864.1 SAP30
NM001558.1 IL10RA
NM003264.1 TLR2
NM014221.1 MTCP1
AV756141 CSF2RB
AI123251 LCP2
NM006433.2 GNLY
NM000861.2 HRH1
NM001870.1 CPA3
NM003586.1 DOC2A
NM004271.1 MD-1
NM014932.1 NLGN1
NM014947.1 KIAA1041
NM000647.2 CCR2
NM002562.1 P2RX7
NM006058.1 TNIP1
NM013447.1 EMR2
NM013416.1 NCF4
NM001776.1 ENTPD1
NM020037.1 ABCC3
NM006135.1 CAPZA1
NM007036.2 ESM1
AF034607.1 CLIC1
BC000915.1 PDLIM1
AL162068.1 NAP1L1
NM006947.1 SRP72
L12387.1 SRI
AF141349.1 N/A
AF263293.1 SH3GLB1
BC000389.1 TM4SF7
AF007162.1 CRYAB
D38616.1 PHKA2
AV717590 ENTPD1
U87967.1 ENTPD1
H23979 MOX2
AF063591.1 MOX2
BC005254.1 CLECSF2
BC000893.1 H2BFT
L22431.1 VLDLR
AI741056 SELPLG
AF084462.1 RIT1
U62027.1 C3AR1
M87507.1 CASP1
J04132.1 CD3Z
M31159.1 IGFBP3
AF257318.1 SH3GLB1
BC001388.1 ANXA2
AF130095.1 FN1
AF022375.1 VEGF
AA807529 MCM5
AK026737.1 FN1
X14355.1 N/A
AK025608.1 KIAA0930
AF183421.1 RAB31
NM002695.1 POLR2E
AF288391.1 C1orf24
NM003730.2 RNASE6PL
NM016359.1 ANKT
NM014164.2 FXYD5
NM022736.1 FLJ14153
NM021158.1 C20orf97
NM017792.1 FLJ20373
NM020142.1 LOC56901
NM016448.1 RAMP
NM005767.1 P2Y5
NM020169.1 LXN
NM022834.1 FLJ22215
NM018460.1 BM046
NM024629.1 FLJ23468
NM018641.1 C4S-2
NM018295.1 FLJ11000
NM024576.1 FLJ21079
NM016582.1 PHT2
NM003116.1 SPAG4
NM018454.1 ANKT
NM018099.1 FLJ10462
NM007072.1 HHLA2
NM022445.1 TPK1
AW173623 TDE1
AB044088.1 BHLHB3
AF043244.1 NOL3
AF133207.1 H11
AF313468.1 CLECSF12
AA191576 NPM1
AI765383 KIAA1466
BC003654.1 SLC27A3
W60806 N/A
AI335263 NETO2
AI378406 EGLN3
BC005400.1 FKSG14
AI761520 CENTA2
BC000771.1 TPM3
BC000190.1 HSPC216
BC002776.1 SEMA5B
AF132203.1 SCD
BC006107.1 ARHGAP9
AK024263.1 N/A
AK024846.1 SET7
BE878463 N/A
AW304786 PTR4
AI769269 N/A
AI935334 N/A
BF437747 SAMHD1
AW300953 N/A
H37811 N/A
AA603344 SAMHD1
AA742310 N/A
AI248208 FLJ25804
AI962367 ECGF1
NM002053.1 GBP1
NM000089.1 COL1A2
NM021105.1 PLSCR1
NM002467.1 MYC
NM001284.1 AP3S1
AI825926 PLSCR1
NM014736.1 KIAA0101
AF161461.1 LEPROTL1
NM014873.1 KIAA0205
AI005043 N/A
NM000416.1 IFNGR1
NM004172.1 SLC1A3
NM004207.1 SLC16A3
AI761561 HK2
Y09216.1 N/A
NM002922.1 RGS1
NM005990.1 STK10
NM014863.1 GALNAC4S-6ST
NM014737.1 RASSF2
NM000418.1 IL4R
BC000658.1 STC2
NM003751.1 EIF3S9
NM002339.1 LSP1
NM004604.1 STX4A
NM006404.1 PROCR
AF275945.1 EVA1
NM004221.1 NK4
NM004556.1 NFKBIE
NM004688.1 NM
NM003332.1 TYROBP
NM015136.1 STAB1
NM006019.1 TCIRG1
NM004877.1 GMFG
NM002317.1 LOX
NM025201.1 PP1628
NM014800.1 ELMO1
L41944.1 IFNAR2
NM007268.1 Z39IG
NM006994.2 BTN3A3
AF091352.1 VEGF
AB035482.1 ICB-1
Z24727.1 TPM1
M19267.1 TPM1
U13700.1 CASP1
M27281.1 VEGF
BC005838.1 N/A
BC005858.1 FN1
BC005926.1 EVI2B
BE513104 YARS
AU147399 CAV1
AK023154.1 HN1L
AK021757.1 KIAA0648
H95344 VEGF
AB023231.1 FNBP4
AL523076 N/A
NM030666.1 SERPINB1
AB018289.1 KIAA0746
AW043713 SULF1
BE880591 EP400
AU158495 NOTCH2
BE965029 N/A
AL564683 CEBPB
AA349595 RAB6IP1
AI809341 PTPRC
AW205215 KIAA0286
BE349017 HA-1
AF070592.1 HSKM-B
AI769685 CARS
AI935123 LOC113146
BG255188 N/A
AI088622 PRKCDBP
BE222709 N/A
AW007573 DKFZP586L151
BG332462 N/A
AI862658 FEM1C
AI934469 KIAA0779
AB018345.1 KIAA0802
W87466 LOC92689
BE908217 ANXA2
NM005615.1 RNASE6
BE300252 K-ALPHA-1
BF740152 MYO1F
AV711904 LYZ
AW072388 N/A
AW190316 SHMT2
NM005412.1 SHMT2
NM006417.1 IFI44
AL008730 C6orf4
L16895 LOC114990
Z21533.1 HHEX
AK022955.1 DKFZp762L0311
BF001267 N/A
AL558987 N/A
AA577672 LOC151636
BE620734 ZAK
AI937446 N/A
H99792 N/A
BE966748 N/A
AI659418 MGC21854
AI990891 DKFZp761K2222
AA827892 N/A
AL135264 N/A
AI375753 N/A
AA573502 TAP2
BG387557 CASP2
AA554833 MAP1B
AK026764.1 N/A
AU146532 PDK1
BE348597 N/A
AL577758 LOC133957
AI133452 FGG
AU157224 N/A
AI742057 N/A
BE500942 N/A
N25631 RFXANK
AU145366 N/A
AW270037 KIAA0779
BF526978 N/A
AW182575 N/A
BF339831 MGC13114
AI056992 N/A
BE222668 N/A
BG165011 N/A
AI188445 MGC14289
BE551416 HAK
AI972498 a1/3GTP
AW662189 N/A
AA142842 N/A
BF939473 N/A
AI681260 N/A
AA551090 AP1S2
AA045175 MS4A6A
W05495 N/A
AI093231 N/A
AI565054 N/A
AL553774 N/A
AK023470.1 MGC15875
AL157377 ENPP3
AL139109 TEX11
AK025631.1 POLH
AI873425 N/A
BF541967 N/A
AI686890 N/A
AI936034 ITGA4
U88964 ISG20
AJ243797 TREX1
D29642 KIAA0053
D87433 STAB1
AI129310 FLJ21562
TABLE 4
Genes With Down-Regulated Expression
In stage II Renal Cell Carcinoma
Genbank ID Gene Symbol
NM012248.1 SPS2
NM002300.1 LDHB
BC000306.1 HADHSC
NM001640.2 APEH
NM005875.1 GC20
NM003365.1 UQCRC1
BF031714 HYA22
NM005808.1 HYA22
AF113129.1 ATP6V1A1
NM002402.1 MEST
NM006844.1 ILVBL
NM004636.1 SEMA3B
NM002496.1 NDUFS8
NM006556.1 PMVK
NM004255.1 COX5A
NM002225.2 IVD
NM004524.1 LLGL2
AI950380 BCL7A
AB020707.1 WASF3
NM000481.1 AMT
NM012317.1 LDOC1
NM006456.1 STHM
NM006614.1 CHL1
NM015393.1 DKFZP564O0823
AV729634 DNAJC6
NM002628.1 PFN2
NM003500.1 ACOX2
NM002655.1 PLAG1
NM004393.1 DAG1
NM003026.1 SH3GL2
NM002010.1 FGF9
NM014033.1 DKFZP586A0522
NM004868.1 GPSN2
BC000649.1 UQCRFS1
S69189.1 ACOX1
AF153330.1 SLC19A2
AF094518.1 ESRRG
M55575.1 BCKDHB
BE044480 MGC32124
BF382393 N/A
AV751731 PNKP
U55984 N/A
BF059512 DNER
AK025934.1 Evi1
AL036088 SEMA6D
BE964222 FLJ38482
AW290940 N/A
AL545998 N/A
AW274874 N/A
AI709389 N/A
BF224092 MGC15854
AU145805 N/A
AW079843 MGC33338
AW138815 N/A
AW242286 N/A
AW025023 N/A
BE672659 N/A
AB019695.1 TXNRD2
M61900.1 PTGDS
BF967998 N/A
BF967998 N/A
AL526243 KIAA0446
NM000532.1 PCCB
BE042354 LDHB
AI587323 ATP5A1
AW195882 ATPW
H71135 ADH6
AV659180 ALDOB
AK027006.1 TNRC9
AV693216 PLXNB1
BG398937 N/A
NM002489.1 NDUFA4
NM003849.1 SUCLG1
NM014019.1 HSPC009
NM024952.1 FLJ20950
NM014185.1 MOG1
NM018013.1 FLJ10159
NM018373.1 SYNJ2BP
NM014067.2 LRP16
NM013261.1 PPARGC1
NM021963.1 NAP1L2
NM018658.1 KCNJ16
NM014553.1 LBP-9
AF112204.1 ATP6V1H
AU145941 CDC14B
AF061264.1 MGC4825
BF941492 FLJ10496
AI984229 HSPC121
N71923 FLRT3
BC005050.1 NICN1
AF172327.1 N/A
AF356515.1 HINT2
BE620739 RHOBTB3
BF435123 N/A
AW149498 BTBD6
AW024437 LOC118491
AW195353 N/A
BE044193 N/A
AI493303 FLJ31709
AI636080 N/A
BF509031 ATP6V1G3
AW242920 N/A
BF002046 ANGPTL1
BF130943 N/A
AW452631 N/A
AI792937 N/A
AI810572 N/A
BG165743 LOC112817
AW466989 N/A
R48991 N/A
BF029215 MSI2
D21851 LARS2
Z83838 ARHGAP8
TABLE 5
Genes With Up-Regulated Expression In both
stage I & stage II Renal Cell Carcinoma
Genbank ID Gene Symbol
NM005566.1 LDHA
NM000291.1 PGK1
NM001219.2 CALU
NM002966.1 S100A10
NM000034.1 ALDOA
NM002627.1 PFKP
NM006082.1 K-ALPHA-1
AI922599 VIM
NM020474.2 GALNT1
NM006406.1 PRDX4
NM015344.1 LEPROTL1
NM014755.1 TRIP-Br2
AI796269 NBS1
NM005783.1 APACD
BF197655 N/A
NM001233.1 CAV2
NM002845.1 PTPRM
NM014302.1 SEC61G
U47924 CD4
NM004106.1 FCER1G
NM015474.1 SAMHD1
NM004915.2 ABCG1
NM002432.1 MNDA
NM005565.2 LCP2
NM005531.1 IFI16
NM005849.1 IGSF6
NM002189.1 IL15RA
NM004353.1 SERPINH1
NM017760.1 FLJ20311
NM022349.1 MS4A6A
NM023003.1 TM6SF1
NM016184.1 CLECSF6
NM031284.1 DKFZP434B195
BC002342.1 CORO1C
AA775177 PTPRE
AL162070.1 CORO1C
AF253977.1 MS4A6A
AF237908.1 MS4A6A
W03103 DDEF1
AK022888.1 FENS-1
AI141784 N/A
NM014812.1 KIAA0470
AF208043.1 IFI16
BC002654.1 TUBB-5
BC006379.1 K-ALPHA-1
BC006481.1 K-ALPHA-1
AF000426.1 LST1
AF000424.1 LST1
BG500301 ITGB1
AL516350 ARPC5
M27487.1 HLA-DPA1
M27487.1 HLA-DPA1
AW517686 ATP2B4
AL581768 K-ALPHA-1
AA524505 TSGA
Z78330 ACTR3
Z78330 ACTR3
BG532690 ITGA4
AW005535 RAP2B
NM007161.1 LST1
AK026577.1 ALDOA
AI091079 SHC1
AV713720 LST1
NM021103.1 TMSB10
NM016337.1 RNB6
NM013260.1 HCNGP
NM021199.1 SQRDL
NM018149.1 FLJ10587
NM016951.2 CKLF1
AB033038.1 FLJ10392
AI184968 C1QG
AL161725 FLJ00026
NM018440.1 PAG
AL553942 FLJ31951
AI394438 N/A
T64884 N/A
T64884 N/A
AW511319 N/A
AI640834 RA-GEF-2
AI655467 N/A
AL161725 FLJ00026
T92908 N/A
TABLE 6
Genes With Down-Regulated Expression In Both
stage I And stage II Renal Cell Carcinoma
Genbank ID Gene Symbol
NM004092.2 ECHS1
NM000270.1 NP
NM002354.1 TACSTD1
AF017987.1 SFRP1
NM003012.2 SFRP1
NM000666.1 ACY1
NM000191.1 HMGCL
NM015254.1 KIF13B
NM000140.1 FECH
U75667.1 ARG2
NM000196.1 HSD11B2
NM014636.1 RALGPS1A
NM001441.1 FAAH
NM005978.2 S100A2
NM001678.1 ATP1B2
NM001099.2 ACPP
NM014731.1 ProSAPiP1
BF343007 N/A
NM000035.1 ALDOB
NM005950.1 MT1G
NM002371.2 MAL
NM006984.1 CLDN10
NM002567.1 PBP
NM000019.1 ACAT1
NM001692.1 ATP6V1B1
X77737.1 N/A
NM006226.1 PLCL1
NM000893.1 KNG
NM000412.2 HRG
NM001963.2 EGF
NM003361.1 UMOD
NM000050.1 ASS
NM001438.1 ESRRG
NM020632.1 ATP6V0A4
AI632015 SLC12A1
NM000701.1 ATP1A1
NM031305.1 DKFZP564B1162
AF130089.1 ALDH6A1
AK025651.1 N/A
W45551 MMP24
W67995 FXC1
AL136566.1 IBA2
AF105366.1 SLC12A6
AF284225.1 DMRT2
AA191708 N/A
AL355708.1 N/A
BE783949 FLJ10101
AL529672 N/A
AL568674 MYBBP1A
AU147564 CLMN
AK000208.1 N/A
AB051536.1 FLJ14957
AI569747 TFDP2
AK025562.1 N/A
AI660243 TMPRSS2
N50413 N/A
AI347918 N/A
AL536553 GRP58
BC000282.1 LOC89894
BF106962 FAM3B
AI051248 FLJ32115
AI928242 N/A
BG236006 N/A
AI653107 N/A
AI824037 FLJ25461
R61322 N/A
AW071744 KCNJ10
BF059276 N/A
BC002449.1 FLJ13612
J02639.1 SERPINA5
BC002571.1 DKFZP564O243
U03884.1 KCNJ1
AF173154.1 HYAL1
AF130103.1 PBP
AL117618.1 PDHB
AF063606.1 N/A
BC005314.1 N/A
BF686267 PBP
AI742553 PRKWNK1
D83782.1 SCAP
AB029031.1 TBC1D1
AK025432.1 KIAA0564
AL117643.1 N/A
AW772192 N/A
NM003944.1 SELENBP1
AL049977.1 CLDN8
AK023937.1 THEA
AK025084.1 TNRC9
X03363.1 ERBB2
AK026411.1 ALDOB
NM016026.1 RDH11
NM016286.1 DCXR
NM019027.1 FLJ20273
BG338251 RAB7L1
NM006113.2 VAV3
NM018075.1 FLJ10375
NM013271.1 PCSK1N
NM017586.1 C9orf7
NM016321.1 RHCG
NM025247.1 MGC5601
BC002449.1 FLJ13612
AI379517 N/A
AA058832 MGC33926
AW274034 N/A
AI580268 NUDT6
AI761947 DKFZP564B1162
AI793201 N/A
AK025898.1 N/A
AB046810.1 C20orf23
AK024204.1 N/A
BF594722 N/A
R88990 N/A
N73742 N/A
AI697028 FLJ90165
BF590528 N/A
AI733359 N/A
H20179 N/A
AA991551 MGC14839
AI758950 SLC26A7
AA911561 N/A
AI769774 N/A
AA669135 N/A
AW136060 SLC13A2
AI733593 N/A
BF739841 N/A
AA600175 N/A
BF477980 N/A
AI934557 N/A
BE326951 KNG
AI632567 N/A
BE300882 N/A
BE855713 N/A
AA485440 DBP
AA915989 FLJ10743
AA085764 SIGIRR
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-3/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-3-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 2.5
(TIEG)
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-t3 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 eceptor (+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-3.
The investigators then examined TGF-13-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 III 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-β receptors 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
NM_003505 FZD1
AL035683 B4GALT5
R56118 N/A
NM_014575 SCHIP1
AI694320 ZNF533
BC031322 N/A
BF346665 N/A
BC004283 LOC283639
AF302786 GNPTAG
AU121975 PAICS
NM_016315 GULP1
AL541302 SERPINE2
BG391217 C9orf80
NM_000700 ANXA1
N30188 N/A
NM_003651 CSDA
AI830227 FLII
U20350 CX3CR1
NM_005692 ABCF2
U34074 AKAP1
AB056106 TARSH
AU151483 CDH6
BC026260 TTC3
AL133001 SULF2
NM_003358 UGCG
NM_001282 AP2B1
AF322067 RAB34
NM_001540 HSPB1
N58363 STATIP1
AF072872 FZD1
BF247552 SLC38A1
X69397 CD24
BC000251 GSK3B
BF691447 B4GALT5
AB046817 SYTL2
AF255647 DKFZP566N034
BF344237 N/A
AW242720 LOC143381
AA115485 MGC3222
NM_006588 SULT1C2
NM_000546 TP53
N92494 JWA
W74580 MGC3222
AF131749 PSK-1
AW026491 CCND2
NM_012410 PSK-1
NM_002800 PSMB9
BF512748 JAK3
AA404269 PRICKLE1
M33376 AKR1C1
AF035321 DNM1
NM_002862 PYGB
AF132000 DKFZP564K1964
L07950 HLA-C
AF114011 TNFSF13
BF674052 VMP1
AI922599 VIM
AF044773 BANF1
NM_015925 LISCH7
NM_001684 ATP2B4
AI123348 CHST11
NM_001304 CPD
NM_006762 LAPTM5
NM_000211 ITGB2
AA995910 ALOX5
NM_018965 TREM2
AL353715 STMN3
BC019612 C20orf75
AF086074 N/A
NM_005045 RELN
AI935123 C14orf78
AL550875 C7orf28B
L27624 TFPI2
AL574096 TFPI2
AA005141 MET
D86983 D2S448
AW439242 C6orf68
AB000221 CCL18
NM_002121 HLA-DPB1
U17496 PSMB8
U05598 AKR1C1
BF342851 D2S448
BF311866 PTGFRN
NM_001449 FHL1
AA954994 N/A
Y13710 CCL18
BG170541 MET
AB037813 DKFZp762K222
D28124 NBL1
NM_021103 TMSB10
AI949772 N/A
AC004382 DKFZP434K046
NM_000248 MITF
NM_022154 SLC39A8
AI436813 N/A
AF007162 CRYAB
NM_015392 NPDC1
AL136585 DKFZp761A132
AB040120 SLC39A8
NM_138473 SP1
AU144387 182-FIP
NM_022763 FAD104
AI093231 APBB1IP
NM_000235 LIPA
AI817079 EXOC7
NM_004385 CSPG2
NM_024801 TARSH
BF218922 CSPG2
BF590263 CSPG2
NM_001233 CAV2
AB020690 PNMA2
AW188198 TNFAIP6
NM_007115 TNFAIP6
AI742838 DOCK11
AW117264 N/A
AF016266 TNFRSF10B
NM_013952 PAX8
AA771779 ZFP90
W72333 FLJ21657
H23979 MOX2
BG542521 PPM2C
AF063591 MOX2
BF247383 BMPR2
NM_005114 HS3ST1
BE466145 N/A
BC005352 TNFAIP8
AC002045 LOC339047
BC040558 D2LIC
U13699 CASP1
NM_002718 PPP2R3A
BF476502 MPPE1
BC034275 LOC253982
AF279145 ANTXR1
AV724216 NDRG4
BG165613 N/A
NM_018205 LRRC20
NM_022083 C1orf24
NM_006169 NNMT
AF141347 TUBA3
NM_000064 C3
AV710838 BCDO2
AI417917 EHD2
AI681260 LILRB1
NM_000389 CDKN1A
AF288391 C1orf24
NM_002627 PFKP
NM_001975 ENO2
NM_030786 SYNCOILIN
NM_006169 NNMT
AI417917 EHD2
NM_006868 RAB31
L03203 PMP22
AF199015 VIL2
AI873273 SLC16A6
NM_017821 RHBDL2
BF740152 MYO1F
AA954994 N/A
AI458735 MGC26717
NM_003254 TIMP1
AI688631 N/A
AK026037 N/A
BG327863 CD24
NM_016008 D2LIC
AI394438 LOC253981
AA947051 D2LIC
AI819043 N/A
AI378044 UGCG
NM_024576 OGFRL1
M76477 GM2A
NM_002214 ITGB8
AI879381 ADCK2
NM_000152 GAA
H15129 MEIS4
L42024 HLA-C
NM_002178 IGFBP6
AI761561 HK2
AA722799 DCBLD2
NM_003255 TIMP2
NM_000107 DDB2
AV699565 CTSC
NM_000861 HRH1
TABLE 9
Genes With Down-Regulated Expression In
stage I Papillary Renal Cell Carcinoma
Genbank ID Gene Symbol
AF232217 N/A
AI823572 MGC45438
AU154994 SLC13A3
AW979271 N/A
AF064103 CDC14A
AI524125 PCDH9
AI733474 GPR155
AI767756 HS6ST2
NM_000412 HRG
NM_021614 KCNN2
M13149 HRG
H17038 N/A
NM_002010 FGF9
AI635774 EMCN
AW007532 IGFBP5
NM_004070 CLCNKA
NM_014621 HOXD4
AI733593 N/A
NM_020632 ATP6V0A4
AI697028 FLJ90165
AA897516 PTGER4
NM_024307 MGC4171
J02639 SERPINA5
NM_000085 CLCNKB
AA058832 MGC33926
BF059276 N/A
BC043647 LOC284578
AL161958 THY1
AL121845 KIAA1847
AY079172 ATP6V0D2
AA928708 CYP8B1
H71135 ADH6
NM_000102 CYP17A1
Z92546 SUSD2
AL558479 THY1
BC005314 ALDOB
NM_173591 FLJ90579
BF510426 N/A
AF331844 SOST
X77737 SLC4A1
NM_004392 DACH1
BC001077 LOC87769
AA218868 THY1
BF478120 RECQL5
BC041158 CYP4A11
AI623321 MTP
AI796189 PAH
NM_021161 KCNK10
NM_000163 GHR
AL136880 ESPN
NM_024426 WT1
M61900 PTGDS
AW963951 SIAT7C
AW340588 MAN1C1
AI263078 SLC23A3
BF130943 PPAPDC1
AI732596 N/A
AA603467 ZNF503
R41565 N/A
AI951185 NR2F1
NM_002609 PDGFRB
NM_006984 CLDN10
BG413612 N/A
D64137 CDKN1C
AK026344 PEPP2
AI670852 PTPRB
AI693153 GABRB3
NM_001393 ECM2
N93191 PR1
BC005090 AGMAT
NM_000717 CA4
D38300 PTGER3
AI650260 N/A
BC024226 IFRG15
BC006294 DHRS10
NM_003039 SLC2A5
AI675836 SORCS1
NM_005276 GPD1
NM_014298 QPRT
M10943 MT2A
NM_005952 MT1X
NM_002450 MT1X
NM_002910 RENBP
BF246115 MT1F
AF078844 MT1F
AF170911 SLC23A1
AF333388 MT1H
NM_003500 ACOX2
AA995925 N/A
NM_001218 CA12
BF432333 FLJ31196
NM_001385 DPYS
NM_003052 SLC34A1
NM_000778 CYP4A11
AL136551 SESN2
NM_000792 DIO1
NM_016725 FOLR1
NM_019101 APOM
NM_014270 SLC7A9
AF124373 SLC22A6
NM_016327 UPB1
NM_024734 CLMN
NM_016527 HAO2
NM_003645 SLC27A2
AB051536 FLJ14957
NM_025149 FLJ20920
BC005939 PTGDS
AL574184 HPGD
NM_000161 GCH1
H57166 N/A
NM_000597 IGFBP2
NM_000790 DDC
NM_004668 MGAM
NM_021027 UGT1A6
AF348078 GPR91
NM_016347 NAT8
AF338650 PDZK3
BE221817 CNTN3
NM_004476 FOLH1
NM_004615 TM4SF2
NM_023940 RASL11B
AI742872 SLC2A12
BC001196 HS6ST1
AW195353 TFCP2L1
NM_003122 SPINK1
NM_144707 PROM2
AI653981 L1CAM
AI796169 GATA3
M96789 GJA4
N74607 AQP3
NM_014059 RGC32
AI572079 SNAI2
AI056877 N/A
NM_006206 PDGFRA
AW771314 MGC35434
NM_016955 SLA/LP
AI569804 LOC375295
NM_001584 C11orf8
BG261252 EVI1
NM_006226 PLCL1
NM_001172 ARG2
AL050264 TU3A
BC003070 GATA3
AL120332 MGC20785
NM_000459 TEK
AW242836 LOC120224
AI926697 Gup1
NM_000486 AQP2
AI870306 IRX1
AW264204 CLDN11
BF431989 THRB
AI459140 N/A
NM_001864 COX7A1
AI471866 SLC7A13
AI653107 NRK
NM_004466 GPC5
BF195936 KRT18L1
NM_022454 SOX17
AW299531 HOXD10
AL137716 AQP6
AI332407 SFRP1
AL565812 PTN
AI452457 LOC199920
AI281593 DCN
M21692 ADH1B
AI660243 TMPRSS2
AI754423 FLJ38507
AA759244 FXYD4
U75667 ARG2
NM_000930 PLAT
AF083105 SOX13
NM_013231 FLRT2
BI825302 PR1
NM_003012 SFRP1
AF138300 DCN
AU155612 N/A
BG435302 EBF
NM_005978 S100A2
NM_000900 MGP
AK026748 DKFZP761M1511
J03208 DBT
NM_002345 LUM
NM_006623 PHGDH
AF063606 my048
NM_001647 APOD
AI935541 N/A
NM_005558 LAD1
AW138125 PRODH2
NM_003877 SOCS2
AI768894 CGN
AW772192 N/A
AF094518 ESRRG
T40942 ANGPTL3
NM_001146 ANGPT1
AI242023 N/A
BF970431 N/A
NM_005670 EPM2A
AW071744 KCNJ10
AI928242 TFCP2L1
AI769774 LOC155006
AW274034 USP2
NM_004633 IL1R2
NM_003289 TPM2
BF512388 C10orf58
BC005830 ANXA9
NM_000362 TIMP3
NM_001438 ESRRG
AU146204 ENPP6
AA775681 FLJ23091
AI393205 ACY-3
AF017987 SFRP1
NM_005951 MT1H
NM_005950 MT1G
NM_021805 SIGIRR
AA557324 CYP4X1
BF528646 DKFZP564I1171
AW340112 LOC401022
R73554 IGFBP5
AI826437 N/A
AV720650 KIAA0888
AA780067 HS3ST3B1
NM_000640 IL13RA2
AI806338 TBX3
NM_003155 STC1
AA931562 N/A
AI694325 N/A
AF205940 EMCN
NM_001290 LDB2
NM_016242 EMCN
AW014927 CALB1
AI758950 SLC26A7
AK024256 KIAA1573
BF726212 ANK2
AI985987 SCNN1G
AW242408 UPP2
NM_000860 HPGD
BF447963 KIAA0962
BF941499 GPR116
AW242409 N/A
BF509031 ATP6V1G3
NM_000934 SERPINF2
BF248364 AF15Q14
AL534095 FLJ23091
NM_004929 CALB1
AI222435 N/A
NM_005397 PODXL
AI090268 N/A
AI300520 STC1
BC006236 MGC11324
NM_024609 NES
NM_002591 PCK1
NM_005410 SEPP1
AB020630 PPP1R16B
AF022375 VEGF
NM_016246 DHRS10
AA873542 SLC6A19
U95090 PRODH2
D26054 FBP1
AI732994 MGC13034
NM_000151 G6PC
AK025651 PNAS-4
AF161441 N/A
AF161454 APOM
NM_022129 MAWBP
AI733515 MGC52019
NM_001443 FABP1
AI433463 MME
AL049313 N/A
BF195998 ALDOB
NM_022829 SLC13A3
NM_000035 ALDOB
NM_007287 MME
NM_003399 XPNPEP2
NM_000196 HSD11B2
BF431313 N/A
NM_004844 SH3BP5
NM_003206 TCF21
AI311917 DPYS
AA843963 PRLR
NM_017753 PRG-3
NM_006633 IQGAP2
NM_001133 AFM
T90064 N/A
BF696216 N/A
NM_004413 DPEP1
Z98443 FLJ38736
NM_018456 EAF2
AW771563 N/A
NM_014495 ANGPTL3
AI074145 KMO
NM_000896 CYP4F3
NM_001072 UGT1A6
AI631993 N/A
NM_000277 PAH
M74220 PLG
AI935789 UMOD
NM_002472 MYH8
BC020873 CLCNKA
NM_000550 TYRP1
AA806965 BTNL9
NM_020163 LOC56920
NM_004490 GRB14
AA788946 COL12A1
AW242315 N/A
AI735586 LOC152573
R88990 N/A
NM_003278 TNA
NM_007180 TREH
AW173045 TBX2
U28049 TBX2
NM_001395 DUSP9
NM_000336 SCNN1B
U43604 N/A
BC029135 N/A
NM_005414 SKIL
BQ894022 PDE1A
NM_013335 GMPPA
NM_003221 TFAP2B
BF057634 HOXD8
AA523172 N/A
AF319520 ARG99
NM_002885 RAP1GA1
NM_003361 UMOD
NM_000142 FGFR3
NM_000893 KNG1
BC029135 N/A
NM_147174 HS6ST2
NM_000218 KCNQ1
U03884 KCNJ1
X83858 PTGER3
BF439270 N/A
AA911235 MST1
NM_000955 PTGER1
NM_022844 MYH11
BC042069 N/A
NM_005518 HMGCS2
NM_001963 EGF
AI632015 SLC12A1
AF339805 N/A
BF106962 FAM3B
NM_005019 PDE1A
AU146305 PDE1A
NM_000663 ABAT
AU119437 LOC144997
BC036095 DRP2
R49295 N/A
AI623202 PRDM16
AW452355 N/A
AA563621 HSPB6
X15217 SKIL
AK095719 N/A
AI056187 N/A
AI668598 N/A
AI700882 SLC13A3
NM_000963 PTGS2
AW051712 N/A
AL832099 MGC33190
AK057337 LOC145820
AW300204 SLC30A8
NM_005856 RAMP3
AI458003 CYYR1
AK026877 N/A
AI632567 N/A
U91903 FRZB
AF352728 FLJ12541
BM128432 IGFBP5
NM_003102 SOD3
BE676272 TACC1
AI692180 PPFIBP2
AL544576 LOC92162
NM_017688 BSPRY
AU146310 N/A
AI912976 RASGRF2
U83508 ANGPT1
L47125 GPC3
NM_000663 ABAT
TABLE 10
Genes With Up-Regulated Expression In Benign Oncocytoma
Genbank ID Gene Symbol
NM_005114 HS3ST1
AA650558 GNAS
BF062244 LIN7A
NM_030674 SLC38A1
NM_014766 SCRN1
BC002471 CPLX1
AF183421 RAB31
AK022100 KIAA0256
BF508244 AKR1C2
BG772511 N/A
AB037848 SYT13
AK055769 N/A
T58048 N/A
NM_012105 BACE2
AA992805 LEF1
AK026420 DMN
NM_024812 BAALC
AI057226 N/A
AW138767 ELOVL7
NM_013233 STK39
AF178532 BACE2
AI521166 LOC283104
AA005023 NOD27
AV725364 GPRC5B
AW195581 GPSM2
BG503479 B4GALT6
BF031829 DSG2
AW975728 SLC16A7
NM_022495 C14orf135
AA703159 N/A
BF247552 SLC38A1
NM_001673 ASNS
NM_024622 FLJ21901
AI565054 N/A
AW058459 LOC134285
NM_001233 CAV2
BC036550 N/A
BE464483 N/A
NM_002512 NME2
AF178532 BACE2
TABLE 11
Genes With Down-Regulated Expression In Benign Oncocytoma
Genbank ID Gene Symbol
BF593625 SYK
AI310001 FLJ22789
NM_006206 PDGFRA
NM_003740 KCNK5
AW138125 PRODH2
NM_000336 SCNN1B
BC005314 ALDOB
AI796189 PAH
NM_013363 PCOLCE2
NM_004466 GPC5
AI627531 N/A
U28055 MSTP9
NM_152759 MGC35140
AW052159 N/A
NM_017712 PGPEP1
AI961231 TOX
AI767962 BNC2
AF350881 TRPM6
AU146418 N/A
BE875072 N/A
AI653981 L1CAM
AI634662 SLC13A3
NM_000486 AQP2
AW206292 AQP2
AI572079 SNAI2
AI694118 N/A
NM_000142 FGFR3
U78168 RAPGEF3
AI913600 UNQ846
W93847 MUC15
NM_004616 TM4SF3
AI935789 UMOD
NM_007180 TREH
AL110152 CD109
AW051599 N/A
AI796169 GATA3
AF017987 SFRP1
BE550027 DKFZp761N1114
AA535065 KIAA1847
NM_003361 UMOD
AI263078 SLC23A3
M13149 HRG
AF278532 NTN4
AI632015 SLC12A1
NM_000412 HRG
NM_000893 KNG1
BG398937 KNG
AL049977 CLDN8
N74607 AQP3
AW071744 KCNJ10
AW015506 AQP2
AI927000 SOSTDC1
AI471866 SLC7A13
NM_001099 ACPP
NM_005074 SLC17A1
AA995925 N/A
AF352728 FLJ12541
BF343007 TFAP2A
NM_016929 CLIC5
AA911235 MST1
AA639753 N/A
NM_004887 CXCL14
AW771565 AIM1
AI264671 N/A
BF510426 N/A
AV728958 TLN2
T90064 N/A
AA218868 THY1
NM_003104 SORD
AJ292204 AGXT2
AI056359 MAPT
AL568422 DZIP1
AF339805 N/A
NM_000163 GHR
AI042017 NPL
AW340457 N/A
BF431199 DEHAL1
BF432254 MGC15937
AI368018 GPD1
AF144103 CXCL14
NM_016725 FOLR1
NM_000050 ASS
AA693817 N/A
NM_004929 CALB1
NM_000592 C4A
AL574184 HPGD
AA676742 DMGDH
AI631993 N/A
AI566130 AK3
AW024233 GLYAT
AA873542 SLC6A19
AK026966 AK3
NM_022829 SLC13A3
NM_005950 MT1G
AV700405 MGC52019
AI733515 MGC52019
NM_000860 HPGD
U95090 PRODH2
NM_001385 DPYS
BG401568 SLC16A9
NM_000846 GSTA1
BF195998 ALDOB
NM_004413 DPEP1
NM_000151 G6PC
NM_006744 RBP4
NM_013410 AK3
NM_000035 ALDOB
AK026411 ALDOB
AL135960 CYP4A11
M74220 PLG
NM_001713 BHMT
AW614558 SLC39A5
Z92546 SUSD2
NM_000778 CYP4A11
NM_000792 DIO1
AI222435 N/A
D26054 FBP1
AW025165 SLC22A8
NM_007287 MME
AW274034 USP2
NM_147174 HS6ST2
AA074145 PRODH
AL049176 CHRDL1
NM_020353 PLSCR4
NM_024803 TUBAL3
D16931 ALB
NM_019076 UGT1A10
AF138303 DCN
D13705 CYP4A11
NM_000587 C7
R49295 N/A
NM_000385 AQP1
AI669229 RARRES1
U36189 C5orf13
AL110135 FLJ14753
AW271605 N/A
BF358386 N/A
NM_016270 KLF2
AA905508 LOC128153
NM_021630 PDLIM2
AA915989 TBC1D13
AL565812 PTN
AI990790 N/A
BC041158 CYP4A11
NM_138474 N/A
NM_002899 RBP1
AK024256 KIAA1573
AW779672 SLC17A1
NM_021161 KCNK10
BF196891 TPMT
AY028896 CARD10
NM_018456 EAF2
NM_017806 FLJ20406
X59065 FGF1
AI650353 DACH1
AW771563 N/A
BF431313 N/A
NM_000896 CYP4F3
BC005090 AGMAT
U24267 ALDH4A1
AI090268 N/A
AW014927 CALB1
AL023553 PIPPIN
AL049313 N/A
AK021539 NCAG1
AI220117 MGST1
NM_020300 MGST1
NM_022568 ALDH8A1
BE874872 FAM20C
NM_004668 MGAM
BF033242 CES2
BC004542 PLXNB2
NM_000204 F
NM_004525 LRP2
AA442149 MAF
NM_000049 ASPA
AI830469 TFEC
NM_003759 SLC4A4
AF169017 FTCD
AF170911 SLC23A1
AA865601 LOC123876
AA863031 MGC32871
AW136060 SLC13A2
NM_003041 SLC5A2
NM_021924 MUCDHL
AW299568 N/A
AI927941 N/A
AI433463 MME
AL365347 SLC7A8
AA502331 PRAP1
NM_024709 FLJ14146
AF289024 FTCD
NM_017614 BHMT2
NM_016347 NAT8
NM_000277 PAH
NM_000316 PTHR1
NM_001091 ABP1
NM_000790 DDC
BF217861 MT1E
BF447963 KIAA0962
NM_001081 CUBN
NM_018484 SLC22A11
AW192692 N/A
BF000045 TINAG
BC005830 ANXA9
NM_025257 C6orf29
NM_020973 GBA3
NM_001977 ENPEP
AI632692 N/A
BI825302 PR1
L12468 ENPEP
AL571375 SCD4
AL136858 ZMYND12
NM_024027 COLEC11
NM_014934 DZIP1
BG496631 FBI4
NM_018265 FLJ10901
AI770035 UPB1
AF177272 UGT2B28
NM_004392 DACH1
N95363 CDKN1C
AF261715 FOLH1
NM_000042 APOH
NM_001393 ECM2
R88990 N/A
AA557324 CYP4X1
AF116645 ALB
BC015993 MGC27169
AL558479 THY1
NM_000785 CYP27B1
AW051926 AMN
AA928708 CYP8B1
BE407830 KIFC3
AI431643 RRAS2
AF001434 EHD1
BC005894 FMO2
NM_006798 UGT2A1
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).
