Background: Renal cell cancer (RCC) rates have been increasing and affect more men than women. The etiology of RCC is not known but based on our preliminary data and prior publication it is clear that estrogen- metabolizing genes (EMG) such as cytochrome P450 (CYP) 1B1 and catechol-O-methyltransferase (COMT) are involved in the pathogenesis of RCC. Single nucleotide polymorphisms (SNPs) of EMG have been shown to alter enzyme levels or catalytic activity and thus, may play a role in renal carcinogenesis. Research Objectives: The main goal of this project is to determine if polymorphisms of EMG are risk factors for RCC. We hypothesize that (1) EMG SNPs contribute to the pathogenesis of RCC, (2) EMG SNPs lead to enzyme levels and activities that are increased for CYP1B1 and decreased for COMT in RCC, and (3) Over- expression of COMT or silencing of CYP1B1 inhibit RCC through cell cycle arrest and apoptosis pathways. Project Design and Methods:
Aim #1. To investigate whether SNPs of EMG are risk factors for RCC. Blood and RCC tissue will be collected from patients as well as blood from age-matched, healthy male volunteers. The following experiments will be performed: (a) Determination of EMG promoter and missense SNPs in various stages and grades of RCC patient's blood by utilizing the techniques of sequence-specific PCR and direct genomic sequencing, (b) For comparison, determine EMG SNPs in blood samples from age-matched healthy controls, and (c) Determine whether EMG SNPs affect EMG expression in RCC tissues and correlates with stage and grade of cancer. Accomplishment of these experiments will determine if EMG SNPs are risk factors for RCC susceptibility and whether they're involved in cancer growth and progression.
Aim #2. To determine the effects of EMG SNPs on gene expression levels and enzyme activity. From the polymorphic variants screened in Aim #1, we will analyze the functional significance of these SNPs. The following experiments will be performed: (a) Development of EMG wild-type plasmids by cloning and generation of polymorphic variants using site-directed mutagenesis, (b) Transfection of promoter region constructs into renal cell lines and determine their effects on gene expression by using a luciferase reporter assay, (c) Transform missense constructs of EMG into E coli cells to express and purify altered proteins and determine their enzyme activity by using biochemical assays and HPLC. Accomplishment of these experiments will determine if EMG SNPs in the promoter region will lead to higher or lower expression levels and whether missense EMG enzymatic variants have increased or decreased catalytic activity.
Aim #3. To investigate the functional significance of EMG in human renal cancer cells. Since SNPs may alter levels of gene expression or activity in cells, we will determine the functional importance of EMG in renal cells. The following experiments will be performed: (a) Analyze EMG expression levels in normal and cancerous renal cell lines, (b) Either over-express or silence EMG in RCC cell lines based on constitutive levels, (c) Measure cell proliferation, apoptosis, cell invasion, and cell cycle distribution in EMG-treated renal cell lines by flow cytometry and biological assays, (d) Evaluate the effects of EMG on cell morphology and expression of various genes by cDNA microarray analyses, (e) Monitor growth characteristics of EMG-modified RCC cells in animal models. Accomplishment of these experiments will determine the functional significance of EMG in RCC and may implicate potential therapeutics for RCC. Clinical Relevance: Successful completion of the proposed research will generate important insights into the role of EMG and its polymorphisms in RCC etiology and progression. Such information can lead to the identification of Veterans with higher risk for RCC. Proposed research may also lead to implications for possible therapy for RCC.
The proposed research seeks to determine the role of EMG SNPs in RCC among Veterans. Based on the published literature, EMG and their byproducts are involved in the pathogenesis of RCC. However, the functional importance of EMG and their SNPs in RCC has not been reported. In this project, we plan to identify potential EMG SNP markers that may place individuals at higher risk for RCC and determine their effects on EMG expression and activity. Also, the functional significances of EMG in RCC are examined. Successful completion of the proposed research will generate important insight into the role of EMG SNPs in RCC etiology and progression, and serve as a step towards identifying risk factors for RCC susceptibility and their effects on cellular mechanisms. Also, results may lead to implicate potential therapeutics for RCC. Since the goal of this proposal is to determine genetic risk factors for RCC, we believe that the work proposed is highly relevant to Veterans health and the VA mission.
