Genistein, microRNAs and kidney cancer

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DESCRIPTION (provided by applicant): Background: Renal cell carcinoma (RCC) is one of the most common malignancies among our Veterans. The major barrier to progress in the management of RCC is the high toxicity of the drugs used for the treatment. The present proposal will help to address this problem by utilizing dietary methods for the management of RCC using in vitro and in vivo models. The rationale for this project is that recent studies have shown significant effects of diet on modulation of miRNAs using both in vitro and in vivo models. However, such studies are lacking in kidney cancer. Research Objectives: The main goal of this project is to investigate whether genistein can inhibit kidney cancer growth through activation of tumor suppressor microRNAs (miRNAs) using both in vitro and in vivo models. We hypothesize that genistein can activate a set of tumor suppressor miRNAs thereby regulating kidney cancer progression through two different pathways. First, genistein induced tumor suppressor miRNAs can repress oncogene expression by binding to the 3' untranslated region of mRNA (3'UTR). Second genistein induced tumor suppressor miRNAs can activate the transcription of tumor suppressor genes by binding to the 5' upstream region of the gene or activate translation by binding to the 5' untranslated region of mRNA (5'UTR). Project Design and Methods: Specific Aim #1. To investigate whether genistein mediated inhibition of kidney cancer is through activation of tumor suppressor miRNAs. Under this specific aim, we will treat kidney cancer cell lines with genistein and then analyze whether genistein mediated inhibition of kidney cancer cells is through activation of tumor suppressor miRNAs. To investigate the functional significance of these tumor suppressor miRNAs, we will activate these miRNAs by genistein treatment and then target genes of these miRNAs will be analyzed.The miRNA-mediated repression of oncogenes or activation of tumor suppressor genes will be analyzed by luciferase assays using 3'UTR or 5'UTR sequence constructs, respectively. Alterations in cell growth will be assessed by monitoring cell proliferation, cell cycle distribution, apoptosis and in vitro invasion. Assays include cell proliferation, flow cytometry, migration, clonogenic survival, in vitro invasion and apoptosis assays. Specific Aim #2: To investigate the molecular mechanisms of genistein mediated activation of tumor suppressor miRNAs in kidney cancer cells. We will investigate epigenetic mechanisms of genistein mediated activation of tumor suppressor microRNAs in kidney cancer cells. We will treat kidney cancer cells with genistein and then analyze hypomethylation of CpG Islands in putative promoter regions of miRNAs using sodium bisulfite methylation techniques and direct DNA sequencing. We will investigate whether the mechanisms of activation of these tumor suppressor miRNAs are through modulation of histone acetylation and chromatin remodeling. The expression and activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in miRNA transfected kidney cancer cells will be analyzed. Specific Aim #3: Investigate whether genistein mediated activation of tumor suppressor miRNAs can inhibit growth and proliferation of kidney tumors in a nude mouse model. We will investigate whether genistein mediated activation of miRNAs can inhibit growth and proliferation of kidney tumors in nude mice. Kidney cancer cells will be injected sub-cutaneously (xenograft) or orthotopically (kidney) into nude mice treated with genistein and tested for their growth and progression compared to controls. Clinical Relevance: The main goal of this project is to investigate the role of genistein in the inhibition of RCC through activation of tumor suppressor miRNA. This information can lead to novel strategies for treatment and management of RCC. Since the goal of this proposal is to investigate the dietary mediated inhibition of RCC through activation of tumor suppressor miRNAs, we believe that the work proposed is highly relevant to Veterans health and the VA mission.