Mechanistic Studies of Renal Cell Carcinoma: from Rhoa-Mediated Expression of Cd44-Targeting Micrornas to the Development of Biomarkers and Therapies

Project: A - Government Institutionb - Ministry of Science and Technology

Description

Renal cell carcinoma (RCC) is highly vascular and metastatic neoplasm, and early diagnosis can significantly increase survival rate. We aim to untangle the mechanism underlying the onset and progression of RCC from epithelial to mesenchymal transition (EMT) to renal tumors, with particularly focuses on an inverse relationship of RhoA and CD44, and CD44-targeting and other significantly expressed micro (mi) RNAs, and to seek for reliable RCC miRNA biomarkers and therapeutic intervention. We observed that reduced RhoA and upregulated CD44 expression are critical for transformation and tumorigenesis in various RCC cells (RCCs) and in human renal epithelial cells (HREs)-treated with 3-methylcholanthrene (3MC; an AhR agonist, carcinogen). We have characterized the specific features of RCCs, such as reduced RhoA, upregulated CD44, and increased EMT markers (i.e., N-cadherin, vimentin, fibronectin and Slug), and increased cell proliferation, migration and invasion. The adverse effects can be restored in RCCs with the gain-of-function of RhoA by overexpressing constitutive active (CA)RhoA. In year 1, we aim to explore the effect of a reciprocal relationship of reduced RhoA and increased CD44 expression on RCC onset and metastasis. Because HREs exposed to 3MC exhibited similar features as RCCs, this cell model is developed to reveal the mechanism of how RhoA is inhibited and what its relations with the key molecules (i.e. Von-Hippo Lindow (VHL), and HIF-1a) of RCC are. We demonstrate that 3MC-mediated RhoA inhibition are due to HDAC1/3 upregulation and altered p190RhoGAP/p190RhoGEF expression, because similar to SAHA (a HDAC inhibitor), simvastatin rescued RhoA function by downregulating HDAC1/3 and corrected p190RhoGAP/p190RhoGEF levels. Therefore, therapeutic intervention of RhoA restoration (i.e., CARhoA, calpeptin and simvastatin), and CD44 inhibition (i.e., neutralizing antibody (HCAM), siCD44, and target miRNA mimics) will be examined. In year 2, we hypothesize that RhoA reduces CD44 expression through miRNA expression, because the inverse relationship of RhoA and CD44 is independent on the presence of actinomycin D, cycloheximide and MG132. Thus, we aim to seek for the differential upregulated CD44-targeting miRNAs from miRNA microarray analysis of HREs or RCCs transfected with CARhoA versus those with DNRhoA, with QPCR validation in RCCs or patient specimens. Additionally, the therapies used in Year 1 will be validated in RCC nude mice model with subcutaneous and orthotopic xenograft of RCC cell lines. In year 3, conversely, we seek for differentially downregulated miRNAs in RCCs transfected with CARhoA, but upregulated in RCCs overexpressing DNRhoA, to develop reliable RCC miRNA biomarkers for an early non-invasive detection (i.e., patients’ urine). After integrating the results of miRNA and gene array analysis, the gene targets of these miRNAs will be identified and functionally annotated in their involvement of RCC progression. Whether reduced ratio of RhoA to CD44, and increased extent of AhR agonist exposure correlate with high grades of RCC progression will be determined by IHC staining or AhR transactivational activity using dioxin-responsive element (DRE)-driven luciferase assay in patient specimens. Furthermore, another advanced RCC nude mice model derived from xenograft transplantation of human RCC primary cells will be developed to ultimately validate the therapeutic efficacies. Altogether, we intend to shed light on the mechanisms underlying the onset and progression of RCC in relation to a reciprocal relationship of RhoA and CD44, and AhR agonist exposure, and to identify CD44-targeting and other significantly altered miRNAs, and to develop reliable RCC miRNA biomarkers. We anticipate coming up with feasible RCC therapeutic approaches in the future.
StatusFinished
Effective start/end date8/1/167/31/17