Metabolic Advantages Of Urea Cycle Misregulation In Renal Cell Carcinoma

Background: The urea cycle is the central pathway involved in the detoxification of free ammonia species in mammals, which takes place at two sites: the liver and the kidney. Renal cell carcinoma is the most common type of kidney malignancy and the main subtype, clear cell renal cell carcinoma (ccRCC), makes up 75% of these cancers. ccRCC is distinguished by both radio- and chemo-resistance and treatment options for this disease are limited. Argininosuccinate synthase 1 (ASS1) and arginase II (ARG2) are two key enzymes in the urea cycle, the former being responsible for the synthesis of arginine while the latter hydrolyzes arginine to ornithine in the last step of the cycle. We have shown that in vitro, both normal kidney and ccRCC cell lines require exogenous arginine for growth and this suggests that increased arginine availability contributes to the pathogenesis of ccRCC. In this study, we measured expression changes of ASS1 and ARG2, as well as other enzymes involved in arginine metabolism, in ccRCC versus normal kidney tissue from patient samples to determine the metabolic advantages of an altered urea cycle. Our findings highlight the applicability of targeting cancer metabolism of a specific nutrient to reduce the proliferative capacity of ccRCC tumors. Methods: This study utilized both normal kidney and ccRCC cell lines to analyze the expression levels of four enzymes involved in arginine metabolism and the urea cycle. These results were confirmed in matched normal and ccRCC primary patient samples obtained from the Cooperative Human Tissue Network (CHTN, n=26) using both qPCR analysis for mRNA and immunohistochemistry for the levels of protein in these tissues. Moreover, our results were validated on a large-scale analysis of gene expression in ccRCC tumors via bioinformatic analysis of data available from The Cancer Genome Atlas (TCGA, n=480). We are currently manipulating the levels of ARG2 and ASS1 in normal kidney and ccRCC cell lines to elucidate metabolic advantages in proliferation and colony formation. Furthermore, we are working toward metabolomic analysis of primary patient samples of ccRCC tissue to discover additional altered metabolites of the urea cycle in this setting. Results: The expression levels of ASS1 and ARG2 are decreased 4-fold and 7-fold, respectively, based on analysis of ccRCC TCGA data. Additionally, analysis of matched tissue obtained from the CHTN mirrors this data, with the majority of ccRCC samples (>85%) under-expressing at least one of these enzymes. Moreover, the arginine metabolizing enzyme glycine amidinotransferase is 3-fold under-expressed and the arginine transporter CAT-1 is 10-fold over-expressed in ccRCC. Cumulatively, these data point to an altered metabolism of arginine in ccRCC, with the modified enzyme levels leading to an increased level of arginine to support growth and biosynthesis of ccRCC tumors. Conclusion: Our data demonstrate that arginine metabolism is altered in ccRCC tumors versus normal kidney tissue and that this increased arginine availability likely enables sustained proliferation of the tumor tissue. This study highlights the applicability of targeting cancer metabolism of a specific amino acid, arginine, as a possible therapeutic intervention for ccRCC. Citation Format: Joshua Ochocki, Nan Lin, Bo Qiu, M. Celeste Simon. Metabolic advantages of urea cycle misregulation in renal cell carcinoma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C32.