Abstract 4363: Effects of the small GTPase RhoC on inflammatory breast cancer metabolism

Abstract

Abstract Inflammatory breast cancer (IBC) is an extremely aggressive and rare form of cancer that disproportionally affects African American and younger women. While IBC represents 1-5% of breast cancers, it accounts for 10% of all breast cancer deaths annually in the United States. We have previously shown that the metabolic characteristics of IBC, specifically in the triple negative (TN) and inflammatory breast cancer cell line SUM149, are significantly altered from those of normal breast cancer cells. We have shown previously that RhoC, a member of the Ras superfamily of GTPases, contributes to the metastatic IBC phenotype and acts as a regulator of the metabolite N-acetyl aspartate (NAA) in SUM149 cells. NAA is the second most abundant metabolite in the brain (only exceeded by glutamate) and has been used for the diagnosis of neurodegenerative disorders such as the fatal genetic disorder Canavan’s disease, which is characterized by toxic NAA levels due to a deleterious mutation in aspartoacyclase(ASPA). ASPA is the enzyme involved in the catabolism of NAA into aspartate and acetate. NAA has previously been considered a brain specific metabolite and the fundamental role of NAA outside of the central nervous system, especially in the context of cancer, remains elusive but highly intriguing. We have previously shown that NAA levels are significantly higher in the IBC-derived TN SUM149 cells than TN cancer cell line MDA-MB-231. Interestingly, and independently, recent studies have also implicated high levels of tumoral NAA with significantly worse survival rates in ovarian cancer patients. The synthesis of NAA from acetyl-CoA and aspartate is catalyzed by the enzyme Asp-NAT, which is encoded for by the gene NAT8L. Knockdown of RhoC in SUM149 cells using shRNA significantly decreases both NAT8L expression and NAA metabolite levels. To further understand RhoC’s role in the modulation of NAA, we generated RhoC and RhoA knockout cell lines using CRIPSR-Cas9 in a set of inflammatory and noninflammatory breast cancer cell lines. RNA-seq analysis of these show that SUM149 RhoC knockout cells produce the largest amount of differentially regulated genes when compared to wild-type cells. The differentially regulated genes include many of the key genes involved in NAA associated pathways. Furthermore, metabolic studies reveal that the most downregulated metabolites in the SUM149 RhoC knockout cell line are N-acetyl derivates including NAA, N-acetylglutamate(NAG), and N-acetylaspartylglutamate (NAAG). Overexpression and siRNA cell lines for both NAT8L and ASPA have been generated to further elucidate the altered metabolism seen in these cells. Additional molecular studies are ongoing to determine the specific role of NAA in the adapted metabolic pathways of inflammatory breast cancer that may shed light on IBC’s increased metastatic potential and decreased survival. Citation Format: Laura E. Goo, Joel A. Yates, Andrew C. Little, Daniel Kremer, Ilya Kovalenko, Christopher R. Oliver, Trisha Westerhof, Zhifen Wu, Nathalie Vandecan, Liwei Bao, Peter J. Ulintz, Costas A. Lyssiotis, Sofia D. Merajver. Effects of the small GTPase RhoC on inflammatory breast cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4363.