Abstract 4364: Oncometabolite L-2-hydroxyglutarate blocks differentiation of renal proximal tubule cells in matrigel

Abstract

Abstract Previously, the Sudarshan laboratory has identified elevations in the level of the oncometabolite L-2-hydroxyglutarate (L-2HG) in human renal cell carcinomas (RCCs). Of particular interest in these regards, both L-2HG inhibits 2-oxoglutarate-dependent dioxygenases (2-OGDs), including histone demethylases, which regulate the epigenetic landscape of cells, and cellular differentiation. Thus, it was of interest to determine whether the accumulation of L-2HG alters the differentiation of the normal renal proximal tubule (RPT), ultimately affecting the phenotype of the RCC. Towards these ends, L-2HG dehydrogenase (L2HGDH) was knocked down in primary cultures of normal rabbit RPT cells, and their capacity for cellular differentiation was examined. Initially, we examined the ability of primary RPT cells to form tubules in matrigel following an L2HGDH knockdown (KD). While tubulogenesis was stimulated by Epidermal Growth Factor (EGF) in primary RPT cells transduced with a control lentiviral vector, tubulogenesis in matrigel was dramatically impaired in primary RPT cells when L2HGDH was knocked down by lentiviral L2HGDH shRNA. In order to determine whether the expression of differentiated transport functions was affected, RealTime PCR (RTPCR) was conducted. The results indicated that an L2HGDH knockdown (80%) resulted in a reduction in the expression of the Na+/Pi cotransporter NaPi2a (81%), the Na+/glucose cotransporter SGLT2 (88%), the water transporter Aquaporin 1 (AQP1) (95%), and the Na,K-ATPase ß1 subunit (atp1b1) (43%), whereas the expression of the p-Aminohippurate transporter OAT1 was not significantly affected. Similar results were obtained when using L2HGDH siRNA and lentiviral shRNA. Not only is tissue architecture critical for the maintenance of functional differentiation, but alterations in tissue architecture are a necessary component of tumor formation. Thus, we are examining the underlying causes for the reduced tubulogenesis in matrigel cultures with an L2HGDH KD. In our initial studies, we examined the expression of differentiated transport functions in matrigel vs. monolayer cultures. Notably, while the expression such transporters as AQP2 increased dramatically in matrigel (as opposed to monolayer cultures), the expression of other transporters such as NaPi2a, SGLT2 and atp1b1 was affected to a much smaller extent. Of particular interest in these regards, is the known role of AQP1 in the migration of renal proximal tubule cells, tumor spread, and wound healing. We are currently studying the effects of an L2HGDH KD on the expression of such genes as AQP1 during tubulogenesis in matrigel.This work has been funded by Grant # 1RO1CA200653-01A1 to Dr. Sunil Sudarshan (PI) with a subaward to Dr. Mary Taub. Citation Format: Mary L. Taub, Sunil Sudarshan. Oncometabolite L-2-hydroxyglutarate blocks differentiation of renal proximal tubule cells in matrigel [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 4364.