Abstract 1047: Role of endocytosis in NM23 mediated motility suppression
Authors: Imran Khan, Patricia S. Steeg
Journal: Cancer Research (2017)

Abstract NM23 has been identified as a metastasis suppressor gene and is known to inhibit motility of cancer cells and suppress metastasis in multiple in vivo model system. Biochemically, NM23 is a nucleoside diphosphate kinase (NDPK). As an NDPK, NM23 is first autophosphorylated on its histidine 118 before transferring its phosphate to an NDP. NM23 is also known to trans-phosphorylate other proteins to act as a protein histidine kinase (PHK) using same histidine H118 phosphorylation intermediate. In spite of many studies on NM23 in different cancers, the molecular mechanism of NM23 action has not been well worked out. Studies on Drosophila homologue of the NM23, awd, highlights its role in regulating cell differentiation and motility by influencing endocytosis. We now hypothesize that NM23 mediated motility/metastasis suppression could be due to increased endocytosis of cell surface receptors, needed for cellular motility. We over expressed NM23 (human H1 and H2 and murine h1) in MDA-MB-231T breast cancer cells and observed that the motility suppression phenotype (4 fold, p&amp;lt;0.0001) of NM23 correlates with increase in NM23 histidine auto phosphorylation, as observed by using a new anti-histidine 1-phosphate (1-pHis) antibody. NM23 over expressing cells were studied for the level of endocytosis using Transferrin-Alexa-594 and pHrodo-Red-EGF labelled dyes. It was observed that NM23 over expression causes increased endocytosis of both the above labelled ligands. Based on the literature, Dynamin is known to be involved in endocytosis and is also an interacting partner of NM23. We validated NM23 and Dynamin interactions by pulldown studies (using both anti-NM23 and anti-Dynamin antibodies) in NM23 over expressing cells. We also observed reduced endocytosis with Dynamin inhibitors. Further, when a series of Dynamin inhibitors (MiTMAB, OcTMAB, Dynole-34-2 and Iminodyn-22) were used in motility assays, they were found to be inhibitory to motility. However, no further decrease in motility was observed upon NM23 overexpression, highlighting the reversal of NM23 mediated motility suppression in absence of Dynamin. To further address the role of endocytosis in NM23 mediated motility suppression, two crucial mutants of NM23 namely; P96S (has NDPK activity with very low HPK activity) and H118F (has no NDPK and HPK activity) were made and stable cells were generated. We observed that the motility suppression phenotype of NM23 was reverted to normal in both the mutants, highlighting a possible role of HPK activity in motility suppression. Similarly, endocytosis studies on these cells shows loss of increased endocytosis in both the mutants. To date, the data indicate that NM23 phosphohistidine levels, motility suppression and increased endocytosis are well correlated and is being influenced by Dynamin signaling. Different approaches to further understand the physiological meaning of NM23-dynamin interactions are underway. Citation Format: Imran Khan, Patricia S. Steeg. Role of endocytosis in NM23 mediated motility suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1047. doi:10.1158/1538-7445.AM2017-1047