Abstract 728: Enhancement of survival and migration of neutrophils by tumor associated a2 isoform vacuolar ATPase
Authors: Ibrahim Safaa A. , Kulshrestha Arpita , Jaiswal Mukesh K. , Katara Gajendra K. , Amin Magdy A. , Beaman Kenneth D. 
Published: July 2016

Tumors associated neutrophils (TAN) regulate the tumor environment by directly promoting tumor progression and represent a potential therapeutic target for cancer treatment. Identifying tumor associated factors that promote neutrophil recruitment and survival will uncover new targets for cancer therapy. In tumor cells, a secreted peptide from the N­terminal domain of a2­ isoform vacuolar ATPase (a2NTD) promotes the protumorigenic properties of neutrophils which in turn, enhance tumor progression. In order to investigate the regulatory role of a2NTD on neutrophils survival, we stimulated freshly isolated neutrophils from healthy volunteers with recombinant a2NTD. a2NTD treatment delayed apoptosis in neutrophils and led to 2.1 fold increase in the percentage survival (P &lt; 0.001). In addition, a2NTD treated neutrophils (a2Neuφ) showed a significant decreased gene expression of caspase-3, -6, -7 and -8, that was associated with decreased activity of these caspases. Further analysis of the apoptosis related molecules revealed that a2NTD treatment upregulates the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL, c-FLIP, G-CSF and downregulates the gene expression of pro-apoptotic factors; BAX and Apaf-1. Since NF-kB pathway is an important regulator of apoptosis, here we showed by immunofluorescence analysis that a2NTD induces NF-kB p65 activation in neutrophils by nuclear translocation in a reactive oxygen species generation dependent manner. Additionally, treatment of a2Neuφ with specific NF-kB inhibitor; parthenolide showed a significant abrogation in neutrophil survival as well as downregulation of the gene expression of Bcl2-A1, Bcl-xL, G-CSF and upregulation of BAX. Suggesting that a2NTD enhances neutrophil survival by activating NF-kB pathway in neutrophils. Interestingly, live imaging revealed that a2NTD stimulates neutrophil polarization which was confirmed by the increased assembly of F-actin. Also, flow cytometry analysis revealed that a2NTD treatment stimulates surface expression and activation of Mac-1 integrin. Moreover, important regulators of neutrophil migration; FAK and Src Kinases were activated in a2Neuφ. Previous studies showed that IL-8; potent neutrophil chemoattractant is secreted by neutrophils. Here, we showed that a2NTD treatment led to upregulation of the gene expression and the secreted protein levels of IL-8 in a time dependent manner. This enhanced secretion of IL-8 was NF-kB dependent. Furthermore, to evaluate the influence of a2NTD on neutrophil migration we did trans-well migration assay. a2NTD stimulation caused 2.8 fold increase in neutrophil migration which decreased to 1.5 fold upon neutralizing IL-8 (P &lt; 0.05). Together, these data demonstrate a novel role of the tumor associated a2-vacuolar ATPase on regulating neutrophil survival and migration, by the action of a2NTD.

Citation Format: Safaa A. Ibrahim, Arpita Kulshrestha, Mukesh K. Jaiswal, Gajendra K. Katara, Magdy A. Amin, Kenneth D. Beaman. Enhancement of survival and migration of neutrophils by tumor associated a2 isoform vacuolar ATPase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 728.