Abstract 3357: Breast cancer-associated a2 isoform vacuolar ATPase regulates neutrophils: potential association for tumor invasion

Abstract

Abstract Breast cancer is the most common cancer worldwide and it is a prominent cause of cancer death in developing countries and the second leading cause of cancer death in American women. Breast cancer infiltrating immune cells like macrophages and neutrophils, TAM and TAN respectively are highly influencing tumor progression. Factors modulating TAN to produce pro-tumorigenic factors are not well understood. Vacuolar ATPases are overexpressed on many tumors and their expression highly correlates to the tumors metastatic potential. In tumor cells a cleaved peptide from the N-terminal domain of the a2-Isoform vacuolar ATPase (a2NTD) is secreted in the microvesicles and promotes pro-tumorigenic properties of macrophages as in TAM. In the current study we show by immunofluorescent staining that human breast cancer cell lines MDA-MB-231 and MCF-7 overexpress a2V in comparison to the non-tumorigenic cell line MCF-10A. Also, immunohistochemical analysis showed that a2NTD is highly expressed on infiltrative ductal carcinoma (IDC, represents 80% of all breast cancers) sections as compared to normal breast tissue. In order to investigate the regulatory effect of a2NTD on neutrophils we stimulated freshly isolated neutrophils from healthy volunteers with recombinant a2NTD. a2NTD treatment up-regulates the gene expression of anti-inflammatory mediators as IL-10 and interleukin 1 receptor antagonist (IL-1RA) and pro-inflammatory cytokines and chemokines like TNFα, IL-1β, IL-1α, IL-6, CCL-2, CXCL-1 and CXCL-2 in neutrophils. The multiplex luminex assay was done confirming the up-regulation of these mediators at the protein level. Moreover, a2NTD stimulated neutrophils enhanced the secretion of angiogenic and metastatic factors such as IL-8, VEGF, MMP-9 and elevated the gelatinase activity of MMP-9, which was detected by zymography. To evaluate the influence of a2NTD treated neutrophils on tumor progression we did an in vitro angiogenesis assay using Human Umbilical Vein Endothelial Cells (HUVECs). a2NTD treated neutrophils supernatant caused a 4 fold increase in the number of the capillary tubes formed by HUVECs and increased the branching of these tubes as well as the formation of closed structures. Furthermore, we tested the potential of a2NTD treated neutrophils on the invasiveness of the MDA-MB231 and MCF-7 breast cancer cell lines using in vitro trans-well invasion assay. Interestingly, a2NTD treated neutrophils supernatant promoted the MDA-MB-231 and MCF-7 invasion by 20 and 8 percent respectively as compared with untreated neutrophil supernatant. These data demonstrate the potential effect of tumor associated a2V and its soluble peptide a2NTD in regulating the sterile inflammation present in tumor microenvironment by modulating neutrophils, which promotes angiogenesis and tumor invasion. Citation Format: Safaa A. Ibrahim, Magdy Amin, Arpita Kulshrestha, Sahithi Pamarthy, Kenneth D. Beaman. Breast cancer-associated a2 isoform vacuolar ATPase regulates neutrophils: potential association for tumor invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2015-3357