Abstract P2-05-16: Tumor-associated glycans as key molecules to promote growth of triple-negative breast cancer cells

Abstract

Enhanced invasion and migration into the surrounding tissues are hallmarks of the malignancy of tumor cells. To successfully metastasize, a cancer cell has to detach from the primary tumor, invade into surrounding tissues, and intravasate into blood or lymphatic vessels. These processes are composed of complex mechanisms involving tumor recognition, degradation of extracellular matrix (ECM) proteins and migration into tissue. Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. It is widely recognized that TN breast cancers have a poorer prognosis than any other subtype of breast cancer. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of migration and invasion of these tumors will provide insight into developing novel approaches to lower the mortality from TN breast cancer. Previous studies demonstrated that NEDD9 plays a key role facilitating progression and metastasis of various tumor cells including breast. We previously demonstrated that NEDD9 plays a critical role in promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, we established stable cell lines expressing NEDD9 using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that enzymes (CHST11, CHST15, and CSGALNACT1) involved in biosynthesis of chondroitin sulfate (CS) but not heparan sulfate (HS) were markedly upregulated in HCC38(NEDD9) compared to control HCC38(Vector) cells. These results suggest that NEDD9 regulates specific structures of tumor-associated glycans such as chondroitin sulfate. Core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to Serglycin and CD44 core proteins. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent growth of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggest that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression metastasis and possibly stem cell phenotypes. The opinion and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense. Citation Format: Iida J, Dorchak J, Clancy R, Slavik J, Cutler ML, Shriver CD. Tumor-associated glycans as key molecules to promote growth of triple-negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-16.