Abstract A20: Globo H ceramide acts as immune checkpoint and angiogenic factor in tumor microenvironment
Authors: J Yu , JY Cheng , SH Wang , YC Tsai , JC Wu , JT Hung , J Lin , KT Yeh , AL Yu 
Published: October 2016

Abstract
Since aberrant glycosylation is a feature of cancer cells, targeting tumor associated carbohydrate antigen (TACA) for cancer immunotherapy is gaining international attention. Globo H, the most prevalent TACA over-expressed in cancers of breast, lung, pancreas, stomach, colon, etc., is a hotly pursued target for cancer vaccine. Both Globo H and its pentasaccharide precursor, Gb5, are expressed in breast cancer stem cells. Notably, Globo H ceramide (GHCer) acts as an immune checkpoint molecule, as reflected by uptake of GHCer shed from cancer cells by tumor infiltrating lymphocytes Addition of GHCer to human PBMC, mouse splenocytes or purified CD4+ T cells inhibited their proliferative response to anti-CD3/CD28, and reduced the secretion of IL-2, IFN-γ and IL-4. GHCer also significantly suppressed the proliferation of splenocytes or CD19+ B cells in response to LPS or LPS + IL4 +CD40 ligand and their production of IgG and IgM, with negligible generation of plasma cells. Ceramide displayed no such inhibitory effects. On the other hand, GHCer failed to raise the number of regulatory T cells, or their expression of FOXP3/CTLA4, nor did it increase apoptosis. Molecularly, GHCer significantly suppressed the Notch1 signaling during activation of human PBMC and murine T and B cells. This was linked to GHCer-induced upregulation of expression of ID3 and itch, leading to ID3-dependent downregulation of Notch1 and itch-mediated Notch1 degradation. Furthermore, GHCer increased the expression of egr2 and egr3 at 2hrs after activation which preceded itch upregulation. These results provide the first evidence for GHCer to facilitate the escape of cancer cells from immune surveillance via down-regulation of Notch1 signaling at transcriptional level by ID3, and protein level by egr2/3 controlled itch expression. In addition, GHCer is also incorporated into endothelial cells, through the absorption of microvesicles shed from tumor cells. In endothelial cells, GHCer addition induces migration, tube formation, and intracellular Ca2+ mobilization in vitro and angiogenesis in vivo. Clinically, Globo H+ breast cancer specimens contained Globo H+ tumor infiltrating lymphocytes, and higher vessel density than Globo-H- tumors. Mechanistic investigations linked its angiogenic effects to its binding to TRAX, GHCer is also incorporated into endothelial cells, enhancing angiogenesis. Clinically, Globo H+ breast cancer specimens contained Globo H+ tumor infiltrating lymphocytes, and higher vessel density than Globo-H- tumors. Mechanistic investigations linked the angiogenic effects of GHCer to its binding to TRAX, with consequent release of PLCb1 from TRAX to trigger Ca2+ mobilization. The binding of GHCer to TRAX was validated by Biacore system with immobilized TRAX protein on sensor chip CM5. At concentrations ranging from 9.77 nM to 5 μM, GHCer binds TRAX with a fast association rate, followed by slow dissociation. The KD of the GHCer binding with TRAX was estimated to be about 4.09 x10-8 M, by fitting the binding curves with BIAevaluation 4.1. On the other hand, galactosyl-ceramide, lactosyl-ceramide or Gb4-ceramide showed negligible binding to TRAX. Preliminary data showed that recombinant C-terminal domain of PLCB1 bound to TRAX and was competed by GHCer in a concentration-dependent manner. Taken together, these data support the notion that GHCer plays triple roles in serving as a cancer antigen (including breast cancer stem cells), as an immune checkpoint and as an angiogenic factor, thereby propelling the ongoing multi-national phase II/III clinical trial of globo H vaccine in breast cancer and ovarian cancer.
Citation Format: Yu J, Cheng JY, Wang SH, Tsai YC, Wu JC, Hung JT, Lin J, Yeh KT, Yu AL. Globo H ceramide acts as immune checkpoint and angiogenic factor in tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A20.