Galectins play a role in mammary morphogenesis and are expressed in all breast cancer subtypes.Galectins bind with glycoconjugates, as well as carbohydrate-independent targets in both the cytosolic and nuclear subcellular fractions. All tissues express galectins and galectins bind to numerous targets localized in different subcellular domains, including cell membrane, nucleus, mitochondria, and extracellular matrix. Galectin-3 and galectin-1 are associated with breast cancer progression and metastasis and will be the focus of this chapter. The chapter highlights mechanisms involved in galectins' role in modulating metastatic breast cancer phenotype.Immunocytochemistry analysis of galectin-3 in human breast cancer cells is used to illustrate galectin expression in the metastatic phenotype.Note to the Reader: This chapter is part of the book Breast Cancer (ISBN: 978-0-6453320-3-2), scheduled for publication in July 2022. The book is being published by Exon Publications,

γ-Tocotrienol is a rare natural isoform of vitamin E that displays potent apoptotic and anti-metastatic activity against breast cancer. Galectin-3 is a protein that plays an important role in metastasis by binding to glycoconjugates on receptors and extracellular matrix proteins. Studies were conducted to investigate the effects of γ-tocotrienol on galectin-3 expression and activity in highly malignant mouse +SA mammary tumor cells. Computer-aided molecular modeling studies were carried out within the active site of galectin-3 based on the crystal structure (5EXO) obtained from the protein data bank with a known ligand, and anticancer agents (salirasib, γ-tocotrienol, and β-lactose) galectin-3 docking scores and amino acid interactions were determine. Treatment effects on galectin-3 expression and distribution was determined by Western blot analysis and immunocytochemical fluorescent staining, respectively. Treatment effects on +SA cellular migration and invasion was also determined. Results show that salirasib and β-lactose (known inhibitors of galectin-3) that share some pharmacophoric properties with γ- tocotrienol, with protein docking studies showing that salirasib (active binding -2.8) and γ- tocotrienol (active binding -2.87) had a similar binding score and binding moieties. Western blot analysis shows that γ-tocotrienol treatment induces a downregulation of galectin-3 expression, whereas immunocytochemistry studies shows that galectin-3 is highly expressed in +SA and TS/A cells, and treatment with 5 μM & 6 μM γ-tocotrienol greatly reduced galectin-3 expression in these cells, respectively. Furthermore, galectin-3 was found to form globular structures on the outer membrane of +SA and TS/A cells. In treated and control +SA cells, fibronectin and galectin-3 were present on the outside of cells, and γ-tocotrienol greatly attenuated galectin-3 and fibronectin distribution. In TS/A cells, galectin-3 distribution changed in control and treatment groups. Furthermore, treatment with 1.5 μM histamine significantly stimulated +SA mammary tumor cell migration, whereas combined treatment with 5 μM γ-tocotrienol completely blocked histamine-induced +SA cell migration. In summary, these results demonstrate for the first time that γ-tocotrienol treatment inhibits extracellular galectin-3 expression and disrupts galectin-3 carbohydrate binding and activation of +SA and TS/A mammary tumor cells. These findings also suggest that γ-tocotrienol may provide significant benefits in the prevention and/or treatment of metastatic breast cancer. Citation Format: Jessie J. Grazier, Paul W. Sylvester. γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2889.

γ-Tocotrienol is a rare natural isoform of vitamin E that displays potent apoptotic and anti-metastatic activity against breast cancer. Galectin-3 is a protein that plays an important role in metastasis by binding to glycoconjugates on receptors and extracellular matrix proteins. Studies were conducted to investigate the effects of γ-tocotrienol on galectin-3 expression and morphology in highly malignant +SA, TS/A MDA-MB-231 tumor cells. Computer-aided molecular modeling studies compared anticancer agents, salirasib and γ-tocotrienol, with the active site of galectin-3 and β-lactose (a known ligand). Results showed that γ-tocotrienol displays similar galectin-3 amino acid interactions as β-lactose and salirasib. Protein docking show that salirasib (active binding -2.8) and γ- tocotrienol (active binding -2.87) had similar binding scores and binding moieties. Immunocytochemical fluorescent staining show that γ-tocotrienol treatment disrupted galectin-3 expression and distribution, and a corresponding reduction in +SA cellular migration similar to that induced by β-lactose. Western blot analysis shows that γ-tocotrienol treatment induces a downregulation of galectin-3 expression. Immunocytochemistry studies shows that galectin-3 is highly expressed in +SA and TS/A cells, and γ-tocotrienol greatly reduced galectin-3 expression and distribution Furthermore, galectin-3 was found to form extracellular structures on the outer membrane of +SA, TS/A, and MDA-MB-231 cells, and γ-tocotrienol greatly attenuated galectin-3 and fibronectin distribution. γ-Tocotrienol treatment decreased f-actin stress fibers distribution in all cell lines. Furthermore, treatment with 1.5 μM histamine significantly stimulated +SA mammary tumor cell migration, whereas combination treatment with 5 μM γ-tocotrienol completely blocked histamine-induced +SA cell migration. In TS/A cells, 6-15 μM tocotrienol induced a dose-responsive increase in doxorubicin staining inside the nucleus. In MDA-MB-231 cells, syncytial cells were associated with galectin-3 expression and this was suppressed by 5-15 μM γ-tocotrienol. In summary, these results demonstrate that γ-tocotrienol treatment inhibits extracellular galectin-3 expression and disrupts galectin-3 carbohydrate binding and activation of +SA, TS/A and MDA-MB-231 breast cancer cells. These findings also suggest that γ-tocotrienol may provide significant benefits in the prevention and/or treatment of metastatic breast cancer. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Paul W. Sylvester, Jessie J. Grazier. γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 997.