Abstract 2133: 24R,25(OH)2D3 is differentially tumorigenic in ER+ and ER- breast cancer
Authors: Verma Anjali , Cohen David J. , Muktipaty Chandana , Jacobs Thomas W. , Koblinski Jennifer , Boyan Barbara D. , Schwartz Zvi 
Published: August 2019

Abstract
Vitamin D3 (VD3) has long been associated with improved breast cancer prognoses. However, recent clinical trials examining the efficacy of VD3 supplementation on patient prognoses have been largely inconclusive. One reason for this may be that most clinical trials ignore the complex VD3 metabolome and focus solely on 25(OH)D3, the circulating form of VD3. Our study focuses on 24R,25(OH)2D3 (24,25D), a naturally-occurring metabolite of VD3. The aim of the present study was to examine the effect of 2425D on breast cancer tumorigenicity, and to determine if this effect is dependent on estrogen receptor expression in vitro and in vivo.In vivo, MCF7 (an estrogen receptor positive [ER+] breast cancer cell [BCC] line) or HCC38 (ER-) BCCs were implanted in adult female NOD SCID gamma IL2R mice. Mice were supplemented with 24,25D (0, 25, or 100 ng/ IP injection 3X/week). Tumor growth was monitored throughout the study. In vitro, both cells were treated with 0-100nM 24,25D for 15 min or 24h and assayed for phospholipase D (PLD) activity, proliferation (DNA synthesis), apoptosis (BAX, BCL2, p53, TUNEL), epithelial-to-mesenchymal transition (EMT) (SNAI1, MMP1, ERBB2), migration (scratch test), and metastasis (CXCR4/CXCL12, OPG/RANKL). Inhibitors to PLD, caveolin-1, and palmitoylation were used to examine the mechanism of 24,25D. Data were analyzed with one or two-way ANOVA with Tukey’s post-tests.In vivo, mice with ER+ tumors given 24,25D showed decreased tumor burden, metastasis, and increased survival as compared to vehicle-treated controls. Conversely, mice with ER- tumors given 24,25D showed a dose-dependent increase in tumor burden compared to controls. In vitro, both ER+ and ER- cells showed increased proliferation at 24 hours after exposure to 24,25D for 15min but not after exposure for 24h. In ER+ cells, 2425D increased apoptosis and decreased EMT, migration, metastasis, and PLD activity; while in ER- cells, the opposite trend was observed, with 24,25D decreasing apoptosis and increasing EMT, migration, metastatic markers, and PLD activity. The proliferative and apoptotic effects of 24,25D in both cell lines were inhibited by pre-treatment with inhibitors to PLD, caveolin-1, and palmitolyation.24,25D had opposing effects on ER+ and ER- tumor burden in vivo, suggesting that 24,25D is differentially tumorigenic, and observed differences in tumorigenicity are dependent on the expression of ER. In vitro, this ER-associated differential effect was confirmed, with 24,25D decreasing tumorigenic markers in ER+ BCCs and enhancing them in ER- BCCs. Furthermore, 24,25D appears to act at the membrane through a PLD-dependent caveolae-associated mechanism using a palmitoylated transmembrane receptor(s) in both cell types. This study indicates the important role of 24,25D in breast cancer and suggests that ER-status may be an important prognostic marker to consider before prescribing vitamin D3 supplementation to breast cancer patients.
Citation Format: Anjali Verma, David J. Cohen, Chandana Muktipaty, Thomas W. Jacobs, Jennifer Koblinski, Barbara D. Boyan, Zvi Schwartz. 24R,25(OH)2D3 is differentially tumorigenic in ER+ and ER- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2133.