Vitamin K2 enhances the tumor suppressive effects of 1,25(OH)2D3 in triple negative breast cancer cells

Abstract

K vitamins are well known as essential cofactors for hepatic γ-carboxylation of coagulation factors, but their potential role in chronic diseases including cancer is understudied. K2, the most abundant form of vitamin K in tissues, exerts anti-cancer effects via diverse mechanisms which are not completely understood. Our studies were prompted by previous work demonstrating that the K2 precursor menadione synergized with 1,25 dihydroxyvitamin D3 (1,25(OH)₂D₃) to inhibit growth of MCF7 luminal breast cancer cells. Here we assessed whether K2 modified the anti-cancer effects of 1,25(OH)₂D₃ in triple negative breast cancer (TNBC) cell models. We examined the independent and combined effects of these vitamins on morphology, cell viability, mammosphere formation, cell cycle, apoptosis and protein expression in three TNBC cell models (MDA-MB-453, SUM159PT, Hs578T). We found that all three TNBC cell lines expressed low levels of the vitamin D receptor (VDR) and were modestly growth inhibited by 1,25(OH)₂D₃ in association with cell cycle arrest in G0/G1. Induction of differentiated morphology by 1,25(OH)₂D₃ was observed in two of the cell lines (MDA-MB-453, Hs578T). Treatment with K2 alone reduced viability of MDA-MB-453 and SUM159PT cells but not Hs578T cells. Co-treatment with 1,25(OH)₂D₃ and K2 significantly reduced viable cell number relative to either treatment alone in Hs578T and SUM159PT cells. The combination treatment induced G0/G1 arrest in MDA-MB-453 cells, Hs578T and SUM159PT cells. Combination treatment altered mammosphere size and morphology in a cell specific manner. Of particular interest, treatment with K2 increased VDR expression in SUM159PT cells suggesting that the synergistic effects in these cells may be secondary to increased sensitivity to 1,25(OH)₂D₃. The phenotypic effects of K2 in TNBC cells did not correlate with γ-carboxylation suggesting non-canonical actions. In summary, 1,25(OH)₂D₃ and K2 exert tumor suppressive effects in TNBC cells, inducing cell cycle arrest leading to differentiation and/or apoptosis depending on the specific cell line. Further mechanistic studies to clarify common and unique targets of these two fat soluble vitamins in TNBC are warranted.

Keywords: Breast cancer; Menaquinone-4; TNBC; Vitamin D; Vitamin K2.

Figure 1.. Effect of 1,25(OH)₂D₃ and K2 on TNBC cell morphology.

TNBC cells were plated in their respective media and treated the next day for 72h (MDA-MB-453) or 96h (SUM159PT, Hs578T) with 100nM 1,25(OH)₂D₃ ± 10μM K2 or vehicle. Images were obtained with the 10x (SUM159PT) and 20x (Hs578T, MDA-MB-453) objectives on a Nikon Eclipse TS100 microscope. Scale bars: 100μM. After imaging, cultures were processed for MUSE analysis.

Figure 2.. Cell viability and caspase activity in TNBC cells treated with 1,25(OH)₂D₃ ± K2.

Cells plated in their respective media were treated for 72h (MDA-MB-453) or 96h (SUM159PT, Hs578T) with 100nM 1,25(OH)₂D₃ ± 10μM K2 or vehicle. Cell proliferation data (top panels) and caspase activity (bottom panels) were assessed on the MUSE analyzer. Data represent Mean ± SD (n=2) and are representative of at least three independent experiments. Bars annotated with different letters are significantly different (p<0.05) as assessed by one-way ANOVA.

Figure 3.. Immunoblot analysis of TNBC cells treated with 1,25(OH)₂D₃ ± K2.

Cells plated in their respective media were treated for 48h with 100nM 100nM 1,25(OH)₂D₃ ± 10μM K2 or vehicle. VDR protein, Gla modified proteins, and proteins involved in cell proliferation and differentiation (c-Myc and Cyclin D1) were examined. Representative blots of at least two independent experiments. GAPDH was used as loading control.

Figure 4.. Effect of 1,25(OH)₂D₃ and K2 on mammosphere formation, VDR and stem cell marker CD44.

(A) Representative images of primary mammospheres. SUM159PT (4,000 cells/well) and Hs578T (6,000 cells/well) were plated in 6-well ultra low attachment plates and incubated with 100nM 1,25(OH)₂D₃ ± 10μM K2 or vehicle for 10 days. Scale bars: 100 μM. (B) Representative images of secondary mammospheres. Primary mammospheres were dissociated and re-plated in 6-well ultra low attachment plates and incubated with vehicle or 1,25(OH)₂D₃ + K2 for an additional 10 days. Scale bars: 100 μM. (C) Primary and secondary mammospheres collected after 10 days of treatment with vehicle or 1,25(OH)₂D₃ + K2 and analyzed for VDR, CD44 and GAPDH by immunoblotting.