Elsevier

Biochemical Pharmacology

Volume 72, Issue 3, 28 July 2006, Pages 332-343
Biochemical Pharmacology

Gene expression profiling changes induced by a novel Gemini Vitamin D derivative during the progression of breast cancer

https://doi.org/10.1016/j.bcp.2006.04.030Get rights and content

Abstract

We investigated gene expression changes induced by a novel Gemini Vitamin D3 analog, RO-438-3582 (1α,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-cholecalciferol, Ro3582), in a unique human breast MCF10 model. We used two breast epithelial cell lines from this model, namely MCF10AT1 (Ha-ras oncogene transfected MCF10A, early premalignant) and MCF10CA1a (fully malignant and metastatic derived from the MCF10AT1 line). We analyzed gene expression changes induced by Ro3582 using GeneChip technology, quantitative RT-PCR, Western blot analysis, or a gene transcription assay. Interestingly, we found distinct gene expression profile differences between Ro3582-induced response of the early premalignant MCF10AT1 and the malignant and metastatic MCF10CA1a cell lines. Moreover, while the Gemini Vitamin D3 analog Ro3582 modulated the expression of several Vitamin D target genes such as the 24-hydroxylase, CD14, osteocalcin, and osteopontin in both cell lines, Ro3582 regulated many genes involved in cell proliferation and apoptosis, cell adhesion, invasion, angiogenesis as well as cell signaling pathways, such as the BMP and TGF-β systems, differently in the two cell lines. The Gemini Vitamin D3 analog Ro3582 induced more significant gene changes in the early premalignant MCF10AT1 cells than in the malignant metastatic MCF10CA1a cells, suggesting that Gemini Vitamin D3 analogs may be more effective in preventing the progression of an early stage of breast carcinogenesis than in treating late stage breast cancer.

Introduction

The hormonally active metabolite of Vitamin D3, 1α,25(OH)2D3, functions in the maintenance of calcium/phosphate homeostasis through regulation of genes in intestine, kidney and bone, and it also controls immune cells and hormone secretion [1], [2]. Most of the effects of 1α,25(OH)2D3 are mediated through the Vitamin D receptor (VDR)-regulated transcription [3], [4]. The VDR is a member of the nuclear receptor superfamily, and functions as a heterodimer with the master dimerization partner, retinoid X receptor (RXR) [5]. When 1α,25(OH)2D3 or its analogs bind to the VDR, the receptor interacts with the RXR, as well as with other transcription factors including coactivators and corepressors, to activate the selective transcription of VDR-dependent genes [4].

The VDR is present in normal and lactating mammary gland, in breast tumors, and in cell lines derived from human breast cancers [6], [7], [8], and 1α,25(OH)2D3 and certain Vitamin D3 analogs have been shown to exert potent growth inhibitory effects on breast cancer cells. For example, 1α,25(OH)2D3 and its classic synthetic analogs have been shown to induce apoptosis and to arrest the cell cycle in the G1 phase by increasing the level of cyclin-dependent kinase inhibitors, such as p21 and p27 [9], [10]. In addition, 1α,25(OH)2D3 and certain Vitamin D3 analogs have been shown to inhibit invasion, angiogenesis, and metastasis [3], [10], [11], [12]. Although the growth inhibitory effect of 1α,25(OH)2D3 and several of its classic analogs in cell culture may be important for the treatment and prevention of cancer, the hypercalcemic effect of 1α,25(OH)2D3 has limited their use for the treatment and prevention of breast cancer [2]. Therefore, many different classes of synthetic Vitamin D3 analogs have been developed to overcome the hypercalcemic toxicity. Among these, Gemini analogs of Vitamin D3 have been reported to have considerably less toxicity than 1α,25(OH)2D3 in animals [13].

In this study, we have investigated more than 25 different Gemini analogs of Vitamin D3 that contain two six carbon side-chains, combining a C-20-normal with a C-20-epi side chain (see Fig. 1 for some selected analogs) [14], [15], [16]. Certain Gemini analogs of Vitamin D3 have shown low hypercalcemic toxicity profiles, due to increased metabolic stability of the analogs and different properties of the liganded VDR facilitating VDR action, resulting in more cofactor binding and elevated levels of transcription [17], [18], [19]. Although certain Gemini analogs of Vitamin D3 were shown to treat and prevent colon cancer [13], Gemini analogs of Vitamin D3 have not been investigated in breast cancer models.

Here, we report for the first time the effects of Gemini analogs of Vitamin D3 on proliferation and gene expression in a unique MCF10 estrogen receptor negative breast cancer model. The series of cell lines of the MCF10 model have the same origin (MCF10A; normal immortalized). All established MCF10 breast epithelial cell lines were initiated after transfecting Ha-ras oncogene into MCF10A (designated as MCF10AT1), and further passaged in mice to select more aggressive and malignant cell lines [20], [21]. The establishment of MCF10 cell lines, such as MCF10AT1 (early premalignant), MCF10DCIS (invasive potential, isolated from MCF10AT1), MCF10CA1h (aggressive malignant, isolated from MCF10AT1) or MCF10CA1a (fully malignant and metastatic, isolated from MCF10AT1), has been reported [22], [23], [24]. Among the several MCF10 cell lines available, we have tested novel Gemini Vitamin D3 derivatives in two cell lines, MCF10AT1 (forms premalignant lesions in immunodeficient mice) and MCF10CA1a (produces undifferentiated carcinomas with metastatic potential in immunodeficient mice). Although several gene profiling studies with 1α,25(OH)2D3 have been reported for prostate cancer and breast cancer cell lines [25], [26], [27], gene changes in a progression model of estrogen receptor negative breast cancer by 1α,25(OH)2D3 or its analogs have not been investigated. We report here that a novel Gemini Vitamin D3 analog (Ro3582; see Fig. 1 for structure) regulates gene expression differently in cell lines representing two different stages of breast cancer, namely MCF10AT1 (early premalignant) and MCF10CA1a (malignant and metastatic).

Section snippets

Reagents

1α,25(OH)2D3 and all Gemini Vitamin D3 analogs including Ro3582 [1α,25-dihydroxy-20S,21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluorocholecalciferol] (>95% purity) were synthesized at Hoffmann-La Roche Inc. (Nutley, NJ). Fugene6 and Trizol® solution were obtained from Roche Diagnostics (Indianapolis, IN) and Invitrogen (Carlsbad, CA), respectively. 1α,25(OH)2D3 and Vitamin D3 analogs were dissolved in dimethylsulfoxide (DMSO) before addition to cell cultures; final concentrations of DMSO

Growth inhibitory effect of 1α,25-dihydroxyvitamin D3 and the Gemini Vitamin D3 analog Ro3582 in MCF10 breast epithelial cells

In a series of MCF10 breast epithelial cell lines, we selected MCF10AT1 cells (early premalignant) and MCF10CA1a (fully malignant and metastatic) for growth inhibition and microarray assays. However, the parental MCF10A cell line was not included because normal immortalized MCF10A cells do not have Ha-ras oncogene and Gemini Vitamin D3 analogs only very weakly inhibited growth of these cells (data not shown). The growth rate of MCF10AT1 cells is similar to MCF10CA1a cells using the same medium.

Discussion

In the present study, we utilized an oligo microarray approach to evaluate changes in the gene expression profile after treatment of MCF10AT1 and MCF10CA1a cell lines with the Gemini Vitamin D3 analog, Ro3582. Although several studies have shown gene expression profile changes after treatment with 1α,25(OH)2D3 in prostate, colon, ovarian, and breast cancer cells [25], [26], [27], [33], [34], [35], [36], [37], [38], our study is unique because it reports on the effect of a novel Gemini Vitamin D3

Acknowledgments

This work was supported by NIH K22 CA 99990, NIH R03 CA112642, and a CINJ new investigator award to N.S, and Cancer Center Support Grant (5 P30 CA 072720-10) to D.N. We thank Dr. Allan Conney for helpful advice on our work. The MCF10 cell lines were established and provided by Dr. Fred Miller's group at the Karmanos Cancer Institute. The authors thank the Department of Chemical Biology and Shared Microarray Resource at the Cancer Institute of New Jersey for technical help with this project.

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