Gemini vitamin D analog suppresses ErbB2-positive mammary tumor growth via inhibition of ErbB2/AKT/ERK signaling

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Abstract

Numerous synthetic vitamin D analogs have been studied for their effects on the prevention and treatment of breast cancer. However, the inhibitory effects of naturally occurring 1α,25-dihydroxyvitamin D3 or its synthetic analogs on ErbB2 overexpressing mammary tumorigenesis have not been reported. Gemini vitamin D analogs are novel synthetic vitamin D derivatives with a unique structure of two six-carbon chains at C-20. We have previously shown that Gemini vitamin D analogs significantly inhibited carcinogen-induced estrogen receptor (ER)-positive mammary tumorigenesis and reduced ER-negative MCF10DCIS.com xenograft tumor growth without hypercalcemic toxicity. In the present study, we have determined the inhibitory effect of a potent Gemini vitamin D analog BXL0124 (1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol) on the ErbB2/Her-2/neu overexpressing mammary tumorigenesis. The Gemini BXL0124 inhibits ErbB2-positive mammary tumor growth and down-regulates the phosphorylation of ErbB2, ERK and AKT in tumors of MMTV-ErbB2/neu transgenic mice. These effects of Gemini BXL0124 in vivo were confirmed by using the ErbB2 overexpressing tumor cells derived from the mammary tumors of MMTV-ErbB2/neu mice. In conclusion, the Gemini vitamin D analog BXL0124 inhibits the growth of ErbB2 overexpressing mammary tumors through regulating the ErbB2/AKT/ERK signaling pathways, suggesting that Gemini vitamin D analog may be considered for translational studies.

Introduction

The hormonally active ligand for vitamin D receptor (VDR), 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), formed from vitamin D3 predominantly through hydroxylation by a 25-hydroxylase in the liver and a 1α-hydroxylase in the kidney, is known to play a major role in maintaining calcium/phosphate homeostasis [1], [2]. 1α-Hydroxylase in non-renal tissues, such as breast, colon and prostate, also produces the active form 1α,25(OH)2D3, which is important for the inhibition of cell proliferation by cell cycle arrest in the G0/G1 phase, induction of apoptosis and increased cell differentiation in cancer cells [3], [4], [5], [6]. Despite the anti-cancer activities of 1α,25(OH)2D3, the use of 1α,25(OH)2D3 for the prevention and treatment of cancer is limited due to its hypercalcemic toxicity. Therefore, numerous synthetic vitamin D analogs have been developed for better efficacy with decreased calcemic toxicity [2].

Gemini vitamin D analogs are novel synthetic vitamin D analogs that have a unique structure of two six-carbon chains at C-20 (see Fig. 1). In colon cancer models where MC-26 mouse colorectal cancer cells are implanted in BALB/c mice, certain Gemini vitamin D analogs were reported to exert greater activity than 1α,25(OH)2D3 in inhibiting tumor growth and in reducing the invasive spread of tumor cells into surrounding muscle without inducing hypercalcemic toxicity [7], [8]. We have previously reported that certain Gemini vitamin D analogs exert better growth inhibitory effect than 1α,25(OH)2D3 in MCF10 breast cancer cells [9]. In addition, we have demonstrated that Gemini vitamin D analogs inhibit tumor growth in ER-positive N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis and in ER-negative MCF10DCIS.com xenograft breast cancer, where cyclin-dependent kinase inhibitor, p21, and insulin-like growth factor binding protein 3 (IGFBP3) were shown as common molecular targets of certain Gemini vitamin D analogs [10], [11]. These findings have led us to study Gemini vitamin D analogs on another major subtype, ErbB2 overexpressing breast cancer.

ErbB2, also known as Her-2/neu, is an important molecular target in breast cancer prevention and therapy because ErbB2 is known to be amplified and overexpressed in 20–30% of human breast primary tumors [12]. To evaluate the efficacy of drugs and investigate the mechanism of action, the MMTV-ErbB2 transgenic mouse model overexpressing wild-type neu protooncogene has been widely used for the last two decades [12], [13]. Thus far, the potential activities of 1α,25(OH)2D3 and classic synthetic vitamin D analogs on ErbB2 overexpressing models have not been reported. In the present study, we have tested Gemini vitamin D analog BXL0124 (Fig. 1) in the MMTV-ErbB2 transgenic mouse model to determine whether the Gemini vitamin D analog BXL0124 exhibits potential suppressive effects on ErbB2 overexpressing mammary tumor growth. The detailed mechanisms of action whereby the Gemini vitamin D analog BXL0124 regulates the ErbB2-related downstream signaling pathway are investigated in vitro and in vivo.

Section snippets

Reagents and cell culture

1α,25(OH)2D3 and Gemini vitamin D analog (BXL0124, >95% purity) (Fig. 1) were provided by BioXell, Inc. (Nutley, NJ) and dissolved in DMSO. For in vivo animal experiments, BXL0124 was diluted in cremophore/PBS (1:8, v/v). The ErbB2/Her-2/neu overexpressing cells (E18-9A-42) were derived from MMTV-ErbB2 mammary tumors [14], [15] and kindly provided by Dr. Powel Brown at Baylor College of Medicine, Houston, TX. The cells were maintained in 10% fetal bovine serum (FBS)/DMEM medium supplemented

The Gemini vitamin D analog BXL0124 inhibits the growth of mammary tumors of MMTV-ErbB2/neu transgenic mice

We investigated the effect of Gemini vitamin D analog BXL0124 on MMTV-ErbB2/neu transgenic mice. Because of the close association between overexpression of ErbB2 and human breast cancer, the MMTV-ErbB2 transgenic mouse model is one of the most widely used hormone-independent animal models in breast carcinogenesis [17]. The treatment with BXL0124 at the dose of 0.3 μg/kg BW had no effect on body weight (Fig. 2A). As shown in Fig. 2B, the tumor volume (cm3) in the BXL0124 treatment group was 0.54 ± 

Discussion

In human breast and colon cancer, Sjoblom et al. describe 189 genes’ mutation at significant frequency [18], and breast cancer is considered to be a highly heterogeneous disease resulting from genetic changes, histopathological variation, and clinical outcomes [17]. These genetic and histological complexities of breast cancer indicate that multi-functional drugs need to be developed and used for the prevention and treatment of cancer [19]. Among different animal models, the MMTV-ErbB2

Acknowledgments

The authors thank Maria Hyra and Lamberto R. Navoa of the Animal Facility in the Department of Chemical Biology for their technical assistance in taking care of the animals.

This work was supported in part by NIH R03 CA112642, NIH R01 CA127645 and the Trustees Research Fellowship Program at Rutgers, The State University of New Jersey.

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    Special issue selected article from the 14th Vitamin D Workshop held at Brugge, Belgium on October 4–8, 2009.

    1

    Current address: Department of Food Science & Technology, Chung-Ang University, Anseong 456-756, South Korea.

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