The alkynylphosphonate analogue of calcitriol EM1 has potent anti-metastatic effects in breast cancer

Highlights

• EM1 reduces the number of lung metastases in an animal model of breast cancer.

• EM1 inhibits cellular migration and invasion in a breast carcinoma cell line.

• EM1 upregulates E-cadherin expression in a breast carcinoma cell line.

• EM1 does not affect the migration of a non-malignant mammary cell line.

• EM1 maintains plasma calcium within the normal levels at a 50 μg/kg body weight dose.

Abstract

The active form of vitamin D₃, calcitriol, plays a major role in maintaining calcium/phosphate homeostasis. In addition, it is a potent antiproliferative and prodifferentiating agent. However, when effective antitumor doses of calcitriol are employed, hypercalcemic effects are observed, thus precluding its therapeutic application. To overcome this problem, structural analogues have been designed with the aim at retaining or even increasing the antitumor effects while decreasing its calcemic activity. This report shows the biological evaluation of an alkynylphosphonate vitamin D less-calcemic analogue in a murine model of breast cancer. We demonstrate that this compound has potent anti-metastatic effects through its action over cellular migration and invasion likely mediated through the up-regulation of E-cadherin expression. Based on the current in vitro and in vivo results, EM1 is a promising candidate as a therapeutic agent in breast cancer.

Introduction

The active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (calcitriol), is mainly known for its effects on calcium and phosphate homeostasis. In addition, non-classical effects have been demonstrated over the last 30 years, including pro-differentiating and antiproliferative effects in cancer cells [1], [2], [3], [4]. Moreover, calcitriol has been shown to regulate almost all the processes a tissue undergoes in its progression to malignancy (the hallmarks of cancer) including cellular proliferation and differentiation, cell death, angiogenesis, metastasis, DNA repair, energy metabolism reprogramming, immunomodulation and inflammation [5], [6].

Breast cancer (BC) cells have also been shown to be targets of vitamin D compounds [7], [8], [9], [10]. Furthermore, numerous in vivo studies demonstrated that vitamin D compounds suppress BC tumor growth and metastasis [4], [10], [11], [12], [13], [14], [15], [16]. On the basis of this overwhelming evidence of the antitumor action of vitamin D compounds, some clinical trials evaluated the effects of these compounds in the treatment of various malignancies including BC, either alone or in combination. However, important clinical antitumor effects are infrequently observed and dose-limiting toxicity appeared in some cases which precluded the optimal dose for antitumor activity [4]. In other clinical trials, no toxicities have been observed but systemic stable levels of the drug could not be achieved due to the current available formulations of calcitriol [4], [17].

The above data indicate the need for designing and synthesizing novel vitamin D analogues with the intention of retaining the potent anticancer effects of calcitriol while being less calcemic. We had previously reported [18] the synthesis of a novel phosphonate analogue of vitamin D (diethyl [(5Z,7E)-(1S,3R)-1,3-dihydroxy-9,10-secochola-5,7,10(19)-trien-23-in-24-yl] phosphonate analogue, named EM1 hereafter). This analogue combines the low calcemic properties of phosphonates [19] with decreased metabolic inactivation due to the presence of a triple bond in C-24 [20] and proved to exert antiproliferative effects on various tumor cell lines while lacking hypercalcemic activity in mice [18]. In the present work we demonstrate that EM1 exerts inhibitory effects on the metastatic process in a syngeneic murine model of hormone-independent breast cancer, through its action on cellular migration and invasion.