Breast Tumor Inhibition by Vitamin D in a Mouse Model

Institution: Stanford University
Investigator(s): David Feldman, M.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12IB-0074 Award: $234,388
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2006)
This project will examine the efficacy of the active form of vitamin D, called “calcitriol”, in the treatment of breast cancer using a mouse model. In previous studies calcitriol has shown only modest activity. However, calcitriol has several new actions that we recently discovered that indicate it would be extremely active in combination with non-steroidal anti-inflammatory drugs (NSAIDs) and aromatase inhibitors (AIs). Prostaglandins (PGs) have been implicated in causing cancers in general including breast cancer and simulating progression and metastasis. We discovered that calcitriol inhibits PGs by three different molecular pathways. This would be useful for therapy, because PGs are major stimulators of aromatase activity in breast cancer. By inhibiting PGs, calcitriol would indirectly reduce aromatase, the enzyme critical for estrogen synthesis. In addition, we found that calcitriol directly inhibits the synthesis of aromatase thus acting by two pathways to reduce estrogens in breast cancer. Also, calcitriol decreases the levels of the estrogen receptor, reducing the ability of estrogens to stimulate breast cancer growth. Our preliminary experiments in cultured breast cancer cells demonstrate that calcitriol in combination with NSAIDs or AIs show a synergistic activity to inhibit breast cancer cell growth.

The general methodology will use molecular biology techniques to study changes in gene expression and protein levels in breast cancer xenografts in immunodeficient (“nude”) mice. These studies will focus on therapy with calcitriol in combination with NSAIDs and/or with AIs.

Calcitriol is an orally active, available drug that has been shown in clinical trials to have the ability to delay progression and prolong survival in prostate cancer patients without causing serious side-effects. Calcitriol reduced the toxicity of taxanes when given in combination. We postulate similar activity in breast cancer patients and propose combination therapy with NSAIDs or AIs. We envision these studies to rapidly pave the way to clinical trials in breast cancer patients.

Final Report (2008)
This project examined the beneficial effects of calcitriol (1,25-dihydroxyvitamin D3), the active form of vitamin D, in a mouse model of breast cancer (BCa). Our initial studies using cultured BCa cells have shown that calcitriol inhibits the synthesis and biological activity of estrogens, major stimulators of BCa growth. Calcitriol selectively decreases the expression of aromatase, the enzyme that converts androgenic precursors to estrogens. Calcitriol also down-regulates the expression of estrogen receptor alpha, the mediator of estrogen actions. Thus, calcitriol inhibits estrogen signaling and stimulation of BCa growth. We have also recently discovered that calcitriol inhibits the synthesis and biological activity of prostaglandins (PG), known stimulators of aromatase and potent mediators of cancer cell growth. Therefore, inhibition of the PG pathway provides an additional mechanism for indirectly down regulating aromatase by calcitriol in BCa cells as well as blocking the proliferative stimulus by the PGs. The goals of this grant were to evaluate our hypothesis that calcitriol in combination with either an aromatase inhibitor (AI) or a non-steroidal anti-inflammatory drug (NSAID) would be synergistic and more effective in treating BCa than either agent alone.

In this study we examined the effect of calcitriol and combination therapy on these molecular pathways in vivo in female nude mouse bearing xenografts of MCF-7 human BCa cells. In a short-term study calcitriol was injected intraperitonealy (IP) into the tumor bearing mice for 3 consecutive days. In the long-term studies calcitriol (low and high dose) was injected either alone or in combination with anastrozole (an AI) or naproxen (a NSAID) for 4 weeks. Body weight and tumor volume were monitored weekly. Animals were euthanized ~14 h after the last injection of calcitriol and vehicle. Blood was collected for serum calcium measurements. Tumors were excised, weighed and processed for isolation of RNA.

Gene expression profiles of critical genes involved in the estrogen synthesis and PG pathway were analyzed using real-time PCR. Both short-term and long-term studies with calcitriol resulted in a significant decrease in the expression of COX-2, the key enzyme involved in the synthesis of PGs. Expression of the EP2 PG receptor was also decreased. Additionally both aromatase and the estrogen receptor alpha were reduced significantly by calcitriol. Overall these results indicate that calcitriol elicited gene changes within the BCa tumors resulting in decreased synthesis and biological effects of both PGs and estrogens, both potent stimulators of BCa growth and progression. We also demonstrated the effects of calcitriol to be tissue specific, with the BCa showing decreased synthesis of estrogens and PGs while there was increased expression of COX-2 and aromatase in the bone. In in vivo studies using a combination of calcitriol and anastrozole (an AI) we showed that the combination was more effective in inhibiting tumor growth than either agent alone. No toxicity was found. Gene expression changes also revealed that the combination of calcitriol with anastrozole enhanced the actions of calcitriol in most cases. Combination with naproxen (NSAID) however did not produce impressive results.

In conclusion, the results of this study demonstrate that the combination of calcitriol and an AI is better than either agent alone, even at reduced doses. We envision that these findings will pave the way to clinical trials in BCa patients using a combination of calcitriol and an AI (anastrozole or other AI).