Abstract LB-024: Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer

Purpose : The hormonally active form of vitamin D [1,25(OH)2D3], has several antitumor effects including antiproliferative, prodifferentiative and proapoptotic functions in tissues expressing and binding the vitamin D receptor. 1,25(OH)2D3 is synthesized from its precursor 25-hydroxyvitamin D [25(OH)D] via the catalytic action of the mitochondria cytochrome P450 enzyme 25(OH)D-1α-hydroxylase (CYP27B1). In the current study we investigated the role of CYP27B1 ablation on vitamin D signaling using the mouse mammary tumor virus promoter-driven polyoma middle T oncoprotein (MMTV-PyMT) mouse, an oncogene-driven model of highly aggressive spontaneous mammary tumors that closely mimics the estrogen receptor negative breast cancer in human disease. Methods: Tumors from animals were digested and primary cells were obtained from both CYP27B1 ablated AOH93Cre+ cells and wild type MT1107 Cre- control cells. The cells were next grown in complete DMEM medium, transfected with either a GFP-tagged human VDR or RXRα and treated with 1,25(OH)2D3 (10−7 mol/L) or 25(OH)D3 (10−7 mol/L) before data acquisition. Results: Using live and fixed cell fluorescence imaging, in situ proximity ligation assay and immunoblotting techniques, we show that the nuclear localization of both endogenous and exogenously tagged VDR and RXR were impaired in CYP27B1 ablated cells when compared to non-ablated cells. Furthermore, the intranuclear shuttling of VDR and its interaction with RXR were significantly reduced in CYP27B1 ablated cells as shown by both Florescence Recovery After Photobleaching and Fluorescence Resonance Energy Transfer techniques. Our results also show that CYP27B1 ablation disrupts VDR function by significantly impairing its nuclear localization, intranuclear transport, interaction with adaptor proteins, hVDR/hRXR, and this correlated with impaired binding to DNA and chromatin. Lastly, we demonstrate impaired induction of 53BP1, p65 and γ-H2AX DNA double-strand breaks in the CYP27B1 ablated cells compared to non-ablated cells. Conclusion: These results suggest that ablation of CYP27B1 results in dysregulation in vitamin D signaling which may impair its anticancer functions. Citation Format: Sylvester Jusu, John Presley, Bertrand Jean-Claude, Ursula Stochaj, Richard Kremer. Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-024.