In addition, alteration of multiple signaling pathways also affected CYP24 splicing and cellular sensitivity in response to vitamin D appeared to correlate with the induction of CYP24 splicing. These results suggest that 1,25(OH) 2 D 3 not only regulates CYP24 transcription, but also plays an important role in posttranscriptional modulation of CYP24 by inducing its splicing. Our findings reveal an additional regulatory step that makes the vitamin D mediated action more prompt and efficient.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687-694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER- patients.

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Peroxisome proliferator-activated receptor gamma (PPARδ) are ligand activated nuclear receptor and binds to natural and synthetic ligands. Earlier we had reported that PPARδ agonist troglitazone inhibited the development of precancerous lesions induced by DMBA in mouse mammary gland organ culture (MMOC). In the present study we evaluated the effects of PPARδ agonist GW7845 and antagonist GW9662 on the development of 7,12 dimethylbenz(a)anthracene (DMBA)-induced mammary alveolar lesions (MAL) in ER negative mammary glands in MMOC. The results showed that both GW7845 and GW9662 inhibited development of ER- MAL in MMOC. Since PPARδ and ER cross talk has been reported in the literature, we determined the effects of GW9662 on the induction of ER and PR in this model. Real-time PCR studies and immunohistochemical analyses showed that GW9662 induced ERα in the absence of estradiol, whereas GW7845 did not induce ERα under these conditions. Both GW7845 and GW9662 failed to suppress mRNA expression of PPARδ in the mammary glands that are otherwise ER negative. However GW9662 suppressed the expression of PPARδ response genes adipsin and aP2. Conversely, GW7845 an agonist of PPARδ enhanced the expression of aP2 and adipsin. These results suggested that the effects of these agents are mediated by PPARδ in MMOC. Finally, Tamoxifen does not inhibit MAL in MMOC, however sequential treatments with GW9662 followed by Tamoxifen suppressed development of DMBA-induced MAL in these glands. Since the ER negative breast cancer patients are generally not treated with SERM, inducing ERα in the mammary cells by GW9662 possibly could make them responsive to Tamoxifen or other SERM. [This work was supported by N01 CN433303] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2580. doi:1538-7445.AM2012-2580

Colon cancer cells interact with immune cells in vivo, creating a microenvironment which affects both tumor growth and treatment. Macrophages are often the most abundant immune cells in the tumor microenvironment and are involved in inflammation. Since inflammation is tightly associated with colon carcinogenesis, it is important to understand how vitamin D affects the interaction between colon epithelial cells and macrophages. In this study, we used a co-culture system to examine the role of 25-hydroxyvitamin D3 (25(OH)D3) on the clonogenic growth of colon cancer cells and on the profiling of cytokines/chemokines. 25(OH)D3 at 250 nM had minor effect on clonogenic growth in CaCo-2 cells, co-culture of macrophage-like THP-1 cells with CaCo-2 cells significantly stimulated the clonogenic growth of CaCo-2 cells in the absence of 25(OH)D3. However, in the presence of 25(OH)D3, THP-1 cells failed to stimulate the the clonogenic growth of CaCo-2 cells. Cytokine antibody array demonstrated that several cytokines were differentially expressed in the medium after co-culture of the two cell lines for 2 days in the absence of 25(OH)D3. These include increased expression of GROα, MCP-1 and decreased expression of TNFα, Angiogenin; however, in the presence of 25(OH)D3, MCP-2 and MDC were significantly induced and TNFα level was restored. Proliferation assay showed that MCP-2 and MDC had minor or no effect on cell proliferation in CaCo-2 cells, suggesting that these chemokines mainly target macrophages. 25(OH)D3 treatment of THP-1 cells for 24h also induced CYP24, an immediate vitamin D target gene and enhanced MDC and MCP-2 expression as evaluated by qRT-PCR. These results demonstrate that 25(OH)D3 at 250 nM might selectively target macrophages in a co-culture system with colon cells and block the interaction between colon cancer cells and macrophages, which may result in prevention of inflammation. [Supported by NCI R01 CA121157]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2011-456