br Studies examining the use of OH D as a
Studies examining the use of 1α,25(OH)2D3 as a safe, easy to deliver supplement against breast cancer tumorigenicity have been promising; however, there is a risk of hypercalcemia with excess 1α,25(OH)2D3. Hypercalcemia is a common co-morbidity of cancer, with up to 30% of all cancer patients suﬀering from severe hypercalcemia . It is the leading cause of hospitalization in cancer patients, and those suﬀering from breast cancer are particularly susceptible. Excess 1α,25(OH)2D3 is a documented cause of hypercalcemia , and blood serum levels of free 1α,25(OH)2D3 as low as 200 pmol/L (with ~85 pmol/L approximating normal 1α,25(OH)2D3 levels) can be toxic . Many in vitro studies only observe the anti-tumorigenic eﬀects of 1α,25(OH)2D3 when treating with 1–100 nmol/L, well above the toxi-city threshold for this compound [76–78].
An increasing number of studies have observed connections be-tween CYP24A1, 24,25(OH)2D3, and tumorigenicity. CYP24A1 is the 24-hydroxylase chiefly responsible for 24,25(OH)2D3 production, and its overexpression has been shown to increase 24,25(OH)2D3 in vivo . A recent study in lung cancer demonstrated a correlation between increased CYP24A1 mRNA expression and improved hazard ratios for 5-year survival . 24R,25(OH)2D3 treatment prolonged survival in Lewis lung carcinoma mice  and ovarian cancer SC 236 . A si-milar study showed that in rats, 24R,25(OH)2D3 treatment reduced cancerous and precancerous stomach lesion incidence in a dose-de-pendent manner . Moreover, in the colon, continual dosing with 24R,25(OH)2D3 was observed to reduce colon cancer foci development .
Many of the mechanistic explanations for preliminary results re-garding the anti-tumor eﬀect of 24,25(OH)2D3 have been attributed to the rapid actions of the hormone. In rats, reduction of cancer incidence by 24R,25(OH)2D3 was attributed to the metabolite’s alteration of calcium pharmacodynamics during carcinogenesis . In the colon, 24R,25(OH)2D3 reduced the development of malignant foci by reg-ulating c-myc, c-foc, and c-jun oncogene expression, suggesting that the rapid actions of 24R,25(OH)2D3 were involved .
On the other hand, CYP24A1 overexpression has also been asso-ciated with worsened prognosis for a number of cancers. A study on the ERα66-, ERα36 + breast cancer cell line MDA-MB-231 showed that increased CYP24A1 prevented apoptosis, possibly by interfering with 1α,25(OH)2D3 signaling mechanisms . Similarly, in the ERα66+, ERα36-  prostate cancer cell line PC-3, CYP24A1 inhibition in-creased apoptosis . Other studies have shown that estrogen can regulate the expression of CYP24A1 and subsequent expression of 24R,25(OH)2D3 . Xenografts of the ERα negative colon cancer cell line, HT-29,  showed a marked increase in tumor growth with the overexpression of CYP24A1 . Furthermore, a soy-based diet en-hanced the growth of CYP24A1-overexpressing tumors but not the growth of wild-type tumors . The impact of soy, a potent phy-toestrogen , on the growth of CYP24A1-overexpressing tumors suggests a connection between estrogen and CYP24A1 or its products, such as 24R,25(OH)2D3.
Recent studies in our lab  show that treating with 10–100 nM 24R,25(OH)2D3 (which had no calcemic eﬀect in vivo at these doses [9,75]) can prevent apoptosis and enhance epithelial-to-mesenchymal transition and metastatic markers in ERα66-, ERα46+, ERα36 + HCC38 cells (Fig. 2A). However, the same dose of 24R,25(OH)2D3 has the opposite eﬀect in ERα66+, ERα46+, ERα36 + MCF7 cells. 24R,25(OH)2D3 induces apoptosis, down-regulates the expression of epithelial-to-mesenchymal transition mar-kers, and decreases protein levels of metastatic markers in these cells as measured by snail family zinc finger 1 (SNAI1), matrix metalloprotei-nase 1 (MMP1), HER2, chemokine receptor type 4 over chemokine li-gand type 12 (CXCR4/CXCL12) and osteoprotegerin over nuclear factor kappa-B (NFκB) ligand (OPG/RANKL) ratios (Fig. 2B). 1α,25(OH)2D3 and 24S,25(OH)2D3 were also examined, but were found to have no eﬀect on proliferation in MCF7 cultures in vitro. The eﬀect of 24S,25(OH)2D3 on apoptosis in MCF7 was also examined and was si-milarly found to have no eﬀect on cultures in vitro, suggesting that the observed eﬀects of 24R,25(OH)2D3 on cell cycle regulation are stereo-specific . Similar results were observed in vivo. 100 ng of 24R,25(OH)2D3 given three times weekly over a period of 5–6 weeks increased tumor volume in HCC38 orthotopic mammary fat pad xeno-grafts. In contrast, the same dose of 24R,25(OH)2D3 reduced tumor volume in MCF7 mammary fat pad xenografts (Fig. 3).