br While a consensus receptor
While a consensus receptor for 24R,25(OH)2D3 remains elusive, membrane signaling by 24R,25(OH)2D3 has been described. In costo-chondral resting zone chondrocytes, 24R,25(OH)2D3 binds to an as yet unidentified membrane-bound receptor and inhibits phospholipase A2 activity (PLA2) . The resulting signaling pathway alters fatty SC 560 turnover and subsequently inhibits the release of arachidonic acid and prostaglandin E2 (PGE2) production . This inhibition of arachidonic acid turnover alters membrane fluidity and calcium flux within the cell, modulating signal transduction downstream and stimulating PKC ac-tivity [48,49]. The eﬀect of 24R,25(OH)2D3 is stereospecific; 24S,25(OH)2D3 does not activate this pathway, providing further evi-dence that the response to the hormone is receptor mediated.
Fig. 4. Meta-analyses of data taken from the total cancer genome atlas. 5-year percent survival of (A) estrogen receptor negative or (B) estrogen receptor positive breast cancer patients with higher or lower than median CYP24A1.
Fig. 5. 24R,25(OH)2D3 is pro-apoptotic, pro-proliferative, anti-epithelial-to-mesenchymal, and anti-metastatic in breast cancer cells that overexpress ERα66 with a net-positive eﬀect on tumorigenicity. Conversely, 24R,25(OH)2D3 has the opposite eﬀect in cells that do not express ERα66, inhibiting apoptosis, promoting pro-liferation, epithelial-to-mesenchymal transition, and metastasis. r> 24R,25(OH)2D3 binding to the membrane receptor also stimulates PLD, which increases diacylglycerol production (DAG), to in turn ac-tivate PKCα [50,51]. This increase in PKCα has been observed in whole cell lysate and cell membrane fractions, while 24R,25(OH)2D3 has been shown to increase PKCα in resting zone matrix vesicles .
Although 24,25(OH)2D3 stimulation of PLD2 activates DAG to sti-mulate PKC, this subsequent activation of PKC does not coincide with the translocation of PKC to the plasma membrane . This suggests that 24R,25(OH)2D3 stimulation of PKC activity via PLD may not be a direct result of 24R,25(OH)2D3 binding to a membrane receptor and is instead a result of downstream signaling by other secondary messen-gers. Furthermore, although resting zone cells have active phospholi-pase C (PLC) isoforms – namely PLCβ1 and PLCβ3 – neither PLC is
activated by 24R,25(OH)2D3, and inhibition of PLC does not aﬀect 24R,25(OH)2D3 stimulation of PKC . Together the inhibition of PLA2 and stimulation of DAG act to enhance PKC activity to stimulate the phosphorylation of multiple proteins in multiple anti-apoptotic downstream pathways, including MAPK and ERK, which are also sti-mulated by 17β-estradiol through both genomic and non-genomic pathways [53–56].
1α,25(OH)2D3 also acts via membrane-associated mechanisms, but they diﬀer from those activated by 24R,25(OH)2D3. 1α,25(OH)2D3 membrane signaling involves a protein complex including protein dis-ulfide isomerase family A 3 (Pdia3), which is not involved in 24R,25(OH)2D3-dependent PKCα activation. Antibodies to Pdia3 do not block the stimulatory eﬀect of 24R,25(OH)2D3 on PKCα or its
downstream eﬀects [57,58]. This suggests that 24R,25(OH)2D3 signals through a membrane-associated mechanism independent from the mechanisms attributed to 1α,25(OH)2D3. This is distinct from the ability of 24R,25(OH)2D3 to stimulate the classical 1α,25(OH)2D3-VDR genomic signaling pathway at high concentrations, which is attributed to its weak binding capability to the ligand-binding pocket of VDR [59,60].
5. Vitamin D3 supplementation in cancer
Preclinical studies and meta-analyses have suggested that 1,25(OH)2D3 and 25(OH)D3 may have potential as a preventive therapy for breast cancer. Despite this, clinical studies investigating vitamin D supplementation on breast cancer prognosis have been largely incon-clusive [61–63]. Oral vitamin D3 has been shown to increase cell turnover, autophagy, and reduce tumor growth in the mammary glands of normal mice [64–66], and intravenous 1α,25(OH)2D3 has been shown to reduce tumor growth in estrogen receptor positive breast cancer xenografts . In vitro, 1α,25(OH)2D3 has been shown to re-duce angiogenic, metastatic, and apoptotic markers in estrogen re-ceptor positive breast cancer cells, but not in normal cultured human mammary epithelial cells [66–68]. This could be a function specific to the interactions of 1α,25(OH)2D3 with the VDR, which regulates 1α,25(OH)2D3′s genomic actions by dimerizing with the retinoic acid receptor to form a transcription complex . Dimerized VDR bound to 1α,25(OH)2D3 has been shown to suppress transcription of the ESR1 gene , which could inhibit the feed-forward loop of estrogen-ERα signaling . Alternatively, 1α,25(OH)2D3′s anti-tumorigenicity could be a function of its non-genomic actions, like its analogs 1,24,25-tri-hydroxyvitamin D3 and 1,25,26-trihydroxyvitamin D3, which are in-capable of binding to VDR, have also been shown to inhibit the growth of breast cancer cells in vitro .