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  • br ER mediates the anti apoptotic e ect of


    ERα36 mediates the anti-apoptotic effect of E2 by activating PLD at the membrane leading to activation of LPA signaling and PI3K, which in turn leads to attenuation of the caspase cascade that promotes apoptosis [57]. E2-ERα36 signaling has also been implicated in the activation of other rapid pathways such as ERK-MAPK, PI3K-Akt, and PKC-ERK, and may have downstream effects on the employment of IGF-IR, EGFR, HER2 [128], resulting in a decrease in apoptosis and an increase in proliferation and metastatic potential in a variety of cancer cells, in-cluding breast cancer cells [42,129]. These pathways have been re-ported to stimulate tumorigenicity in both ER positive and negative breast cancer tumors [73,96]. These rapid signaling mechanisms mediated by ERα36 are insensitive to anti-estrogens and have even been shown to employ agonist activities in response to anti-estrogens, such as tamoxifen and ICI-182,780, which are both known inhibitors of the classical ERα66 [126]. Both Fasudil that block the ERα36 mediated effect of E2 and ICI 182,780 (which blocks cytosolic and nuclear ERs) abolished the pro-apoptotic effect of 24R,25(OH)2D3. However, only the antibody to ERα36 blocked the proliferative effect. This implies that 24R,25(OH)2D3 and both the canonical and mem-brane-associated signaling pathways of E2 share a common downstream pathway, the exact mechanism of which remains to be revealed [Fig. 8].
    Upon activating a signaling cascade at the membrane, 24R,25(OH)2D3 stimulates the secondary messenger PLD. PLD stimu-lates the LPAR1/3 receptor, and downstream signaling stimulates PI3K, upregulates Bax, and downregulates Bcl2 expression to increase p53 production and induce apoptosis in MCF7 cells. A similar mechanism is responsible for the proliferative actions of 24R,25(OH)2D3, which is regulated by a membrane-mediated receptor in the caveolae niche via a PLD-dependent mechanism. 24R,25(OH)2D3 also decreases invasion and metastatic markers through an as yet unidentified mechanism.
    5. Conclusion
    Like other steroid hormones, 24R,25(OH)2D3 has been found to initiate multiple rapid signaling pathways from the cell membrane in different types of cells, and a membrane receptor has been implicated, which remains the subject of active investigation. Our results suggest that the rapid actions of 24R,25(OH)2D3 in MCF7 breast cancer cells may be modulated by a membrane-mediated, caveolae-localized pathway employing a palmitoylated nuclear receptor to stimulate PLD with downstream regulatory effects on apoptosis, proliferation, migra-tion, and metastasis. Our work also suggests that these effects may be modulated by cross-talk between the as yet undiscovered 24R,25(OH)2D3 receptor and E2 signaling via membrane-associated ERα isoforms. It is clear that 24R,25(OH)2D3 confers a chemoprotective effect against breast cancer cells in vivo. However, the mechanism by which it mediates this effect and the crosstalk with other pertinent pathways needs to be further evaluated. However, this work provides a foundation for the further investigation of 24R,25(OH)2D3 as an anti-tumorigenic agent in breast cancer, and suggests that 24R,25(OH)2D3, as a naturally-occurring, non-calcemic, alternative to traditional vi-tamin D3, should be investigated as an alternative vitamin D3 supple-ment for breast cancer patients.
    Mouse models created in support of this research were generated by the VCU Massey Cancer Center Mouse Models Core Laboratory, sup-ported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059.
    This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.