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  • br Materials and methods br Results

    2021-04-07


    Materials and methods
    Results
    Discussion We have evaluated the breast cancer prevention potential of PR blockade under conditions that included exposure to progestogens that are relevant to women (progesterone and MPA) and are known to differ in their receptor-binding and down-stream effects [25], [26], [27]. In our mouse study, we observed that TPA opposed the pro-hyperplastic effects of MPA and prevented mammary intraepithelial neoplasia in the virgin mice. We then tested the anti-cancer efficacy of TPA in a progestogen-stimulated MNU-induced mammary carcinogenesis model in rats, and confirmed that MPA and VX-765 synthesis increased MNU-induced tumor formation, which is inhibited by TPA. In addition to increasing tumor latency and decreasing tumor burden, TPA decreased the tumor cell proliferation (pHH3), and angiogenesis (CD34), and significantly reduced ER and PR expressions in the presence of MPA and progesterone, respectively. Our data are in agreement with previous studies. These include experiments testing the therapeutic value of TPA (10 mg/kg) or mifepristone in DMBA-induced rat mammary tumors, where growth suppression was observed [28], and a study testing tumor prevention in a rat-MNU model, where a subcutaneous TPA pellet (30 mg) significantly reduced mammary tumor formation [29]. In DMBA-treated mice, we did not observe mammary tumors prior to euthanasia for massive skin tumors, but we observed an increase in mammary epithelial hyperplasia with MPA that was significantly reduced with TPA treatment. Currier et al. have reported that forced breeders of DMBA treated FVB mice were susceptible to DMBA-induced mammary carcinogenesis [43]. This suggests that the oscillating hormonal environment of continuous pregnancies may render the mammary glands more susceptible to carcinogenesis than the VX-765 synthesis steady exposure of MPA in virgin mice. Another possibility is that dose of MPA (25 mg) might not be sufficient for mammary tumor formation. Furthermore, MPA and progesterone have been shown to promote the growth of T47D and BT474 xenograft tumors with estradiol supplementation in mice; mifepristone inhibited tumor growth indicating involvement of PR [44]. The prevention benefits of PR blockade may also extend to BRCA-associated tumorigenesis; in a study of nulliparous BRCA1/p53-deficient mice, mifepristone treatment completely prevented mammary tumor formation [45]. In humans, mifepristone given pre-operatively to premenopausal women reduced breast epithelial proliferation [46]. However, mifepristone has significant GR binding, whereas the second-generation agents TPA and UPA are highly selective for the PR, and a better safety profile has been demonstrated in studies of women with uterine fibroids [18], [47], making them excellent candidates for further development as breast cancer prevention agents. One avenue of breast tumor inhibition by TPA therapy may relate to the decreased serum progesterone concentrations observed in TPA-treated women (R. Wiehle, personal communication). We did not observe these expected decreases, but our result is consistent with a previous report in DMBA-induced carcinogenesis rat model [28], which showed that TPA increased serum progesterone in the presence of exogenous progesterone. However, in an MNU-carcinogenesis model, in the absence of exogenous progesterone, TPA treatment has been shown to decrease endogenous progesterone level in a dose dependent manner [29]. This variable effect of TPA on serum progesterone level may be due to the TPA action on pituitary gland [48], [49], [50]. Relatively low doses of mifepristone (2–5 mg), TPA (12.5–50 mg) and UPA (5–10 mg) block ovulation in healthy women [51]. Therefore, TPA action on the pituitary gland is likely to be similar to that of mifepristone. We also examined ER and PR expressions in MNU-induced mammary tumors and did not see a significant effect of MPA or progesterone on ER and PR expressions. However, ER expression was significantly decreased in MPA plus TPA treated animals, whereas PR expression was significantly decreased with the addition of TPA, similar to a previous study where TPA was used without exogenous progesterone [29]. Benakanakere et al. reported that both MPA and progesterone accelerated DMBA-induced mammary carcinogenesis in rats, but MPA significantly increased ER expression without altering PR expression while progesterone slightly lowered ER and PR expressions compared to DMBA controls in mammary tumors [11]. Thus in both DMBA and MNU-induced mammary tumors, progesterone suppresses PR expression, and MPA is more potent for tumorigenesis than progesterone [11], but their effects on ER and PR expressions vary. These differential effects between MPA and progesterone can be partially explained by their pharmacokinetics and hormone-receptor binding specificity: MPA is readily bioavailable, has a longer half-life than progesterone, and binds to AR and GR besides PR [52]. The PR down-regulation by progesterone seen in these rodent models is similar to the results from T47D breast cancer model in which R5020 treatment disrupts the expression of PRA and PRB isoforms through extensive post-translational PR modifications [53], [54], [55], [56], [57]. In addition, several recent studies suggested that PRA determines responsiveness to anti-progestins [58], [59], [60], and the PR expression is differentially modulated by each anti-progestin: UPA up-regulates PR but a novel antagonist APR19 suppresses it [61].