Oestradiol Contributes to Differential Antitumour Effects of Adjuvant Zoledronic Acid Observed Between Pre- and Post-Menopausal Women

Abstract

Clinical trials have demonstrated that adding zoledronic acid (Zol) to (neo)adjuvant standard of care has differential antitumour effects in pre- and post-menopausal women: Both benefit from reduced recurrence in bone; however, while postmenopausal women also incur survival benefit, none is seen in premenopausal women treated with adjuvant bisphosphonates. In the current study, we have used mouse models to investigate the role of oestradiol in modulating potential antitumour effects of Zol. Pre-, peri-, and post-menopausal concentrations of oestradiol were modelled in BALB/c wild-type, BALB/c nude, and C57BL/6 mice by ovariectomy followed by supplementation with oestradiol. Mice also received 40 mg/kg/day goserelin to prevent ovariectomy-induced increases in follicle-stimulating hormone (FSH). Metastasis was modelled following injection of MDA-MB-231, 4T1, or E0771 cells after ovariectomy and saline or 100 μg/kg Zol administered weekly. Supplementing ovariectomised mice with 12.5 mg/ml, 1.38 mg/ml, and 0 ng/ml oestradiol, in the presence of goserelin, resulted in serum concentrations of 153.16 ± 18.10 pg/ml, 48.64 ± 18.44 pg/ml, and 1.00 ± 0.27 pg/ml oestradiol, which are equivalent to concentrations found in pre-, peri-, and post-menopausal humans. Osteoclast activity was increased 1.5–1.8-fold with peri- and post-menopausal compared with premenopausal oestradiol, resulting in a 1.34–1.69-fold reduction in trabecular bone. Zol increased trabecular bone in all groups but did not restore bone to volumes observed under premenopausal conditions. In tumour-bearing mice, Zol reduced bone metastases in BALB/c (wild-type and nude), with greatest effects seen under pre- and post-menopausal concentrations of oestradiol. Zol did not affect soft tissue metastases in immunocompetent BALB/c mice but increased metastases 3.95-fold in C57BL/6 mice under premenopausal concentrations of oestradiol. In contrast, Zol significantly reduced soft tissue metastases 2.07 and 4.69-fold in immunocompetent BALB/c and C57BL/6 mice under postmenopausal oestradiol, mirroring the results of the clinical trials of (neo)adjuvant bisphosphonates. No effects on soft tissue metastases were observed in immunocompromised mice, and differences in antitumour response did not correlate with musculoaponeurotic fibrosarcoma (MAF), macrophage capping protein (CAPG), or PDZ domain containing protein GIPC1 (GIPC1) expression. In conclusion, oestradiol contributes to altered antitumour effects of Zol observed between pre- and post-menopausal women. However, other immunological/microenvironmental factors are also likely to contribute to this phenomenon.

Considerations of Timing Post-ovariectomy in Mice and Rats in Studying Anxiety- and Depression-Like Behaviors Associated With Surgical Menopause in Women

“…Despite few studies on endocrine changes occurring in ovariectomized mice, it has been reported that after 4 days and 1 week post-ovariectomy, the concentrations of FSH are significantly increased with respect to intact cycling mice (Bronson, 1976;Chandrashekar and Bartke, 1996;Xu et al, 2000), but greater effects are detected after 2-4 weeks post-ovariectomy (Rodin et al, 1990), as it occurs in ovariectomized rats. Accordingly, after 2-14 weeks postovariectomy in mice, serum estradiol levels were significantly decreased by approximately 31% with respect to the shamoperated mice, which was accompanied by a significantly increased concentration of FSH (Park et al, 2014;Lee et al, 2020;Canuas-Landero et al, 2021). These data clearly show the importance of considering and standardizing the postovariectomy time frame to identify behavioral and hormonal changes associated with this surgical manipulation, thus avoiding erroneous interpretation of results and compare results from different laboratories.…”

MiR-662 is associated with metastatic relapse in early-stage breast cancer and promotes metastasis by stimulating cancer cell stemness

“…Experiments were conducted as previously described [ 11 , 30 ]. Briefly, tumour growth was monitored in anesthetised mice using an IVIS Lumina II system (Caliper Life Sciences, UK), following subcutaneous injection of d -Luciferin (Invitrogen, UK) to animals 4 min before imaging.…”

“…Histological analysis was carried out on paraffin-embedded medial sections of tibial metaphysis as previously described [ 11 , 30 ]. The in situ detection of osteoclasts was performed on tartrate-resistant acid phosphatase (TRAP)-stained bone tissue sections using a commercial kit (Sigma).…”

“…For in vivo experiments, power calculations for animal experiments are based on our previous work [ 11 , 18 , 32 ]. In experiments conducted with NW1 cells or 4T1 cells, 60–70% of animals have metastasis.…”

“…So far, miR-662 has been found to be a poor prognosis factor in patients with early-stage squamous cell lung carcinoma (SCC) and to promote motility and chemoresistance of SCC cells in vitro [ 10 ]. Here, miR-662 functions in BC cells were investigated using several in vitro cell-based assays (proliferation, migration, cancer cell stemness, osteoclastogenesis), RNA sequencing, and metastatic outgrowth in bones of established animal models of BC metastasis [ 11 ]. Overall, our results show a pro-metastatic role for miR-662 in animal models, through enhancement of malignant traits (proliferation, migration, stemness) in BC cells.…”