Funk, Janet L.

Abstract Text While the importance of estrogen in driving ER+ breast cancer progression is clear, data from pre-clinical studies exploring the role of tumoral progesterone (PR) and androgen (AR) receptors are conflicting, likely due in part to the promiscuity of steroid receptor ligands, as well as relative levels of steroid hormone receptor expression and of steroid hormones within the tumor milieu. While bone is usually the first site of metastases in ER+ metastatic breast cancer, the roles of tumoral PR and AR (present in 58 percent and 86 percent of clinical ER+ bone metastases [BMET], respectively) in ER+ breast cancer progression at this site have not been well studied. Studies were undertaken to examine the effects of ovariectomy (OVX), a model of surgical menopause that significantly reduces endogenous progesterone and androgen levels, in a pre-clinical model of human ER+ bone metastases that is dependent on exogenous 17ß-estradiol (E2) supplementation. Effects of OVX (at 9-weeks of age) were ascertained using skeletally mature female nude mice supplemented with a modest dose of E2 (0.36 mg 60 day pellets) prior to intracardiac inoculation (at 10 weeks of age) with bone metastasis-derived MCF7 cells (43.4M cells) that form robust E2- and TGFß-dependent osteolytic metastasis in vivo, and express PR and AR in vitro. Osteolytic bone metastatic lesions in E2-supplemented mice were reduced in both incidence and size in OVX (vs ovary intact) mice. Anti-tumoral effects of OVX were not limited to bone; E2-driven orthotopic tumor progression was similarly reduced. Histologic assessment of cytokeratin-positive breast cancer tumor cells within bone revealed a small but statistically significant reduction in Ki67-positivity in OVX (vs ovary -intact) mice that correlated with osteolytic lesion size. In addition, PR and AR expression in BMET lesions, which was readily detected by IHC in ovary-intact mice, was markedly reduced in OVX mice. While additional experiments are underway to query possible roles of specific reproductive hormones altered in OVX mice, these results are consistent with the postulate that tumoral AR and PR signaling may promote ER+ bone metastases progression in this model. In addition, while ovariectomy is a common pre-clinical means of reducing estrogen to model natural menopause in women, resultant changes in steroid receptor signaling are clearly much more complex. Funding: NIH 5R25CA275753, 5P30CA023074 Date of Presentation October 17, 2024

Abstract Text While the importance of estrogen in driving ER+ breast cancer progression is clear, data from pre-clinical studies exploring the role of tumoral progesterone (PR) and androgen (AR) receptors are conflicting, likely due in part to the promiscuity of steroid receptor ligands, as well as relative levels of steroid hormone receptor expression and of steroid hormones within the tumor milieu. While bone is usually the first site of metastases in ER+ metastatic breast cancer, the roles of tumoral PR and AR (present in 58 percent and 86 percent of clinical ER+ bone metastases [BMET], respectively) in ER+ breast cancer progression at this site have not been well studied. Studies were undertaken to examine the effects of ovariectomy (OVX), a model of surgical menopause that significantly reduces endogenous progesterone and androgen levels, in a pre-clinical model of human ER+ bone metastases that is dependent on exogenous 17ß-estradiol (E2) supplementation. Effects of OVX (at 9-weeks of age) were ascertained using skeletally mature female nude mice supplemented with a modest dose of E2 (0.36 mg 60 day pellets) prior to intracardiac inoculation (at 10 weeks of age) with bone metastasis-derived MCF7 cells (43.4M cells) that form robust E2- and TGFß-dependent osteolytic metastasis in vivo, and express PR and AR in vitro. Osteolytic bone metastatic lesions in E2-supplemented mice were reduced in both incidence and size in OVX (vs ovary intact) mice. Anti-tumoral effects of OVX were not limited to bone; E2-driven orthotopic tumor progression was similarly reduced. Histologic assessment of cytokeratin-positive breast cancer tumor cells within bone revealed a small but statistically significant reduction in Ki67-positivity in OVX (vs ovary -intact) mice that correlated with osteolytic lesion size. In addition, PR and AR expression in BMET lesions, which was readily detected by IHC in ovary-intact mice, was markedly reduced in OVX mice. While additional experiments are underway to query possible roles of specific reproductive hormones altered in OVX mice, these results are consistent with the postulate that tumoral AR and PR signaling may promote ER+ bone metastases progression in this model. In addition, while ovariectomy is a common pre-clinical means of reducing estrogen to model natural menopause in women, resultant changes in steroid receptor signaling are clearly much more complex. Funding: NIH 5R25CA275753, 5P30CA023074 Date of Presentation October 17, 2024

Skeletal muscle is a highly plastic tissue, adapting its structure and metabolism in response to various stimuli like exercise. This study aimed to determine the plasticity of skeletal muscle DNA methylation in response to an acute exercise bout in volunteers with a range of insulin sensitivities. We studied 14 sedentary participants (7 male/7 female): age = 34.5 ± 3.1 years, body mass index = 29.3 ± 1.9 kg/m2 and HbA1c = 5.3 ± 0.1 %. We performed VO2peak tests to determine exercise capacity (VO2peak = 25.5 ± 1.6 mL per Kg per minute) and euglycemic hyperinsulinemic clamps (Rd value = 5.9 ± 0.8 mg per Kg per minute) with vastus lateralis muscle biopsies before and 30 minutes after a single 48-minute exercise bout. We extracted DNA from the muscle biopsies and performed reduced representation bisulfite sequencing. After performing quality control on the sequencing data, 1,034,947 sites were included for downstream analyses. MethylSig analysis revealed 17,030 differentially methylated cytosines (DMC), 9,020 increased and 8,010 decreased. To identify biologically meaningful DMCs, we used a mean change of >10%, which resulted in 1,179 and 1,436 increased and decreased sites, respectively. Pathway analysis of the increased DMCs revealed an enrichment in efferocytosis, leukocyte transendothelial migration, and FOXO-mediated transcription of oxidative stress pathways. In addition, pathway analysis of the decreased DMCs revealed an enrichment in metabolic (type 2 diabetes/ PI3K-Akt signaling), actin cytoskeleton, integrin/extracellular matrix and calcium signaling pathways. Our study reveals that an acute exercise bout induces alterations in the DNA methylation of skeletal muscle, impacting specific pathways in individuals with varying degrees of insulin sensitivity. Notably, our findings underscore the remarkable plasticity of skeletal muscle tissue. This research was funded by the National Institutes of Health, grant number R01DK094013 (Dawn Coletta). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.