Julia Cheng Abstracts
Julia Cheng
Ph.D. Candidate
Cancer Biology GIDP
American Association for Cancer Research
Atlanta, Georgia
March 29-31, 2019
Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming clinically evident bone metastases (BMETs). To query ERα’s role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. Female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, & 0.72 mg E2). Osteolytic BMET formation was assessed radiographically and E2 effects on bone were determined by DXA and microCT at various time points. The BMET size and proliferation were assessed by immunohistochemical (IHC) analyses at the end of the experiment (42 days). The effect of E2 on tumoral secretion of the osteolytic factor, parathyroid hormone related protein (PTHrP), was determined using a commercially available radioimmunoassay.
The incidence and size of osteolytic BMET, which were not evident in the absence of E2 supplementation, were E2 dose-dependent in young mice. In contrast, E2 effects on the bone microenvironment were not dosedependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV). In skeletally immature (young) mice vs skeletally mature mice treated with an identical E2 dose (0.72 mg), which caused significantly different effects on bone turnover, progression of E2-dependent BMET was identical. These results suggest that E2 effects on the tumor, rather than bone, were driving E2-dependency of BMET progression. IHC analysis demonstrated that neither the size of human cytokeratin-positive tumors nor the proportion of Ki67-positive proliferating tumor cells in bone were E2 dose-dependent, suggesting that proliferative effects of E2 could not explain E2-dose dependent differences in osteolytic BMET formation. PTHrP, an osteolytic factor expressed in most clinical BrCA BMET, was E2-inducible ex vivo and secreted in higher levels by tumor cells isolated from BMETs. These results suggest that during ER+ BrCA BMET progression, tumoral effects of E2 not only support ER+ BMET proliferation, but may also have bone-specific effects due to the induction of PTHrP, which may explain the marked osteolytic capacity of the ER+ tumor cells in this model despite a microenvironment where bone volume is markedly increased.
Abstract for Lay Audience
The most common subtype of breast cancer is one where tumor cells express the estrogen receptor (ER) on their surface (denoted as “ER+”). Advanced-staged ER+ breast cancer tumors have a high rate of spreading to the skeleton, a process known as bone metastasis, which currently has no cure. When ER+ cells spread to bone, they create osteolytic lesions where the bone is lysed or “chewed up”, causing pain or fractures. ER+ breast cancer tumors are more likely to spread to the bone than tumors that do not express ER (denoted as “ER-“), however the reason for this is unknown. Since the major difference between ER+ and ER- breast cancer tumors is the expression of the ER, our laboratory hypothesizes that ER signaling may drive ER+ breast cancers to form more bone metastases.
To study the multiple steps that ER signaling may be involved during bone metastasis, our laboratory developed and characterized a mouse model of human ER+ breast cancer bone metastasis using a human ER+ breast cancer tumor cell line commonly used for research, called the MCF-7 cell line. Because we experimented with a human cell line, we used athymic mice with compromised immune systems to prevent any immune system rejection of the tumor. Before we injected a mouse with tumor cells, it was implanted with an extended-release estrogen (17β-estradiol) pellet to mimic optimal human growth conditions for ER+ tumor cells. Imaging of the hind legs, using both weekly x-rays and end-point microCT, allowed us to observe the formation of osteolytic lesions and any changes to bone density. We also sectioned and stained tissue slices containing the bone metastases to measure tumor size and the number of proliferating tumor cells. Lastly, we tested whether treating MCF-7 cells in a petri dish (outside of the mouse) with estrogen could induce the secretion of factors that could stimulate osteolysis within the bone.
We observed that the rate of formation and size of osteolytic lesions within our mouse models was dependent on the dose of the supplemented estrogen pellets. In other words, the higher the estrogen pellet concentration given to a mouse, the larger the osteolytic lesions, and vice versa. Estrogen also dramatically changed the bones of the mice by increasing bone density, but these changes were not dose-dependent-regardless of the estrogen dose, the bone density increased the same amount. We then compared ER+ breast cancer bone metastasis progression between mice with different bone environments: skeletally young mice whose bones were still growing, and skeletally mature mice whose bone growth were stabilized. We supplemented both age groups with the same dose of estrogen pellet, and inoculated them with the same number of MCF-7 tumor cells. The resulting bone metastasis progression was identical. These results suggest that ER+ bone metastasis progression in the mice was due to estrogen directly affecting the tumor, and not the surrounding bone.
Estrogen could be affecting the ER+ tumor’s ability to form bone metastasis in a variety of ways. For example, estrogen can signal through the ER to induce cell growth or secretion of factors. Therefore, we assessed estrogen’s effects on tumor proliferation within the bone, and tumor secretion of factors that would enhance osteolysis. The data showed that ER+ tumor size and how much the tumors proliferated within the bone were not dependent on the estrogen dose, suggesting that the estrogen dose-dependent effects of ER+ tumor osteolysis we observed was not simply due to estrogen’s ability to stimulate tumor growth. We then treated MCF-7 cells in petri dishes with estrogen and observed that this stimulated the secretion of parathyroid hormone-related protein (PTHrP), an osteolytic factor expressed in most clinical breast cancer bone metastases. Furthermore, PTHrP was secreted at even higher amounts in ER+ tumor cells that have been re-isolated from the bone metastases of mice injected with MCF-7 cells. These results suggest that during ER+ breast cancer bone metastasis progression, estrogen can not only promote tumor cell growth via the ER, but can stimulate the tumor to secrete osteolysis factors, such as PTHrP, which may explain the augmented ability of ER+ breast cancer tumors to form higher rates of bone metastases than ER- tumors.
The results from my research could lead to a better understanding of how ER+ breast cancer bone metastases progress, and thus advancements in biomarkers for early metastasis detection and targeted therapies.