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Page 14 of 18 Cheng et al. J Cancer Metastasis Treat 2021;7:17 https://dx.doi.org/10.20517/2394-4722.2021.27

mice treated with the same E dose appeared attributable to greater E -mediated anabolism in young mice
2
2
since tumor cell dissemination and proliferation were otherwise the same. Increased osteolytic BMET lesion
size in E -treated (vs. control) mice inoculated with ER- breast cancer cells further confirmed a role of
2
anabolic bone microenvironmental effects of E in driving osteolytic breast cancer BMET progression,
2
independent of tumoral ER signaling, consistent with previous similar reports in ER- BMET models [35,36] .
Because these experiments provide the first evidence, to our knowledge, that bone anabolic effects of E
2
promote ER+ BMET progression subsequent to tumor cell dissemination to bone (as bone seeding was E -
2
and age-independent), this finding may have clinical implications when estrogens and/or other anabolic
agents are used to treat osteoporosis in post-menopausal women , an age where breast cancer incidence is
[75]
[76]
the highest and silent bone micrometastases may already be present prior to a ER+ breast cancer
diagnosis [77-80] . However, additional studies are required to explore this more specifically for both ER+ and
ER- BMET, as, for example, studies evaluating anabolic effects of parathyroid hormone (PTH) on ER-
BMET progression have yielded mixed results to date [28,81,82] . Additionally, it should be noted that the
absence of an E effect on ER+ tumor cell dissemination to bone confirms previous reports [20,27] and is
2
consistent with the clinical observation of similar incidences of bone micrometastases in clinical series of
patients with ER+ or ER- breast cancers [77-80] .

Lastly, the study of only a single ER+ breast cancer cell line in these pre-clinical experiments is a limitation.
However, it should be noted that studies using breast cancer cells derived from a single ER- cell line (MDA-
MB-231), which shares fewer attributes with clinical breast tumors than the MCF-7 cells used here ,
[83]
account for a large portion of pre-clinical breast cancer BMET research, but have still yielded important
clinical insights, including the now standard therapeutic use of bisphosphonates for BMET . Because of
[84]
the reported low take-rates and rare formation of BMETs by ER+ patient derived xenografts (PDX) [85-88] ,
ER+ MCF-7 cells were initially chosen for these studies given their well-described ability to form osteolytic
BMETs in E -supplemented mice [20-26] . In addition, inoculation of other commonly used ER+ cell lines
2
known to disseminate to bone (T47D and ZR-75-1) [18,89] did not result in osteolytic BMET formation, with
or without E supplementation (data not shown). However, this difference in osteolytic BMET potential
2
between ER+ tumor cells provides evidence that the pro-osteolytic effects of E signaling in bone-
2
disseminated ER+ breast cancer cells are likely also interdependent on other cellular transformations and
signaling pathways present in ER+ tumor cells within the bone microenvironment - a postulate that awaits
further testing.

In conclusion, while the study of ER+ breast cancer BMETs is complicated by the duality of ERα signaling
effects in bone vs. bone-disseminated ER+ tumor cells, the experiments reported here, by taking advantage
of differential dose-dependent effects of E on bone vs. ER+ tumor-associated osteolysis, suggest that ER+
2
osteolytic BMET progression may be specifically promoted by tumoral ERα signaling via the induction of
osteolysis. Thus, additional bone-specific molecular targets downstream of ERα, in addition to those that
drive proliferation, may complement existing therapeutics for the treatment of osteolytic ER+ BMETs,
particularly for HT-resistant metastatic ER+ breast cancer, while potentially providing a mechanistic basis
for the long-standing clinical observation of the association of tumoral ERα expression with breast cancer
metastatic risk specific to bone.

DECLARATIONS
Acknowledgements
We would like to acknowledge Andrea Grantham in memoriam for her many years of artful histological
processing of our murine bone samples at the University of Arizona and for reminding us that a life outside
the lab lived in the service of others is the most rewarding. We would also like to thank Alfred Li at the San