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Cheng et al. J Cancer Metastasis Treat 2021;7:17 https://dx.doi.org/10.20517/2394-4722.2021.27 Page 13 of 18

PTHrP, an osteolytic factor expressed in most clinical BMETs [62,63] , from BMET-derived ER+ breast cancer
cells further supports this postulate and provides possible mechanistic insights for specific pathways
downstream of tumoral ERα activation that may contribute to ER+ BMET-associated osteolysis. The
enhanced secretion of PTHrP regulated by ERα from BMET-derived tumor cells, in particular, suggests: (1)
ERα expression in ER+ cells metastatic to bone - rather than being just a biomarker for BMETs - may also
be a potential molecular driver of osteolysis and metastatic progression in bone; and/or (2) either a
subpopulation of highly PTHrP-expressing cells preferentially formed BMETs and/or the bone
microenvironment altered the phenotype of bone-disseminated tumor cells to favor PTHrP-mediated
osteolysis. Either of these possibilities is consistent with clinical observations that PTHrP-positivity in breast
[62]
cancer is greater in BMET than in other metastatic sites or in primary tumors , a finding also verified in
pre-clinical murine studies documenting greater PTHrP expression in human breast cancer cells
spontaneously forming metastases in bone vs. other sites . The possible mechanistic importance of
[73]
tumoral PTHrP secretion in promoting tumor-associated osteolysis and, in turn, osteolytic BMET
progression, has already been established in one commonly studied pre-clinical ER- human BMET model,
where osteolytic BMET progression does not occur in the absence of tumoral PTHrP bioactivity [8,42] . Also of
particular relevance to the current studies, while E -regulation of PTHrP expression in ER+ MCF7 cells has
2
not, to our knowledge, been examined by laboratories other than our own , overexpression of PTHrP by
[74]
stable transfection in MCF-7 cells has been demonstrated to increase osteolysis specifically, in concert with
[26]
a significant increase in osteolytic BMET progression (as compared to wild-type cells) . Thus, existing
evidence supports the postulate that enhanced secretion of PTHrP mediated by ERα in ER+ tumor cells
disseminated to bone, as documented here, may be one specific pathway driving E dose-dependent tumor
2
osteolysis and osteolytic ER+ BMET progression documented in vivo.

Clearly, though, these studies have limitations. Indeed, while a bone-specific hypothesis for tumoral ERα
signaling driving BMET progression via mediation of tumor-associated osteolysis is straightforward, testing
in pre-clinical models, where E supplementation is necessary to support robust progression of osteolytic
2
BMET and a syngeneic mouse model is not available, is difficult since E has anabolic effects on the bone
2
microenvironment and also clearly drives ER+ breast cancer cell proliferation, which is not unique to the
bone microenvironment. Thus, while prior experiments utilizing E -driven ER+ human breast cancer
2
xenograft models and a single dose of E have demonstrated tamoxifen-inhibition of ER+ BMET following
2
intracardiac tumor cell inoculation, or a role of zoledronic acid or tumor cell PREX1 expression in
regulating dissemination of ER+ cells from primary orthotopic tumors ultimately home to bone [18,21,27] , none
have been able to elucidate the relative importance of bone vs. tumor effects of E , or other agents with dual
2
bone vs. tumor effects, such as zoledronic acid. In the experiments described here, which are the first, to our
knowledge, to test E dose dependency in an ER+ BMET model, the constancy across doses of E -driven
2
2
bone anabolism - an anticipated effect given E ’s known direct and/or indirect (via T and B lymphocytes,
2
with only the latter being present in the model used here) stimulatory effects on osteoblasts and inhibition
of myeloid-derived osteoclasts [32,60] - could not account for the E dose-dependency of tumor-associated
2
osteolysis. The osteolytic capacity of the ER+ tumors to overcome the marked increase in bone occurring in
E -treated mice, yielding osteolytic lesions similar in size and incidence to those reported in ER- models
2
[41]
where anabolic increases in bone do not occur , was also notable. However, the possibility that bone
anabolism may have played a permissive, albeit constant, role in BMET progression in this ER+ model
cannot be ruled out.

While the E dose-dependency of ER+ osteolytic BMET progression was not attributable to anabolic E bone
2
2
effects given the constancy of this tumor microenvironment effect across doses, E -driven bone anabolism
2
clearly had independent pro-metastatic effects as well. Larger osteolytic lesion sizes in young (vs. mature)