The Roles of Estrogen and Transforming Growth Factor-β Signaling Pathways in Estrogen Receptor-Positive Breast Cancer Osteolytic Bone Metastasis Progression

Abstract

Tumors that express the estrogen receptor alpha (ERa) are the most commonly diagnosed breast cancer subtype (>70%), and also have the highest predilection to form bone metastases (BMETs). These incurable and primarily osteolytic ERa-positive (ER+) BMETs often recur decades after cessation of hormone therapy and/or in conjunction with ligand-independent ER mutations. In addition, despite similar rates of both ER- and ER+ tumors disseminating to the bone, ER+ tumors are more likely to develop into overt BMETs. Taken together, this suggests that tumoral ERa signaling within the bone microenvironment may act as a molecular driver, analogous to the role of transforming growth factor β (TGFβ), a bone-derived factor released during osteolysis that drives preclinical models of ER- BMETs by mediating tumor-secretion of pro-osteolytic factors such as parathyroid hormone-related protein (PTHrP), an important osteolytic factor expressed in >90% of clinical BMETs. However, a bone-specific role for tumoral ERa in driving ER+ BMET, including the possible induction of tumoral secretion of osteolytic factors—analogous to TGFβ—during ER+ BMET, is unknown due to the lack of a well-characterized preclinical model. Development of murine ER+ BMET models has proven challenging due to the absence of a syngeneic murine ER+ BMET model and the necessity of 17β-estradiol (E2)-supplementation for optimal human tumor growth and osteolytic BMET progression in mice, which also markedly alters the bone microenvironment, making it difficult to distinguish tumoral vs. microenvironmental effects of E2. These studies aim to characterize the dose-dependent skeletal impact of E2 supplementation in immunocompromised athymic mice used for ER+ breast cancer BMET models, including an assessment of tumoral vs. bone microenvironmental effects of E2. After identifying bone metastatic human ER+ breast cancer cell lines and characterizing the model, the roles of tumoral ER and TGFβ signaling pathways, including their possible crosstalk, will be elucidated, with the postulate that while TGFβ signaling can contribute to ER+ breast cancer BMET osteolytic progression, effects are abated in an estrogenic milieu, as suggested by published literature, and that tumoral ERa signaling, independent of proliferative effects, can drive ER+ tumor progression within the bone microenvironment. The studies described herein have successfully delineated between E2’s direct effects on the tumor during ER+ BMET vs. anabolic effects on the bone in a preclinical model and have elucidated the roles of tumoral ER and TGFβ signaling during ER+ BMET progression. ER+ BMET was demonstrated to be E2 dose-dependent, specifically through ERa-mediated osteolytic events, including PTHrP-secretion, without involvement of other ERs expressed by breast cancer cells (i.e. ERβ and G-protein coupled ER [GPER]), and were also independent of ER-driven effects on tumor proliferation. Contrary to previous reports suggesting opposing roles of ER and TGFβ signaling in ER+ breast cancer cells, TGFβ was also demonstrated to mediate tumoral PTHrP secretion in vitro, with additive effects when combined with E2. Furthermore, the bone metastatic potential of various ER+ tumor cell lines was associated with TGFβ-inducible Smad signaling, and treatment with a TGFβ-neutralizing antibody significantly blocked in vivo progression of ER+ BMETs. Finally, mice supplemented with estetrol (E4), an estrogen that drives ER+ cell proliferation and orthotopic tumor growth in mice due to exclusive signaling through canonical (cytoplasmic) ER, while antagonizing E2’s effects on non-canonical (membrane-bound) ER, did not form osteolytic BMETs, despite evidence of tumor cell dissemination to the bone and additive effects in secreting PTHrP with E2 and/or TGFβ in vitro. This suggests that, though their specific roles may be more complex, both cytoplasmic and membrane-bound ERa are important during ER+ BMET progression. Taken together, these novel findings provide the first proof of a bone-specific role for ERa in driving ER+ BMET progression, and additive effects with TGFβ signaling pathways that are present in ER- BMETs, providing a mechanistic explanation for the increased propensity of ER+ tumors to form clinical evident BMET, as well as an opportunity to identify additional drug targets for a disease that, despite hormone therapy and bone-specific agents, remains incurable.