Identification of an estrogen-regulated circadian mechanism necessary for breast acinar morphogenesis

Abstract

Altered estrogen receptor α (ERA) signaling and altered circadian rhythms are both features of breast cancer. By using a method to entrain circadian oscillations in human cultured cells, we recently reported that the expression of key clock genes oscillates in a circadian fashion in ERA-positive breast epithelial cells but not in breast cancer cells, regardless of their ERA status. Moreover, we reported that ERA mRNA oscillates in a circadian fashion in ERA-positive breast epithelial cells, but not in ERA-positive breast cancer cells. By using ERA-positive HME1 breast epithelial cells, which can be both entrained in vitro and can form mammary gland-like acinar structures in three-dimensional (3D) culture, first we identified a circuit encompassing ERA and an estrogen-regulated loop consisting of two circadian clock genes, PER2 and BMAL1. Further, we demonstrated that this estrogen-regulated circuit is necessary for breast epithelial acinar morphogenesis. Disruption of this circuit due to ERA-knockdown, negatively affects the estrogen-sustained circadian PER2-BMAL1 mechanism as well as the formation of 3D HME1 acini. Conversely, knockdown of either PER2 or BMAL1, by hampering the PER2-BMAL1 loop of the circadian clock, negatively affects ERA circadian oscillations and 3D breast acinar morphogenesis. To our knowledge, this study provides the first evidence of the implication of an ERA-circadian clock mechanism in the breast acinar morphogenetic process.

Harnessing 3D models of mammary epithelial morphogenesis: An off the beaten path approach to identify candidate biomarkers of early stage breast cancer

“…As we reported in previous studies [2, 7, 28, 29], when grown in reconstituted basement membrane 3D culture (Matrigel), HME1 non-tumorigenic human mammary epithelial cells develop into acinar structures that are hollow (see DAPI-stained nuclei lining the lumen), lined by polarized cells (see localization of Golgi and integrin), and growth-arrested (see lack of EdU-positive, proliferating cells) (Figure 1A, left). In contrast, five HME1-derived stable clonal lines carrying distinct genetic factors (collectively referred to as HME1 mutant lines) develop into “DCIS-like” morphologically aberrant 3D acini with a lumen filled with proliferating cells (Figure 1A, right).…”

“…HME1-derived stable clonal lines HME1-DNC4, HME1-shERA, HME1-shPER2, HME1-shMTG16, and HME1-MYC (in this study collectively referred to as HME1 mutant lines) were previously described [2, 28, 29]. HME1 clones with stable ANXA2 knock down were generated by stable transfection with pSUPER-shANXA2 sequence B or pSUPER-shANXA2 sequence C targeting both ANXA2 transcript variant 1 and 2.…”

“…By using a human, non-tumorigenic HME1 mammary epithelial model, we previously reported that stable distinct genetic mutations, by interfering with different functions or signaling pathways, affect the 3D HME1 cell morphogenetic potential [2, 7, 28, 29]. Specifically, regardless of the initiating mutation, all HME1 clonal lines when seeded in basement membrane culture, develop into 3D amorphous and proliferative “DCIS-like” acini.…”

Estrogen’s effects in chronic autoimmune/inflammatory diseases and progression to cancer

“…The concomitant circadian increase of growth factors during the late night in cancer patients, as well as some estrogen-regulated circadian mechanisms necessary for breast acinar morphogenesis, suggest further important similarities between both pathological conditions [66,67].…”

Night Shifts and Melatonin: Relevance to Age and Breast Cancer

“…In fact expression of clock genes oscillates in a circadian fashion in ER-alpha mRNA-positive breast epithelial cells but not in ER-alpha-positive breast cancer cells. Downregulation of two circadian clock genes either Per2 or BMAL1 negatively affects ER-alpha circadian oscillations and breast acinar morphogenesis (Rossetti et al 2012a ). Not only ER-alpha-negative human breast cancer cells display a defective non-circadian oscillations clock but also ER-alpha-positive breast cancer cells do not display circadian oscillations in any of the clock genes.…”