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.

Figure 1. ERA knockdown affects estrogen-mediated regulation of PER2 and BMAL1 transcription. (A) Schemes showing the PER2 and BMAL1 promoter regions of the luciferase constructs used in these experiments (left). Estrogen (1 µM E2, 24 h) significantly induces PER2 and BMAL1 transcription in HME1 cells, as shown by both luciferase assay (middle) and quantification of PER2 and BMAL1 mRNAs by qRT-PCR (right). (B) Stable ERA-knockdown in HME1 cells (HME1-shERA) significantly decreases the ERA protein level (left) as well as the transcriptional response to estrogen (1µM E2, 24 h), as shown by the lack of induction of both ERE-luciferase (middle) and the ERA-target RARA (right). (C) Luciferase assay (left) and qRT-PCR (right) showing that estrogen (1 µM E2, 24 h) fails to induce PER2-luciferase and BMAL1-luciferase as well as endogenous PER2 and BMAL1 transcription in HME1-shERA cells.

Figure 2. ERA-knockdown affects the amplitude of PER2 and BMAL1 mRNA oscillation under entrainment conditions. The estrogen present in the entrainment medium can induce significantly more ERE-luciferase (A) and PER2 and BMAL1 mRNA (B) in HME1-Ctrl, relative to HME1-shERA cells. (C) The amplitude of PER2 and BMAL1 mRNA oscillations are significantly attenuated in HME1-shERA, relative to HME1-Ctrl cells.

Figure 3. ERA-mediated estrogen signaling controls the BMAL1-PER2 loop. (A) Transient PER2 transfection significantly promotes BMAL1-luciferase expression in HME1 cells. (B) Stable PER2-knockdown in HME1 cells significantly decreases PER2 mRNA level in both non-entrained and serum shock-entrained HME1-shPER2 cells (clones 16 and 18), relative to control HME1 cells. (C) PER2-knockdown leads to downregulation of both BMAL1-luciferase (Left) and BMAL1 mRNA (Right) before and after estrogen treatment (1 µM E2, 24h). (D) PER2 knockdown decreases BMAL1 mRNA level both in non-entrained and entrained cells. (E) Stable BMAL1 knockdown in HME1 cells significantly decreases BMAL1 mRNA level in both non-entrained and serum shock-entrained HME1-shBMAL1 cells (clones 5 and 9). (F) Entrained HME1-shBMAL1 clones display reduced PER2 mRNA oscillations relative to HME1-Ctrl cells.

Figure 4. Influence of PER2 and BMAL1 downregulation on ERA mRNA circadian oscillation. (A) ERA mRNA oscillates in a circadian fashion in entrained HME1-Ctrl cells (left). The estrogen content in the entrainment medium can induce the transcription of the ERA-direct target gene RARA in HME1-Ctrl, but not in HME1-shERA cells (middle). Upon entrainment, RARA mRNA oscillates in a circadian fashion (right). (B) The circadian oscillation of both ERA (left) and RARA (right) mRNAs is impaired in HME1-shPER2 and HME1-shBMAL1 cells. (C) RARA mRNA oscillation is impaired also in T47D and MCF7 breast cancer cells, where ERA mRNA does not undergo circadian oscillation.

Figure 6. The ERA-PER2-BMAL1 circuit. In the presence of an intact ERA-PER2-BMAL1 circuit, whereby ERA-mediated estrogen signaling affects the oscillations of PER2 and BMAL1, and the PER2-BMAL1 regulatory loop in turn affects ERA oscillations, HME1 cells undergo proper acinar morphogenesis. Interference with any of the components of the ERA-PER2-BMAL1 circuit has a rebound on the circadian oscillation of the other components and results in impaired HME1 acinar morphogenesis.

Figure 5. Evidence that circadian components of the estrogen-regulated circuit are implicated in breast acinar morphogenesis. (A) Immunocytochemistry followed by confocal microscopy showing control HME1 acini after 12 d of three-dimensional (3D) culture on reconstituted basement membrane (Matrigel). In the top panel, the organization of the DAPI-stained nuclei (blue) indicates whether the acini are hollow, the presence of an outer layer of integrin (green) indicates baso-lateral polarity, while the presence of the Golgi apparatus (red) in the inner portion of the acinus is a sign of apico-basal polarity. The percentage of acini with the displayed morphology is indicated in the upper left corner. In the bottom panel, immunostaining with anti-ERA (green) shows ERA expression both in the cytoplasm and, to a lesser extent, in the DAPI-stained nuclei (blue). The lower inset shows an enlargement of the framed area. (B) The top panels show the aberrant morphology of the HME1-shERA (left), HME1-shPER2 (middle) and HME1-shBMAL1 (right) acini, with the relative percentage. The bottom panels show that HME1-shERA, HME1-shPER2 and HME1-shBMAL1 aberrant acini differ in ERA expression.