Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

<p>Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, <i>P</i> < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G₂/M phases, whereas the G₁ phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G₂/M phases (by 9.1 hours, <i>P</i> < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G₂/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G₂/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.</p

Indole-3-carbinol and its N-alkoxy derivatives preferentially target ER<b>α</b>-positive breast cancer cells

<div><p>Indole-3-carbinol (I3C) is a natural anti-carcinogenic compound found at high concentrations in <i>Brassica</i> vegetables. I3C was recently reported to inhibit neutrophil elastase (NE) activity, while consequently limiting the proteolytic processing of full length cyclin E into pro-tumorigenic low molecular weight cyclin E (LMW-E). In this study, we hypothesized that inhibition of NE activity and resultant LMW-E generation is critical to the anti-tumor effects of I3C. LMW-E was predominately expressed by ERα-negative breast cancer cell lines. However, ERα-positive cell lines demonstrated the greatest sensitivity to the anti-tumor effects of I3C and its more potent N-alkoxy derivatives. We found that I3C was incapable of inhibiting NE activity or the generation of LMW-E. Therefore, this pathway did not contribute to the anti-tumor activity of I3C. Gene expression analyzes identified ligand-activated aryl hydrocarbon receptor (AhR), which mediated sensitivity to the anti-tumor effects of I3C in ERα-positive MCF-7 cells. In this model system, the reactive oxygen species (ROS)-induced upregulation of ATF-3 and pro-apoptotic BH3-only proteins (e.g. NOXA) contributed to the sensitivity of ERα-positive breast cancer cells to the anti-tumor effects of I3C. Overexpression of ERα in MDA-MB-231 cells, which normally lack ERα expression, increased sensitivity to the anti-tumor effects of I3C, demonstrating a direct role for ERα in mediating the sensitivity of breast cancer cell lines to I3C. Our results suggest that ERα signaling amplified the pro-apoptotic effect of I3C-induced AhR signaling in luminal breast cancer cell lines, which was mediated in part through oxidative stress induced upregulation of ATF-3 and downstream BH3-only proteins.</p></div

Combination drug treatment prevents induction of aberrant acinar development by LMW-E.

<p>(A) Cells were seeded on Matrigel for 24 hours and then treated with rapamycin, sorafenib, and roscovitine as indicated. Medium containing drugs was replaced every 4 days, and lysates were collected on day 15 for Western blot analysis with the indicated antibodies. (B) On day 15 of Matrigel culture, cells grown as in (A) were fixed and stained with E-cadherin (red) and Ki67 (green), and nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. (C) The diameters of the acini were measured and averaged from three independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). (D) The number of Ki67-positive cells per acinus was counted and averaged from three independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). Kaplan-Meier survival plots demonstrating association between full length and high LMW-E on disease-specific survival. Association of:</p

LMW-E is tumorigenic.

<p>Athymic mice were injected subcutaneously with 1×10⁷ 76NE6 cells stably transfected with empty vector, EL, LMW-E and MDA-MB-468 cells. After 10 weeks, the tumors were removed for expansion in culture for further <i>in vivo</i> passaging and also for IHC analysis. Tumor incidence rate was estimated with exact 95% confidence intervals and Fisher's exact tests were used to compare tumor incidence rate between/among groups (*p<0.0001: 76NE6-vector vs. 76NE6-LMW-E; 76NE6-EL vs. 76NE6-LMW-E; 76NE6-vector vs. TDCs; 76NE6-EL vs. TDCs).</p

LMW-E renders hMECs tumorigenic, and LMW-E expression is selected with increased <i>in vivo</i> passaging.

<p>(A) Schematic of the generation of <i>in vivo</i> passaged clones with 3 successive injections (T1G2, T1G3, and T1G4). (B) Tumors from <i>in vivo</i> passaging were removed from mice, minced, and cultured on monolayer plates. Lysates were extracted and subjected to Western blot analysis with antibodies against cyclin E, elafin, and β-actin. EL (C), LMW-E (D), and elafin (E) protein levels were quantified by densitometry and compared between different generations of <i>in vivo</i> passaged cells (Student <i>t</i> test, *p<0.05). (F) Paraffin-embedded slides of 4 representative tumors were stained with hematoxylin and eosin (top panel) and cyclin E antibody (bottom panel).</p

High LMW-E expression is associated with activated b-Raf-ERK1/2-mTOR pathway <i>in vitro</i> and in patient tissues.

<p>(A) Hierarchal cluster analysis of protein expression in 76NE6, 76NE6-LMW-E and all of the LMW-E-expressing tumor clones grown on 2D (red) and 3D (green) cultures and 276 breast cancer patient samples (blue). (B) Proteins whose expression was associated with high LMW-E levels. Red indicates that high LMW-E along with high protein expression was associated with poor prognosis; grey indicates that high LMW-E along with low protein expression was associated with poor prognosis. (C) The lysates from 3D culture were subjected to Western blot analysis to validate the RPPA data. The cell lines are 76NE6-parental (P) and with stable expression of vector (V), EL, and LMW-E and the tumor-derived cells (TDCs). (D) Linear regression analysis of the RPPA data and the densitometry values of all the proteins analyzed by Western blot analysis in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002538#pgen-1002538-g005" target="_blank">Figure 5C</a>. The same antibodies were used for the two types of analysis.</p

LMW-E induces formation of large and highly proliferative acini.

<p>(A) 76NE6, MCF-10A, HS 578T, and MDA-MB-231 cells were seeded at a density of 70 cells/mm² on 1-mm-thick Matrigel. After 15 days in 3D culture, cells were fixed and immunostained with GM-130 and α6-integrin antibodies. Nuclei were counterstained with DAPI. Scale bar = 50 µm. (B) Lysates from these cells were isolated at days 0 (d0) and 15 (d15) of acinar morphogenesis and subjected to Western blot analysis with the indicated antibodies. (C and D) 76NE6 cells stably transfected with vector, EL, and LMW-E and tumor-derived cells (TDCs) were subjected to analysis similar to that in (A) (V = vector). Scale bar = 50 µm. The diameters of the acini were measured and averaged from 3 independent experiments. Values underneath each figure represents mean (µm) ± SEM. Error bars = SEM (Student <i>t</i> test, *p<0.05). (E) Lysates from these cells were isolated at day 15 and subjected to Western blot analysis with the indicated antibodies. <i>In vitro</i> kinase assay was performed by immunoprecipitation of lysates from 3D culture using polyclonal cyclin E antibody and incubation with (γP32)ATP and GST-Rb. (F and G) Cells cultured on Matrigel for 15 days were fixed and immunostained with E-cadherin and Ki67 antibodies. Nuclei were counterstained with DAPI. Scale bar = 50 µm. The number of Ki67-positive cells per acinus was counted and averaged from 3 independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). (H) Linear regression of the correlation between acinar diameter and percentage of Ki67-positive cells.</p

CDK2-associated kinase activity is required for LMW-E-mediated tumorigenesis and deregulation of acinar morphogenesis.

<p>(A) 76NE6-TetR cells were cultured with or without doxycycline induction, and the lysates were extracted and subjected to Western blot analysis with antibodies against cyclin E, CDK2, and β-actin. <i>In vitro</i> kinase assay was performed by immunoprecipitation with FLAG antibody and histone H1 and GST-Rb were added as substrates. Doxycycline was administered to achieve approximately 1× and 2× cyclin E protein levels. (The doxycycline concentrations for the 76NE6-TetR-V and wild-type EL and LMW-E cells were 0, 0.2, and 0.4 ng/ml, and the doxycycline concentrations for the EL^R130A and LMW-E^R130A cells were 0, 1, and 2 ng/ml.) (B) 76NE6-TetR cells were cultured on Matrigel for 15 days with or without doxycycline induction. Bright-field images were taken at day 15. Values underneath each figure represent mean diameter (µm) ± SEM. (C) The diameters of at least 100 acini from 3 different experiments were measured. Error bars = SEM (Wilcoxon rank-sum test, *p<0.05). (D) Multi-acinar complexes were counted. Error bars = SEM (Wilcoxon rank-sum test, *p<0.05). Multi-acinar complexes were defined as complexes with more than 2 acini growing on top of each other. Logistic regression models were used to compare the rate of formation of multi-acinar complexes between/among groups (*p<0.05).</p

Patient protein expression based on low and high LMW-E and EL levels by RPPA analysis.

*<p>Wilcoxon ranksum test.</p

The tumorigenicity of LMW-E requires CDK2–associated kinase activity.

<p>Athymic mice were injected with 1×10⁷ 76NE6-TetR cells with inducible expression for empty vector, EL, LMW-E, EL^R130A, and LMW-E^R130A. Doxycycline was added to drinking water containing 1% sucrose 24 hours after injection and replaced twice weekly. The diameter of the tumors were measured and recorded weekly. Tumor incidence rate was estimated with exact 95% confidence intervals and Fisher's exact tests were used to compare tumor incidence rate between/among groups (*p<0.0001: LMW-E 0 vs. LMW-E 500; vector 500 vs. LMW-E 500; EL 500 vs. LMW-E 500; LMW-E 500 vs. EL^R130A 500; LMW-E 500 vs. LMW-E^R130A).</p