Estrogen-Mediated Upregulation of Noxa Is Associated with Cell Cycle Progression in Estrogen Receptor-Positive Breast Cancer Cells

Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimental model, we show that Noxa is upregulated by E2 via p53-independent processes that involve c-Myc and ERα. Experiments using small interfering ribonucleic acids (siRNA) to specifically knock down p53, c-Myc, and ERα demonstrated that c-Myc and ERα, but not p53, are involved in the transcriptional upregulation of Noxa following E2 treatment. Furthermore, while E2 promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP) assays, E2 did not induce p53 recruitment. Interestingly, E2-mediated upregulation of Noxa was not associated with apoptosis. However, siRNA-mediated knockdown of Noxa resulted in cell cycle arrest in G0/G1-phase and significantly delayed the G1-to-S-phase transition following E2 treatment, indicating that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells

Noxa knock-down arrests cells in G₁/G₀ phase and delays E2-induced S-phase entry.

<p>(<b>A & B</b>) MCF7 cells were transfected with 50 nM of non-silencing control (NS) siRNA or Noxa (S1, S2, and S3) siRNA for 24 hr, after which Noxa mRNA expression was analyzed by qPCR (A), and Noxa protein expression was analyzed by western blotting (B). S1, S2 and S3 are three different siRNA sequences that target different regions of Noxa mRNA. (<b>C</b>) MCF7 cells were transfected with non-silencing control (NS) siRNA or Noxa siRNA (a pool of S1, S2, and S3) for 0, 1, or 2 days, as indicated. The number of viable cells was determined using the “Cell Titer-Glo Assay” kit. (<b>D</b>) MCF7 cells were transfected with 50 nM of non-silencing control (NS) siRNA or Noxa siRNA (a pool of S1, S2, and S3) for 48 hr. Cell cycle distribution was analyzed by flow cytometry using the PI-staining method. (<b>E</b>) MCF7 cells were transfected with non-silencing control siRNA (NS) or Noxa siRNA (a pool of S1, S2, and S3) for 32 hr, followed by E2 (10 nM) or vehicle (veh) treatment for 16 hr. The S-phase population was analyzed by flow cytometry using the PI-staining method. (<b>F</b>) Dose titration and timecourse experiments were performed to determine the effect of E2-induced Noxa expression on apoptosis, using doxorubicin treatment as a positive control. MCF7 cells were treated with vehicle (Veh), 1.7 µM doxorubicin (Doxo), or increasing concentrations of E2 (10 nM, 50 nM, and 100 nM) for 16 hr or 48 hr, as indicated. Cleaved and uncleaved poly-ADP-ribose polymerase (PARP) protein expression was analyzed by western blotting. Graphical data points in A, C, D, and E are means ± S.D. of three independent experiments (*** <i>P</i><0.001).</p

E2F1 mediates ERα-binding to the NOXA promoter in the presence of E2.

<p>(<b>A</b>) Schematic representation of the <i>NOXA</i> promoter. Horizontal arrows indicate the primers used for PCR in site-specific ChIP assays. Note that the figure is not drawn to scale. BS: binding site. TSS: transcription start site. ERE: estrogen response element. KB: distance in kilobases from the TSS. BP: distance in base pairs from the TSS. (<b>B</b>) ChIP assays were performed on MCF7 cell lysates to detect ERα binding to the <i>NOXA</i> promoter. Anti-ERα antibody or normal rabbit IgG (R IgG; control antibody) were used to immunoprecipitate ChIP DNA, and ChIP PCR was performed using primers that amplify a putative ERE site at −3.7 kb (lanes 1–3), a non-specific (NS) negative control site where no binding is expected to occur (lanes 4–6), and the proximal region of the <i>NOXA</i> promoter (lanes 7–9). PCR products were resolved on agarose gels. (<b>C</b>) Co-immunoprecipitation (co-IP) of protein complexes containing ERα and E2F1. Left panel: normal rabbit IgG (R IgG; antibody control) or anti-E2F1 antibody was used for immunoprecipitation (IP) and anti-E2F1 and anti-ERα antibodies were used for immunoblotting (IB). Right panel: R IgG or anti-ERα antibody was used for IP and anti-ERα and anti-EF21 antibodies were used for IB. (<b>D</b>) MCF7 cells were transfected with non-silencing control (NS) or ERα siRNA for 24 hr, followed by treatment with E2 (10 nM) or vehicle (veh) for 4 hr. The recruitment of ERα, E2F1, and RB to the proximal region of the <i>NOXA</i> promoter was analyzed by ChIP assays, as in B. (<b>E</b>) Left panel: MCF7 cells were transfected with non-silencing control (NS) siRNA or E2F1 siRNA for 48 hr, and E2F1 protein levels were monitored by western blotting. Right panel: MCF7 cells were transfected with E2F1 siRNA for 24 hr and then treated with vehicle (veh) or E2 (10 nM) for 4 hr. Recruitment of ERα to the proximal region of the <i>NOXA</i> promoter was analyzed by ChIP assays, as in B. (<b>F</b>) MCF7 cells were transfected with non-silencing control (NS) siRNA or E2F1 siRNA for 24 hr and then treated with vehicle (veh) or E2 (10 nM) for 8 hr. Noxa mRNA expression was assayed by qPCR. Graphical data points in F are means ± S.D. of three independent experiments (** <i>P</i><0.01).</p

E2-induced Noxa transcription is ERα-dependent and p53-independent.

<p>(<b>A</b>) MCF7 cells were treated with vehicle (veh), 10 nM E2 alone, 10 nM E2 in combination with 1 µM tamoxifen (Tam), or 10 nM E2 in combination with 1 µM ICI 182780 (ICI) for various times. Four, 8, 16 and 24 hr after treatment, cells were harvested, and Noxa mRNA expression levels were analyzed by qPCR. (<b>B, C, & D</b>) MCF7 cells were transfected with non-silencing control (NS), ERα, or p53 siRNA for 24 hr, followed by treatment with vehicle (veh) or E2 (10 nM) for 8 hr, and then harvested for analyses of ERα, p53, and β-actin (internal control) protein expression by western blotting (B), Noxa mRNA expression by qPCR (C), and pS2 mRNA expression by qPCR (D). (<b>E</b>) MCF7 cells were treated with vehicle (veh), E2 (10 nM), doxorubicin (DOX; 1.70 µM), adozelesin (ADO; 4 nM), or left without treatment (NT) for 16 hr. Occupancy of p53 on the proximal region of the <i>NOXA</i> promoter was determined by ChIP assays, using anti-p53 antibody or normal mouse IgG (M IgG; control antibody) for immunoprecipitation. Graphical data points in A, C, and D are means ± S.D. of three independent experiments (*** <i>P</i><0.001).</p

A Novel ALDH1A1 Inhibitor Targets Cells with Stem Cell Characteristics in Ovarian Cancer

A small of population of slow cycling and chemo-resistant cells referred to as cancer stem cells (CSC) have been implicated in cancer recurrence. There is emerging interest in developing targeted therapeutics to eradicate CSCs. Aldehyde-dehydrogenase (ALDH) activity was shown to be a functional marker of CSCs in ovarian cancer (OC). ALDH activity is increased in cells grown as spheres versus monolayer cultures under differentiating conditions and in OC cells after treatment with platinum. Here, we describe the activity of CM37, a newly identified small molecule with inhibitory activity against ALDH1A1, in OC models enriched in CSCs. Treatment with CM37 reduced OC cells&#8217; proliferation as spheroids under low attachment growth conditions and the expression of stemness-associated markers (OCT4 and SOX2) in ALDH+ cells fluorescence-activated cell sorting (FACS)-sorted from cell lines and malignant OC ascites. Likewise, siRNA-mediated ALDH1A1 knockdown reduced OC cells&#8217; proliferation as spheres, expression of stemness markers, and delayed tumor initiation capacity in vivo. Treatment with CM37 promoted DNA damage in OC cells, as evidenced by induction of &#947;H2AX. This corresponded to increased expression of genes involved in DNA damage response, such as NEIL3, as measured in ALDH+ cells treated with CM37 or in cells where ALDH1A1 was knocked down. By inhibiting ALDH1A1, CM37 augmented intracellular ROS accumulation, which in turn led to increased DNA damage and reduced OC cell viability. Cumulatively, our findings demonstrate that a novel ALDH1A1 small molecule inhibitor is active in OC models enriched in CSCs. Further optimization of this new class of small molecules could provide a novel strategy for targeting treatment-resistant OC

TP53 status as a determinant of pro- versus anti-tumorigenic effects of estrogen receptor-beta in breast cancer

BACKGROUND: Anti-tumorigenic versus pro-tumorigenic roles of estrogen receptor-beta (ESR2) in breast cancer (BC) remain unsettled. We investigated the potential of TP53 status to be a determinant of the bi-faceted role of ESR2 and associated therapeutic implications for triple negative BC (TNBC). METHODS: ESR2-TP53 interaction was analyzed with multiple assays including in situ proximity ligation assay (PLA). Transcriptional effects on TP53-target genes and cell proliferation in response to knocking down or overexpressing ESR2 were determined. Patient survival according to ESR2 expression levels and TP53 mutation status was analyzed in the Basal-like/ TNBC subgroup in METABRIC (n = 308) and Roswell (n = 46) patient cohorts by univariate Cox regression and log-rank test. All statistical tests are two-sided. RESULTS: ESR2 interaction with WT and mutant TP53 caused pro-proliferative and anti-proliferative effects, respectively. Depleting ESR2 in cells expressing WT TP53 resulted in increased expression of TP53-target genes CDKN1A (control group mean = 1 [SD = 0.13] vs ESR2 depletion group mean =2.08 [SD = 0.24]; p=.003) and BBC3 (control group mean = 1 [SD = 0.06] vs ESR2 depleted group mean =1.92 [SD = 0.25]; p=.003); however expression of CDKN1A (control group mean = 1 [SD = 0.21] vs ESR2 depleted group mean =0.56 [SD = 0.12];p=.02) and BBC3 (control group mean = 1 [SD = 0.03] vs ESR2 depleted group mean =0.55 [SD = 0.09]; p = .008) was decreased in cells expressing mutant TP53. Overexpressing ESR2 had opposite effects. Tamoxifen increased ESR2-mutant TP53 interaction leading to reactivation of TP73 and apoptosis. High levels of ESR2 expression in mutant TP53- expressing Basal-like tumors is associated with better prognosis (METABRIC cohort: log-rank p = 0.001; HR = 0.26, 95% Confidence interval= 0.08 to 0.84, univariate Cox p = 0.02). CONCLUSIONS: TP53 status is a determinant of the functional duality of ESR2. Our study suggests that ESR2-mutant TP53 combination prognosticates survival in TNBC revealing a novel strategy to stratify TNBC for therapeutic intervention potentially by repurposing tamoxifen

ESR1 and p53 interactome alteration defines mechanisms of tamoxifen response in luminal breast cancer

Summary: The canonical mechanism behind tamoxifen’s therapeutic effect on estrogen receptor α/ESR1+ breast cancers is inhibition of ESR1-dependent estrogen signaling. Although ESR1+ tumors expressing wild-type p53 were reported to be more responsive to tamoxifen (Tam) therapy, p53 has not been factored into choice of this therapy and the mechanism underlying the role of p53 in Tam response remains unclear. In a window-of-opportunity trial on patients with newly diagnosed stage I–III ESR1+/HER2/wild-type p53 breast cancer who were randomized to arms with or without Tam prior to surgery, we reveal that the ESR1-p53 interaction in tumors was inhibited by Tam. This resulted in functional reactivation of p53 leading to transcriptional reprogramming that favors tumor-suppressive signaling, as well as downregulation of oncogenic pathways. These findings illustrating the convergence of ESR1 and p53 signaling during Tam therapy enrich mechanistic understanding of the impact of p53 on the response to Tam therapy