NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications - PubMed (original) (raw)
NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications
Anthony G Clementz et al. Breast Cancer Res. 2011.
Abstract
Introduction: Women with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells.
Methods: Real-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both.
Results: Herein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo.
Conclusions: Taken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.
Figures
Figure 1
PEA3 positively regulates Notch-1 and Notch-4 transcription. MDA-MB-231 cells were transfected with one of two unrelated scrambled siRNA (SCRBia or SCRBib) or one of two unrelated PEA3 siRNA (PEA3ia or PEA3b). (A) Relative mRNA levels of Notch receptors (NOTCH-1, NOTCH-2, NOTCH-3 and NOTCH-4) were measured by quantitative PCR. Relative mRNA levels of PEA3 were measured as knocked-down controls using quantitative PCR. (B) Protein levels of Notch-1 and Notch-4 were detected by Western blot analysis. For Notch-4 Western blot analysis, cells were treated with or without lactacystin for 24 hours. The Western blots are representative of three independent experiments. (C) Downstream targets of PEA3 (MMP-9) and Notch (HEY-1) were measured by quantitative PCR. Transcripts were normalized to HPRT, and mean values were plotted. Error bars represent standard deviations. The statistical significance of three or more experiments was determined by performing a two-tailed, unpaired Student's _t_-test for two comparisons.
Figure 2
PEA3 regulates Notch-1 and Notch-4 in different subtypes of breast cancer. (A) through (C) MDA-MB-231, MCF-7, BT474 and SKBr3 cells were transfected with either scrambled or PEA3 siRNAa. Once normalized to MDA-MB-231 cells, the transcript levels of PEA3 (A), Notch-1 (A) and Notch-4 (C) were compared and measured by quantitative real-time PCR. BT474 cells (D) and SKBr3 cells (E) were transfected with either empty vector or PEA3 expression plasmid. Notch-1 and Notch-4 mRNA levels were normalized to empty vector and measured by quantitative PCR. Means and standard deviations of three experiments were plotted. Statistical significance was determined by performing a two-tailed, unpaired Student's _t_-test.
Figure 3
PEA3 is enriched on the Notch-1 and Notch-4 promoters. (A) MDA-MB-231 cells were transfected with pcDNA3.1 alone or pcDNA3.1-PEA3 expression plasmid. Lysates were immunoprecipitated (I.P.) with IgG control or PEA3-specific antibody. Western blot analysis was performed to detect PEA3. Heavy and light chains of IgG were used for loading. The Western blots are representative of three independent experiments. (B) and (C) MDA-MB-231 cells transfected with pcDNA3.1 or pcDNA3.1-PEA3 expression plasmid were lysed, and lysates were subjected to chromatin immunoprecipitation using either PEA3-specific antibody or an isotype control IgG on the Notch-1 promoter (B) or the Notch-4 promoter (C), respectively. PEA3 enrichment was measured by quantitative PCR and normalized to pcDNA3.1 and IgG control. Schematic representations of Notch-1 and Notch-4 promoter regions and the primers used in the studies flanking the specific sites are shown. Error bars represent standard deviations. Statistical significance of three or more experiments was determined by performing a two-tailed, unpaired Student's _t_-test for two comparisons.
Figure 4
PEA3 is required for enrichment in Notch-1 and Notch-4 promoter regions. (A) MDA-MB-231 cells were cotransfected with pcDNA3.1-PEA3 expression plasmid and control siRNAa or PEA3 siRNAa. Lysates were subjected to chromatin immunoprecipitation using either PEA3-specific antibody or an isotype control IgG on the Notch-1 and Notch-4 promoters, respectively. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Statistical comparisons were performed between PEA3 siRNAa and control siRNAa using a two-tailed, unpaired Student's _t_-test for two comparisons. (B) Lysates from the experiment described in (A) were subjected to SDS-PAGE followed by Western blot analyses to detect PEA3 protein and actin protein as a loading control. The Western blots are representative of three independent experiments. Error bars represent standard deviations. The statistical significance of three or more experiments was determined by performing a two-tailed, unpaired Student's _t_-test for two comparisons.
Figure 5
The requirement of c-JUN for PEA3 enrichment on the Notch-4 promoter. (A) MDA-MB-231 cells were cotransfected with pcDNA3.1-PEA3 and phMB vector alone or with phMB-TAM-67 expression plasmid. Lysates were subjected to chromatin immunoprecipitation using either PEA3-specific antibody or an isotype control IgG on the Notch-1 or Notch-4 promoter, respectively. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. In an independent experiment, cells were cotransfected with an AP-1 luciferase reporter, Renilla luciferase and phMB or phMB-TAM-67. Dual Firefly and Renilla luciferase assays were performed to measure AP-1 activity. Statistical comparisons were performed between phMB-vector and phMB-TAM-67 using a two-tailed, unpaired Student's _t_-test for two comparisons. (B) MDA-MB-231 cells were cotransfected with pcDNA3.1-PEA3 expression plasmid and control siRNA or c-JUN siRNA. Lysates were subjected either to chromatin immunoprecipitation and quantified as described above or to SDS-PAGE followed by Western blot analyses to detect c-JUN and actin (loading control). Statistical comparisons were performed between control siRNA and c-JUN siRNA using a two-tailed, unpaired Student's _t_-test for two comparisons. (C) PEA3 was immunoprecipitated from MDA-MB-231 lysates exogenously expressing PEA3 followed by Western blot analysis to detect PEA3 and c-JUN. The light chain of IgG was detected for loading purposes. The Western blots are representative of three independent experiments. Error bars represent standard deviations. The statistical significance of three or more experiments was determined by performing a two-tailed, unpaired Student's _t_-test for two comparisons.
Figure 6
PEA3 is required for Notch-4 reporter activity. Schematic representation of the Notch-4 luciferase construct (-650 to +1 region) indicating the wild-type (wt) and mutated (mut) AP-1 consensus sites and the wild-type Ets site. MDA-MB-231 cells were cotransfected with AP-1 luciferase and either scrambled (SCRBia) or PEA3 siRNA (PEA3ia). Cells were cotransfected with either wild-type AP-1 or mutated AP-1 Notch-4 luciferase and either scrambled or PEA3 siRNAa. Firefly luciferase was measured normalized to Renilla luciferase as an internal transfection control. Means and standard deviations of three or more experiments were plotted. Statistical significance was determined by performing a two-tailed, unpaired Student's _t_-test.
Figure 7
Differential regulation of Notch-4 by AP-1 members. (A) MDA-MB-231 cells were transfected with control, PEA3, Fra-1, c-JUN or c-FOS siRNA for 48 hours as described previously. Real-time PCR was performed to detect Notch-4 and HPRT transcripts. Transcript expression relative to control siRNA was calculated after normalization for HPRT transcripts. (B) Real-time PCR and Western blot analysis were performed to determine the efficacy of the specific siRNA. Means and standard deviations of three or more experiments were plotted. Statistical significance was determined by performing a two-tailed, unpaired Student's _t_-test for two comparisons and analysis of variance for multiple comparisons.
Figure 8
Dual inhibition of Notch and PEA3 inhibits cell proliferation and induces apoptosis. MDA-MB-231 cells were transfected with scrambled or PEA3 siRNA alone or were treated with vehicle (DMSO) or a γ-secretase inhibitor (MRK-003 GSI), or a combination thereof, for 48 hours. Cell cycle analysis (A) and annexin V staining (B) were performed by using flow cytometry. (C) Percentages of viable cells were measured by the trypan blue exclusion method using a standard light microscope (×10 original magnification). The mean percentage and standard deviation of cells in each experiment were plotted. Statistical significance was determined by performing analysis of variance for multiple comparisons. The error bars represent standard deviations of the mean for three independent experiments.
Figure 9
Inhibition of Notch and PEA3 inhibits anchorage-dependent growth in vitro and tumor growth in vivo. (A) and (B) MDA-MB-231 cells were transfected with either scrambled or PEA3 siRNAa and treated with vehicle (DMSO) or γ-secretase inhibitor (MRK-003 GSI) independently or in combination for 48 hours. A colony formation assay was performed using methylcellulose, and the colonies were incubated for 14 days. (A) Colonies were photographed using a standard light microscope (×40 original magnification). The photomicrographs are representative of three independent experiments. (B) Twelve fields per well were counted, and the means plus or minus standard deviations of three independent experiments were plotted. Statistical significance was determined by performing analysis of variance for multiple comparisons. (C) Cells were injected into each of two mammary fat pads of female athymic nude mice which were number-tagged prior to surgery to monitor each tumor. The mice were randomized to vehicle or MRK-003 GSI, which was fed orally by gavage on a schedule of three days on, four days off. Tumor areas (length × width) were measured twice per week using vernier calipers for up to 3.5 weeks. The _y_-axis is cross-sectional in centimeters squared, which is calculated using the formula [(area × π)/4]. The _x_-axis is weeks of treatment. The graph shows the mean cross-section of the tumors plus or minus the standard deviations of 20 tumors/10 mice/group. Linear regression analysis was performed on individual tumors to calculate the slope of a line with a correlation coefficient >0.85. Statistical analysis was performed using a two-tailed, unpaired Student's _t_-test on the slopes of each line. *Denotes statistical differences between vehicle + PEA3i and vehicle + Controli. **Denotes statistical differences between MRK-003 GSI + Controli and vehicle + Controli. (D) MDA-MB-231 cells were transfected with control or PEA3 siRNAa for 24 hours. Real-time PCR was performed on MDA-MB-231 cells prior to injection to detect PEA3 transcripts.
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