Cross-talk between notch and the estrogen receptor in breast cancer suggests novel therapeutic approaches

Abstract

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor alpha-negative (ERalpha(-)), Her2/Neu nonoverexpressing cells. In ERalpha(+) cells, estradiol inhibited Notch activity and Notch-1(IC) nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERalpha(-)) cells, Notch-1 knockdown or gamma-secretase inhibition decreased cyclins A and B1, causing G(2) arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERalpha(+)) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, gamma-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERalpha(+) breast cancers and that Notch signaling is a potential therapeutic target in ERalpha(-) breast cancers.

Figure 1

Breast cancer cell lines have variable levels of CBF-1 transcriptional activity; estrogen up-regulates Notch-1 but decreases Notch-4 expression. A, Western blots showing expression of Notch-1 and Notch-4 in a panel of breast cancer cell lines as well as HMEC controls. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, CBF-1 luciferase activities of the breast cancer cell lines shown in A and HMEC controls. Cells were transfected simultaneously (n = 3) at comparable levels of confluence and tested side by side. Data are normalized for efficiency of transfection using a Renilla luciferase plasmid. Columns, mean; bars, SD. Data are representative of at least three independent experiments. RLU, relative luciferase unit. C and D, Western blots. C, 10^–9 mol/L 17β-estradiol (E2) in steroid-free medium for 72 h up-regulated Notch-1 in T47D:A18 but not T47D:C42 cells. Results represent five independent experiments. D, 10^–9 mol/L 17β-estradiol in steroid-free medium for 72 h up-regulated Notch-1 but decreased Notch-4 in either MCF-7 or T47D:A18. 4-OH-TAM (1 μmol/L) antagonized both effects. Results represent three independent experiments. EtOH, ethanol.

Figure 2

Estradiol decreases Notch transcriptional activity and Notch nuclear localization in breast cancer cells. A, luciferase assay showing inhibition of CBF-1 transcriptional activity by 48-h treatment with 10^–9 mol/L 17β-estradiol in T47D:A18:CBF-1Luc (stably transfected with the firefly luciferase gene under the control of six CBF-1–responsive elements). 4-OH-TAM (10^–6 mol/L), raloxifene (10^–6 mol/L), or fulvestrant (10^–7 mol/L) did not inhibit CBF-1 activity. B, luciferase assay showing inhibition by 24-h treatment with 10^–9 mol/L 17β-estradiol of CBF-1 transcriptional activity in MCF-7 transiently transfected with a plasmid containing the firefly luciferase gene under the control of four CBF-1–responsive elements. Renilla luciferase was used to normalize for transfection efficiency. 4-OH-TAM (10^–6 mol/L), raloxifene (10^–6 mol/L), and fulvestrant (10^–7 mol/L) did not inhibit CBF-1 activity. C, real-time RT-PCR showing the relative amount of HEY1 mRNA in MCF-7 cells cocultured for 5 d with OP9 cells expressing Delta-1 with or without 10 nmol/L 17β-estradiol for 2 and 24 h. Data are expressed as fold increase compared with MCF-7 cells cocultured in parallel with control OP9-GFP cells. D, MCF-7 (top) and T47D:A18 (bottom) cells were transfected with a construct coding for full-length Notch-1 fused to Renilla luciferase. Twenty-four hours after transfection, the medium was replaced with hormone-free medium and cells were treated with 10^–9 mol/L estradiol for 24 h. Nuclei were isolated and the ratio of Notch-Renilla luciferase in the nuclei versus total cellular Notch-Renilla luciferase was measured. Columns, mean; bar, SD. Data represent three independent experiments.

Figure 3

Estradiol regulates the cellular localization of Notch-1. A, confocal immunofluorescence showing total cellular Notch-1 staining (green) in MCF-7 and T47D:A18 after treatment with 10^–9 mol/L 17β-estradiol, 10^–6 mol/L 4-OH-TAM, or vehicle. All pictures were taken at the same magnification. B, left, Western blot showing the amount of biotinylated Notch-1 present on the cell membrane of T47D:A18 with or without 48-h treatment with 10^–7 mol/L 17β-estradiol. Similar results were obtained with 10^–9 mol/L estradiol and in MCF-7 cells with both treatments. Results of one of two independent experiments are shown. Right, Western blot showing the total amount of Notch-1 in the same cells with or without estradiol treatment. C, T47D:A18 cells were grown in charcoal-stripped medium for 3 d and then transfected by electroporation with GAL4 luciferase reporter and DNA plasmid coding fusion protein GAL4VP16-Notch1ΔE. After transfection, cells were treated for 48 h with hormones. Twenty-four hours before lysing the cells, 25 μmol/L lactacystin was added to inhibit Notch degradation. The experiment was repeated at least thrice. Columns, mean; bars, SD. D, T47D:A18 and MCF-7 cells were grown in charcoal-stripped medium for 3 d and then transfected by electroporation with GAL4 luciferase reporter and DNA plasmid encoding fusion protein GAL4VP16-APPc99 (24). After transfection, cells were treated for 48 h with 10^–9 mol/L 17β-estradiol and UAS luciferase activity was measured. The experiment was repeated at least thrice. Columns, mean; bars, SD.

Figure 4

Notch signaling is required for proliferation and survival in breast cancer cell lines and regulates cyclin A, cyclin B1, and NOXA. A, left, Western blots showing that RNAi silencing of Notch-1 in MDA-MB231 cells also down-regulated Notch-4, whereas nearly quantitative silencing of Notch-4 by RNAi had no effects on Notch-1. Control was a siRNA with no homology with known mammalian genes. Right, RNAi silencing of Notch-1 (which also affects Notch-4) or silencing of Notch-4 alone inhibited the proliferation of MDA-MB231 cells. Data are representative of three experiments each conducted in triplicate. SDs where not visible were smaller than data point symbols. B, silencing of either Notch-1 or Notch-4 in T47D:A18 cells inhibits proliferation and potentiates the effects of 4-OH-TAM. T47D:A18 cells were transfected with siRNA to Notch-1 or Notch-4 or scrambled control siRNA. Twenty-four hours after transfection, cells were seeded in a 96-well plate (8,000 per well) and treated with decreasing concentrations of 4-OH-TAM (25–0.38 μmol/L) in 0.5% ethanol for 24 h. At the end of treatment, cytotoxicity was evaluated by crystal violet staining. C, RNase protection assay showing that mRNA levels for cyclins A and B1 were reduced in MDA-MB231 cells transfected with Notch-1 siRNA. L32 (rRNA) and glyceraldehyde-3-phosphate dehydrogenase were internal controls. Additional controls were untreated cells and tRNA (nonspecific protection control). D, Western blots showing that cells transfected with Notch-1 siRNA had dramatically reduced levels of cyclin B1 and cyclin A proteins, as well as increased NOXA expression, compared with controls. Left, MDA-MB231 cells (48 h after transfection); right, T47D:A18 cells (72 h after transfection).

Figure 5

Inhibition of γ-Secretase has antineoplastic effects on breast cancer cell lines and affects cyclins A and B1 as well as NOXA. A, dose-response curve of growth inhibition by GSI alone in T47D:A18 breast cancer cells. B, dose response of 4-OH-TAM in the same cells in the absence of GSI and in the presence of two sub-IC₅₀ concentrations of GSI. Growth inhibition assays showed that 4-OH-TAM at a concentration as low as 0.8 μmol/L caused significant growth inhibition in T47D:A18 cells in the presence of 0.2 and 0.4 μmol/L of GSI (IC₅₀ of GSI alone, 0.84 μmol/L). The IC₅₀ of 4-OH-TAM alone in this assay is 11 μmol/L. C, Western blots of MDA-MB231 cells treated for 24 h with GSI (1 μmol/L). This treatment decreased cyclins A and B1 and induced NOXA. D, Western blots of T47D:A18 cells treated for 48 h, showing that 0.4 μmol/L GSI alone, 10 μmol/L 4-OH-TAM alone, or a combination inhibited expression of cyclins A and B1. Only the combination caused induction of NOXA at 24 h.

Figure 6

GSI has antineoplastic effects in vivo in two breast cancer models. A, MDA-MB231 established xenografts were treated with GSI alone (see Supplementary Materials and Methods). GSI caused statistically significant decrease of tumor volume at all time points, virtually abolishing tumor growth. B, T47D:A18 established xenografts receiving menopausal estrogen levels were treated with GSI, tamoxifen, or a combination thereof. Either drug alone virtually arrested tumor growth. *, statistically significant differences between controls and each individual agent. **, the combination GSI/tamoxifen caused tumor regression and was significantly different not only from controls but also from each individual treatment. C, histologic aspects of MDA-MB231 tumors. Final magnification, ×400. Note nuclear pyknosis (green arrows), necrosis, and hemorrhage (yellow arrow) in GSI-treated tumor. D, histologic aspects of T47D:A18 tumors. Magnification, ×400. Note that GSI, either alone or with tamoxifen, caused widespread cell death with nuclear pyknosis (green arrows), edema (red arrows), and hemorrhage (yellow arrows).