Notch1 signaling regulates the epithelial-mesenchymal transition and invasion of breast cancer in a Slug-dependent manner

Abstract

Background: The epithelial-mesenchymal transition (EMT) is crucial for the invasion and metastasis of breast cancer. However, how Notch signaling regulates the EMT process and invasion in breast cancer remains largely unknown.

Methods: The impact of Notch1 silencing by specific shRNAs on the EMT and invasion of human breast cancer MCF-7 and MDA-MB-231 cells as well as xenografts was tested by western blot, real-time polymerase chain reaction (RT-PCR), immunofluorescence, transwell, and immunohistochemistry assays. The effect of Slug silencing or upregulation on the EMT and invasion of breast cancer cells was analyzed, and the effect of Notch1 signaling on Slug expression was determined by the luciferase reporter assay.

Results: The Notch1 intracellular domain (N1ICD) and Jagged1 were expressed in breast cancer cells. Notch1 silencing reversed the spontaneous EMT process and inhibited the migration and invasion of breast cancer cells and the growth of xenograft breast cancers. The expression of N1ICD was upregulated significantly by Jagged1-mediated Notch signaling activation. Moreover, Jagged1-mediated Notch signaling promoted the EMT process, migration, and invasion of breast cancer cells, which were abrogated by Notch silencing. Furthermore, the N1ICD positively regulated the Slug expression by inducing Slug promoter activation. Importantly, the knockdown of Slug weakened the invasion ability of breast cancer cells and reversed the Jagged1-induced EMT process with significantly decreased expression of vimentin and increased expression of E-cadherin. In addition, Slug overexpression restored the Notch1 knockdown-suppressed EMT process.

Conclusions: Our novel data indicate that Notch signaling positively regulates the EMT, invasion, and growth of breast cancer cells by inducing Slug expression. The Notch1-Slug signaling axis may represent a potential therapeutic target for breast cancer therapy.

Figure 1

The expression of Notch1 and Jagged1 in human breast cancer cell lines. (A) The protein expression of N1ICD in a panel of breast cancer cells (MDA-MB-231, T47D, MCF-7, ZR-75-1, and SK-BR-3), HMECs, and MCF-10A cells was evaluated by western blot. Protein samples (150 μg) were separated by 10% SDS-PAGE. β-Actin was used as a loading control. (B) The mRNA expression of Notch1 and Jagged1 was estimated by real-time PCR. GAPDH was used as a normalization control for quantifying the expression of each target gene. Experiments were performed three times. Column: mean; bar: SD.

Figure 2

Inhibition and activation of Notch signaling, respectively, in both MCF-7 and MDA-MB-231 cells. Normal group: MCF-7 or MDA-MB-231 cells were incubated under normal conditions; shNC group: MCF-7 or MDA-MB-231 cells were stably transfected with NC shRNA; shNotch1 group: MCF-7 or MDA-MB-231 cells were stably transfected with Notch1 shRNA; normal + Jagged1: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h; shNC + Jagged1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h after stable shNC transfection. Jagged1 + shNotch1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h and then Notch1 shRNA was transiently transfected for an additional 48 h. (A), (C), and (E) Total protein was isolated for western blot analysis using Notch1, Hey1, Hes1, and NF-κB65 antibodies. β-Actin was used as a loading control. (B), (D), and (F) Total RNA was extracted, and the expression levels of Notch1, Hey1, Hes1, and NF-κB65 were assayed by real-time PCR. The expression of every target gene was quantified using GAPDH as a normalization control. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P < 0.05).

Figure 3

Characteristic of EMT under conditions of Notch1 knockdown or Jagged1 induced Notch signaling activation in breast cancer cells. (A) and (B) Morphological changes of MCF-7 and MDA-MB-231 cells. (C) Total protein was extracted for immunoblotting analysis of the EMT-related markers E-cadherin, occludin, N-cadherin, and vimentin in MCF-7 and MDA-MB-231 cells. β-Actin was used as a loading control. (D) Real-time PCR analysis of the EMT-related markers E-cadherin, occludin, N-cadherin, and vimentin in MCF-7 and MDA-MB-231 cells. GAPAH was used as a normalization control. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P < 0.05).

Figure 4

The effects of Notch1 knockdown and Jagged1 on breast cancer EMT. (A) Nuclear protein was extracted for western blot analysis, and a β-catenin antibody was used to observe the changes of β-catenin in the nucleus. LaminB1 was used as a loading control for nuclear lysate. (B) Immunofluorescence analysis of E-cadherin and vimentin in MDA-MB-231 cells. DAPI staining was used to detect nuclei and is merged with E-cadherin and vimentin in their respective panels. The cells were observed under an immunofluorescence microscope at 400× magnification. The data are from three independent experiments.

Figure 5

Jagged1-mediated activation of Notch signaling induces EMT and cannot rescue the changes caused by Notch1 interference. shNotch1 + Jagged1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h after stable shNotch1 transfection. (A) Immunofluorescence analysis of E-cadherin and vimentin in MCF-7 cells. DAPI staining was used to detect nuclei and is merged with E-cadherin and vimentin in their respective panels. The cells were observed under an immunofluorescence microscope at 400× magnification. (B) Western blot analysis of the EMT-related markers E-cadherin, occludin, N-cadherin, and vimentin in MCF-7 and MDA-MB-231 cells. (C) The mRNA expression levels of E-cadherin, occludin, N-cadherin, and vimentin were estimated by real-time PCR. (D) Western blot analysis of nuclear β-catenin in MCF-7 and MDA-MB-231 cells. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P < 0.05).

Figure 6

Notch1 knockdown inhibits Jagged1-enhanced breast cancer migration and invasion. (A) and (B) The cells were seeded into a migration chamber or a Matrigel-coated invasion chamber and incubated for 24 h. The number of migrated cells was quantified by counting the numbers of cells from six random fields at 100× magnification. The data are from three independent experiments. There are similar numbers of migrated and invaded breast cancer cells between the Normal + Jagged1 and the shNC + Jagged1 groups of cells (data not shown). Column: mean; bar: SD. The symbol * represents a significant difference (P < 0.05).

Figure 7

Notch1 regulates Slug expression by enhancing its promoter activity. (A) and (B) The level of Slug expression was evaluated by western blot and real-time PCR after MDA-MB-231 cells were stably transfected with shNC or shNotch1. (C) and (D) The Slug expression level following treatment with Jagged1 ligand for 48 h was estimated by western blot and real-time PCR. (E) The schematic diagram shows the construction of the pGL3-Slug promoter and its negative control pGL3-basic. (F) Luciferase reporter assays were carried out in MDA-MB-231 cells, which were cotransfected with the pGL3-Slug promoter or its negative control pGL3-basic and N1ICD overexpression plasmid pcDNA3.1(+) or its negative control plasmid pcDNA3.1. Each independent experiment was repeated three times. Column: mean; bar: SD. The symbol * represents a significant difference (P < 0.05).

Figure 8

Slug serves as a mediator for Notch1-induced EMT, migration, and invasion. (A) and (B) Western blot analysis and real-time PCR assays were carried out to evaluate the expression of Slug when MDA-MB-231 cells were transfected with negative control siRNA (siNC) or Slug siRNA (siSlug) for 48 h. (C) MDA-MB-231 cells were transfected with siNC or siSlug for 48 h and then treated with Jagged1 for an additional 48 h. E-cadherin and vimentin protein levels were evaluated by western blot. (D) and (E) MDA-MB-231 cells were transfected with siNC or siSlug for 48 h and then treated with Jagged1 for an additional 48 h. The cells were seeded into a migration chamber or a Matrigel-coated invasion chamber and incubated for 24 h. The number of migrated cells was counted under a light microscope. (F) MDA-MB-231-shNC or MDA-MB-231-shNotch1 cells were transfected with negative control vector (pcDNA3.1) or Slug overexpression vector (pcDNA3.1-Slug), respectively. Forty-eight hours later, western blot analysis was performed to assess the expression levels of E-cadherin and vimentin. (G) and (H) MDA-MB-231-shNotch1 cells were transfected with pcDNA3.1 or pcDNA3.1-Slug for 48 h and then the migration and invasion abilities were evaluated. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P < 0.05).

Figure 9

Downregulation of Notch1 reverses EMT in vivo . (A) Tumor growth curve. The tumor volume in nude mice injected with MDA-MB-231-shNotch1 cells was significantly smaller than that of the control MDA-MB-231-shNC cells. Tumor volumes represent means ± SD, n = 4 per group, the symbol * represents a significant difference (P < 0.05). (B) In vivo metastasis assays of MDA-MB-231 cells with or without Notch1 downregulation. (C) Representative images of immunohistochemical staining for the EMT-related markers E-cadherin and N-cadherin as well as hematoxylin and eosin staining (magnification: 400×). The data are from three independent experiments.