Cross-talking Between Notch-1 And NF-κB In Breast Cancer Cell Migration And Its Molecular Mechanisms

Cancer is a major public health problem in the word. The latest statistics report showed that lung cancer, colon cancer, prostate cancer and breast cancer are still the most common causes of cancer death according to the American Cancer Society in 2015. The number of cancer incidence is about 3.6 million in 2012 in China, and the cancer death is about 2.2 million, accounting for about a quarter of global cancer death. The main reason for the high mortality from cancer is due to the highly invasive behavior of cancer cells, which usually results in cancer progression and metastasis. Progression towards a metastatic phenotype requires a concerted effort between different molecules that have been implicated in advancing of the metastatic. It is a complex process controlled by multigene. Blockade of metastasis can benefit for cancer treatment, however, the underling mechanism of metastasis remains poorly understood.Notch signaling pathway, which is highly conserved and plays an important role in cell-cell communication, controls multiple cell differentiation processes during the embryonic and adult periods. Accumulated evidence shows that Notch signaling is dysregulated in many cancers. Overexpression of Notch-1 and its ligands has been shown to be associated with poor prognosis in breast cancer. However, the specific role and the underling mechanisms of Notch-1 signaling pathway on the malignant behavior of breast cancer are poorly understood. In the present study, using multiple cellular and molecular approaches, we demonstrated that activation of Notch-1 signaling pathway promotes the malignant behavior of MDA-MB-231 cells, and the related mechanisms were elucidated. The results are shown as follows:1. To get the Notch-1 overexpression and Notch-1 knockdown cells. Full length of Notch-1 intracellular domain(NICD) c DNA was attained through reverse transcription-PCR from human glioma cell line of U251, then cloned into pc DNA3.1(+) vector to form a recombinant pc DNA3.1-NICD. Notch-1 activated MDA-MB-231 cells were obtained by pc DNA3.1-NICD transfection. The overexpression of NICD was confirmed by real-time PCR and western blot. The Notch-1 sh RNA was transfected into MDA-MB-231 cells. The knockdown of Notch-1 was also confirmed by real-time PCR and western blot.2. The role of Notch-1 in the malignant of breast cancer cells. 1) The cell proliferation assay, colony formation assay and soft agar assay were performed to detect cell growth. These results showed that overexpression of NICD significantly promoted, while knockdown of Notch-1 strikingly inhibited, the proliferation of MDA-MB-231 cells. 2) Cell cycle was tested by flow cytometer and the results showed that NICD overexpression led to an increase of S phase cell number, while Notch-1 knockdown decreased the S phase cell number. Moreover, NICD overexpression inhibited cell apoptosis, while Notch-1 silencing induced cell apoptosis. 3) Cell adhesion assay, Transwell test and monolayer wound healing assay showed that activation of Notch-1 by NICD overexpression facilitated cell adhesion, migration and invasion. In contrast, Notch-1 silencing inhibited cell adhesion, migration and invasion. Together, these results indicated that Notch-1 activation promotes the malignant behavior of MDA-MB-231 cells.3. Notch-1 induced the expression of MMP-2, MMP-9 and VEGF. Real-time PCR was used to test the expression levels of MMP-2, MMP-9 and VEGF. The results showed that Notch-1 activation induced high expression of these genes, while Notch-1 silencing inhibited their expression. What’s more, the gelatin zymography test showed that Notch-1 activation also increased MMP-9 activity.4. Notch-1 activates NF-κB pathway. 1) Immunofluorescence staining and western blot showed that activated Notch-1 induced the degradation of IκBα and promoted the nuclear translocation of p65. However, Notch-1 silencing inhibited IκBα degradation and p65 nuclear translocation. EMSA and dual-luciferase reporter assay showed that NICD also increased, whereas Notch-1 silencing decreased, the specific DNA-protein combination and NF-κB transcriptional activity. 2) Notch-1 activation induced the expression of NF-κB target genes, e.g., Cyclin D1, Bcl-x L and surivin, while Notch-1 silencing inhibited their expression. These results indicated that Notch-1 induces NF-κB activation.5. Notch-1 promoting cell invasion is NF-κB dependent. 1) Cell invasion was increased by treating with NF-κB activator LPS, which also antagonized the decrease of MDA-MB-231 cell invasion caused by Notch-1 silencing or inhibition. Moreover, the increase of cell invasion caused by NICD overexpression was restrained by NF-κB inhibitor(BAY11-7082) treatment. 2) The expression of invasion-related genes MMP-2, MMP-9 and VEGF was inhibited by Notch inhibitor DAPT or NF-κB inhibitor BAY11-7082. In contrast, LPS induced their expression and resisted the effect of DAPT. These results indicated that Notch-1 induced MDA-MB-231 cells invasion is NF-κB dependent.6. The mechanism of Notch-1 activates NF-κB. 1) The phosphorylation of p85 subunit of PI3 K, and the phosphorylation of AKT were enhanced in NICD overexpressed cells. Furthermore, Notch-1 silencing decreased the phosphorylation of p85 and AKT. PI3 K inhibitor LY294002 treatment decreased the levels of phospho-p85 and phosphoAKT. Moreover, LY294002 treatment partially blocked NICD overexpression induced phosphorylation of p85 and AKT, and increased the expression of total I?B?. 2) AKT inhibition increased I?B? expression and led to predominantly cytosolic localization of p65, but not nuclear translocation. NICD induced p65 nuclear translocation was completely abolished by AKT inhibitor, and we observed significant cytosolic sequestering of p65. Collectively, these results demonstrated that Notch-1 induces NF-κB activation through PI3K/AKT-dependent pathway. 3) NICD overexpression increased the phosphorylation of PP2 A, indicating PP2 A activity was suppressed, whereas Notch-1 silencing reduced PP2 A phosphorylation, indicating PP2 A activity was enhanced. OA was used to inhibit PP2 A activity, and the results showed that the phosphorylation of PP2 A, p85 and AKT went up under OA treatment. Moreover, the decrease of phosphoAKT caused by Notch-1 silencing was abrogated by OA treatment. Immunoprecipitation and immunofluorescence staining revealed that OA treatment enhanced p65 nuclear localization. What’s more, the effects of Notch-1 activation or inhibition on p65 subcellular distribution were abrogated by OA treatment. Dual-Luciferase reporter assay showed that NICD overexpression enhanced NF-κB transcriptional activity. In contrast, Notch-1 silencing suppressed NF-κB transcriptional activity compared with scrambled Notch-1 sh RNA. However, OA treatment increased NF-κB transcriptional activity to the same level whether Notch-1 was activated or inhibited. Taken together, these data strongly suggest that Notch-1 regulates NF-κB activity through PP2A-dependent PI3K/AKT pathway.In summary, our study revealed the role of Notch-1 in regulating the proliferation, adhesion, migration and invasion of breast cancer. We also clarified a novel mechanism that Notch-1 activates NF-κB through PP2A-dependent PI3K/AKT pathway to facilitate the malignant of breast cancer cell MDA-MB-231. PP2 A may be a potential therapeutic target in breast cancer.