The Role Of Notch1 Intracellular Domain And EGFR Inhibitor On EGFR Positive Breast Cancer Cells

Background:Breast cancer is one of the most common malignancies in women and it remains the second most frequent cause of cancer mortality in women. A continued focus on elucidating the molecular mechanisms underlying this disease is necessary to develop novel synergistic and more effective combination treatments for better management of breast cancer in the clinic. Cell growth and differentiation are controlled by a complex interplay of signaling pathways. Cancer is often perceived as a disease of malfunctioning cell signaling. It is of significant importance to explore the mechanism of tumor genesis and development.The Notch signaling pathway is an evolutionarily conserved intracellular signaling mechanism. Mammals have four Notch receptors (Notch1-4) and five ligands (Delta-like 1,3-4 and Jagged 1-2). These receptors are activated in most contexts by a ligand residing on an adjacent cell. Once activated, Notch receptors are cleaved and release the intracellular domain (Notch-IC), which is then translocated to the nucleus to play a transcriptional role mostly by binding to CSL family of transcription factors.The first indication that Notch signalling might play a role in breast cancer development came from the characterisation of an insertion site for the mouse mammary tumor virus in Czech II mice. The mouse mammary tumor virus insertions into Notch1 and Notch4 have been described, resulting in unregulated Notch signalling, suggesting that leads to tumor formation. Howerever, the role of Notch signalling in human breast cancer is less known. To explore the expression and the role of Notch signalling in human breast cancer is of significant importance, providing evidence for breast cancer therapy.Objective:The aims of the study are to construct the retrovirus vector containing Notch1 intracellular domain, to ransfect the retrovirus into breast cancer cell line MDA-MB-231 and generate stable cell line MDA-MB-231-ICN overexpressing Notchl intracellular domain, to explore the role of exogenous Notchl on breast cancer cells, and to investigate the role of Notchl on breast cancer genesis.Methods:1. Retrovirus packaging and monoclone cell line generating1.1 Retrovirus packagingAlkaline lysis was used to extract plasmid DNA (including pCMV-VSV-G encoding the vesicular stomatitis virus G-glycoprotein, pkat encoding a retroviral packaging plasmid and MSCV-ICN/GFP encoding a constitutively active form of Notchl consisting of the intracellular domain). Calcium acid phosphate-DNA coprecipitation was used to cotransfect HEK293T cells and retrovirus particles were collected.1.2 Monoclone cell line generatingHuman MDA-MB-231 cells were infected with retrovirus stocks and stable cell line MDA-MB-231-ICN overexpressing Notchl intracellular domain was generated by limited dilution.2. The role of Notchl intracellular domain (Notchl-IC) on EGFR positive breast cancer cells2.1 The expression of Notchl signaling after retrovirus infectionReal-time RT-PCR was used to detect the expression of Notchl and its downstream target genes at mRNA levels. Western blot was used to detect the expression of these genes at protein levels. 2.2 The role of Notch1 on breast cancer cells proliferationMTT method was used to detect the growth of breast cancer cells. Cell cycle analysis using PI stain was used to detect the cell cycle distribution. Real-time RT-PCR was used to detect the expression of cell cycle related proteins CycinD, CDK2, and P21 at mRNA levels. Western blot was used to detect the expression of these genes at protein levels. Cell death analysis using Annexin V/ PI stain was used to detect the cell death rate.Results:1. Retrovirus packaging and monoclone cell line generating1.1 Retrovirus packagingHEK293T cells are efficient packaging cells and were successfully cotrasfected with plasmid DNA. The virus particles were collected at 48 and 72 h after transfection, filtered and then frozen for use.1.2 Monoclone cell line generatingHuman MDA-MB-231 cells were infected with retrovirus stocks and cells were collected after 72h. Stable monoclone cell line overexpressing Notch1 intracellular domain was generated by limited dilution and designated as MDA-MB-231-ICN cells.2. The role of Notch1 intracellular domain (Notch1-IC) on EGFR positive breast cancer cells2.1 Notch1 signaling expression was upregulated after retrovirus infectionData showed that MDA-MB-231-ICN cells expressed more Notch1 compared with parental MDA-MB-231 cells, suggesting transcriptional activation of Notch1 gene expression. The result of western blot analysis was in agreement with the Real-time RT-PCR data, showing that the protein level of Notchl was upregulated in MDA-MB-231-ICN cells compared with MDA-MB-231 cells. Hesl expression, a known downstream target of Notch signaling, was upregulated at both mRNA and protein levels compared with MDA-MB-231 cells. Another Notch1 downstream target gene Hey1 was also upregulated at mRNA level, indicating Notch1 signaling activation. 2.2 The role of Notchl on breast cancer cells proliferationMDA-MB-231-ICN cells showed significantly increased cell growth rate as compared with MDA-MB-231 cells. MDA-MB-231-ICN cells showed a significant drop in the fraction of cells at G0/G1 phase compared with MDA-MB-231 cells. The decrease in G0/G1 phase cells observed was significant, suggesting that Notchl signaling induces cell cycle progression. Accordingly, an increase of CycinD and CDK2 expression and a decrease of P21 expression supported the cell cycle distrabution. Cell apoptosis analysis showed that both early and late apoptotic cells in MDA-MB-231-ICN cells were lower than those in MDA-MB-231 cellsConclusion:We constructed the retrovirus vector containing Notchl intracellular domain, transfected the retrovirus into breast cancer cell line MDA-MB-231 and generated stable cell line MDA-MB-231-ICN overexpressing Notchl intracellular domain. We found that the Notchl signaling expression and activation were upregulated. The overexpression of Notchl signaling could promote the proliferation of breast cancer cells, inhibit cell death and promote cell cycle progression, indicating a positive role of Notchl signaling on breast cancer cells.The role of EGFR and Notchl signaling on EGFR positive breast cancer cellsBackground:Cell growth and differentiation are controlled by a complex interplay of signaling pathways. Rather than functioning as completely independent and insulated modules, signaling pathways interface in intricate ways to create a web of specific interactions that the cell integrates and interprets in a spatially and temporally appropriate manner. The evolutionarily conserved EGFR pathway and the Notch pathway represent two such signal transduction mechanisms.Notch and members of its signaling pathway are conserved in evolution, affecting many differentiation processes and cell-fate determination. Upon activation, Notch receptors release the intracellular domain, which is then translocated to the nucleus and acts as a transcriptional activator inducing the expression of members of the Hes family. Hes proteins in turn regulate the expression of downstream target genes.Epidermal growth factor receptor (EGFR) is a transmembrane protein receptor with tyrosine kinase activity that triggers numerous signaling pathways. It has been implicated in the intercellular communication and cell fate specification. The EGFR family includes four members, EGFR (HER1) and HER2-4. Briefly, activation of EGFR by its ligands leads to the relay of signals via the generic Ras/Raf/MEK/MAPK cascade, culminating in MAPK phosphorylation. Modified MAPK enters the nucleus and phosphorylates specific target transcription factors, thus linking signaling with gene expression regulation. EGFR overexpression has been found in many human tumors, including lung, colon, breast, prostate, brain, head and neck, thyroid, ovarian, bladder, gliomas, and renal carcinoma. Overexpression of EGFR signaling are crucial to cancer progression, including angiogenesis, metastatic spread, and the inhibition of apoptosis. Inhibition of EGFR in vitro by antisense RNA has been shown to inhibit proliferation and induce apoptosis. EGFR inhibitor has been shown to inhibit the growth of multiple cell lines and to potentiate the cytotoxic effects of many chemotherapeutic agents on xenografts of human tumors derived from ovarian, colon, lung, vulval, and hormone-refractory prostate cancers.The link between Notch and EGFR signaling in human breast cancer remains unknown. Here we investigated the regulation of Notch expression and activity by a novel cross-talk mechanism with EGFR. We present data supporting the possibility of gene therpy on human breast cancer. Objective:The aims of the study is to investigate the role of EGFR signaling on human breast cancer cells using an EGFR inhibitor gefitinib to inhibit the activity of EGFR signaling, and to reveal the crosstalk between Notchl and EGFR signaling.Methods:1. The role and mechanism of EGFR inhibitor on breast cancer cells1.1 The role of EGFR inhibitor on breast cancer cellsHuman MDA-MB-231 and MCF-7 cells were chosen for the study. EGFR inhibitor gefitinib was used to inhibit the activity of EGFR signaling with DMSO as solvent control. MTT method was used to detect the growth of breast cancer cells. Cell cycle analysis using PI stain was used to detect the cell cycle distribution. Cell death analysis using Annexin V/PI stain was used to detect the cell death rate.1.2 The role of EGFR inhibitor on Notchl and EGFR expressionReal-time RT-PCR was used to detect the expression of Notchl and EGFR expression at mRNA levels.2. The crosstalk between Notchl and EGFR signaling on breast cancer cellsHuman MDA-MB-231 and MDA-MB-231-ICN cells were chosen for the study. EGFR inhibitor gefitinib was used with DMSO as solvent control. MTT method was used to detect the growth of breast cancer cells. Cell cycle analysis using PI stain was used to detect the cell cycle distribution. Real-time RT-PCR was used to detect the expression of Notchl, EGFR and cell cycle related proteins CycinD, CDK2, and P21 at mRNA levels. Cell death analysis using Annexin V/PI stain was used to detect the cell death rate.Results:1. The role and mechanism of EGFR inhibitor on breast cancer cells1.1 The role of EGFR inhibitor on breast cancer cellsAfter EGFR inhibitor administration, the growth rate of breast cancer cells was decreased. Cell cycle analysis showed an increase in G0/G1 phase. Cell death analysis showed that both early and late apoptotic cells death rate were increased.1.2 The role of EGFR inhibitor on Notch1 and EGFR expressionCompared to DMSO solvent control, the expression of EGFR and Notch1 were both decreased in MDA-MB-231 and MCF-7 cells after EGFR inhibitor administration. It indicated that the activity of EGFR signaling was downregulated and it also showed a crosslinked role of EGFR and Notch1 signaling.2. The crosstalk between Notchl and EGFR signaling on breast cancer cellsThe expression levels of Notchl and EGFR after gefitinib administration were analyzed. Real-time RT-PCR results showed that Notchl and EGFR expression were decreased, but the inhibitory effect was higher in MDA-MB-231 than in MDA-MB-231-ICN cells. Gefitinib inhibited cell proliferation in both MDA-MB-231 and MDA-MB-231-ICN cells, and the inhibitory effect was higher in MDA-MB-231 than in MDA-MB-231-ICN cells. In addition, a typical G0/G1 cell cycle arrest pattern was observed in gefitinib treated cells. Consistent with cell cycle arrest, expression of CyclinD and CDK2 were found to be decreased whereas P21 expression was increased. Apoptosis analysis showed that gefitinib induced early apoptosis in breast cancer cells, and the apoptosis rate was higher in MDA-MB-231 than in MDA-MB-231-ICN cells.Conclusion:EGFR inhibitor could inhibit the growth of breast cancer cells, promote cell death and cause cell cycle arrest, indicating that EGFR signaling play a positive role on breast cancer cells. Overexpression of Notchl signaling could partially rescue EGFR inhibitor-induced cell toxicity in breast cancer cells showing that EGFR and Notchl signaling may be positively cross-linked in human breast cancer.