Study Of The Sensitivity To EGFR-TKI Modulated By P53 On Triple-negative Breast Cancer Cells

Purpose and backgroundBreast cancer remains the most frequently diagnosed female cancer worldwide and the leading cause of cancer death. These treatment options are absent in patients diagnosed with tumors lacking estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2). These breast carcinomas are therefore referred to as triple-negative breast cancer (TNBC). Chemotherapy was the most important treatment regimen for TNBC since there were no indications to administrate endocrine therapy and target therapy. Therefore, TNBCs have a poor prognosis.EGFR overexpression in breast cancer is correlated with large tumor size and poor prognosis. Its overexpression is more frequent (70%) than in other subtypes. Gefitinib, approved for lung cancers, is a small-molecule tyrosine kinase inhibitor. Unfortunately, gefitinib showed little efficacy in most clinical studies of breast cancer even though its EGFR overexpression.More than 30% of breast cancer occured p53 mutation and 80% in TNBC. p53 could regulate sensitivity to EGFR-TKI by modulating EGFR downstream signaling. MDA-MB-468 cells, a TNBC cell line, were insensitive to EGFR-TKI. In our experiment, Ad-p53 was used in combination with gefitinib to treat MDA-MB-468 cells in vitro and in vivo.MethodsWe retrospectively analyzed the clinical and pathological data of 264 patients with invasive breast cancer collected from the Pathology Department of Shandong Cancer Hospital and Institute between January 2012 and August 2013. Then, P53 and EGFR expression and their relation was analyzed. MDA-MB-468 cells were treated with Ad-p53 and/or gefitinib. The effect of Ad-p53 and gefitinib on the growth of MDA-MB-468 cells was evaluated by MTT. Colony formation was used to detect cell proliferation. Cell apoptosis and cell cycle analysis were detected by flow cytometry. Western blot analysis was used to determine the change of p53 and EGFR, and expression of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway and apoptosis-related protein. In vivo, Ad-p53 combining with gefitinib was used to detect the effect on tumor xenograft in nude mice.ResultsThe rate of p53 mutation and EGFR overexpression were higher and positively correlated with each other in TNBC, while in the other subtypes, their expressions were lower and had no correlation with each other.The MTT assay and colongenic formation showed that inhibition of gefitinib was more powerful on MDA-MB-468 cells infected with Ad-p53 than cells treated with the control. Cell apoptosis assay revealed that the apoptosis rate of MDA-MB-468 cells in vehicle-treated group, Ad-p53 group, gefitinib group, combination group was 8.5%,17.4%,20.5% and 32.6%, respectively. Gefitinib induced G2/M phase arrest from 21.9% to 45.4% compared to vehicle-treated; G2/M phase increased to no more than 31.5% after exposed to Ad-p53. In comparison, when combined treatment was performed, G2/M arrest was enhanced evidently from 21.9% to 65.3%.The apoptosis rate of MDA-MB-468 cells in combination group was higher than in other groups (P<0.05).Western blot analysis revealed that the expression of p53 was significantly up-regulated in the presence of Ad-p53. Ad-p53 and gefitinib combination significantly down-regulated p-Akt (S473)(P<0.01) and up-regulated caspase-9 and cleaved caspase-3(all P<0.01). Tumor inhibition rate (TIR) in Ad-p53 group, gefitinib group, and combination group was 35.7%, 28.7% and 74.4%, respectively. Ad-p53 and gefitinib combination therapy can significantly rid the bulk of the tumor burden in nude mice (all P<0.05)Conclusions1. The rate of p53 mutation and EGFR overexpression were higher and positively correlated with each other in TNBC, while in the other subtypes, their expressions were lower and had no correlation with each other.2. P53 mutation may be the important factor for those TNBC patients resistant to TKI.3. Wild-type p53 is able to reverse the drug resistance of MDA-MB-468 cells toward gefitinib through the inactivation of the PI3K/Akt pathway by triggering apoptosis dependent to caspase cascade.4. P53 modification can modulate TNBC cells to resense to TKI.