Expression of EGFR,p-mTOR༌p-S6K1 and p-4EBP1 in TNBC. The expression of EGFR༌p-mTOR༌p-S6K1 and p-4EBP1 in BC patients were analysed by immunohistochemistry on 48 patient tissue samples. Overall, the expression of four proteins were positively correlated with lymph node metastasis and clinical stage (P < 0.05). The positive rate of EGFR, p-mTOR, p-S6K1 and p-4EBP1 expression in patients with lymph node metastasis ≥4 were 66.7%, 63.3%, 60.0% and 70% respectively, which were significantly higher than those in patients with lymph node metastasis 0-3 (P<0.05).(Supplement table S1) Moreover, the expression rate of EGFR, p-mTOR, p-S6K1 and p-4EBP1 at stage I to stage Ⅲ were remarkably increased from 0.04%, 20.9%, 14.0% and 25.5%, to 61.5%, 63.6%, 63.6% and 68.2% respectively.(Supplement table S2)According to the expression of ER, PR, HER-2 and Ki-67, the included 111 breast cancer patients were divided into three types: Luminal(42), TNBC(48) and HER-2+ types (21). The expression rate of EGFR, p-mTOR, p-S6K1 and p-4EBP1 in Luminal, TNBC and HER-2+ types were 19.0%, 23.8%, 26.2%, 28.6%; 43.8%, 52.1%, 39.6%, 41.7% and 23.8%, 19.0%, 28.6%, 26.2% respectively. The expression rate of EGFR and p-mTOR in TNBC were significantly higher than that in luminal type and HER-2+ type (P<0.05) (Supplement table S3). The representative images for immunostaining and immunohistochemistry of TNBC tissues and their adjacent tissues are shown in Figure 1༌EGFR showed cytoplasmatic and membranous staining pattern, p-mTOR was expressed cytoplasmatic༌p-S6K1 and p-4EBP1 were expressed in cytoplasm and/or nucleus, whereas four proteins were nagative or weak expressed on adjacent tissues.
Correlation of EGFR, p-mTOR, p-S6K1 and p-4EBP1 expression with TNBC patients Survival. The 48 TNBC patients were followed up to 60 months for analysis the correlation between survival and the expression of EGFR, p-mTOR, p-S6K1 and p-4EBP1. The Kaplan Meier survival analysis of TNBC patients showed the average 5-year DFS in EGFR positive group (43.15 months) and p-4EBP1 positive group (47.16 months) were significantly shorter than those in negative groups (54.66 and 51.78 months respectively), otherwise, there was no significant difference between p-mTOR positive group (49.4 months) and p-S6K1 (48.47months) compared with their negative groups (50.42 and 50.79 months respectively)(Fig. 2). The results indicated that overexpression of EGFR and p-4EBP1 in TNBC patients might predict a poor prognosis.
Expression EGFR, p-mTOR, p-S6K1 and p-4EBP1 in EGFR-mTOR signaling by EGF induction. The MDA-MB-468 is a TNBC cell line. For evaluating the downstream signaling of EGFR-mTOR, MDA-MB-468 cells were treated with two concentrations of EGF in two separate runs. Western blot results showed the expression of mTOR, p-mTOR, p-S6K1 and p-EGFR in 10 ng/ml as well as 100 ng/ml of EGF treatment were significantly increased than those without EGF treatment (p<0.05). On the contrary, the expression of p-4EBP1 in EGF treatment was significantly decreased than those without EGF treatment (p<0.05) (Fig. 3). There was no significant difference between two concentrations and two different tests (p>0.05). This result showed the activation of EGFR-mTOR signaling in MDA-MB-468 cells.
The EGFR and mTOR inhibitors are synergistic in TNBC cell. For evaluating the effects of EGFR and mTOR inhibitor on the growth of TNBC cell line MDA-MB-468, Iressa and Everolimus were used to test cell proliferation individually and collaboratively in CCK-8 assay. The results showed EGFR inhibitor Iressa and mTOR inhibitor Everolimus significantly inhibited the proliferation of MDA-MB-468 cells in a dose-dependent manner used separately or in combination (P<0.05). The growth inhibitory value at 50% (IC50) for Iressa and Everolimus were 20.470±5.23 and 20.192±6.10µM respectively. For evaluating synergy of the two-drug combination, different concentration of combinations were used to test the inhibitory effects on MDA-MB-468 cells. The result showed each combination significantly inhibited cells compared with untreated control (P<0.01)(Fig. 4). According to the formulation: combined effect Q=inhibitory rate of combination/[ inhibitory rate of drug A+(1-inhibitory rate of drug A)×inhibitory rate of drug B], the Q value of 10 µM Iressa combined with 10 µM was 1.097, indicating the synergistic effects.
Effects of EGFR and mTOR inhibitor on the cell cycle and apoptosis. The inhibition of cancer cell generally resulted in cell growth arrest. Subsequently, we examined the cell cycle and apoptosis of MDA-MB-468 cells treated with Iressa and Everolimus by flow cytometry. The results showed that 71.19±1.476% of Iressa treated cells, 73.337±0.55% of Everolimus treated cells and 78.273±0.34% of cells treated in combination are in G0/G1 phase, which is significantly higher than that in untreated cells (52.487±0.416%)(P<0.01), and the proportion of cells in G0/G1 phase treated in combination is significantly higher than that in cells treated individually(P<0.05). The results indicated that both Iressa and Everolimus and its combination arrested MDA-MB-468 cells at G0/G1-phase (Fig. 5).
For evaluating the effects of EGFR and mTOR inhibitors on the cell death, we analyzed apoptosis of MDA-MB-468 cells with Annexin V/PI assay after Iressa and Everolimus were treated individually and in combination. The results showed both Iressa or Everolimus alone and their combination significantly induced apoptosis of MDA-MB-468 cells(P<0.01), the apoptosis rate induced by Iressa or Everolimus and their combination were 12.2%, 11.3% and 10.6% respectively(Fig. 6). However, there was no significant difference between the treatment in combination and the treatment applied individually (p>0.05). This indicates there is no synergistic effect between the two drugs in apoptosis induction.
Effects on EGFR and mTOR pathways after treatment of MDA-MB-468 cells with EGFR and mTOR inhibitors. To assess the effects of iressa and everolimus on the EGFR/mTOR signalling pathways, we analysed the protein expression of total and phosphorylated forms of EGFR, mTOR, S6K1 and 4EBP1 by Western blotting. MDA-MB-468 cell lines were treated with iressa and everolimus as single agents and in combination. There were no significant difference of EGFR expression in each treatment. Compared with untreated, both iressa alone and in combined with everolimus significantly decreased protein expression of mTOR, p-mTOR and p-SK61 (P<0.05). However, everolimus has no effects on mTOR and p-mTOR, but markedly decreased the expression of p-SK61. Compared with single agents, the combination only significantly decreased the expression of p-mTOR. In contrast to predict, the expression of p-4EBP1 significantly increased in single agents and in combination (P<0.05) (Fig. 7, Supplement table S4).