Survivin regulation by HER2 through NF-κB and c-myc in irradiated breast cancer cells

Abstract

Radiotherapy is an important treatment modality against cancer resulting in apoptosis and inhibition of cell growth. Survivin is an important cancer biomarker conferring to tumour cells increased survival potential by inhibiting apoptosis. In the present study, we investigated the implication of breast cancer cells features, as hormone receptors and p53 status, in the radio-resistance of breast cancer cells and in the regulation of survivin's expression by nuclear factor (NF)-κB and c-myc. Six breast cancer cell lines Michigan Cancer Foundation (MCF-7), MCF-7/Human Epidermal Growth Factor Receptor (HER)2, M. D. Anderson - Metastatic Breast (MDA-MB-231), SK-BR-3, BT-474 and Human Breast Lactating (HBL-100) were irradiated and cell viability as well as cell cycle distribution were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Survivin mRNA and protein levels were evaluated by real time PCR and Western blot analysis. Survivin and HER2 gene knockdown was performed with siRNA technology and investigation of transcription factors binding to survivin and c-myc gene promoters was assessed by chromatin immunoprecipitation. Student's t-test and F-statistics were used for statistical evaluation. Our results demonstrated that only HER2(+) breast cancer cells up-regulated survivin upon irradiation, whereas HER2 knockdown in HER2(+) cells led to survivin's down-regulation. Survivin and especially HER2 knockdown abolished the observed G2/M cell cycle checkpoint and reduced the radio-resistance of HER2 overexpressing breast cancer cells. Additionally, HER2 was found to regulate survivin's expression through NF-κB and c-myc transcription factors. This study revealed the significance of HER2 in the radio-resistance of HER2(+) breast cancer cells through induction of transcription factors NF-κB and c-myc, leading to activation of survivin, a downstream target oncogene preventing apoptosis.

Fig 1

Survivin mRNA and protein expression was evaluated by real time PCR and Western blotting in non-irradiated and irradiated breast cancer cell lines respectively. Histograms represent the fold increase or decrease of survivin mRNA expression in irradiated cells, compared to non-irradiated cells. The normalized survivin mRNA expression of non-irradiated cells was set to 1 (*P < 0.05).

Fig 2

Effect of HER2 expression in the regulation of survivin mRNA expression levels. (A) A total of 40 μg of protein from non-irradiated, 10 and 20 Gy irradiated cells were subjected to Western blot analysis of HER2 protein. Analysis for β-actin was performed to show equal loading. For simplicity reasons, only 48 hr blots are represented here (72 hrs for MCF-7/HER2 cells). (B) Effect of HER2 knockdown on HER2 and survivin mRNA expression levels. (Bars: standard errors; *P < 0.05). (C) Effect of HER2 knockdown on HER2 and survivin proteins, as demonstrated by Western blot analysis.

Fig 3

(A) SK-BR-3 cells were transfected with siRNA against HER2, irradiated twenty-four hours after transfection with a single 10 Gy dose and cell viability was assessed by MTT assay 24, 48 and 72 hrs after irradiation. Cell viability in non-transfected, non-irradiated cells was considered to be at 100% (Bars: standard errors; *P < 0.05). (B) SK-BR-3 cells were transfected with siRNA against survivin. Increasing doses of siRNA (33 and 100 nM) reduced survivin mRNA and protein levels. (C) SK-BR-3 cells were transfected with 33 nM of siRNA against survivin, irradiated with a single 10 Gy dose and measured for cell viability using MTT assay 24, 48 and 72 hrs after irradiation (Bars: standard errors; *P < 0.05). (D) SK-BR-3 cells were transfected with siRNA against survivin and subsequently irradiated with a single 10 Gy dose. Cell cycle distribution analysis was performed for non-irradiated/non-transfected (1), non-irradiated/scrambled (2), non-irradiated/survivin knocked down (3), irradiated/non-transfected (4), irradiated/scrambled (5) and irradiated/survivin knocked down (6) cells. For simplicity reasons, only 48 hrs after irradiation histograms are presented here.

Fig 4

Occupancy of the survivin promoter in irradiated and non-irradiated breast cancer cell lines by ChIP analysis.

Fig 5

(A) Non-irradiated, 10 and 20 Gy irradiated cells were harvested at 24, 48, 72 and 96 hrs, lysed and 40 μg of total protein were subjected to Western blot analysis of c-myc protein. Analysis for β-actin was performed to show equal loading. For simplicity reasons, only 48 hr blots are represented here (72 hrs for MCF-7/HER2 cells). (B) Occupancy of the c-myc promoter in irradiated and non-irradiated breast cancer cells by NF-κB transcription factor by ChIP analysis.