Degradation of HER2/neu by ANT2 shRNA suppresses migration and invasiveness of breast cancer cells

Abstract

Background: In breast cancer, the HER2/neu oncoprotein, which belongs to the epidermal growth factor receptor family, may trigger activation of the phosphoinositide-3 kinase (PI3K)/Akt pathway, which controls cell proliferation, survival, migration, and invasion. In this study, we examined the question of whether or not adenine nucleotide translocase 2 (ANT2) short hairpin RNA (shRNA)-mediated down-regulation of HER2/neu and inhibitory effects on the PI3K/Akt signaling pathway suppressed migration and invasiveness of breast cancer cells.

Methods: We utilized an ANT2 vector-based RNA interference approach to inhibition of ANT2 expression, and the HER2/neu-overexpressing human breast cancer cell line, SK-BR3, was used throughout the study.

Results: In this study, ANT2 shRNA decreased HER2/neu protein levels by promoting degradation of HER2/neu protein through dissociation from heat shock protein 90 (HSP90). As a result, ANT2 shRNA induced inhibitory effects on the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling by ANT2 shRNA caused down-regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) expression, decreased matrix metalloproteinase 2 (MMP2) and MMP9 activity, and suppressed migration and invasion of breast cancer cells.

Conclusions: These results indicate that knock-down of ANT2 by shRNA down-regulates HER2/neu through suppression of HSP90's function and inhibits the PI3K/Akt signaling pathway, resulting ultimately in suppressed migration and invasion of breast cancer cells.

Figure 1

Down-regulation of HER2/neu by ANT2 shRNA in HER2/neu overexpressing breast cancer cell lines (SK-BR3). (A) After 24 hours of transfection with scramble shRNA or ANT2 shRNA, expression levels of HER2/neu were analyzed using flow cytometry. (B) For identification of dissociation and degradation of HSP90 from HER2/neu by ANT2 shRNA, cells were transfected with ANT2 shRNA for 24 hours and the cellular extracts were prepared for immuno-precipitation with anti-Her2/neu antibody and subjected to western blotting with anti-HSP90 and anti-ubiquitin antibodies. HSP90 inhibitor, 17-AAG was used as a positive control. (C) After 24 hours of transfection with ANT2 shRNA, cells extracts were prepared for western blotting with anti-Her2/neu and anti-β-actin antibodies.

Figure 2

Down-regulation of Akt activity by ANT2 shRNA in SK-BR3 cells. (A) After 24 hours of transfection with ANT2 shRNA in SK-BR3, cells extracts were prepared for western blotting with anti-phospho Akt and anti-Akt antibodies. 17-AAG was used as a positive control. (B) To investigate if ANT2 plays a role in the regulation of Akt activity (phophorylated Akt level) in SK-BR3, cells were transfected with scramble shRNA, ANT2 shRNA, pcDNA, or pcDNA-ANT2 and cultured for 24 hours, then cells extracts were prepared for western blotting with anti-phospho Akt and anti-Akt antibodies. PI3K inhibitor, LY294002 was used as a positive control to inhibit PI3K/Akt signaling pathway. (C) To evaluate the effect of ANT2 shRNA-mediated HSP90 suppression on the stability and phosphorylation of Akt, cells were transfected with ANT2 shRNA for 24 or 48 hours and the cellular extracts were prepared for immuno-precipitation with anti-HSP90 antibody and subjected to western blotting with anti-phospho-Akt and anti-Akt antibodies.

Figure 3

ANT2 shRNA down-regulates VEGF expression, which is dependent on the PI3K signaling pathway. (A) Cells were transfected with scramble shRNA or ANT2 shRNA and cultured for 24 hours. Total RNA was extracted from respective cell lines and subjected to RT-PCR using specific primers for human VEGF or GAPDH (internal control). Intracellular levels of VEGF were also detected by flow cytometry. (B) Cells were pre-treated with PI3K inhibitor, LY294002. After 2 hours of incubation, cells were transfected with scramble shRNA, ANT2 shRNA, pcDNA, or pcDNA-ANT2 and cultured for 24 hours. Total RNA was extracted from respective cell lines and subjected to RT-PCR using specific primers for human VEGF or GAPDH (internal control).

Figure 4

ANT2 shRNA down-regulates MT1-MMP expression by suppressing PI3K signaling in SK-BR3 cells (A). Cells were transfected with scramble shRNA or ANT2 shRNA. After 24 hours of incubation, total RNA and cell extracts were prepared for RT-PCR using specific primers for human MT1-MMP or GAPDH (internal control) and western blotting with anti-MT1-MMP and anti-β-actin antibodies. (B) Cells were transfected with scramble shRNA or ANT2 shRNA with or without the wild-type PI3K expression vector and cultured for 24 hours. After 24 hours of incubation, total RNA and cell extracts were prepared for RT-PCR using specific primers for human MT1-MMP or GAPDH (internal control) and western blotting with anti-MT1-MMP and anti-β-actin antibodies.

Figure 5

ANT2 shRNA down-regulates MMP2 and MMP9 expression and activity in SK-BR3 cells. (A) Cells were transfected with scramble shRNA or ANT2 shRNA and cultured for 24 hours. Total RNA was extracted from respective cell lines and subjected to RT-PCR using specific primers for human MMP2, MMP9, or GAPDH (internal control). (B) Cells were transfected with scramble shRNA or ANT2 shRNA and cultured in conditioned medium for 24 hours. Conditioned medium and cell lysates were electrophoresed in a polyacrylamide gel containing 1 mg/ml of gelatin. Proteolysis was detected as a white zone in a dark blue field. Band at 92 kDa indicates the active MMP9, 72 kDa the inactive MMP2, and 62 kDa active MMP2.

Figure 6

ANT2 shRNA suppresses invasiveness and migration of SK-BR3 cells in vitro. (A) To evaluate the inhibitory effect of ANT2 shRNA on invasion and migration of SK-BR3 cells, cells were transfected with scramble shRNA or ANT2 shRNA and cultured for 24 hours. Matrigel invasion assays were performed using modified Boyden chambers with polycarbonate Nucleopore membranes. Following incubation for 18 hours at 37°C, noninvaded cells on the upper surface of the filter were wiped out with a cotton swab, and the invaded cells on the lower surface of the filter were fixed and stained with Diff-Quick kit. (B) Transwell migration assays were performed using the same procedure used for performance of the invasion assay, except that the underside of filters was coated with type I collagen. Data were analyzed using the Student's t test. P < 0.005 was considered statistically significant.