Jang et al. BMC Cancer 2010, 10:391 http://www.biomedcentral.com/1471-2407/10/391 surface-localized oncoprotein. These findings suggest that manipulation of HER2/neu may be of substantial value in treatment of breast cancer [6-12]. Heat shock protein 90 (HSP90) is known as a chaper- one, assisting in correct folding of nascent proteins including many kinds of oncoproteins, and therefore is associated with tumor growth. Suppression of HSP90 reveals down regulation of its client proteins including Raf-1, Erk, Pdk-1, Akt, and HER2/neu [13-15]. ATP binding at the N-terminal domain of HSP90 is indispen- sable for the appropriate activity of HSP90 [16,17]. Adenine nucleotide translocase 2 (ANT2), abundantly located in the inner mitochondrial membrane, partici- pates in the formation of mitochondrial permeability transition pore complex, and also plays an important role in the cellular energy metabolism by catalyzing the exchange of mitochondrial ATP for cytosolic ADP and thereby influencing mitochondrial oxidative phosphory- lation [18]. In fact, ANT2 suppression by a DNA vector based RNA interference approach expressing short hair- pin RNA (shRNA) resulted ATP depletion from breast cancer cells and induced cell death [19]. We therefore hypothesized that HSP90 might be one of the main tar- gets which functions are influence by ANT2 shRNA- induced ATP depletion. Breakdown of the extracellular matrix (ECM) by pro- teinases is an essential step in cancer invasion and metastasis [20,21]. Matrix metalloproteinases (MMPs) are a family of ECM degrading proteinases. Owing to their matrix-degrading abilities and high expression in advanced tumors, MMPs were originally implicated in cancer progression, invasion, and metastasis. They are produced by a variety of cells. Production of MMP1, MMP2, MMP9, and MT1-MMP in endothelial cells has been described [22-27]. Of the four known mem- brane type matrix metalloproteinases, MT1-MMP is most often overexpressed one in cancer and is fre- quently detected in association with the activated form of MMP2. Moreover, PI3K/Akt activation contributes to tumor cell invasion through induction of MTI- MMP; selective targeting of the PI3K/Akt signaling pathway significantly blocks the invasive potential of cancer cells [28]. We assumed that ATP depletion by ANT2 shRNA might suppress HSP90 activity, thereby down-regulating HER2/neu expression and inhibiting the PI3K/Akt sig- naling pathway, which might be resulted in down-regu- lation of MMPs expression and subsequent inhibition of migration and invasion of breast cancer cells. Therefore, the purpose of our study was to determine whether or not ANT2 shRNA-mediated HER2/neu inhibition decreased breast cancer cell invasiveness, and to investi- gate the potential of ANT2 shRNA for future use as a Page 2 of 9 therapeutic agent targeting metastasis of human breast cancer. Methods Cell lines and culture The SK-BR3 human breast cancer cell line was used throughout the study. These cells were purchased from the ATCC (Manassas, VA) and cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% FBS (fetal calf serum), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco, Grand Island, NY) in a humidified 5% CO2 /95% air atmosphere at 37°C. Antibodies and reagents Anti-human HER2/neu, ubiquitin, and a-tublin antibo- dies were purchased from Santa Cruz biotechnology (Heidelberg, Germany). Anti-Akt, anti-phospho-Akt, and anti-b-actin were purchased from Cell signaling Tech (Beverly, MA). Anti-MT1-MMP antibodies were pur- chased from Millipore (Billerica. MA). HSP90 inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin) was purchased from A.G. Scientific, Inc (San Diego, CA), and PI3K inhibitor, LY294002 was purchased from Calbiochem (San Diego, CA). Transfection For transfection, cells were plated on either six-well plates (2 × 105 cells per well) or 100 mm dishes (2 × 106 cells) and were allowed to adhere for 24 hours. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used for the transfection. Cells were transfected with either pSilencer™ 3.1-H1 puro ANT2 siRNA vectors or pSilen- cer™ 3.1-H1 puro scramble siRNA vectors (shRNAs). Transfected cells were then cultured for 6 hours; culture media were replaced with fresh media supplemented with 10% FBS. The cells were harvested at 24 - 48 hours after transfection. pcDNA, pcDNA-ANT2, or wild-type PI3K/p110 vectors (kindly provided by Dr. Karin Reif) were transfected into the cells using the same method. Immunoprecipitations The cells were collected and lysed for 30 minutes on ice in 1 mL of a lysis buffer [20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X- 100, and 2 mmol/L EDTA] with protease inhibitor cock- tail (Roche). After removing cell debris by centrifuging at 15,000 g for 15 minutes at 4°C, the cell lysates (800 μl, 15 mg protein) were precipitated with 80 μl of EZview Red anti-Flag M2 Affinity Gel (Sigma) overnight at 4°C. The complexes were subsequently washed five times with TBS buffer [50 mmol/L Tris, 150 mmol/L NaCl (pH 7.4)], suspended in 20 μl of 2× SDS sample buffer [125 mmol/L (pH 6.8), 10% (w/v) SDS, 20% (v/v)