Jang et al. BMC Cancer 2010, 10:391 http://www.biomedcentral.com/1471-2407/10/391 Page 7 of 9 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. little effect on non-neoplastic mammary epithelial cell lines, and also can sensitize cancer cells to a kind of chemotherapeutic agents [31,33]. In the work shown here, knockdown of ANT2 by shRNA inhibited migration and invasion of HER2/neu- overexpressing breast cancer cell lines (SK-BR3). We previously reported that ANT2 shRNA induces apopto- tic cell death in both MCF-7 and MDA-MB-231 cells [34], which cell lines express basal levels of HER2/neu. Here, we further showed that ANT2 shRNA also regu- lates migration and invasion in HER2/neu-overexpres- sing SK-BR3 cells. Although ANT2 knockdown in SK- BR3 cells ultimately induced cell death, its suppressing effect on the cell migration and invasion was observed much earlier after ANT2 shRNA transfection, which further emphasizes the potential of ANT2 shRNA to control breast cancer metastasis. We also observed that ANT2 shRNA suppresses the migration and invasion of MCF-7 and MDA-MB-231 cells expressing basal levels of HER2/neu (data not shown). We demonstrated here for the first time that ANT2 shRNA induces suppression of cell migration and inva- sion, accompanied by down-regulation of HER2/neu through inhibition of HSP90’s function in HER2/neu- overexpressing breast cancer cell lines. Recent studies have reported on enhanced intracellular degradation of HER2/neu, which involved targeting HSP90 by benzo- quinone ansamycins, such as geldanamycin. HSP90 forms complexes with HER2/neu and other client pro- teins. Once geldanamycin has competitively blocked ATP binding to HSP90, the chaperone complex asso- ciated with the client protein is biased toward a degra- dative fate, resulting in poly-ubiquitination and subsequent destruction of the client. The mature HER2/ neu requires association of HSP90 with its kinase domain to maintain the conformation necessary for het- erodimerization with other ligand-activated ErbB pro- teins [35]. In the present study, ANT2 shRNA dissociated the complex of HER2/neu and HSP90 and preceded depletion of HER2/neu. We thus hypothesized that ANT2 shRNA may also disrupt association of the HER2/neu and HSP90 complex through depletion of ATP, which may explain why ANT2 shRNA can deplete HER2/neu protein. Findings from our present study show that ANT2 shRNA-induced degradation of mature HER2/neu involves poly-ubiquitination of HER2/neu and subsequent hydrolysis by the proteasome. Here we have shown that ANT2 shRNA-induced degradation of HER2/neu led to loss of its activation with PI3K, and a rapid decline in Akt activity in HER2/ neu-overexpressing breast cancer cell lines. The PI3K/ Akt pathway is known to be important in cell survival, growth, migration, and invasion. This pathway is a pos- sible target for cancer therapy [36-38]. Findings from studies with HER2/neu-overexpressing breast cancer cell lines have demonstrated constitutive phosphorylation of HER2/neu and highly phosphorylated Akt [39]. Activa- tion of HER2-containing heterodimers results in recep- tor autophosphorylation on COOH-terminal tyrosine residues, which become docking sites for a number of signal transducers and adaptor molecules that initiate a plethora of signaling programs leading to cell prolifera- tion, differentiation, migration, adhesion, protection from apoptosis, and transformation, among other effects. PI3K and Akt kinases operate downstream of EGFR and HER2 in cancer cells, transmitting signals that regulate cell survival and cell migration [40]. EGFR- and/or HER2-triggered PI3K/Akt activation may be involved in regulation of the malignant and metastatic potential. Therefore, these molecules could serve as targets for metastases of breast cancer. It would be possible that ANT2 shRNA-mediate inhibition of HSP90’s activity ultimately result in Akt depletion and subsequent sup- pression of Akt activity. However, time kinetics of the