Jang et al. BMC Cancer 2010, 10:391 http://www.biomedcentral.com/1471-2407/10/391 Page 4 of 9 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-b-actin antibodies. kinds of breast cancer cells [28], ANT2 knockdown by shRNA resulted in decrease of ATP levels in SK-BR3 cells (data not shown). These data suggested that ANT2 shRNA decreased HER2/neu protein levels by promot- ing degradation of HER2/neu protein as a result of dis- sociation from HSP90. ANT2 shRNA inhibits the PI3K/Akt signaling pathway in HER2/neu-overexpressing breast cancer cells HER2/neu-overexpressing breast cancer cells have been shown to make increased use of the signaling pathway mediated by PI3K/Akt. Activated Akt is considered the focal point of a survival pathway known to protect cells from apoptosis by several stimuli. Moreover, PI3K could be involved in regulation of MMP2 and MT1-MMP in response to cytoskeletal remodeling and induction of the potent angiogenic factor, VEGF. We next examined Akt phosphorylation in response to ANT2 shRNA. Notably, a dramatic decrease in Akt phosphorylation was detected in SK-BR3 cells transfected with ANT2 shRNA for 24 hours, whereas ANT2 over-expression by pcDNA vector increased Akt phosphorylation (Fig. 2A and 2B). It is well known that Akt is also a client pro- tein of HSP90. Therefore, to investigate if the decreased Akt activity (phosphorylation) observed in ANT2 shRNA-treated SK-BR3 cells at 24 hours might be a result from a direct suppression of Akt level, we exam- ined the effects of inhibition of HSP90 by ANT2 shRNA on the level of Akt protein. As shown in Fig. 2C, although ANT2 shRNA led to dissociation of Akt from HSP90 after 48 hours of transfection, Akt protein was 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.