Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2

Abstract

Ansamycin antibiotics, such as 17-allylaminogeldanamycin (17-AAG), bind to Hsp90 and regulate its function, resulting in the proteasomal degradation of a subset of signaling proteins that require Hsp90 for conformational maturation. HER2 is a very sensitive target of these drugs. Ansamycins cause RB-dependent G1 arrest that is associated with loss of D-cyclins via a PI3 kinase, Akt dependent pathway. Downregulation of D-cyclin was due, in part, to loss of Akt expression in response to drug. Moreover, in HER2 overexpressing breast cancer cells, 17-AAG caused rapid inhibition of Akt activity prior to any change in Akt protein. Ansamycins caused rapid degradation of HER2 and a concomitant loss in HER3 associated PI3 kinase activity. This led to a loss of Akt activity, dephosphorylation of Akt substrates, and loss of D-cyclin expression. Introduction into cells of a constitutively membrane bound form of PI3 kinase prevented the effects of the drug on Akt activity and D-cyclins. Thus, in breast cancer cells with high HER2, Akt activation by HER2/HER3 heterodimers is required for D-cyclin expression. In murine xenograft models, non-toxic doses of 17-AAG markedly reduced the expression of HER2 and phosphorylation of Akt and inhibited tumor growth. Thus, pharmacological inhibition of Akt activation is achievable with ansamycins and may be useful for the treatment of HER2 driven tumors.

Figure 1

17-AAG induced loss of Akt protein expression and phosphorylated Akt levels. (a) Breast cancer cell lines MCF-7 and MDA-468 were treated with 1 µm 17-AAG; SKBr-3 and BT-474, cells that overexpress HER2, were treated with 50 nm 17-AAG. Levels of Akt and phosphorylated Akt (P-Akt) were analysed by immunoblotting. (b) SKBr-3 cells were treated with 50 nm 17-AAG and Akt and P-Akt were analysed by Western blot. Akt kinase activity was measured by in vitro phosphorylation of GSK-3. Kinase activity was detected by blotting with an anti-P-GSK-3 antibody. (c) SKBr-3 cells were treated with 50 nm 17-AAG and levels of p85, p110, P-PDK1 and PDK1 were detected by immunoblotting. (d) SKBr-3 cells were treated with the indicated doses of 17-AAG for 4 h and levels of Akt and phosphorylated Akt were analysed by immunoblotting

Figure 2

17-AAG induced dephosphorylation of Akt substrates and loss of cyclin D1 and cyclin D3. SKBr-3 cells were transfected with empty vector or p110-CAAX. (a) Transfected cells were treated with 50 nm 17-AAG. 4E-BP1, P-4E-BP1, GSK-3, and P-GSK-3 levels were determined by immunoblotting. (b) Transfected cells were treated with 50 nm 17-AAG or 50 µm LY294002 and levels of cyclin D were assessed

Figure 3

17-AAG induced loss of HER3 associated p85 and PI3K activity. SKBr-3 cells were treated with 50 nm 17-AAG. (a) Cell lysates were immunoblotted for HER2 and P-Her2. Samples were also immunoprecipitated with HER3 or IgG and immunoblotted for HER3, HER2, PY99, and p85 subunit of PI3k. (b) Cell lysates were immunoprecipitated with HER3 and PI3k kinase assays were performed. Kinase reactions were separated by thin layer chromatography and detected by autoradiography. (c) Cells were treated with 17-AAG alone or following 1 h pretreatment with 10 µm lactacystin. Cell lysates were immunoblotted for HER2, P-Her2, p85, Akt, P-Akt, and cyclin D1. Samples were also immunoprecipitated with HER3 or IgG and immunoblotted for HER3, PY99, and p85 subunit of PI3k

Figure 4

p110-CAAX prevented 17-AAG induced loss of phosphorylated Akt. SKBr-3 cells were transfected with empty vector or p110-CAAX. (a) Transfected cells were treated with 50 nm 17-AAG. HER2, Akt, and P-Akt expression were determined by immunoblotting. Akt kinase activity was detected as described earlier. (b) Transfected cells were treated with 50 µm LY294002 and Akt, P-Akt, and Akt activity were assessed

Figure 5

17-AAG induced loss of phosphorylated Akt in mice bearing human breast cancer xenografts and inhibited their growth. (a) Mice with BT-474 xenografts were treated with two cycles of 17-AAG 75 mg/kg/day i.p. × 5 days (n=12) or EPL vehicle control (n=12). Black rectangles represent treatment cycles; error bars represent the standard deviation. (b) Mice with established BT-474 xenograft tumors were treated with one dose of 17-AAG 50 mg/kg i.p. HER2, Akt, and P-Akt, and PI3k (p85 subunit) protein levels were assessed by immunoblotting