Inhibition of Hsp90 chaperone and 26S proteasomal activities by efrapeptins in breast cancer cells

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Efrapeptins, a family of small, naturally occurring peptides, are known inhibitors of F₁F₀-ATP synthase. We recently reported that F₁F₀-ATP synthase is an Hsp90 co-chaperone assisting in the stabilization of a number of clients proteins including estrogen receptor (ER), mutated p53 (mu.p53), Hsp27, and Hsp70. Here, we examine the effects of efrapeptins on breast cancer cell proliferation. Efrapeptin inhibited the growth of two ERα(+) ( MCF-7, T47D) and three ERα(-) (MDA-MB-453, Hs578T, NCI/ADR-RES) breast cancer cell lines with IC₅₀ concentrations ranging from 5 to 50 nM. Two additional ERα(-) cell lines (MDA-MB-231 and MDA-MB-435) were relatively resistant to the antiproliferative effects of efrapeptins exhibiting IC₅₀ values of 1-10 μ. With the exception of MCF-7 cells, efrapeptins did not affect intracellular levels of ATP. However, inhibition of cell growth by efrapeptins correlated well with the dissociation of the Hsp90/F₁F₀-ATP synthase complex and the subsequent downregulation of both proteins. Transfection of MCF-7 cells with the HER2 gene led to enhanced efrapeptin-induced cytotoxicity as determined by lactate dehydrogenase (LDH) release. Immunoprecipitation experiments revealed that a brief (10 min) treatment of HER2-transfected MCF-7 cells with efrapeptins resulted in the disruption of the Hsp90/HER2 complex suggesting a role for F₁F₀-ATP synthase in stabilizing HER2. In contrast to most common Hsp90 inhibitors, overnight treatment of the HER2-transfected MCF-7 cells with efrapeptins led to accumulation of HER2, a phenomenon also seen with ERα. This accumulation was attributed to the ability of efrapeptins to inhibit the chymotrypsin-like (Ch-L) and postglutamyl peptide hydrolase (PGPH) enzymatic activities of 26S proteasome. Proteasomal inhibition in breast cancer cells treated with efrapeptins was reversible and accumulation of ubiquitinated proteins was followed by increased degradation. Consequently, degradation of client proteins mu.p53, HSF-1, Hsp70, and Hsp27 was seen after overnight treatment of breast cancer cells with efrapeptins. Finally, a synergistic effect appears to exist between efrapeptins and other chemotherapeutics such as doxorubicin.