Decoupling the mechanism of action of efrapeptins, dual inhibitors of proteasome and Hsp90 chaperone function: The possible role of endoplasmic reticulum stress

LB-184

Efrapeptins is a family of small, naturally occurring oligopeptides with potent antitumor activity in vitro and in vivo. Although efrapeptins are known for their ability to inhibit F₁F₀-ATPase, the enzyme responsible for the mitochondrial production of ATP, their antitumor actions have been attributed to their ability to inhibit the chymotrypsin-like and caspase-like activities of 26S proteasome and to suppress Hsp90 chaperone function by dissociating the complex of Hsp90 with its co-chaperone F₁F₀-ATPase. Breast cancer cells are particularly sensitive to the inhibitory activities of efrapeptins (IC₅₀ values range between 5 and 100 nM) with the exception of the MDA-MB-231 cell line (IC₅₀ ~4,000 nM). Here, the mechanisms determining cell sensitivity to the antitumor activities of efrapeptins are explored. As detected by Western immunoblotting, accumulation of ubiquitinated proteins after a brief treatment of MDA-MB-231 cells with efrapeptins suggests proteasomal inhibition. Interestingly, no appreciable accumulation of ubiquitinated proteins is observed in the efrapeptin-sensitive MCF-7 cells. Efrapeptin-induced dissociation of the Hsp90:F₁F₀-ATPase complex is detected in both cell lines indicating the presence of a functional F₁F₀-ATPase. However, accumulation and not degradation of mutated p53 after overnight treatment of MDA-MB-231 cells with efrapeptins indicates that Hsp90 client proteins in MDA-MB-231 cells may not be affected by the dissociation of the Hsp90:F₁F₀-ATPase complex as do in MCF-7 cells. Examination of the protein levels of Hsp90 and F₁F₀-ATPase reveal that MCF-7 cells possess 2.2-fold more Hsp90 and 8-fold more F₁F₀-ATPase than MDA-MB-231 cells, which may explain why inhibition of proteasome is the predominant mechanism of action in MDA-MB-231 cells. The reduction in the levels of F₁F₀-ATPase in the MDA-MB-231 cell line is in agreement with the dependence of the cells on glycolysis for ATP synthesis. Consequently, MDA-MB-231 cells possess increased levels of glucose regulated proteins, i.e., 3.2-fold more Grp78 and 1.5-fold more Grp94 than MCF-7 cells and express the glucose transporter Glut-1 while MCF-7 cells do not. Efrapeptin-treatment results in upregulation of Grp94 (both cell lines) and Grp78 (MDA-MB-231 only) without evidence of a concomitant endoplasmic reticulum (ER) stress induction. On the contrary, efrapeptin-treatment leads to downregulation of P-PERK, a known ER stress sensor. Downregulation of P-PERK has been associated with decreased resistance to ER stress suggesting that efrapeptin treatment may render tumor cells vulnerable to environmental conditions that promote ER-stress such as hypoxia and lack of nutrients. This may further explain the antitumor activity of efrapeptins.