Facilitating Akt clearance via manipulation of Hsp70 activity and levels

Abstract

Members of the 70-kDa heat shock family can control and manipulate a host of oncogenic client proteins. This role of Hsp70 in both the folding and degradation of these client proteins makes it a potential drug target for certain forms of cancer. The phenothiazine family of compounds, as well as the flavonoid myricetin, was recently shown to inhibit Hsp70-ATPase activity, whereas members of the dihydropyrimidine family stimulated ATPase function. Akt, a major survival kinase, was found to be under the regulation of Hsp70, and when the ATPase activity of Hsp70 was increased or decreased by these compounds, Akt levels were also increased or decreased. Also, increasing Hsp70 levels concurrent with inhibition of its ATPase function synergistically reduced Akt levels to a greater extent than either manipulation alone, providing new insights about client fate decisions. Akt reductions mediated by Hsp70 inhibitors were prevented when Hsp70 expression was silenced with small interfering RNA. Inhibiting Hsp70 ATPase function produced cytotoxic events only in breast cancer cell lines where Akt dysfunction was previously shown, suggesting therapeutic specificity depending on the Hsp70 client profile. Thus, increasing Hsp70 levels combined with inhibiting its ATPase function may serve to dramatically reduce Akt levels and facilitate cell death in certain types of cancer.

FIGURE 1.

Small scale siRNA screen reveals opposing effects of Hsp70 and Hsc70 on Akt levels. HeLa cells were plated in 96-well plates at ∼40% confluency and transfected with indicated siRNAs for 72 h. siRNAs have been previously validated by Western blotting in this cell line. In-cell Western analysis for Akt (A, red) and GAPDH (B, green) showed an ∼30% increase in Akt levels when Hsp70 or Hsp27 was knocked down and an ∼30% decrease in Akt levels when Hsc70 was knocked down. Hsc70 siRNA decreased Akt levels most potently. An overlaid image of Akt and GAPDH is presented for contrast (C). Triplicate wells were quantified, and all values are shown as a percent of control siRNA (Ctrl) after GAPDH normalization ± S.D. (D).

FIGURE 2.

Silencing and overexpression of Hsp70 and Hsc70 inversely affect Akt and each other; effect is blocked by heat shock transcription inhibition. HeLa cells were transfected with Hsp70 or Hsc70 siRNA or a nonsilencing control siRNA (Ctrl) for 48 h and then analyzed by Western blotting (20 μg protein/lane) (A). HeLa cells at ∼90% confluency were transfected with Hsp70 and Hsc70 expression vectors for 48 h and analyzed by Western blotting (40 μg protein/lane) (B). Akt levels resulting from the overexpression (O.E.) of Hsp70 and Hsc70 data were quantitated ± S.D. (C). HeLa cells, at ∼50% confluency, were transfected with siRNA for Hsc70 or a nonsilencing control; at the time of transfection, cells were treated with either vehicle (Veh) or 100 μm heat shock element inhibitor KNK-437 for 48 h; results were analyzed by Western blotting (20 μg protein/lane) (D).

FIGURE 3.

Hsp70 ATPase inhibitors show reductions in Akt levels in HeLa cells. Structure of MB and myricetin (Myr) pictured at top. A 6-h dose response was performed with MB (A) or myricetin (C) at indicated concentrations in HeLa cells. A time course of 50 μm MB (B) or myricetin (D) was also performed at indicated time points in HeLa cells. Shown is a 6-h myricetin dose response at indicated dosages in HeLa cells, treated at ∼90% confluency. All blots contained 20 μg protein/lane. Veh, vehicle.

FIGURE 4.

Hsp70 ATPase activators display effects opposite those of inhibitors. HeLa cells, at ∼90% confluency, were treated for 24 h with 10 and 50 μm of Hsp70 ATPase inhibitors (MB or AC) or Hsp70 activators (SW02 or 115–7C), and lysates were analyzed by Western blotting (A). Quantitation was performed using pixel density analysis and is shown as a percentage of vehicle (Veh)-treated cells ± S.D. (♦, inhibitor; ■, activator) (B). HeLa cells, at ∼90% confluency, were treated with 50 μm doses of either SW02 or 115-7C and analyzed by Western blotting (C). Quantitation was performed using pixel density analysis and is shown as a percentage of vehicle-treated cells ± S.D. (■, 1157c; ♦, SW02) (D). All blots contained 20 μg protein/lane.

FIGURE 5.

Efficacy of Hsp70 ATPase inhibitors is dependent on levels of Hsp70. HeLa cells, at ∼50% confluency, were transfected with Hsp70 siRNA or a nonsilencing control (Ctrl) and were then treated with increasing doses of MB as indicated. Lysates were analyzed by Western blot (A), and Akt levels as a percent of vehicle (Veh) ± S.D. following pixel density analysis (■, Hsp70 siRNA; ♦, control siRNA) (B). HeLa cells, at ∼50% confluency, were transfected with nonsilencing siRNA (Ctrl), Hsp70 siRNA, or Hsc70 siRNA for 48 h, and then were treated with 50 μm MB, AC, or vehicle (C). Results were analyzed by Western blotting, and quantitation of Akt levels by pixel density analysis is shown as a percentage of corresponding vehicle treatments (gray bar, MB; black bar, AC) (D). All blots contained 20 μg protein/lane.

FIGURE 6.

Hsp70 inhibitors produce Akt-dependent cytotoxicity in breast cancer cell lines. LDH assays were performed on media from Hs578Bst and Hs578T cell lines, both at ∼90% confluency, and treated with 50 μm MB or vehicle (Veh) for 6 h. Levels are shown as a percent of vehicle treated cells ± S.D. (A). LDH assays were performed on media from MDA-MB-468, MDA-MB-231, MDA-MB-453, MCF-7, T47D, and MDA-MB-361 cell lines, all at ∼90% confluency, treated with 50 μm MB or vehicle for 6 h. LDH levels are shown as a percent of vehicle treated cells ± S.D. and estrogen receptor (ER) expression (B). LDH assays were performed on media collected from Hs578T cells transiently transfected at ∼90% confluency with either a 6TR noncoding vector or a myristoylated Akt vector (CA-Akt) and treated with increasing dosages of MB or a vehicle control for 6 h. LDH levels are shown as a percent of cells overexpressing CA-Akt relative to treatment matched 6TR control transfected cells ± S.D. Inset verifies Akt overexpression by pixel density analysis of Western blot run on cells receiving vehicle treatment (C). LDH assays were performed on Hs578T cells, at ∼90% confluency, receiving a 24-h 50 μm dose of Hsp70 ATPase activator SW02 (gray bars) or vehicle (black bars) followed by a 6-h dose response treatment of Hsp70 ATPase inhibitor MB or vehicle. LDH levels are shown as a percent of vehicle ± S.D. (D). p values were determined by Student's t test. All blots contained 20 μg protein/lane.

FIGURE 7.

Model for targeting clients toward destructive or productive pathways via Hsp70. As distinct clients (i.e. Client 1 and Client 2) are bound by Hsp70, there are two distinct fates which can emerge based on the client. These fates can be classified as either profolding or prodegradation. The biochemical nature of the client bound to Hsp70 along with the state of the cellular environment are the key factors that guide the Hsp70-client complex toward a fate: pro-folding for those designated to be lead away from potentially nonproductive misfolding pathways; or prodegradation for those requiring turnover as indicated by internal mechanisms. However, the exact mechanisms contributing to either of these decisions are unknown. Increasing the levels of Hsp70 is more likely to decrease certain clients while preserving others. By chemically modulating the ATPase activity of Hsp70, we are able to dictate client fate. Hsp70 activators can promote the accumulation of clients that would normally be degraded by Hsp70. Hsp70 inhibitors can promote the degradation of clients that would normally be preserved. Thus, Hsp70 overexpression combined with Hsp70 inhibition would synergistically affect clients that typically prefer degradation. Conversely, for clients that are more prone to refolding, overexpression of Hsp70 would have minimal impact on the levels of these clients, but inhibition of Hsp70 ATPase function would cause reductions.