Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70

Abstract

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.

Keywords: Hsp70; chaperone; non-oncogene addiction; proteotoxic stress; rational drug targeting.

Figure 1. Depletion of Hsp70 results in cancer cell senescence. SA-β-gal-positive cells following Hsp70 depletion in MCF7 and HeLa cells. Results shown are the mean ± SEM of 3 independent experiments. Induction of the cell cycle inhibitor p21 and downregulation of the mitosis factor surviving following Hsp70 depletion in MCF7 cells is also shown.

Figure 2. Hsp70 depletion does not alter ubiquitinylated protein levels. Depletion of Hsp70 does not cause apparent change in ubiquitination. MG132 (left panel, MCF7: 5 μM, 4 h; or right panel, HeLa: 10 μM, 4 h) was used to inhibit the proteasome (positive ubiquitination control). All samples prepared in lysis buffer containing N-ethylmaleimide (NEM, 10 mM). Samples were immunoblotted using an anti-ubiquitin antibody (lower panels). Upper panels: efficiency of Hsp70 depletion. Representative blots of triplicate experiments are shown. Quantification of levels of ubiquitinated proteins in HeLa cells is shown at the bottom.

Figure 3. Hsp70 knockdown does not activate stress-sensitive transcription factors, NRF2 or HSF1. (A) Depletion of Hsp70 does not activate NRF2 luciferase reporter. Left panel: luciferase activity was measured at baseline or following hydrogen peroxide treatment (300 μM, 6 h) in MCF7 cells to mimic increased oxidative stress and normalized to baseline control. The means and ± SEM of 3 experiments are shown. (B) Depletion of Hsp70 does not activate Hsf1 luciferase reporter. Luciferase activity was measured at baseline (0 h) or 5 h after heat shock (45 °C, 10 min) and normalized to baseline control. Left panel: MCF7 cells. Right panel: HeLa cells. The means and ± SEM of 3 experiments are shown.

Figure 4. Cancer cell chaperone capacity is not perturbed by Hsp70 depletion. Hsp70 knockdown does not affect luciferase refolding. Cells were heat shocked at 45 °C for 10 min, and luciferase activity was measured over time (left panel, MCF7; right panel, HeLa). Plot shows mean and ± SEM (n = 3).