HSP70 inhibition by the small-molecule 2-phenylethynesulfonamide impairs protein clearance pathways in tumor cells.

The evolutionarily conserved stress-inducible HSP70 molecular chaperone plays a central role in maintaining protein quality control in response to various forms of stress. Constitutively elevated HSP70 expression is a characteristic of many tumor cells and contributes to their survival. We recently identified the small-molecule 2-phenylethyenesulfonamide (PES) as a novel HSP70 inhibitor. Here, we present evidence that PES-mediated inhibition of HSP70 family proteins in tumor cells results in an impairment of the two major protein degradation systems, namely, the autophagy-lysosome system and the proteasome pathway. HSP70 family proteins work closely with the HSP90 molecular chaperone to maintain the stability and activities of their many client proteins, and PES causes a disruption in the HSP70/HSP90 chaperone system. As a consequence, many cellular proteins, including known HSP70/HSP90 substrates, accumulate in detergent-insoluble cell fractions, indicative of aggregation and functional inactivation. Overall, PES simultaneously disrupts several cancer critical survival pathways, supporting the idea of targeting HSP70 as a potential approach for cancer therapeutics.

A small molecule inhibitor of inducible heat shock protein 70.

The multifunctional, stress-inducible molecular chaperone HSP70 has important roles in aiding protein folding and maintaining protein homeostasis. HSP70 expression is elevated in many cancers, contributing to tumor cell survival and resistance to therapy. We have determined that a small molecule called 2-phenylethynesulfonamide (PES) interacts selectively with HSP70 and leads to a disruption of the association between HSP70 and several of its cochaperones and substrate proteins. Treatment of cultured tumor cells with PES promotes cell death that is associated with protein aggregation, impaired autophagy, and inhibition of lysosomal function. Moreover, this small molecule is able to suppress tumor development and enhance survival in a mouse model of Myc-induced lymphomagenesis. The data demonstrate that PES disrupts actions of HSP70 in multiple cell signaling pathways, offering an opportunity to better understand the diverse functions of this molecular chaperone and also to aid in the development of new cancer therapies.

Hepatic IGFBP1 is a prosurvival factor that binds to BAK, protects the liver from apoptosis, and antagonizes the proapoptotic actions of p53 at mitochondria.

Liver is generally refractory to apoptosis induced by the p53 tumor suppressor protein, but the molecular basis remains poorly understood. Here we show that p53 transcriptional activation leads to enhanced expression of hepatic IGFBP1 (insulin-like growth factor-binding protein-1). Exhibiting a previously unanticipated role, a portion of intracellular IGFBP1 protein localizes to mitochondria where it binds to the proapoptotic protein BAK and hinders BAK activation and apoptosis induction. Interestingly, in many cells and tissues p53 also has a direct apoptotic function at mitochondria that includes BAK binding and activation. When IGFBP1 is in a complex with BAK, formation of a proapoptotic p53/BAK complex and apoptosis induction are impaired, both in cultured cells and in liver. In contrast, livers of IGFBP1-deficient mice exhibit spontaneous apoptosis that is accompanied by p53 mitochondrial accumulation and evidence of BAK oligomerization. These data support the importance of BAK as a mediator of p53's mitochondrial function. The results also identify IGFBP1 as a negative regulator of the BAK-dependent pathway of apoptosis, whose expression integrates the transcriptional and mitochondrial functions of the p53 tumor suppressor protein.

The tetramerization domain of p53 is required for efficient BAK oligomerization.

In addition to a well-defined transcriptional activity that is necessary for efficient apoptosis induction, the p53 tumor suppressor also has a direct apoptogenic role at the mitochondria. This direct role in cell death is mediated at least in part by interaction of p53 with BCL2 family members, including the pro-apoptotic protein BAK. Whereas it is currently accepted that the mitochondrial function of p53 contributes to its tumor suppressive role, the regulation of p53 function at this organelle is poorly understood. In this manuscript we examine the role of p53 oligomerization in the regulation of its pro-apoptotic function at the mitochondria, specifically in regard to its ability to induce BAK oligomerization. We find that deletion or mutation of p53's oligomerization domain markedly impairs the ability of this protein to oligomerize BAK. Along these lines, cross-linking studies indicate that the majority of p53 localized to mitochondria is in dimeric or higher-order oligomeric form. In support of the importance of the p53-BAK interaction in the localization of p53 to mitochondria, we find that mouse embryo fibroblasts from the BAK null mouse have greatly reduced mitochondrial p53 compared to wild type fibroblasts. These data indicate that pro-apoptotic BAK, unlike other BCL2 family members, may serve as a major receptor for p53 on the mitochondria.

p53 moves to mitochondria: a turn on the path to apoptosis.

It has been said that no matter which direction cancer research turns, the p53 tumor suppressor protein comes into view. The widespread role of p53 as a suppressor of tumor development is believed to rely on its ability to induce programmed cell death in response to stress, either the replicative stress associated with uncontrolled cellular proliferation, or the environmental stresses that accompany tumor development, such as hypoxia. For some time it has been believed that the role of p53 in inducing apoptosis in response to such stress was as a master regulator coordinating the expression of other molecules whose ultimate role was the execution of the cell. New data, however, suggest that p53 itself also has a direct role in accomplishing cell death, at the mitochondria.

Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex.

The tumour suppressor activity of the p53 protein has been explained by its ability to induce apoptosis in response to a variety of cellular stresses. Thus, understanding the mechanism by which p53 functions in the execution of cell death pathways is of considerable importance in cancer biology. Recent studies have indicated that p53 has a direct signalling role at mitochondria in the induction of apoptosis, although the mechanisms involved are not completely understood. Here we show that, after cell stress, p53 interacts with the pro-apoptotic mitochondrial membrane protein Bak. Interaction of p53 with Bak causes oligomerization of Bak and release of cytochrome c from mitochondria. Notably, we show that formation of the p53-Bak complex coincides with loss of an interaction between Bak and the anti-apoptotic Bcl2-family member Mcl1. These results are consistent with a model in which p53 and Mcl1 have opposing effects on mitochondrial apoptosis by interacting with, and modulating the activity of, the death effector Bak.

The codon 72 polymorphic variants of p53 have markedly different apoptotic potential.

The gene TP53, encoding p53, has a common sequence polymorphism that results in either proline or arginine at amino-acid position 72. This polymorphism occurs in the proline-rich domain of p53, which is necessary for the protein to fully induce apoptosis. We found that in cell lines containing inducible versions of alleles encoding the Pro72 and Arg72 variants, and in cells with endogenous p53, the Arg72 variant induces apoptosis markedly better than does the Pro72 variant. Our data indicate that at least one source of this enhanced apoptotic potential is the greater ability of the Arg72 variant to localize to the mitochondria; this localization is accompanied by release of cytochrome c into the cytosol. These data indicate that the two polymorphic variants of p53 are functionally distinct, and these differences may influence cancer risk or treatment.