Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling, including by modulating the activity of the transcription factors NF-kB, FoxM1 and Hif1α, the translation regulator HuR and the cell cycle regulators p21 and survivin. We also identified a small molecule inhibitor, YM-1, that disrupts Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anti-cancer target.

Polyglutamine (polyQ) disorders, including Huntington's disease (HD), are caused by expansion of polyQ-encoding repeats within otherwise unrelated gene products. In polyQ diseases, the pathology and death of affected neurons are associated with the accumulation of mutant proteins in insoluble aggregates. Several studies implicate polyQ-dependent aggregation as a cause of neurodegeneration in HD, suggesting that inhibition of neuronal polyQ aggregation may be therapeutic in HD patients. We have used a yeast-based high-throughput screening assay to identify small-molecule inhibitors of polyQ aggregation. We validated the effects of four hit compounds in mammalian cell-based models of HD, optimized compound structures for potency, and then tested them in vitro in cultured brain slices from HD transgenic mice. These efforts identified a potent compound (IC 50 ‫؍‬ 10 nM) with long-term inhibitory effects on polyQ aggregation in HD neurons. Testing of this compound in a Drosophila HD model showed that it suppresses neurodegeneration in vivo, strongly suggesting an essential role for polyQ aggregation in HD pathology. The aggregation inhibitors identified in this screen represent four primary chemical scaffolds and are strong lead compounds for the development of therapeutics for human polyQ diseases.high-throughput screen ͉ small-molecule therapeutics ͉ Drosophila ͉ R6͞2 brain slices ͉ genetic disease A t least nine inherited neurodegenerative diseases, including Huntington's disease (HD), are caused by expansion of polyglutamine (polyQ)-encoding repeats within otherwise unrelated proteins (1, 2). In HD, expansion of polyQ repeats within the huntingtin (Htt) protein causes an adult-onset neurodegenerative disease characterized by movement disorder, psychiatric symptoms, and cognitive dysfunction (3-5). As in several major neurological disorders, including Alzheimer's and Parkinson's diseases, the pathology and death of affected neurons in polyQ diseases are associated with accumulation of mutant polypeptides in insoluble aggregates (6-9). These polyQ-containing aggregates, or inclusions, have been found in the nuclei of affected neurons in postmortem patient tissues and brains from HD transgenic mice (10-12) and have emerged as a hallmark of HD pathology.Mutant polypeptides with extended polyQ tracts aggregate in vitro and in vivo in a polyQ length-dependent manner, which closely correlates with the age of onset in HD and other polyQ-expansion diseases (2,(13)(14)(15). Although the precise role of neuronal aggregates in disease pathogenesis is not clear, therapeutic strategies aimed at inhibiting polyQ aggregation have shown some efficacy in vivo in both Drosophila and mouse models of HD (16,17). These and other studies (18, 19) implicate polyQ-dependent aggregation as a cause of neurodegeneration in HD and suggest that inhibition of neuronal polyQ aggregation may be therapeutic in HD patients (8).Chemical compounds that directly target polyQ aggregation have been identified in high-throughput screens using cell-free ...

Novel classes of anticancer drugs, including proteasome inhibitors and Hsp90 inhibitors, potently induce heat shock proteins (Hsps). Because Hsps show antiapoptotic activities, we suggested that suppression of such induction may sensitize cancer cells to these drugs. Here, we knocked out the major heat shock transcription factor HSF-1 in several cancer cell lines using small interfering RNA and showed that such cells, which can no longer induce Hsps in response to proteasome and Hsp90 inhibitors, become more sensitive to these drugs. Furthermore, we developed a high-throughput screen for small molecules that inhibit induction of Hsps. The first step was a cell-based screen for inhibitors of Hsps-mediated luciferase refolding followed by a counterscreen for toxicity. The second step was a direct testing for inhibition of Hsp induction by immunoblotting with anti-Hsp72 antibody. After screening of 20,000 compounds from several diversity libraries, we focused on a compound we called NZ28, which potently inhibited induction of Hsps by heat shock, proteasome, and Hsp90 inhibitors in a variety of cell lines, and showed no significant toxicity. After testing of a set of analogues of NZ28, we identified a structural element that was critical for the activity. We also identified another inhibitor of the Hsp induction that was practically nontoxic. This compound, which we called emunin, strongly sensitized myeloma cells to proteasome and Hsp90 inhibitors and prostate carcinoma cells to proteasome inhibitors. This work indicates that targeting the heat shock response may facilitate use of proteasome and Hsp90 inhibitors for cancer treatment.

In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI ؉ ], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells. Expansion of glutamine repeats (poly-Q)1 in certain proteins is responsible for neurodegenerative disorders. The hallmark of poly-Q diseases is the formation of insoluble cytosolic and nuclear inclusions (1). Huntington disease is one of the best known poly-Q disorders (2). It is caused by an expansion of the poly-Q stretch in the essential protein called huntingtin (Htt) to more than 37 amino acids. The length of the poly-Q stretch inversely correlates with the time of onset of the disease and the time of formation of Htt aggregates (3). The role of various types of aggregates in cell toxicity remains a matter of debate (4). Recent models propose that toxicity of poly-Q Htt arises from sequestration of certain essential proteins by Htt aggregates (5-7). The poly-Q stretch is located within the N-terminal (exon 1) region of Htt, which is involved in numerous proteinprotein interactions (8). The N-terminal fragment of Htt with poly-Q extensions, when expressed in mice, aggregated and was sufficient to cause Huntington disease-like neurodegeneration (9, 10). This indicates that at least some parameters of poly-Q associated aggregation and toxicity could be reproduced in the experimental assays using only N-terminal poly-Q expanded fragments of Htt.Several models for studying poly-Q aggr...

The small heat shock protein Hsp27 is expressed at high levels in many tumors and provides protection against anticancer drugs. Here, we show that expression of

Previously we demonstrated that the heat shock transcription factor Hsf1 is indispensable for transformation of mammary epithelial cells by the Her2 oncogene. Since Hsf1 affects oncogene-induced senescence (OIS), these findings suggest that Hsf1 affects tumor initiation when OIS plays a role. Indeed, here we report that Hsf1 knockout suppressed mammary hyperplasia in Her2-expressing mice and reduced tumor emergence. On the other hand, Hsf1 expression increases with advanced breast cancer, indicating that there is an additional role of Hsf1 in tumor progression. We studied rare tumors that developed in Hsf1-knockout mice and found that these tumors grew slower than tumors in control animals and showed suppressed angiogenesis. Similarly, in the xenograft model, knockdown of Hsf1 suppressed angiogenesis, which was associated with suppression of the HIF-1 pathway. Suppression of HIF-1 was at the level of translation due to downregulation of the RNA-binding protein HuR. Importantly, besides HIF-1, HuR controls translation of other major regulators of cancer progression, many of which were suppressed in Hsf1-knockdown cells. Therefore, in addition to OIS, Hsf1 regulates the HuR-HIF-1 pathway, thus affecting both cancer initiation and progression.

The major heat shock protein Hsp72 is expressed at elevated levels in many human cancers and its expression correlates with tumor progression. Here we investigated the role of Hsp72 in Her2 oncogene-induced neoplastic transformation and tumorigenesis. Expression of Her2 in untransformed MCF10A mammary epithelial cells caused transformation, as judged by foci formation in culture and tumorigenesis in xenografts. However, expression of Her2 in Hsp72-depleted cells failed to induce transformation. The anti-tumorigenic effects of Hsp72 downregulation were associated with cellular senescence due to accumulation of p21 and depletion of survivin. Accordingly, either knockdown of p21 or expression of survivin reversed this senescence process. Further, we developed an animal model of Hsp72-dependent breast cancer associated with expression of Her2. Knockout of Hsp72 almost completely suppressed tumorigenesis in the MMTVneu breast cancer mouse model. In young Hsp72 KO mice, expression of Her2 instead of mammary tissue hyperplasia led to suppression of duct development and blocked alveolar budding. These effects were due to massive cell senescence in mammary tissue, which was associated with upregulation of p21 and downregulation of survivin. Therefore Hsp72 plays an essential role in Her2-induced tumorigenesis by regulating oncogene-induced senescence pathways.

The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).