No abstract

Inhibition of the major cytosolic protease, proteasome, has been reported to induce programmed cell death in several cell lines, while with other lines, similar inhibition blocked apoptosis triggered by a variety of harmful treatments. To elucidate the mechanism of proand antiapoptotic action of proteasome inhibitors, their effects on U937 lymphoid and 293 kidney human tumor cells were tested. Treatment with peptidyl aldehyde MG132 and other proteasome inhibitors led to a steady increase in activity of c-Jun N-terminal kinase, JNK1, which is known to initiate the apoptotic program in response to certain stresses. Dose dependence of MG132-induced JNK activation was parallel with that of apoptosis. Furthermore, inhibition of the JNK signaling pathway strongly suppressed MG132-induced apoptosis. These data indicate that JNK is critical for the cell death caused by proteasome inhibitors. An antiapoptotic action of proteasome inhibitors could be revealed by a short incubation of cells with MG132 followed by its withdrawal. Under these conditions, the major heat shock protein Hsp72 accumulated in cells and caused suppression of JNK activation in response to certain stresses. Accordingly, pretreatment with MG132 reduced JNK-dependent apoptosis caused by heat shock or ethanol, but it was unable to block JNK-independent apoptosis induced by TNF␣. Therefore, proteasome inhibitors activate JNK, which initiates an apoptotic program, and simultaneously they induce Hsp72, which suppresses JNK-dependent apoptosis. A balance between these two effects might define the fate of cells exposed to the inhibitors.

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.

The heat shock transcription factor HSF1 was recently demonstrated to play a key role in the development of tumors associated with activation of Ras or inactivation of p53. Here we show that HSF1 is required for cell transformation and tumorigenesis induced by HER2 oncogene responsible for aggressive breast tumors. Upon expression of HER2, untransformed human mammary epithelial cells MCF-10A underwent neoplastic transformation, formed foci in culture and tumors in nude mouse xenografts. However, expression of HER2 in MCF-10A cells with knockdown of HSF1 did not cause either foci formation or tumor growth in xenografts. The anti-tumorigenic effect of downregulation of HSF1 was associated with HER2-induced accumulation of the CDK inhibitor p21 and decrease of the mitotic regulator survivin, which resulted in growth inhibition and cell senescence. In fact, either knockout of p21 or overexpression of survivin alleviated these effects of HSF1 knockdown. Proliferation of certain human HER2-postitive breast cancer lines also requires HSF1, since its knockdown led to upregulation of p21 and/or drop of survivin, precipitating growth arrest. Similar effects were observed with a small molecular weight inhibitor of the heat shock response NZ28. Effects of HSF1 knockdown on growth arrest and senescence of HER2-expressing cells were associated with downregulation of Hsp72 and Hsp27. Therefore, HSF1 is critical for proliferation of HER2-expressing cells, most likely since it maintains levels of HSPs, which in turn control regulators of senescence p21 and survivin.

Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out the SEK1 gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylation in unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72.Exposure of eukaryotic cells to various stresses stimulates the stress-activated kinases JNK (c-Jun N-terminal kinase) and p38 (for a review, see reference 22). JNK activates the transcription factor AP-1, thus regulating cell proliferation, immune responses, inflammation, and programmed cell death, or apoptosis (6,19,22,35). UV irradiation, osmotic stress, as well as certain cytokines and mitogens activate JNK via a signal transduction pathway which involves small GTP-binding proteins (7, 29) and a cascade of protein kinases. This kinase cascade includes MEKKs (30) followed by the dual-specificity kinases SEK1 (MKK4) (39, 44) and MKK7 (41), both of which phosphorylate JNK at the vicinal threonine and tyrosine residues, thus activating this kinase (23).Heat shock, ethanol, oxidative stress, and certain other stresses, on the other hand, activate JNK through a pathway which has not yet been established. Furthermore, recent data indicate that heat shock and UV irradiation activate JNK via distinct pathways (1). Consistent with this notion, the yeast Spc1 kinase, a homolog of p38 and JNK, is activated by heat shock through a pathway different from that used by other stresses (38,40). This pathway was reported to involve inhibition of Spc1 dephosphorylation by its phosphatase Pyp1 (although this report has recently been disputed [40]). Phosphatases were also implicated in activation of JNK in mammalian cells. For example, the protein-damaging agent arsenite was demonstrated to activate JNK through specific inhibition of a constitutively active JNK phosphatase (2).Certain stresses including heat shock and ethanol not only activate JNK and p38 but also induce synthesis of heat s...

Harmful conditions including heat shock, oxidative stress, UV, and so forth cause programmed cell death, whose triggering requires activation of the Jun N-terminal kinase, JNK. High levels of Hsp72, a heat-inducible member of Hsp70 family, protect cells against a variety of stresses by a mechanism that is unclear at present. Here we report that elevated levels of Hsp72 inhibit a signal transduction pathway leading to programmed cell death by preventing stress-induced activation of JNK. Stress-induced activation of another stress-kinase, p38 (HOG1), is also blocked when the level of Hsp72 is increased. Similarly, addition of a purified recombinant Hsp72 to a crude cell lysate reduced p38 kinase activation, while depletion of the whole family of Hsp70 proteins with a monoclonal antibody enhanced such activation. In addition, we have found that accumulation of abnormal proteins in cells upon incubation with amino acid analogs causes activation of JNK and p38 kinases, which can be prevented by overproduction of Hsp72. Taken together, these data suggest that, in regulation of JNK and p38 kinases, Hsp70 serves as a "sensor" of the build-up of abnormal proteins after heat shock and other stresses. The inhibitory effect of an increased level of Hsp70 on JNK appears to be a major contributor to acquired thermotolerance in mammalian cells.

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.

Mechanistic studies from cell culture and animal models have revealed critical roles for the heat shock protein Hsp70 in cancer initiation and progression. Surprisingly, many effects of Hsp70 on cancer have not been related to its chaperone activity, but rather to its role(s) in regulating cell signaling. A major factor that directs Hsp70 signaling activity appears to be the co-chaperone Bag3. Here, we review these recent breakthroughs, and how these discoveries drive drug development efforts.