Targeting heat shock protein 27 (HspB1) interferes with bone metastasis and tumour formation in vivo.

BACKGROUND: The small stress heat shock protein 27 (Hsp27) has recently turned as a promising target for cancer treatment. Hsp27 upregulation is associated with tumour growth and resistance to chemo- and radio-therapeutic treatments, and several ongoing drugs inhibiting Hsp27 expression are under clinical trial. Hsp27 is now well described to counteract apoptosis and its elevated expression is associated with increased aggressiveness of several primary tumours. However, its role in the later stage of tumour progression and, more specifically, in the later and most deadly stage of tumour metastasis is still unclear. METHODS/RESULTS: In the present study, we showed by qRT-PCR that Hsp27 gene is overexpressed in a large fraction of the metastatic breast cancer area in 53 patients. We further analysed the role of this protein in mice during bone metastasis invasion and establishment by using Hsp27 genetically depleted MDA-MB231/B02 human breast cancer cell line as a model. We demonstrate that Hsp27 silencing led to reduced cell migration and invasion in vitro and that in vivo it correlated with a decreased ability of breast cancer cells to metastasise and grow in the skeleton. CONCLUSION: Altogether, these data characterised Hsp27 as a potent therapeutic target in breast cancer bone metastasis and skeletal tumour growth.

Hsp27 negatively regulates cell death by interacting with cytochrome c.

Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

Dithiocarbamate fungicide thiram detection: comparison of bioluminescent and fluorescent whole-cell bioassays based on hsp22 stress promoter induction.

Detection of toxic substances interfering with endocrine system is one of the major preoccupations of the European community. A whole-cell bioassay for pollution detection based on stress induction has been designed. Well characterized toxicants, cadmium chloride and thiram (a dithiocarbamate fungicide), were used to optimize the detection conditions such as time-course conditions, cell line and reporter gene to be used. HeLa cells containing the firefly luciferase (luc) reporter gene under the control of the Drosophila melanogaster hsp22 promoter were compared to liver cells (HepG2) containing the same stress gene promoter fused either to the luc or the EGFP (Enhanced-Green Fluorescent Protein) gene. The sensitivity of the obtained bioassay was found to be enhanced by the concomitant use of liver cells and EGFP reporter gene. The detection limits of the toxicants were then lowered from 1 to 0.1 microM and from 1 to 0.01 microM for CdCl(2) and thiram, respectively.

p53/T-antigen complex disruption in T-antigen transformed NIH3T3 fibroblasts exposed to oxidative stress: correlation with the appearance of a Fas/APO-1/CD95 dependent, caspase independent, necrotic pathway.

Simian Virus 40 Large T-antigen expressed in NIH3T3 cells increases p53 level and interacts with this tumor suppressor to form large nuclear complexes. We show here that T-antigen sensitizes NIH3T3 cells to low doses of the oxidative stress inducer menadione. This oxidant increased p53 accumulation and disrupted p53/T-antigen interaction, but not T-antigen/pRb, T-antigen/Hsc70 and p53/Hsc70 complexes; a phenomenon inhibited by the anti-oxidant N-acetyl-cysteine. Analysis of several p53 downstream gene products revealed that the level of Fas receptor, which was sharply reduced by T-antigen expression, was drastically increased in response to menadione treatment. Menadione also induced a T-antigen dependent cleavage of Fas ligand. Analysis performed with Fas receptor antagonist antibody and metalloproteinases inhibitor revealed that menadione triggers a Fas-dependent death of a fraction of T-antigen expressing cells. This Fas pathway does not activate caspase 8 or 3, probably because of the inhibition induced by T-antigen, and leads to a necrotic cell death which contributes at least in part to the hypersensitivity of T-antigen transformed cells to oxidative stress.

Transformation by T-antigen and other oncogenes delays Hsp25 accumulation in heat shocked NIH 3T3 fibroblasts.

We have recently reported that transformation of murine NIH 3T3 cells by v-fos oncogene interfered with Hsp70 and Hsp25 accumulation after heat shock. Here, we have investigated the effect mediated by other oncogenes on the accumulation of these stress proteins. We report that T-antigen transformation of NIH 3T3 cells delayed and reduced the accumulation of Hsp25 after heat shock and decreased the heat-mediated phosphorylation of this protein. This decreased level of Hsp25 correlated with a reduced accumulation of the corresponding mRNA and was related to T-antigen level. In contrast, T-antigen had no effect on the expression of the major stress protein Hsp70 nor did it interfere with the level of Hsp90 or Hsp60. We report also that v-src or Ha-ras oncogenes delayed Hsp25 accumulation after heat shock but that only v-src reduced the heat-induced phosphorylation of this protein. v-src, but not Ha-ras, interfered with Hsp70 expression and none of these oncogenes had an effect on Hsp60 or Hsp90 levels. Taken together, these observations suggest that an altered accumulation of Hsp25 after heat shock is a common characteristic of NIH 3T3 fibroblasts transformed by different oncogenes.

Herpes simplex virus Us11 protein enhances recovery of protein synthesis and survival in heat shock treated HeLa cells.

One of the herpes simplex virus type 1 (HSV-1) true late gene products, Us11 protein, is brought into the cell by the infecting virion and may play a role in the virally-induced post-transcriptional control of gene expression. Us11 protein forms large oligomers, exhibits RNA binding features, concentrates into the nucleolus and is able to replace Rex protein in post-transcriptional control of human T-cell leukemia/lymphoma virus type I (HTLV-I) expression. As heat shock drastically alters protein synthesis, and because HSV-1 infection stimulates heat shock protein (Hsp) expression, we analyzed the consequence of heat shock in HeLa cells expressing Us11 alone, either transiently or constitutively. No detectable modification of the overall pattern of protein synthesis was observed in cells growing at normal temperatures, including no induction of Hsp expression or accumulation. However, Us11 protein expression induced an enhanced recovery of protein synthesis after heat shock. Moreover, the level of Us11 protein-mediated protection of protein synthesis was similar to that observed for cells made thermotolerant, but only when submitted to a mild heat shock. Finally, Us11 protein expression induced in cells an enhanced survival to heat shock.

Inhibition of heat shock protein 27 (HspB1) tumorigenic functions by peptide aptamers.

Human heat shock protein 27 (Hsp27, HspB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. Hsp27 biochemical properties rely on a structural oligomeric and dynamic organization. Downregulation by small interfering RNA or inhibition with dominant-negative mutant have proven their efficiency to counteract the anti-apoptotic and protective properties of Hsp27. In this study, we report the isolation and characterization of Hsp27-targeted molecules interfering with its structural organization. Using the peptide aptamer (PA) strategy, we isolated PAs that specifically interact with Hsp27 and not with the other members of the small heat shock protein family. In mammalian cell cultures, PAs expression perturbed the dimerization and oligomerization of Hsp27, and acted as negative regulators of the anti-apoptotic and cytoprotective activities of this protein. Further studies analyzing SQ20B cell xenografts in immunocompromised mice showed that PAs strongly reduced tumor development through cell cycle arrest. Our data suggest that PAs could provide a potential tool to develop strategies for the discovery of Hsp27 chemical inhibitors.

Protection against heat and staurosporine mediated apoptosis by the HSV-1 US11 protein.

US11 protein, one of herpes simplex virus type 1 (HSV-1) true late gene products, plays a role in the virally induced post-transcriptional control of gene expression. In addition, US11 expression also interferes with the cellular response to HSV-1 infection that can lead to apoptosis. We have previously shown that US11 expression enhanced the recovery of cellular protein synthesis and increased cell survival in response to thermal stress. Since heat shock can activate apoptosis, we tested for a possible anti-apoptotic behavior of US11. Here, we show that, in HeLa cells, US11 expression strongly reduced heat induced apoptosis, a phenomenon independent of Hsp expression and characterized by a delayed cytochrome c efflux from mitochondria and reduced caspase 3 activation. Moreover, US11 expression also protected against staurosporine induced apoptosis. Hence, our results favor an anti-apoptotic activity of US11 polypeptide that appears to be located at the level of mitochondria or upstream signaling pathways.

Thermal sensitivity in NIH 3T3 fibroblasts transformed by the v-fos oncogene. Correlation with reduced accumulation of 68-kDa and 25-kDa stress proteins after heat shock.

The effect of v-fos transformation on the cellular response to heat shock has been investigated. NIH 3T3 fibroblasts were transfected with the FBR p75gag-fos gene fusion under the control of the long terminal repeat (LTR) promoter of Finkel-Biskin-Reilly (FBR) murine sarcoma virus and with the gene encoding hygromycin resistance. Several hygromycin-resistant clone isolates, that expressed various levels of p75gag-fos oncoprotein, were analyzed as they displayed properties of transformed cells, such as altered morphology, shorter doubling time, serum-independent growth and foci formation in soft agar. The thermal response of these clones was compared to that of the control cells expressing the hygromycin-resistance gene only. Here, we report that the v-fos-transformed clones displayed an enhanced thermosensitivity which resulted in a reduced tolerance to thermal stress. Heat-treated v-fos-transformed cells displayed a decreased expression and accumulation of the major stress proteins Hsp68 (68-kDa heat-shock protein) and Hsp25 which probably resulted of a reduced accumulation of the corresponding mRNAs. This effect was particularly intense at the level of Hsp25. These alterations in cell survival and stress-protein expression appeared correlated to the level of p75gag-fos. At least for Hsp68, the transcription of this gene was not found altered by v-fos expression suggesting that this oncogene increases the turn-over of Hsp68 mRNA. After the heat-shock treatment, v-fos transformation also reduced the time period during which the constitutively expressed stress protein Hsc70 redistributes inside the nucleus. Since Hsp68 and Hsp25 are molecular chaperones that in vivo protect cells against the deleterious effects of heat shock, it is conceivable that their reduced accumulation and altered cellular distribution following heat shock may contribute, at least in part, to the thermosensitivity of v-fos-transformed NIH 3T3 fibroblasts.

Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27-kDa heat-shock protein.

The Drosophila melanogaster small heat-shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to alpha-crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. Two internal and adjacent deletion mutants in the conserved alpha-crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian-Virus-40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen-peroxide stresses as judged by Trypan-blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the 5' end of the conserved alpha-crystallin domain and covers the first 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N-terminal half of the conserved alpha-crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the alpha-crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus.