Function Of Hsp27 Research Articles

HSP27 Promotes Mantle Cell Lymphoma Growth and Mediates Resistance to Venetoclax

Introduction Drug resistance has been a major challenge for the treatment of patients with relapsed or refractory mantle cell lymphoma. Venetoclax is a highly potent, Bcl2-selective inhibitor that is effective in refractory/relapsed MCL patients both as monotherapy or in combination with ibrutinib, but the development of acquired resistance remains inevitable. The heat shock protein HSP27 (encoded by HSPB1) is normally upregulated under conditions of protein folding alteration, such as heat shock. However, it has been demonstrated that HSP27 can promote the growth and metastasis of cancer cells and enable resistance to many anticancer therapies. Accordingly, HSP27 has been targeted for therapy of cancer progression and treatment resistance. We hypothesize that HSP27 can induce therapeutic resistance, and that targeting of HSP27 may overcome venetoclax resistance in MCL. Methods Primary cells from patients' with or without therapeutic resistance as well as ibrutinib or venetoclax-sensitive and -resistant MCL cell lines were used in this study. We established a HSPB1- knockout (KO) human MCL JeKo-1 cell line using the CRISPR-Cas9 knockout system. Cell viability and cell proliferation assays were tested in triplicate for 72 h. The annexin V/propidium iodide-binding assay was utilized to evaluate apoptosis following venetoclax treatment. The underlying resistance mechanisms were verified by immunoblotting. Results HSP27 was highly upregulated in primary cells from patients resistant to BTK inhibitors compared to cells from those sensitive to BTK inhibitors. In addition, HSP27 was significantly increased in the Rec-Ven-R MCL cell line, which has acquired venetoclax resistance, compared to the parental Rec-1 cell line. To further investigate the function of HSP27 in regulating growth and therapeutic resistance, we knocked out the HSPB1 gene in JeKo-1 cells using a CRISPR-Cas9 knockout system. We first evaluated the impact of HSP27 on MCL cell growth. HSP27 knockout in the HSP27-high JeKo-1 cells greatly inhibited their growth and proliferation in vitro. Next, we carried out cell viability assays and found that genetic ablation of HSPB1 enhanced the sensitivity to venetoclax. Furthermore, treatment of HSPB1-knockout JeKo-1 cells with 1 uM venetoclax for 24 h and 48 h induced greater apoptosis than in the parental JeKo-1 cells. Western blot analysis of whole cell lysates showed that knockout of HSPB1 triggered an increase in the pro-apoptotic markers cleaved PARP and cleaved caspase 3 after venetoclax treatment for 6 h. Together, these results suggest that HSP27 potentiates the growth and proliferation of MCL cells and mediates the acquired resistance to venetoclax. Conclusions Targeting HSP27 may overcome venetoclax resistance in MCL cells, which provide impressive insights for therapeutic development to treat refractory/relapsed MCL patients and improve patient prognosis.

The protective role of HSP27 in ocular diseases.

Heat shock proteins (HSPs) are stress-induced proteins that are important constituents of the cell's defense system. The activity of HSPs enhances when the cell undergoes undesirable environmental conditions like stress. The protective roles of HSPs are due to their molecular chaperone and anti-apoptotic functions. HSPs have a central role in the eye, and their malfunction has been associated with the manifestation of ocular diseases. Heat shock protein 27 (HSP27, HSPB1) is present in various ocular tissues, and it has been found to protect the eye from disease states such as retinoblastoma, uveal melanoma, glaucoma, and cataract. But some recent studies have shown the destructive role of HSP27 on retinal ganglionic cells. Thus, this article summarizes the role of heat shock protein 27 in eye and ocular diseases and will focus on the expression, regulation, and function of HSP27 in ocular complications.

HSP27 protects against ferroptosis of glioblastoma cells.

Ferroptosis, as an new form of non-apoptotic regulated cell death, plays an important role in human cancers. Although it is reported that HSP27 is an novel regulator of ferroptosis in cancer, it remains unknown how HSP27 affects ferroptosis in glioma. In this study, we examined the effect of HSP27 on the ferroptosis of glioblasotma. HSP27 overexpression protects glioblastoma cells from erastin-induced ferroptosis while HSP27 depletion promotes erastin-induced ferroptosis of glioblastoma. Notably, HSP27 phosphorylation is required for the protective function of HSP27 in erastin-induced ferroptosis. Overall, our study reveal novel molecular mechanisms of ferroptosis in glioma and also identify HSP27 as a negative regulator of ferroptosis and a potential target for the treatment of glioma.

Could Small Heat Shock Protein HSP27 Be a First-Line Target for Preventing Protein Aggregation in Parkinson's Disease?

Small heat shock proteins (HSPs), such as HSP27, are ubiquitously expressed molecular chaperones and are essential for cellular homeostasis. The major functions of HSP27 include chaperoning misfolded or unfolded polypeptides and protecting cells from toxic stress. Dysregulation of stress proteins is associated with many human diseases including neurodegenerative diseases, such as Parkinson’s disease (PD). PD is characterized by the presence of aggregates of α-synuclein in the central and peripheral nervous system, which induces the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and in the autonomic nervous system. Autonomic dysfunction is an important non-motor phenotype of PD, which includes cardiovascular dysregulation, among others. Nowadays, the therapies for PD focus on dopamine (DA) replacement. However, certain non-motor symptoms with a great impact on quality of life do not respond to dopaminergic drugs; therefore, the development and testing of new treatments for non-motor symptoms of PD remain a priority. Since small HSP27 was shown to prevent α-synuclein aggregation and cytotoxicity, this protein might constitute a suitable target to prevent or delay the motor and non-motor symptoms of PD. In the first part of our review, we focus on the cardiovascular dysregulation observed in PD patients. In the second part, we present data on the possible role of HSP27 in preventing the accumulation of amyloid fibrils and aggregated forms of α-synuclein. We also include our own studies, highlighting the possible protective cardiac effects induced by L-DOPA treatment through the enhancement of HSP27 levels and activity.

P390 New features of molecular diagnostics of the regulation of molecular apoptotic pathway in ulcerative colitis

Abstract Background The incidence rate of ulcerative colitis (UC) in Russia is 5–30 cases per 100,000 per year. Molecular pathways of the UC development are not clear. The purpose of the work was to study the molecular interactions of the apoptotic pathway in patients with UC. Methods The clinical trial included 92 patients with UC. The clinical presentation of UC depends on the extent of involvement: distal colitis, extensive colitis; the severity of the disease. Criteria of the diagnosis of UC corresponded to ECCO Consensus. Mucosal specimens were graded according to grades 0, 1, 2, 3. The separation of proteins of colon mucosa was based on technologies of IEF, SDS-PAGE, 2DPAGE, by standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, Bruker, USA). The getting of mass-spectrogram was determined by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, Bruker, USA). Statistical analysis was performed using the software Statistica 10.0 (Statsoft). Results HSP2 controls the apoptosis of colonocytes and immune response in damaged colon mucosa by Bcl-2 and IL-17, and is also responsible for the resistance to therapeutic strategies. Anti-apoptotic functions of HSP27 is possible through the interaction with DAXX7, the activation of Akt and the inhibition of the apoptosis. HSP47 interacts with collagen I, II, III, IV and V types, that contributes to the launch of autoimmune process in UC. Caspase 8 protects colonocytes from TNFα-induced cell death through necroptosis mechanism via the blockade of the expression of RIP3. Significant decrease of the expression of PPARγ promotes the activation of STAT and AP-1 signalling pathways, that promotes the increase of the synthesis of IL-2, -6, -8, -12, TNFα, matrix metalloproteinases, the activity of immune and inflammation processes in the colon mucosa. Results of qualitative proteomic analysis in patients with UC (Tab.) Conclusion Proteomic analysis has demonstrated the cascade with caspases 8, 9, 10, the release of cytochrome C from mitochondria, which interacts with Apaf-1, causing activation of caspase-9 in UC colon mucosa. TNF-α activates of initiator caspase-2, -8, and -10 in the apoptotic pathway. The NF-kB pathway induces cellular inhibitors of apoptosis, which function as specific caspase inhibitors in UC colonic mucosa.

The effects of HSP27 against UVB-induced photoaging in rat skin

Skin photoaging refers to the phenomenon of skin aging or accelerated aging as a result of long-term UV exposure. Ultraviolet radiation can lead to DNA damage, cell apoptosis, cell growth inhibition and carcinogenic effects. Evidence suggests that hsp27 can protect cells from apoptosis induced by various stimuli in vivo and in vitro. However, modulation in hsp27 expression toward skin protection against UVB treatment has not been investigated clearly. In this study, we aimed to investigate the effects of hsp27 against UVB-induced photoaging in rat skin and to explore the underlying mechanisms. In the present study, we identified that the level of hsp27 increased after UVB irradiation induced chronic photoaging rat model. In order to investigate the function of hsp27 in UVB-induced skin photoaging, we used adeno-associated virus (AAV) to specificity reduce the expression of hsp27 in rat skin. In contrast to UVB group, we found that collagen fibers were disorganized and elastic fibers were thickened and twisted in UVB-AAV group. In the UVB-AAV group, reduced hsp27 enhanced the oxidative stress. Aging markers (SA-β-Gal staining and the protein levels of p16, p53, p21) were significantly changed in the hsp27 decreased group. However, in hsp27 deletion group, the expression of antiapoptotic factor bcl-2 was decreased, while the apoptosis factor bax was increased after UVB irradiation. These findings suggested that hsp27 was involved in oxidative stress, aging and apoptosis of skin after UV exposure. Management the expression of hsp27 can be used as a potential intervention method to alleviate UVB-induced skin damage.

Local unfolding of the HSP27 monomer regulates chaperone activity

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.

Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau

Small heat shock proteins (sHSPs) are a class of oligomeric molecular chaperones that limit protein aggregation. However, it is often not clear where sHSPs bind on their client proteins or how these protein-protein interactions (PPIs) are regulated. Here, we map the PPIs between human Hsp27 and the microtubule-associated protein tau (MAPT/tau). We find that Hsp27 selectively recognizes two aggregation-prone regions of tau, using the conserved β4-β8 cleft of its alpha-crystallin domain. The β4-β8 region is also the site of Hsp27–Hsp27 interactions, suggesting that competitive PPIs may be an important regulatory paradigm. Indeed, we find that each of the individual PPIs are relatively weak and that competition for shared sites seems to control both client binding and Hsp27 oligomerization. These findings highlight the importance of multiple, competitive PPIs in the function of Hsp27 and suggest that the β4-β8 groove acts as a tunable sensor for clients.

Hsp27 regulates epithelial mesenchymal transition, metastasis and proliferation in colorectal carcinoma.

The primary factor associated with poor survival rate in patients with colorectal carcinoma (CRC) is the presence of metastasis. The underlying molecular mechanisms of CRC metastasis are yet to be fully elucidated. The present study investigated the function of heat shock protein 27 (Hsp27) on the invasion and proliferation of CRC cells. The clinical significance of Hsp27 was evaluated using tissue microarray analysis (n=81). Invasion and metastasis assays were used to determine the function of Hsp27 in CRC metastasis in vitro and in vivo using RNA interference and the ectopic expression of Hsp27. The upregulation of Hsp27 has been frequently identified in CRC tissues. Patients with CRC and a high expression level of Hsp27 have a reduced overall survival rate. Silencing Hsp27 inhibited the growth and invasion of CRC cells in vitro and in vivo, whereas ectopic overexpression of Hsp27 promoted the proliferation and invasion of CRC cells in vitro. Furthermore, depletion of Hsp27 expression inhibited the epithelial-to-mesenchymal transition (EMT), whilst ectopic overexpression of Hsp27 induced EMT. The results of the present study indicated that Hsp27 serves an important function in the aggressiveness of CRC through inducing EMT. Hsp27 suppression may represent a potential therapeutic option for the suppression of CRC progression.

Induction of heat shock protein 27 by bicyclol attenuates d-galactosamine/lipopolysaccharide-induced liver injury

Heat shock proteins (Hsps) are critical for cell survival under adverse environmental conditions. Bicyclol is a novel hepatoprotectant that has been shown to protect against liver injury by inducing Hsps, including Hsp27 and Hsp70. Although the role of Hsp70 in protecting against acute hepatic failure has been clearly explored, the precise function of Hsp27 in this setting is poorly defined. This study was undertaken to evaluate the role of Hsp27 in bicyclol-mediated hepatoprotection. Both primary hepatocytes and bone marrow-derived macrophages were subjected to bicyclol treatment, followed by detection of Hsp27 expression. Adenoviruses containing the mouse Hsp27 coding sequence or shRNA interference sequence targeting Hsp27 were used to manipulate Hsp27 expression in the liver before the mice were treated with bicyclol and/or confronted with D-galactosamine/lipopolysaccharide (Galn/LPS)-induced acute liver damage. Only hepatocytes increased their Hsp27 expression after bicyclol treatment and the time course of bicyclol-induced Hsp27 expression in hepatocytes was in line with the in vivo results. Although high-dose bicyclol could protect against liver failure without Hsp27, the effect of bicyclol given at a low dose was dependent on Hsp27 induction. Adenovirus-mediated transduction of Hsp27 protected against acute liver damage and partially replicated the protective effect afforded by bicyclol. These results demonstrated that bicyclol induced Hsp27 expression in hepatocytes, which was essential to bicyclol-mediated hepatoprotection. Overexpression of Hsp27 in hepatocytes could confer remarkable protection against acute liver damage.

Overexpression of Hsp27 ameliorates symptoms of Alzheimer's disease in APP/PS1 mice

Hsp27 belongs to the small heat shock protein family, which are ATP-independent chaperones. The most important function of Hsp27 is based on its ability to bind non-native proteins and inhibit the aggregation of incorrectly folded proteins maintaining them in a refolding-competent state. Additionally, it has anti-apoptotic and antioxidant activities. To study the effect of Hsp27 on memory and synaptic functions, amyloid-β (Aβ) accumulation, and neurodegeneration, we generated transgenic mice overexpressing human Hsp27 protein and crossed with APPswe/PS1dE9 mouse strain, a mouse model of Alzheimer's disease (AD). Using different behavioral tests, we found that spatial learning was impaired in AD model mice and was rescued by Hsp27 overexpression. Electrophysiological recordings have revealed that excitability of neurons was significantly increased, and long-term potentiation (LTP) was impaired in AD model mice, whereas they were normalized in Hsp27 overexpressing AD model mice. Using anti-amyloid antibody, we counted significantly less amyloid plaques in the brain of APPswe/PS1dE9/Hsp27 animals compared to AD model mice. These results suggest that overexpression of Hsp27 protein might ameliorate certain symptoms of AD.

Abstract 281: Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer.

Abstract Introduction: Prostate cancer (PCa) is the most common cancer and the second leading cause cancer death in North American men. An important determinant for metastasis is epithelial to mesenchymal transition (EMT) which is known to be correlated to castrate resistant prostate cancer (CRCP). Previous report demonstrated the overexpression of Hsp27 and its phosphorylated form in poor diagnosis prostate cancer. In addition, both epidermal growth factor (EGF) and its receptor (EGFR) are up–regulated in PCa during progression to CRPC. However, the role of EGF and EGFR in PCa progression remains unclear. Our objective is to establish that Hsp27 is a central “node” for several signaling pathways activated by EGF and known to modulate EMT. Methods: To assess the role of Hsp27 in EMT, gain and loss of Hsp27 function has been conducted in LNCaP cells with or without EGF stimulation. EMT markers expression has been evaluated at the protein (WB) and mRNA level (qRT–PCR). Furthermore, invasion, migration assays in addition with MMP–9 activity have been done to evaluate the migratory capacity of those cells. Also, to better understand the molecular mechanism involved in EMT induction, transactivation and Chip assays have been performed. The in vitro results have been confirmed with an in vivo study using an Hsp27 antisense oligonucleotide (OGX-427) in a murine model. Results: We found that Heat shock protein 27 (Hsp27) is up–regulated with CRPC progression and drives EMT in PCa cells by decreasing epithelial cell markers, up–regulating mesenchymal markers and enhancing cell migration and MMP–9 activity. Hsp27 knockdown induces sequestration of beta–catenin in the cytoplasm by increasing its binding to GSK–3beta and abrogating EGF induced beta–catenin nuclear translocation. Additionally, by decreasing the nuclear translocation of beta-catenin, Hsp27 knockdown decreases the binding of beta–catenin to slug promoter thereby lessening its transcriptional activity. As Slug is known to be a critical key in EMT induction, we confirmed the role of EGF and Hsp27 in EMT induction and our capacity to abrogate the EGF-induced EMT effect by Hsp27 knockdown. Finally, targeting Hsp27 using antisense oligonucleotide OGX-427 in vivo, significantly reduced tumor cell metastasis in a PCa murine model. Conclusion: This study shows that EGF through Hsp27 induces modifications in gene expression and morphology toward a mesenchymal phenotype. The mechanism involving Hsp27, β-catenin, nuclear β-catenin/TCF and EGF signaling cascades described in the present study contribute to the comprehension of mechanisms driving metastasis in aggressive castration-resistant prostate cancer. This study reveals that Hsp27 is a crucial effector of EGF–dependent and independent EMT and suggests that targeting Hsp27 holds promises for a new strategy in metastatic PCa. Citation Format: Thomas D. Cordonnier, Jennifer L. Bishop, Masaki Shiota, Ario Takeuchi, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 281. doi:10.1158/1538-7445.AM2013-281

High Levels of Anti-Heat Shock Protein 27 Antibody in Pemphigus Vulgaris

High Levels of Anti-Heat Shock Protein 27 Antibody in Pemphigus Vulgaris

Downregulation of Hsp27 (HSPB1) in MCF-7 human breast cancer cells induces upregulation of PTEN

Hsp27 (HSPB1) is usually overexpressed in breast cancers affecting the disease outcome and the sensitivity of tumors to chemotherapy and radiotherapy. Hsp27 interacts with other proteins such as β-catenin, histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that is deleted in many human tumors. The PI3K/Akt signaling pathway is negatively regulated by PTEN. Hsp27 is described as a key component of the Akt signaling cascade: Akt, BAD, Forkhead transcription factors, Hsp27, mitogen-activated protein kinase kinase-3 and -6. Here, we have examined whether the downregulation of Hsp27 by siHsp27 affects the PTEN levels in the MCF-7 human breast cancer cell line. PTEN was detected with two different antibodies using western blots and immunocytochemistry. p-Akt was also evaluated by western blot. In addition, Hsp27 and PTEN were immunoprecipitated to know whether these proteins interact. Intracellular colocalization studies were carried out by confocal microscopy. A significant reduction in the Hsp27 levels was noted in the siHsp27 transfected cells. These Hsp27 downregulated cells showed a significant increased expression of PTEN. The MW 76 and 55kDa PTEN forms were upregulated as revealed by two different antibodies. The phosphatase activity of PTEN seems to be active because p-Akt levels were reduced. Hsp27 immunoprecipitation was bringing PTEN and vice versa, these two proteins seem to interact at cytoplasmic level by FRET. Downregulation of Hsp27 stabilized PTEN protein levels. Chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) levels were not significantly influenced by Hsp27 downregulation. In conclusion, we report a novel function of Hsp27 modulating the PTEN levels in human breast cancer cells suggesting an interaction between these two molecules.

Decreased heat shock protein 27 expression and altered autophagy in human cells harboring A8344G mitochondrial DNA mutation

Mitochondrial DNA (mtDNA) mutations are responsible for human neuromuscular diseases caused by mitochondrial dysfunction. Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, A8344G mutation in mtDNA, has been associated with severe defects in protein synthesis. This defect impairs assembly of complexes in electron transport chain and results in decreased respiratory function of mitochondria. In this study, we showed a significant decrease of the heat shock protein 27 (Hsp27) in lymphoblastoid cells derived from a MERRF patient and in cybrid cells harboring MERRF A8344G mutation. However, normal cytoplasmic distributions of Hsp27 and normal heat shock responses were observed in both wild type and mutant cybrids. Furthermore, overexpression of wild type Hsp27 in mutant MERRF cybrids significantly decreased cell death under staurosporine (STS) treatment, suggesting a protective function of Hsp27 in cells harboring the A8344G mutation of mtDNA. Meanwhile, reverse transcriptase PCR showed no difference in the mRNA level between normal and mutant cybrids, indicating that alterations may occur at the protein level. Evidenced by the decreased levels of Hsp27 upon treatment with proteasome inhibitor, starvation and rapamycin and the accumulation of Hsp27 upon lysosomal inhibitor treatment; Hsp27 may be degraded by the autophagic pathway. In addition, the increased formation of LC3-II and autophagosomes was found in MERRF cybrids under the basal condition, indicating a constitutively-activated autophagic pathway. It may explain, at least partially, the faster turnover of Hsp27 in MERRF cybrids. This study provides information for us to understand that Hsp27 is degraded through the autophagic pathway and that Hsp27 may have a protective role in MERRF cells. Regulating Hsp27 and the autophagic pathway might help develop therapeutic solutions for treatment of MERRF syndrome in the future.

Altered Cross-Linking of HSP27 by Zerumbone as a Novel Strategy for Overcoming HSP27-Mediated Radioresistance

HSP27 or HSP25 negatively regulates apoptosis pathways after radiation or chemotherapeutic agents. Abrogation of HSP27 function may be a candidate target for overcoming radio- and chemoresistance. Zerumbone (ZER), a cytotoxic component isolated from Zingiber zerumbet smith. Clonogenic survival assay and flow cytometry after Annexin V staining were performed to determine in vitro sensitization effects of ZER with ionizing radiation. A nude mouse xenografting system was also applied to detect in vivo radiosensitizing effects of ZER. ZER produced cross-linking of HSP27, which was dependent on inhibition of the monomeric form of HSP27. ZER was directly inserted between the disulfide bond in the HSP27 dimer and modified normal HSP27 dimerization. Pretreatment with ZER before radiation inhibited the binding affinity between HSP27 and apoptotic molecules, such as cytochrome c and PKCδ, and induced sensitization in vitro and in an in vivo xenografted nude mouse system. Structural analogs lacking only the carbonyl group in ZER, such as α-humulene (HUM) and 8-hydroxy-humulen (8-OH-HUM), did not affect normal cross-linking of HSP27 and did not induce radiosensitization. We suggest that altered cross-linking of HSP27 by ZER is a good strategy for abolishing HSP27-mediated resistance.

Abstract 3392: Heat shock protein 27 increases metastasis of human head and neck squamous cancer cells in vitro

Abstract The small heat shock protein 27 (Hsp27) is a molecular chaperone that is involved in a variety of cellular functions in cancer cells. The purpose of this research is to study the function of Hsp27 in metastasis of head and neck squamous cancer cells. The expression of Hsp27 in primary and metastatic head and neck cancer cells was determined using real-time PCR and western blotting. The siRNA knockdown of Hsp27 was performed in metastatic head and neck cancer cells using in vitro transfection. The proliferation of the primary and metastatic head and neck cancer cells were evaluated using MTS proliferation assay. The metastatic potential was determined using both migration assay and invasion assay in metastatic head and neck cancer cells after siRNA silencing of Hsp27. MTS proliferation showed that both primary (UM-SCC-22A) and metastatic (UM-SCC-22B) head and neck cancer cells have similar proliferation rate from 24-48 hrs. However, the metastatic head and neck cancer cells (UM-SCC-22B) showed more than 2-fold migration ability and more than 3-fold invasion ability than primary head and neck cancer cells (UM-SCC-22A). Real-time PCR demonstrated that Hsp27 mRNA is 22-fold higher in metastatic UM-SCC-22B than primary UM-SCC-22A. Similarly, Western blotting exhibited that primary UM-SCC-22A did not show any detectable levels of Hsp27, while metastatic UM-SCC-22B showed significant high levels (> 40-fold) of Hsp27. siRNA of Hsp27 knocked down expression of Hsp27 by more than 5-fold in mRNA level and 23-fold in protein levels in metastatic head and neck cancer cells UM-SCC-22B. Furthermore, siRNA knockdown of Hsp27 decreased metastasis of UM-SCC-22B by more than 50% in cell invasion assay. These data suggest that Hsp27 may regulate metastatic potential of head and neck squamous cancer cells. Silencing Hsp27 may decrease metastatic potential of head and neck squamous cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3392.

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

The small heat shock protein Hsp27 is a molecular chaperone and an anti-apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti-apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27(C137A)) and comparing it to wild-type protein (Hsp27(WT)). Both proteins were multi-subunit oligomers, but subunits of Hsp27(WT) were disulfide-linked unlike those of Hsp27(C137A), which were monomeric. Hsp27(C137A) was indistinguishable from Hsp27(WT) with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S-thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27(WT). The anti-apoptotic function of Hsp27(C137A) and Hsp27(WT) was studied by overexpressing them in CHO cells. No difference in the caspase-3 or -9 activity was observed in staurosporine-treated cells. The rate of apoptosis between Hsp27(C137A) and Hsp27(WT) overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild-type protein in preventing caspase-3 and caspase-9 activation and apoptosis induced by 1 mM H(2)O(2) in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti-apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti-apoptotic function against oxidative stress.

Activation of Gene Transcription by Heat Shock Protein 27 May Contribute to Its Neuronal Protection

Heat shock proteins are up-regulated as a physiological response to stressful stimuli and generally function as molecular chaperones for improperly folded protein substrates. The small heat shock protein HSP27 (or HSPB1) has multiple cytoplasmic roles. HSP27 also can translocate to the nucleus in response to stress, but the functional significance of this nuclear distribution has not been elucidated. We have previously implicated HSP27 as a genetic modifier of spinocerebellar ataxia 17 (SCA17), a neurological disease caused by a polyglutamine expansion in the TATA-binding protein (TBP). Altered expression of HSP27 is also found in cell models of other polyglutamine diseases, including Huntington disease as well as SCA3 and SCA7. Here, we show that Hsp27, unlike Hsp70, is not detected in mutant TBP aggregates in primary cerebellar granule neurons from transgenic SCA17 mice. Although HSP27 overexpression does not reduce the aggregation of cotransfected mutant TBP containing 105 glutamines, it potentiates activated transcription from both TATA-containing and TATA-lacking promoters. Neither HSP40 nor HSP70 elicits the same transcriptional effect. Moreover, HSP27 interacts with the transcription factor SP1, and coexpression of SP1 and nuclear localization signal-tagged HSP27 synergistically activates reporter constructs for the SP1-responsive neurotrophic receptor genes Ngfr(p75) and TRKA. Overexpression of nuclear localization signal-tagged HSP27 also rescues mutant TBP-mediated down-regulation of TrkA in a PC12 cell model of SCA17. These results indicate that nuclear HSP27 can modulate SP1-dependent transcriptional activity to promote neuronal protection.

Hsp27 associates with the titin filament system in heat-shocked zebrafish cardiomyocytes

Injury to muscle tissue plays a central role in various cardiovascular pathologies. Overexpression of the small heat shock protein Hsp27 protects muscle cells against thermal, oxidative and ischemic stress. However, underlying mechanisms of this protection have not been resolved. A distinctive feature of muscle cells is the stress-induced association of Hsp27 with the sarcomere. The association of Hsp27 with the cytoskeleton, in both muscle and non-muscle cells, is thought to represent interaction with Z-line components or filamentous actin. Here, we examined the association of Hsp27 with myofibrils in adult zebrafish myocardium subjected to hyperthermia and mechanical stretching. Consistent with previously published results, Hsp27 in resting length myofibrils localized to narrowly defined regions, or bands, which colocalized with Z-line markers. However, analysis of stretched myofibrils revealed that the association of Hsp27 with myofibrils was independent of desmin, alpha-actinin, myosin, and filamentous actin. Instead, Hsp27 maintained a consistent relationship with a marker for the titin A/I border over various sarcomeric lengths. Finally, extraction of actin filaments revealed that Hsp27 binds to a component of the remaining sarcomere. Together, these novel data support a mechanism of Hsp27 function where interactions with the titin filament system protect myofibrils from stress-induced degradation.