Stress-inducible Molecular Chaperone Research Articles

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

Deciphering the mechanism and function of Hsp100 unfoldases from protein structure.

Hsp100 chaperones, also known as Clp proteins, constitute a family of ring-forming ATPases that differ in 3D structure and cellular function from other stress-inducible molecular chaperones. While the vast majority of ATP-dependent molecular chaperones promote the folding of either the nascent chain or a newly imported polypeptide to reach its native conformation, Hsp100 chaperones harness metabolic energy to perform the reverse and facilitate the unfolding of a misfolded polypeptide or protein aggregate. It is now known that inside cells and organelles, different Hsp100 members are involved in rescuing stress-damaged proteins from a previously aggregated state or in recycling polypeptides marked for degradation. Protein degradation is mediated by a barrel-shaped peptidase that physically associates with the Hsp100 hexamer to form a two-component system. Notable examples include the ClpA:ClpP (ClpAP) and ClpX:ClpP (ClpXP) proteases that resemble the ring-forming FtsH and Lon proteases, which unlike ClpAP and ClpXP, feature the ATP-binding and proteolytic domains in a single polypeptide chain. Recent advances in electron cryomicroscopy (cryoEM) together with single-molecule biophysical studies have now provided new mechanistic insight into the structure and function of this remarkable group of macromolecular machines.

Higher Intensity of Cell Surface Glucose-Regulated Protein 78 (csGRP78) Expression Is Seen in Patients with Early Progressive Disease/Mortality in a Cohort of Relapsed, Refractory Multiple Myeloma Patients Treated with Carfilzomib, Thalidomide and Dexamethasone

Background: GRP78, an endoplasmic reticulum stress-inducible molecular chaperone, is up-regulated at times of cellular stress to limit proteotoxicity and promote cell survival. Translocation of GRP78 to the cell surface (csGRP78) is emerging as a critical step providing tumour cells with a survival advantage. Here we quantified, monitored and correlated plasma cell (PC) csGRP78 expression in patients (pts) with relapsed/refractory multiple myeloma (RRMM) treated with carfilzomib, thalidomide and dexamethasone (KTd). Method: Patients enrolled in the single arm, multicentre, phase II Australasian Leukaemia & Lymphoma Group MM018/Asian Myeloma Network 002 study were treated with KTd as described previously (Quach et al. Blood 2018 132:1955). Formalin-fixed, paraffin-embedded BM trephine sections collected at baseline (n=29), after 6-months (mo) of KTd (n=19) and at time of disease progression (PD; n=5) were stained for CD138 and GRP78 by multiplex immunofluorescence histochemistry using the OpalTM workflow. Membrane expression of CD138 and GRP78 was extracted using inForm® software, compared across timepoints and correlated to disease characteristics and treatment outcomes. Descriptive statistics, paired/unpaired two-tailed t-test, Pearson's or Spearman's correlation were applied as appropriate. Results: Correlative BM biopsies were collected for 29 pts [male = 18, mean age = 65.0 years (range 41.9-83.2), 2 median prior lines of therapy (range 1-3)] at baseline, 21 pts after 6 months of KTd (7 had PD/died prior to cycle 6, 1 came off study due to grade 4 AE after 5 cycles) and 5 pts at time of PD. There was no difference in the number of fields, BM cellularity (%) or number of nucleated cells (NCs) assessed at baseline and 6mo (p=0.927, 0.331 and 0.491 respectively). PC burden (%; mean±SD) reduced significantly following 6mo KTd (28.3±28.1 vs. 2.12±2.37; p=0.0007). The number of plasma cells expressing csGRP78 (% of all NCs) was reduced following 6mo KTd treatment (27.04±26.83 vs. 2.05±2.32; p=0.0007) while the % of CD138-ve BM cells expressing csGRP78 (% of all NCs; mean±SD) increased (62.61±27.55 vs. 87.46±10.11; p=0.0005). Globally, there was a trend for reduced intensity of csGRP78 expression after 6mo KTd (H-score 70.79±62.16 vs. 53.53±51.45; p=0.2073). There was no correlation between baseline BM NC GRP78 H-score and baseline paraprotein level, involved/uninvolved serum free light chain ratio or depth of response to KTd. Pts with early (<6mo) disease progression/mortality (n=7) had a significantly higher baseline H-score (136±78.8 vs. 75.1±65.2; p=0.049). There was a separation of survival curves, but no significant difference regarding risk of early PD/mortality based a baseline H-score >75th percentile of the cohort (>152); p=0.1472 (Figure 1). Conclusion: Here we demonstrate that cell surface expression of GRP78 is prominent in both the plasma cells and cells of the tumour microenvironment in patients with RRMM and persists in the TME cells in patients on treatment. Early (<6mo) disease progression or mortality is associated with higher baseline intensity of global cell surface GRP78 expression. Additional studies are being performed to evaluate the promise of cell surface GRP78 expression on plasma cells as a potential biomarker of response to therapy. Disclosures Harrison: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: investigator on studies, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding. Quach:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees.

Cell surface glucose-regulated protein 78 (GRP78) is upregulated in plasma cells of patients with multiple myeloma compared to monoclonal gammopathy of uncertain significance

Multiple myeloma (MM) is characterised by paraprotein (PP) and/or serum free light chain (SFLC) production, a hypoxic tumour microenvironment (TME) and increased cellular stress. Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum stress-inducible molecular chaperone, is upregulated at times of cellular stress and acts to limit proteotoxicity and promote cell survival. Translocation of GRP78 to the cell surface (csGRP78) and interaction with cell signalling and survival pathways is emerging as a critical step providing tumour cells with a survival advantage.

The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli

Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify ∼1,500 soluble proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.

Molecular cloning, cellular expression and characterization of Arabian camel (Camelus dromedarius) endoplasmin

Endoplasmin, or GRP94, is an ER-located stress inducible molecular chaperone implicated in the folding and assembly of many proteins. The Arabian one-humped camel lives in an environment of thermal stress, nevertheless is able to encounter the risk of misfolded proteins. Here, the cDNA encoding camel GRP94 was isolated by rapid amplification of cDNA ends. The isolated cDNA contained an open reading frame of 2412 bp encoding a protein of 803 amino acids with predicted molecular mass of 92.5 kDa. Nucleotide and protein BLAST analysis of cGRP94 revealed strong conservation between camel and other domestic mammals. Overexpression of cGRP94 in COS-1 cells revealed multiple isoforms including one N-glycosylated species. Immunofluorescence colocalized cGRP94 with the ER resident protein calnexin. Interestingly, none of the cGRP94 isoforms expressed in CHO cells was N-glycosylated, presumably due to folding determinants that mask the N-glycosylation sites as proposed by in silico modelling. Surprisingly, isoforms of cGRP94 were detected in the culture media of transfected cells indicating that the protein, although an ER resident, also is trafficked and secreted into the exterior milieu. The overall striking structural homologies of GRP94s among mammalian reflect their pivotal role in the ER quality control and protein homeostasis.

Utility of 17-(allylamino)-17-demethoxygeldanamycin treatment for skeletal muscle injury.

Repeated eccentric contractions can injure skeletal muscle and result in functional deficits that take several weeks to fully recover. The 70-kDa heat shock protein (Hsp70) is a stress-inducible molecular chaperone that maintains protein quality and plays an integral role in the muscle's repair processes following injury. Here, we attempted to hasten this recovery by pharmacologically inducing Hsp70 expression in mouse skeletal muscle with 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) (40mg/kg) both prior to and throughout the first 7days after an injurious bout of 150 maximal eccentric contractions. Hsp70 content in the injured skeletal muscle was strongly induced following the eccentric contractions and remained elevated over the next 7days as the muscle underwent repair. Treatment with 17-AAG increased Hsp70 content ∼fivefold; however, this was significantly less than that induced by the injury. Moreover, 17-AAG treatment did not recover the decrements to in vivo isometric torque production following the bout of eccentric contractions. Together, these findings demonstrate that although Hsp70 content was induced in the uninjured skeletal muscle, treatment of 17-AAG (40mg/kg) was not a preventive measure to either reduce the severity of skeletal muscle damage or enhance functional recovery following a bout of maximal eccentric contractions.

Sulfatide-Hsp70 interaction promotes Hsp70 clustering and stabilizes binding to unfolded protein.

The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well as the biological relevance, underlying the glycolipid-Hsp70 interaction is unknown. Here we report that sulfatide promotes Hsp70 oligomerization through the N-terminal ATPase domain, which stabilizes the binding of Hsp70 to unfolded protein in vitro. We find that the Hsp70 oligomer has apparent molecular masses ranging from 440 kDa to greater than 669 kDa. The C-terminal peptide-binding domain is dispensable for the sulfatide-induced oligomer formation. The oligomer formation is impaired in the presence of ATP, while the Hsp70 oligomer, once formed, is unable to bind to ATP. These results suggest that sulfatide locks Hsp70 in a high-affinity state to unfolded proteins by clustering the peptide-binding domain and blocking the binding to ATP that induces the dissociation of Hsp70 from protein substrates.

Validation of the Hsp70-Bag3 protein-protein interaction as a potential therapeutic target in cancer.

Hsp70 is a stress-inducible molecular chaperone that is required for cancer development at several steps. Targeting the active site of Hsp70 has proven relatively challenging, driving interest in alternative approaches. Hsp70 collaborates with the Bcl2-associated athanogene 3 (Bag3) to promote cell survival through multiple pathways, including FoxM1. Therefore, inhibitors of the Hsp70-Bag3 protein-protein interaction (PPI) may provide a noncanonical way to target this chaperone. We report that JG-98, an allosteric inhibitor of this PPI, indeed has antiproliferative activity (EC50 values between 0.3 and 4 μmol/L) across cancer cell lines from multiple origins. JG-98 destabilized FoxM1 and relieved suppression of downstream effectors, including p21 and p27. On the basis of these findings, JG-98 was evaluated in mice for pharmacokinetics, tolerability, and activity in two xenograft models. The results suggested that the Hsp70-Bag3 interaction may be a promising, new target for anticancer therapy.

MALDI‐MSI and label‐free LC‐ESI‐MS/MS shotgun proteomics to investigate protein induction in a murine fibrosarcoma model following treatment with a vascular disrupting agent

Tumour vasculature is notoriously sinusoidal and leaky, and is hence susceptible to vascular disruption. Microtubule destabilising drugs such as the combretastatins form the largest group of tumour vascular disrupting agents and cause selective shutdown of tumour blood flow within minutes to hours, leading to secondary tumour cell death. Targeting the tumour vasculature is a proven anticancer strategy but early treatment response biomarkers are required for personalising treatment planning. Protein induction following treatment with combretastatin A4-phosphate was examined in a mouse fibrosarcoma model (fs188), where tumour cells express only the matrix-bound isoform of vascular endothelial growth factor A (VEGF188). These tumours are relatively resistant to vascular disruption by combretastatin A4-phosphate and hence a study of protein induction following treatment could yield insights into resistance mechanisms. The distribution of a number of proteins induced following treatment were visualised by MALDI-mass spectrometry imaging. Responses identified were validated by LC-ESI-MS/MS and immunohistochemical staining. Significant changes in proteins connected with necrosis, cell structure, cell survival and stress-induced molecular chaperones were identified. Protein-protein interactions were identified using STRING 9.0 proteomic network software. These relationship pathways provided an insight into the activity of the active tumour milieu and a means of linking the identified proteins to their functional partners.

AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12skeletal muscle cells

5'-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICAR-treated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPKα knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPKα knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICAR-induced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPKα or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr(389) was downregulated by AICAR, whereas this was attenuated in AMPKα, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells.

Tu1928 Tissue Expression of Heat Shock Protein 27, 70 and 90 Might Be Utilized As a Diagnostic and Prognostic Biomarker in Patients With Gastric Adenocarcinoma

Background and aim: Intestinal-type gastric carcinomas progress through chronic inflammation and atrophic gastritis. Heat shock protein 27(HSP27), a stress-induced molecular chaperone, inhibit reactive oxygen species (ROS) accumulation. In previous reports have been suggested that HSP27 plays an important role in inflammation and carcinogenesis. We investigated HSP27 expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis. Methods:We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and in gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues. Results: HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of those without gastric neoplasia(P= 0.004).In tumor necrosis factor -treated gastric cancer cell,HSP27 knockdown increased cell death and ROS accumulation that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiated of cancer cells is associated with an epithelial-mesenchymal transition signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells. Conclusion: HSP27 expression in gastric mucosae is negatively correlated with intraepithelial neoplasia, a probable precursor to gastric cancer and HSP27 expression in cancer is positively correlated with poor differentiation.

Tu1932 Endoscopic Features of Food Protein-Induced Proctocolitis in Neonates and Infants

Background and aim: Intestinal-type gastric carcinomas progress through chronic inflammation and atrophic gastritis. Heat shock protein 27(HSP27), a stress-induced molecular chaperone, inhibit reactive oxygen species (ROS) accumulation. In previous reports have been suggested that HSP27 plays an important role in inflammation and carcinogenesis. We investigated HSP27 expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis. Methods:We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and in gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues. Results: HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of those without gastric neoplasia(P= 0.004).In tumor necrosis factor -treated gastric cancer cell,HSP27 knockdown increased cell death and ROS accumulation that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiated of cancer cells is associated with an epithelial-mesenchymal transition signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells. Conclusion: HSP27 expression in gastric mucosae is negatively correlated with intraepithelial neoplasia, a probable precursor to gastric cancer and HSP27 expression in cancer is positively correlated with poor differentiation.

Tu1927 Heat Shock Protein 27 Expression Is Inversely Correlated With Intraepithelial Neoplasia and Positively Correlated With Poor Differentiation of Gastric Cancer

Background and aim: Intestinal-type gastric carcinomas progress through chronic inflammation and atrophic gastritis. Heat shock protein 27(HSP27), a stress-induced molecular chaperone, inhibit reactive oxygen species (ROS) accumulation. In previous reports have been suggested that HSP27 plays an important role in inflammation and carcinogenesis. We investigated HSP27 expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis. Methods:We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and in gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues. Results: HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of those without gastric neoplasia(P= 0.004).In tumor necrosis factor -treated gastric cancer cell,HSP27 knockdown increased cell death and ROS accumulation that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiated of cancer cells is associated with an epithelial-mesenchymal transition signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells. Conclusion: HSP27 expression in gastric mucosae is negatively correlated with intraepithelial neoplasia, a probable precursor to gastric cancer and HSP27 expression in cancer is positively correlated with poor differentiation.

Abstract IA28: Targeting stress-induced molecular chaperones CLU and Hsp27 to inhibit EMT and metastases

Abstract Stress-activated adaptive pathways help drive cancer cell survival, metastases, and acquired treatment resistance. Heat shock and other molecular chaperones play central roles in these stress responses, regulating protein homeostasis (proteostasis) as well as many signaling and transcriptional survival networks to facilitate cell survival and treatment resistance. Clusterin (CLU) and Hsp27 are stress-activated chaperones that treatment-induced and associated with metastases and treatment resistance in many cancers, functioning to protect cells from many varied therapeutic stressors that induce apoptosis. Both CLU and HSP27 levels increase after androgen deprivation and become highly expressed in metastatic CRPC. While studies have defined CLU and Hsp27 as key mediators of adaptive survival pathways and treatment resistance, they also regulate epithelial plasticity and epithelial-to-mesenchymal transition (EMT). Defining mechanistic linkages between treatment stress and effectors of EMT will yield new insights into metastasis and treatment strategies. EMT endows malignant cells with enhanced migratory and survival attributes that facilitate cancer metastases. EMT can be initiated by a wide variety of signals in the tumor environment that activate the transcription factors Twist and Snail, leading to repression of epithelial cell markers like E-cadherin and cytokeratins, and gain of mesenchymal markers like N-cadherin and vimentin. We recently defined roles for both CLU and Hsp27, downstream of TGF-β and IL-6, respectively, as key mediators of EMT and metastases. We found that factors such as TGF-β and IL-6 known to induce EMT, up-regulate molecular chaperones CLU and Hsp27. We then set out to define molecular mechanisms by which TGF-β and IL-6 regulate CLU and Hsp27 expression, respectively, and whether CLU and Hsp27 mediate TGF-β and IL-6 regulated EMT and metastasis. CLU is a mediator of TGF-β-induced EMT. Our data show that the transcription factor Twist1 mediates TGF-β-induced CLU expression, binding to E-boxes in the distal promoter region of CLU gene to regulate basal and TGF-β-induced CLU transcription. TGF-β induced stimulation of Twist1 as well as CLU transcript expression was potently suppressed by Twist1 silencing. CLU silencing reduced TGF-β induction of the mesenchymal markers N-cadherin and fibronectin, and inhibited migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, these findings identify CLU as a mediator of TGF-β-induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression. Hsp27 facilitates IL-6 mediated EMT via modulation of STAT3/Twist. In addition to a role for CLU in EMT, we found that Hsp27 overexpression drives, while its knockdown reverses, EMT, with congruent effects on cell migration, invasion and matrix metalloprotease activity. Mechanistically, silencing Hsp27 decreased IL-6 dependent STAT3 phosphorylation, nuclear translocation, and abrogated STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6 mediated EMT via modulation of STAT3/Twist signaling. Elevated expression of Hsp27 and Twist were also found in high grade PCa tumors. Overall, these data define a role of Hsp27 as a critical regulator of IL-6 dependent and independent EMT and highlights Hsp27 as an attractive anti-cancer target for metastatic CRPC. CLU and Hsp27 as Anti-Cancer Targets. Since Hsp27 and CLU are ATP-independent chaperones and not amenable to inhibition by small molecules, antisense strategies have been used to therapeutically target this chaperones, with evidence of anti-cancer activity in clinical trials. We developed OGX-011 as a CLU inhibitor and demonstrated pre-clinical activity and dose-dependent target suppression in Phase I studies. Moreover, we led a randomized Phase II trial that reported a 7-month-survival benefit when the CLU inhibitor, OGX-011, was combined with docetaxel in patients with CRPC; OGX-011 is now in phase III trials in metastatic CRPC and NSCLC. We also developed OGX-427 as an Hsp27 inhibitor and demonstrated pre-clinical activity and tolerability in Phase I studies. A randomized phase II study of OGX-427 plus Prednisone (P) vs. P alone enrolled 64 mCRPC patients. At 12 weeks, the proportion of patients progression free was 71% in OGX-427+P treated patients and 40% in patients on P. A ≥50% PSA decline occurred in 50% of patients on OGX-427+P and 20% patients treated with P. Measurable disease response occurred in 44% on OGX-427+P and 0 patients on P. CTC conversion from ≥5 to <5/7.5 ml occurred in 55% evaluable patients on OGX-427+P and 41% treated with P. These results confirm, for the first time, single agent activity for an Hsp27 inhibitor in cancer, and Phase II trials in metastatic bladder and CRPC are ongoing. Citation Format: Martin Gleave, Amina Zoubeidi, Kim Chi. Targeting stress-induced molecular chaperones CLU and Hsp27 to inhibit EMT and metastases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA28.

Pathology-Dependent Effects Linked to Small Heat Shock Proteins Expression: An Update

Small heat shock proteins (small Hsps) are stress-induced molecular chaperones that act as holdases towards polypeptides that have lost their folding in stress conditions or consequently of mutations in their coding sequence. A cellular protection against the deleterious effects mediated by damaged proteins is thus provided to cells. These chaperones are also highly expressed in response to protein conformational and inflammatory diseases and cancer pathologies. Through specific and reversible modifications in their phospho-oligomeric organization, small Hsps can chaperone appropriate client proteins in order to provide cells with resistance to different types of injuries or pathological conditions. By helping cells to better cope with their pathological status, their expression can be either beneficial, such as in diseases characterized by pathological cell degeneration, or deleterious when they are required for tumor cell survival. Moreover, small Hsps are actively released by cells and can act as immunogenic molecules that have dual effects depending on the pathology. The cellular consequences linked to their expression levels and relationships with other Hsps as well as therapeutic strategies are discussed in view of their dynamic structural organization required to interact with specific client polypeptides.

Hsp12.6 Expression Is Inducible by Host Immunity in Adult Worms of the Parasitic Nematode Nippostrongylus brasiliensis

Heat shock proteins (Hsp) are a family of stress-inducible molecular chaperones that play multiple roles in a wide variety of animals. However, the roles of Hsps in parasitic nematodes remain largely unknown. To elucidate the roles of Hsps in the survival and longevity of nematodes, particularly at the 2 most critical stages in their lifecycle, the infective-L3 stage and adult stage, which is subjected to host-derived immunological pressure, we examined the temporal gene transcription patterns of Hsp12.6, Hsp20, Hsp70, and Hsp90 throughout the developmental course of the nematode Nippostrongylus brasiliensis by reverse transcriptase real-time PCR. Nb-Hsp70 and Nb-Hsp90 expression were observed throughout the nematode's lifecycle, while the expression of Nb-Hsp20 was restricted to adults. Interestingly, Nb-Hsp12.6 showed a biphasic temporal expression pattern; i.e., it was expressed in infective-L3 larvae and in adults during worm expulsion from immunocompetent rats. However, the activation of Nb-Hsp12.6 in adult worms was aborted when they infected permissive athymic-rnu/rnu rats and was only marginal when they infected mast-cell-deficient Ws/Ws rats, which exhibited a low response of rat mast cell protease (RMCP) II and resistin-like molecule (Relm)- β expression compared to those observed in immunocompetent rats. Moreover, the activation of Nb-Hsp12.6 was reversed when adult worms were transplanted into the naive rat intestine. These features of Nb-Hsp12.6, the expression of which is not only stage-specific in infective-L3, but is also inducible by mucosal immunity in adults, have implications for the survival strategies of parasitic nematodes in deleterious environmental conditions both outside and inside the host.

NF-kB Complex Activation and Association of αB-Crystallin and Vimentin with Poor Prognosis in Primary Breast Cancer.

Abstract Background: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is a protein complex found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, free radicals, cytokines et.c. NF-kB plays a key role in regulating the immune response and incorrect regulation of NF-kB has been linked to the development of cancer, several inflammatory and autoimmune diseases. The role of NF-kB, its subunits and pathways in breast cancer and in the surrounding stromal tissues remains uncertain. αB-crystallin is a heat shock protein, which function as stress-induced molecular chaperones to inhibit the aggregation of denatured proteins. Previous studies have identified αB-crystallin as a marker of poor prognosis for breast cancer and have suggested that it is an excellent marker for tumours of basal origin. The aim of this study is to examine, for the first time, NF-kB and αB-crystallin, vimentin and HSPs in the same set of breast cancers and stroma and relate this to clinical parameters including outcome.Methods: Tissue Micro Arrays of 0.6mm cores of 246 breast cancers were stained with antibodies to αB-crystallin, vimentin, HSP27 (antibody ERD5) and HSP27 82P and 12 subsequent antibodies for NF-kB (IKKa, Ser276, Ser32/36, 180/181, P52, 1kkygmma, P65, RELB, P100/P52, IkBa, cREL, and P50) and scored using the Quick Score Method.Results: Positive associations have been found between αB-crystallin and IKBA (P=0.001 Fishers exact test (FET)), RELB (P<0.001 (FET)) and SER32/36 (P=0.03 (FET)) whereas Vimentin was positively associated IKBA (P=0.003 (FET)), IKKA (P=0.001 (FET)), P50 (P=0.002 (FET)), RELB (P<0.001 (FET)) and SER32/36 (P=0.01 (FET)). Positive associations have also been found between HSP27 and P50 (P=0.002 (FET)), SER32/36 (P<0.001 (FET)). Antibodies such as 180/181 (P=0.04 Kaplan Meier Log Rank (KM), cREL (P=0.03 (KM)), IKBA (P=0.03 (KM)) and IKKA (P=0.01 (KM)) was associated with good survival and P50 (P=0.002 (KM)) and P65 (P=0.01 (KM)) to poor survival. Expression of αB-crystallin was associated with vimentin (P<0.001 (FET)). Its expression was linked to a low expression of the estrogen receptor. Vimentin expression was associated with estrogen receptor (ER) negative cancers and poor survival (P <0.001 (FET)), P=0.002 (KM Log Rank) respectively). In contrast to αB-crystallin, low expression of HSP27 was associated with low ER and progesterone receptor (PGR).Conclusions: Associations were found between various antibodies within the NF-kB pathway as well as the heat shock proteins αB-crystallin, vimentin and HSP27 with varying implications for overall survival. We aim to clarify the mechanisms to prevent the aggregation of stress-accumulated misfolded proteins and apoptosis resulting better survival. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2143.

Targeting HSP70 for Cancer Therapy

In a recent issue of Molecular Cell, Leu et al. (2009) demonstrate that a chemical inhibitor of HSP70 exerts prominent tumor-selective cytotoxic effects, thereby lending further support to the notion that non-oncogene addiction constitutes a promising target for anticancer therapy.

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.