Highlights • Low dose irradiation induces the synthesis of heat shock proteins (HSPs). • Low dose irradiation increases the cell surface expression of HSPs on the membrane of tumor cells. • Low dose ...irradiation induces the release of HSPs from tumor cells. • Membrane-bound and extracellular HSPs can elicit T- and NK-cell mediated anti-tumor immune responses.
Immunization of mice with a 14-mer peptide TKDNNLLGRFELSG, termed "TKD," comprising amino acids 450–461 (aa 450–461 ) in the C terminus of inducible Hsp70, resulted in the generation of an IgG1 mouse ...mAb cmHsp70.1. The epitope recognized by cmHsp70.1 mAb, which has been confirmed to be located in the TKD sequence by SPOT analysis, is frequently detectable on the cell surface of human and mouse tumors, but not on isogenic cells and normal tissues, and membrane Hsp70 might thus serve as a tumor-specific target structure. As shown for human tumors, Hsp70 is associated with cholesterol-rich microdomains in the plasma membrane of mouse tumors. Herein, we show that the cmHsp70.1 mAb can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70⁺ mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2. Three consecutive injections of the cmHsp70.1 mAb into mice bearing CT26 tumors significantly inhibited tumor growth and enhanced the overall survival. These effects were associated with infiltrations of NK cells, macrophages, and granulocytes. The Hsp70 specificity of the ADCC response was confirmed by preventing the antitumor response in tumor-bearing mice by coinjecting the cognate TKD peptide with the cmHsp70.1 mAb, and by blocking the binding of cmHsp70.1 mAb to CT26 tumor cells using either TKD peptide or the C-terminal substrate-binding domain of Hsp70.
Summary Background Proliferation markers and profiles have been recommended for guiding the choice of systemic treatments for breast cancer. However, the best molecular marker or test to use has not ...yet been identified. We did this study to identify factors that drive proliferation and its associated features in breast cancer and assess their association with clinical outcomes and response to chemotherapy. Methods We applied an artificial neural network-based integrative data mining approach to data from three cohorts of patients with breast cancer (the Nottingham discovery cohort (n=171), Uppsala cohort (n=249), and Molecular Taxonomy of Breast Cancer International Consortium METABRIC cohort; n=1980). We then identified the genes with the most effect on other genes in the resulting interactome map. Sperm-associated antigen 5 ( SPAG5 ) featured prominently in our interactome map of proliferation and we chose to take it forward in our analysis on the basis of its fundamental role in the function and dynamic regulation of mitotic spindles, mitotic progression, and chromosome segregation fidelity. We investigated the clinicopathological relevance of SPAG5 gene copy number aberrations, mRNA transcript expression, and protein expression and analysed the associations of SPAG5 copy number aberrations, transcript expression, and protein expression with breast cancer-specific survival, disease-free survival, distant relapse-free survival, pathological complete response, and residual cancer burden in the Nottingham discovery cohort, Uppsala cohort, METABRIC cohort, a pooled untreated lymph node-negative cohort (n=684), a multicentre combined cohort (n=5439), the Nottingham historical early stage breast cancer cohort (Nottingham-HES; n=1650), Nottingham early stage oestrogen receptor-negative breast cancer adjuvant chemotherapy cohort (Nottingham-oestrogen receptor-negative-ACT; n=697), the Nottingham anthracycline neoadjuvant chemotherapy cohort (Nottingham-NeoACT; n=200), the MD Anderson taxane plus anthracycline-based neoadjuvant chemotherapy cohort (MD Anderson-NeoACT; n=508), and the multicentre phase 2 neoadjuvant clinical trial cohort (phase 2 NeoACT; NCT00455533 ; n=253). Findings In the METABRIC cohort, we detected SPAG5 gene gain or amplification at the Ch17q11.2 locus in 206 (10%) of 1980 patients overall, 46 (19%) of 237 patients with a PAM50-HER2 phenotype, and 87 (18%) of 488 patients with PAM50-LumB phenotype. Copy number aberration leading to SPAG5 gain or amplification and high SPAG5 transcript and SPAG5 protein concentrations were associated with shorter overall breast cancer-specific survival (METABRIC cohort copy number aberration: hazard ratio HR 1·50, 95% CI 1·18–1·92, p=0·00010; METABRIC cohort transcript: 1·68, 1·40–2·01, p<0·0001; and Nottingham-HES-breast cancer cohort protein: 1·68, 1·32–2·12, p<0·0001). In multivariable analysis, high SPAG5 transcript and SPAG5 protein expression were associated with reduced breast cancer-specific survival at 10 years compared with lower concentrations (Uppsala: HR 1·62, 95% CI 1·03–2·53, p=0·036; METABRIC: 1·27, 1·02–1·58, p=0·034; untreated lymph node-negative cohort: 2·34, 1·24–4·42, p=0·0090; and Nottingham-HES: 1·73, 1·23–2·46, p=0·0020). In patients with oestrogen receptor-negative breast cancer with high SPAG5 protein expression, anthracycline-based adjuvant chemotherapy increased breast cancer-specific survival overall compared with that for patients who did not receive chemotherapy (Nottingham-oestrogen receptor-negative-ACT cohort: HR 0·37, 95% CI 0·20–0·60, p=0·0010). Multivariable analysis showed high SPAG5 transcript concentrations to be independently associated with longer distant relapse-free survival after receiving taxane plus anthracycline neoadjuvant chemotherapy (MD Anderson-NeoACT: HR 0·68, 95% CI 0·48–0·97, p=0·031). In multivariable analysis, both high SPAG5 transcript and high SPAG5 protein concentrations were independent predictors for a higher proportion of patients achieving a pathological complete response after combination cytotoxic chemotherapy (MD Anderson-NeoACT: OR 1·71, 95% CI, 1·07–2·74, p=0·024; Nottingham-ACT: 8·75, 2·42–31·62, p=0·0010). Interpretation SPAG5 is a novel amplified gene on Ch17q11.2 in breast cancer. The transcript and protein products of SPAG5 are independent prognostic and predictive biomarkers that might have clinical utility as biomarkers for combination cytotoxic chemotherapy sensitivity, especially in oestrogen receptor-negative breast cancer. Funding Nottingham Hospitals Charity and the John and Lucille van Geest Foundation.
We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.
Optical imaging ...of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.
These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.
We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single ...cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro.
The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.
This study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70.
Hypoxia is well known to limit curability of tumors by ionizing radiation. Here, we show that hypoxia treatment of tumor cells causes coexpression of heat shock protein 70 (Hsp70) and ...phosphatidylserine (PS) on the cell surface. Colocalization of Hsp70 and PS, as determined by confocal microscopy, also occurs when exogenous FITC-labeled Hsp70 protein is added to normoxic and hypoxic tumor cells. Moreover, the interaction of Hsp70 with PS was demonstrated in artificial unilamellar phosphatidylcholine/ phosphatidylserine (PC/PS) liposomes at the physiological ratio of 8/2. Indeed, the Hsp70-liposome interaction gradually increased with elevating PS molar ratios (8/2greater-than-or-equal7/3<5/5<4/6<3/7<2/8). In contrast, only a weak Hsp70 interaction was detected in phosphatidylcholine/phosphatidylglycerol (PC/PG) liposomes, thus demonstrating that the interaction was not a charge-related effect. The interaction of Hsp70 with surface PS significantly reduces clonogenic cell survival in normoxic (EC₅₀ of Hsp70=85 μg/ml) and hypoxic (EC₅₀ of Hsp70=55 μg/ml) tumor cells. The radiation-induced tumor cell killing was significantly enhanced by the addition of Hsp70 protein (50 μg/ml). Since apoptosis was not significantly enhanced in normoxic and hypoxic tumor cells by the addition of Hsp70, we hypothesize that the Hsp70 protein-induced reduction in clonogenic cell survival might be through necrosis rather than apoptosis.--Schilling, D., Gehrmann, M., Steinem, C., De Maio, A., Pockley, A. G., Abend, M., Molls, M., Multhoff, G. Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells.
Although heat shock proteins (Hsp's) are present in the sera of healthy individuals and at elevated levels in subjects with early cardiovascular disease, their physiologic role in and value for ...predicting the development and/or progression of atherosclerosis have not been evaluated. Serum was obtained from 218 subjects with established hypertension (diastolic pressure >95 mm Hg) before their enrollment in the European Lacidipine Study on Atherosclerosis. Hsp60 and Hsp70, and anti-human Hsp60, anti-human Hsp70, and anti-mycobacterial Hsp65 antibody levels were measured by enzyme immunoassay. As an indicator of the presence/progression of atherosclerosis, the means of the maximum intima-media (I-M) thicknesses in the far walls of common carotid arteries and bifurcations (CBMmax) were determined by ultrasonography at the time of enrollment and 4 years afterward. Increases in I-M thicknesses at follow-up were less prevalent in subjects having high serum Hsp70 levels (75th percentile) at the time of enrollment (odds ratio, 0.42; 95% confidence interval CI, 0.22 to 0.8, P=0.008). Although a similar trend was observed for serum Hsp60 levels, this was not statistically significant (odds ratio, 0.6; 95% CI, 0.32 to 1.11, P=0.10). There was no relation between anti-Hsp antibody levels and changes in I-M thicknesses. The relation between Hsp70 levels and changes in I-M thickness was independent of age, atenolol or lacidipine treatment, smoking habits, and blood lipid levels. These findings indicate that circulating Hsp70 levels predict the development of atherosclerosis in subjects with established hypertension, and an intriguing possibility is that Hsp70 protects against or modifies the progression of atherosclerosis in this subject group.
Leptin, the satiety hormone, appears to act as a link between nutritional status and immune function. It has been shown to elicit a number of immunoregulatory effects, including the promotion of T ...cell proliferative responses, and the induction of proinflammatory cytokines. Leptin deficiency is associated with an increased susceptibility to infection. As polymorphonuclear neutrophils (PMN) play a major role in innate immunity and host defense against infection, this study evaluated the influence of leptin on PMN activation. The presence of leptin receptor in human PMN was determined both at mRNA and protein levels, and the effect of leptin on PMN activation, as assessed by CD11b expression, was evaluated using flow cytometry. In contrast to monocytes, which express both the short and long forms of the leptin receptor (Ob-Ra and Ob-Rb, respectively), PMN expressed only Ob-Ra. Leptin up-regulated the expression of CD11b, an early marker of PMN activation, on PMN in whole blood, yet it had no effect on purified PMN, even those treated by submaximal doses of TNF-alpha or PMA. The kinetics of leptin-induced activation in whole blood were consistent with an indirect effect mediated by monocytes, and 71% of the leptin-stimulatory effect on PMN was blocked by a TNF-alpha inhibitor. Leptin-mediated induction of CD11b expression was observed when purified PMN were coincubated with purified monocytes. In conclusion, although leptin activates PMN, it does so indirectly via TNF-alpha release from monocytes. These findings provide an additional link among the obesity-derived hormone leptin, innate immune function, and infectious disease.
The presence of circulating tumor cells (CTCs) in the peripheral blood is a pre-requisite for progression, invasion, and metastatic spread of cancer. Consequently, the enumeration and molecular ...characterization of CTCs from the peripheral blood of patients with solid tumors before, during and after treatment serves as a valuable tool for categorizing disease, evaluating prognosis and for predicting and monitoring therapeutic responsiveness. Many of the techniques for isolating CTCs are based on the expression of epithelial cell surface adhesion molecule (EpCAM, CD326) on tumor cells. However, the transition of adherent epithelial cells to migratory mesenchymal cells (epithelial-to-mesenchymal transition, EMT)-an essential element of the metastatic process-is frequently associated with a loss of expression of epithelial cell markers, including EpCAM. A highly relevant proportion of mesenchymal CTCs cannot therefore be isolated using techniques that are based on the "capture" of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)-cmHsp70.1-can be used for the isolation of viable CTCs from peripheral blood of tumor patients of different entities in a more quantitative manner. In contrast to EpCAM, the expression of mHsp70 remains stably upregulated on migratory, mesenchymal CTCs, metastases and cells that have been triggered to undergo EMT. Therefore, we propose that approaches for isolating CTCs based on the capture of cells that express mHsp70 using the cmHsp70.1 mAb are superior to those based on EpCAM expression.
The serine protease granzyme B (grB) has previously been shown to induce perforin-independent apoptosis in membrane Hsp70 positive tumor cells in a number of in vitro experimental systems. Ongoing ...studies that are investigating the in vivo relevance of these findings by assessing the therapeutic potential of grB in a human xenograft tumor mouse model required the development of an expression system for the production of high yields of enzymatic and biologically active human grB. In order to maintain potentially important posttranslational modifications that occur in mammalian cells, human embryonal kidney cells (HEK293) were stably transfected with human grB. The HEK293 host cells were protected from apoptotic cell death by fusing an inactivation site coupled to a (His)6 tag to the gene sequence of GrB. Inactive grB which was actively released from HEK293 cells by insertion of a Igκ leader sequence was purified on a nickel column utilizing the (His)6 tag. After enterokinase digestion and heparin affinity chromatography, high yields of enzymatic and biologically active human grB were obtained. The perforin-independent interaction of grB with membrane Hsp70 positive tumor cells appeared to be associated with mammalian glycosylation and mediated by the oligosaccharide moiety of neuraminic acid (NANA).
► Eukaryotic expression system for the production of inactive human granzyme B. ► Enterkinase digestion oft he inactivation site results in high yields of active human granzyme B. ► Mammalian glycosylation mediates uptake and biological activity of granzyme B.
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