Molecular chaperones play important roles in all cellular organisms by maintaining the proteome in an optimally folded state. They appear to be at a premium in cancer cells whose evolution along the malignant pathways requires the fostering of cohorts of mutant proteins that are employed to overcome tumor suppressive regulation. To function at significant rates in cells, HSPs interact with cochaperones, proteins that assist in catalyzing individual steps in molecular chaperoning as well as in posttranslational modification and intracellular localization. We review current knowledge regarding the roles of chaperones such as heat shock protein 90 (Hsp90) and Hsp70 and their cochaperones in cancer. Cochaperones are potential targets for cancer therapy in themselves and can be used to assess the likely prognosis of individual malignancies. Hsp70 cochaperones Bag1, Bag3, and Hop play significant roles in the etiology of some cancers as do Hsp90 cochaperones Aha1, p23, Cdc37, and FKBP1. Others such as the J domain protein family, HspBP1, TTC4, and FKBPL appear to be associated with more benign tumor phenotypes. The key importance of cochaperones for many pathways of protein folding in cancer suggests high promise for the future development of novel pharmaceutical agents.
Antigen cross presentation is an important mechanism for CD8+ T cell activation by antigen presenting cells (APC). We have investigated mechanisms involved in Hsp90 chaperone mediated cross presentation of ovalbumin (Ova) derived antigens. Hsp90-Ova peptide complexes bound to scavenger receptor expressed by endothelial cells (SREC-I) on the surface of APC. SREC-I then mediated internalization of Hsp90-Ova polypeptide complexes through a Cdc42 regulated, dynamin independent endocytic pathway known as the GPI-AP enriched early endosomal compartment (GEEC) to recycling endosomes. Peptides that did not require processing could then be loaded directly onto MHC-I in endosomes, whereas longer peptides underwent endosomal and cytosomal processing by aminopeptidases and proteases. Cross presentation of Hsp90 chaperoned peptides through this pathway to CD8+ T-cells was highly efficient compared with processing of free polypeptides. In addition, Hsp90 also activated c-src kinase associated with SREC-I, an activity that we determined to be required for effective cross presentation. Extracellular Hsp90 can thus convey antigenic peptides through an efficient endocytosis pathway in APC and facilitate cross presentation in a highly regulated manner.
antigen cross presentation; dendritic cell; Hsp90; SREC-I
Heat shock proteins (HSPs) have been linked to the therapy of both cancer and inflammatory diseases, approaches that utilize contrasting immune properties of these proteins. It would appear that HSP family members Hsp60 and Hsp70, whether from external sources or induced locally during inflammation, can be processed by antigen-presenting cells and that HSP-derived epitopes then activate regulatory T cells and suppress inflammatory diseases. These effects also extend to the HSP-rich environments of cancer cells where elevated HSP concentrations may participate in the immunosuppressive tumor milieu. However, HSPs can also be important mediators of tumor immunity. Due to their molecular chaperone properties, some HSPs can bind tumor-specific peptides and deliver them deep into the antigen-processing pathways of antigen-presenting cells (APCs). In this context, HSP-based vaccines can activate tumor-specific immunity, trigger the proliferation and CTL capabilities of cancer-specific CD8+ T cells, and inhibit tumor growth. Further advances in HSP-based anticancer immunotherapy appear to involve improving the properties of the molecular chaperone vaccines by enhancing their antigen-binding properties and combating the immunosuppressive tumor milieu to permit programming of active CTL capable of penetrating the tumor milieu and specifically targeting tumor cells.
The molecular chaperone heat-shock protein 70 (Hsp70) possesses immune stimulatory properties that have been employed in the preparation of anticancer vaccines. Hsp70 binds antigenic peptides in the cytoplasm of cancer cells. Hsp70 thus serves as a convenient, non-discriminating transporter of antigens and can protect the peptides during internalization by APC and cross presentation to T lymphocytes. We describe a method for purifying Hsp70–peptide complexes that can be used to prepare molecular chaperone-based vaccines, involving sequential gel fi ltration, ion exchange, and af fi nity chromatography
Affinity chromatography; Chaperone; Heat-shock protein; HSP70; Lipopolysaccharide; Vaccine
Molecular chaperone-based vaccines offer a number of advantages for cancer treatment. We have discussed the deployment of a vaccine prepared by gentle isolation of Hsp70 from tumor dendritic cell fusions (Hsp70 fusion vaccine). The vaccine was highly effective in triggering specific T cell immunity and in the treatment of tumor bearing mice and the preparation was shown to retain an increased amount of tumor antigens compared to other chaperone-based isolates. This approach has the further advantage that tumor sub-populations could be used to prepare the Hsp70 fusion vaccine. Cellular fusion vaccines were made to specifically target drug resistant cancer cells and tumor cell populations enriched in ovarian cancer stem cells (CSC). Such vaccines showed enhanced capacity to trigger T cell immunity to these resistant ovarian carcinoma populations. We have discussed the potential of using the cellular and Hsp70 fusion vaccine approaches in therapy of treatment resistant cancer cells and its deployment in combination with ionizing radiation or hyperthermia to enhance the effectiveness of both forms of therapy.
We aim to explore the role of macroautophagy in cellular responses to hyperthermia. Protein damage incurred during hyperthermia can either lead to cell death or may be repaired by polypeptide quality control pathways including: (1) the deterrence of protein unfolding by molecular chaperones and (2) proteolysis of the denatured proteins within the proteasome. A third pathway of protein quality control is triggered by formation of protein aggregates in the heat shocked cell. This is the macroautophagy pathway in which protein aggregates are transported to specialized organelles called autolysosomes capable of degrading the aggregates. The consequences for cell viability of triggering this pathway are complex and may involve cell death, although under many circumstances, including exposure of cells to hyperthermia, autophagy leads to enhanced cell survival. We have discussed mechanisms by which cells detect protein aggregates and recruit them into the macroautophagy pathway as well as the potential role of inhibiting this process in hyperthermia.
Directed macroautophagy, with its key role in protein quality control, seems an attractive target for a therapy such as hyperthermia that functions principally through denaturing the proteome. However, much work is needed to decode the mechanisms of thermal stress-mediated macroautophagy and their role in survival / death of cancer cells before recommendations can be made on targeting this pathway in combination with hyperthermia.
Hyperthermia; protein aggregate; autophagy
HSF1 is an essential factor in the acute response to proteotoxic stress, in which it causes rapid transcription of heat shock protein (HSP) genes in order to permit survival of cells and restoration of global protein quality. In addition to this property however, HSF1 is chronically activated or overexpressed in a wide range of cancers and is essential for multiple pathways of malignant transformation. Studies in recent years indicate a remarkable pleiotropy in the properties of HSF1 in cancer. HSF1 functions as a transcription factor for HSP genes, reminiscent of its role in the stress response, and the resultant elevation in HSP levels leads to a reduction in programmed cell death and senescence and permits overexpression of mutated oncogenic protein clients required to fuel tumor growth. In addition HSF1 plays a role as a signal modulator, stimulating kinase activity, regulating energy metabolism and permitting the development of polyploidy in cancer cells. HSF1 can also function as an inhibitor of transcription and in cooperation with NuRD family factors can repress genes that oppose metastasis. Inhibitors of HSF1 are undergoing selection and future studies may see the testing of HSF1 as a target in cancer therapy.
Heat shock factor 1 (HSF1) is the transcriptional activator of heat shock protein (HSP) genes in both cell stress and cancer. The studies of Santagata et al clearly establish that HSF1 levels are increased in the nuclei of mammary cancer cells both at the in situ and invasive stages, and that these levels are closely correlated with increased mortality. HSF1 levels were elevated in ER-positive cells as well as HER2 expressing and triple negative breast cancer cells and higher levels of nuclear HSF1 were associated with a poor prognosis. These studies establish a clear role for HSF1 in human mammary carcinoma and suggest the potential for targeting HSF1 in breast cancer treatment.
Heat shock factor 1; nuclear; human mammary carcinoma; patient survival; prognosis
Heat shock proteins (HSP) are a subset of the molecular chaperones, best known for their rapid and abundant induction by stress. HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1). During the progression of many types of cancer, this heat shock transcriptional regulon becomes co-opted by mechanisms that are currently unclear, although evidently triggered in the emerging tumor cell. Concerted activation of HSF1 and the accumulation of HSPs then participates in many of the traits that permit the malignant phenotype. Thus cancers of many histologies exhibit activated HSF1 and increased HSP levels that may help to deter tumor suppression and evade therapy in the clinic. We review here the extensive work that has been carried out and is still in progress aimed at: (1) understanding the oncogenic mechanisms by which HSP genes are switched on, (2) determining the roles of HSF1 / HSP in malignant transformation and, (3) discovering approaches to therapy based on disrupting the influence of the HSF1 controlled transcriptome in cancer.
Heat shock proteins; heat shock factor; cancer; carcinogenesis; drug resistance; apoptosis; metastasis; prognosis
Extracellular heat shock proteins (HSP) play important roles in cell signaling and immunity. Many of these effects are mediated by cell surface receptors expressed on a wide range of cell types. We have investigated the nature of such proteins by cloning candidate receptors into cells (CHO-K1) with the rare property of being null for HSP binding. Using this approach we have discovered that Hsp70 binds to a least two classes of receptor: c-type lectin receptors (CLR) and scavenger receptors (SR). However the nature of the receptor-ligand interactions is not yet clear. Hsp70 can bind to LOX-1 (a member of both the CLR and SR), with the c-type lectin binding domain (CTLD) as well as the SR family members SREC-I and FEEL-1/CLEVER-1/STABILIN-1, which by contrast have arrays of EGF-like repeats in their extracellular domains. In this chapter we discuss (1) methods for determining HSP receptors, (2) approaches to study of individual receptors in cells that contain multiple such receptors and (3) methods for investigating HSP receptor function in vivo.
extracellular; heat; shock; protein; scavenger; receptor; immunity
Heat shock protein (HSP) levels are elevated in breast cancer and are molecular targets for novel therapies. HSPs were first observed as proteins induced in massive amounts in normal cells exposed to stresses that lead to protein denaturation. Their expanded expression in mammary carcinoma appears to be largely due to the proliferation of malfolded mutant proteins and overexpressed oncoproteins that trigger transcription of HSP genes. HSPs play major roles in malignant transformation and progression mediated through their intrinsic molecular chaperone properties. These permit the emergence of new malignant traits through the facilitated accumulation of altered oncoproteins. The elevation of HSP concentrations in mammary carcinoma is at least partially dependent on heat shock transcription factor 1 (HSF1), a protein that responds to unfolded proteins and leads to HSP transcription. HSF1 activation has additional downstream activities, crucial for emergence of the breast cancer phenotype and these include activated cell signaling, HSP-mediated ability to evade apoptosis and senescence and an HSF1-dependent bias in transcriptional activity towards a metastatic phenotype. The HSPs are currently being targeted in breast cancer therapy and effective drugs for Hsp90 have been synthesized and evaluated in clinical trial. Mammary carcinoma cells also contain abundant quantities of HSP-tumor antigen complexes and these complexes are being used to develop effective tumor vaccine approaches that provide personalized therapy for each individual’s cancer.
Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.
heat shock protein; vaccine; inflammation; antigen presentation
Heat shock proteins (HSPs) are molecular chaperones that bind tumor antigens and mediate their uptake into antigen presenting cells. HSP–antigen complexes are then directed toward either the MHC class I pathway through antigen cross presentation or the conventional class II pathway, leading to activation of T cell subsets. Uptake of HSP-chaperoned polypeptides can involve both receptor-mediated and receptor-independent routes, and mechanisms of antigen sorting between the Class I and II pathways after uptake are currently under investigation. The processes involved in internalization of HSP–antigen complexes differ somewhat from the mechanisms previously determined for (unchaperoned) particulate and free soluble antigens. A number of studies show that HSP-facilitated antigen cross presentation requires uptake of the complexes by scavenger receptors (SR) followed by processing in the proteasome, and loading onto MHC class I molecules. In this review we have examined the roles of HSPs and SR in antigen uptake, sorting, processing, cell signaling, and activation of innate and adaptive immunity.
heat shock proteins; antigen cross presentation; CTL response; scavenger receptor; antigen presenting cells; soluble vs. particulate antigen; anti-cancer vaccine; tumor immunity
Heat shock proteins (HSP) and heat shock factor 1 (HSF1), key factors in the heat shock response (HSR) have been implicated in the etiology of breast cancer. At least two members of the HSP family, Hsp27 and Hsp70 undergo significant increases in cellular concentration during the transformation of mammary cells. These changes result in HSP-mediated inhibition of tumor cell inactivation through blockade of the apoptosis and replicative senescence pathways. The increases in HSP thus mediate two of the common hallmarks of cancer and favor cell birth over cell death. In addition, Hsp90 plays a role in facilitating transformation by stabilizing the mutated and overexpressed oncoproteins found in breast tumors, and permitting the activation of growth stimulatory and transforming pathways in the absence of growth factors. HSF1 appears to play a similar role as a facilitator of transformation in mammary carcinoma. Induction of some facets of the HSR in breast cancer cells therefore leads to growth stimulation and inhibits cell death. Pharmacological targeting of HSP and HSF1 is therefore indicated and in the case of Hsp90, inhibitory drugs are undergoing clinical trial for treatment of breast carcinoma and other cancers.
heat; shock; protein; HSF1; breast; cancer
In the present study, exposure of mammary tumor cells derived from mice transgenic for the polyomavirus middle T (PyMT) oncogene to ionizing radiation resulted in the generation of a tumor cell population that preferentially expressed cancer stem cell markers. In addition, these cells were more resistant to further radiation treatments and appeared to acquire enhanced capacity for dissemination to the lungs of mice. We therefore tested an immunotherapy approach to treatment of local and disseminated mammary tumor cells in a murine model, employing a recently developed molecular chaperone-based vaccine that specifically targets the radioresistant subpopulation of tumor cells. Heat shock protein 70-peptide complexes (Hsp70.PC-F) were extracted from fusions of dendritic cells (DC) and radiation enriched tumor cells and the resulting chaperone vaccines used to treat mice with pre-existing lung metastases. Immunization of mice with the Hsp70.PC-F vaccine resulted in a T-cell-mediated immune response including a significant increase in CD4 and CD8 T cell proliferations and the induction of effector T cells capable of targeting radioresistant tumor cells. Importantly, the growth of primary tumors was inhibited and the number of tumor cells metastasizing to lung significantly reduced by combining chaperone vaccine with radiotherapy. These results indicate that Hsp70.PC-F vaccine can induce specific immunity to radioresistant populations of mammary tumor cells and can thus compliment radiotherapy, leading to synergistic killing.
Radioresistance; Cancer stem cells; Immunotherapy
We have developed an enhanced molecular chaperone-based vaccine through rapid isolation of Hsp70 peptide complexes after the fusion of tumor and dendritic cells (Hsp70.PC-F). In this approach, the tumor antigens are introduced into the antigen processing machinery of dendritic cells through the cell fusion process and thus we can obtain antigenic tumor peptides or their intermediates that have been processed by dendritic cells. Our results show that Hsp70.PC-F has increased immunogenicity compared to preparations from tumor cells alone and therefore constitutes an improved formulation of chaperone protein-based tumor vaccine.
Heat shock proteins70 (Hsp70); Dendritic cells (DC); Cell fusion; Extraction of Hsp70 peptide complexes (Hsp70.PC); Tumor vaccine
Heterogeneity within the cell population is a feature of many tumors. This lack of cellular homogeneity may originate from a number of sources, including differential nutrient status due to the de novo microcirculations of tumors, to infiltration of normal cells into the tumor, and to the hierarchical natures of the cell populations from which cancers arise. Tumors are thought to arise from one or more tumor initiating cells (TIC) within the population and to found hierarchies of progenitors and more differentiated cancer cells. TIC are often derived from tissue stem cells and these cancer stem cells are characterized by resistance to most cytotoxic treatments and by a high metastatic rate. Many of the properties of tumor populations, including the ability to express mutated oncogenes and to evolve new features such as treatment resistance and invasive and metastatic potential appear to depend on the molecular chaperone Hsp90. We discuss the potential of targeting the heterogeneous cell population with Hsp90 inhibitory drugs and its potential ability to inactivate TIC and to block the evolution of new phenotypes in cancer.
Heat shock proteins (HSP) are molecular chaperones and have been implicated in longevity and aging in many species. Their major functions include, chaperoning misfolded or newly synthesized polypeptides, protecting cells from proteotoxic stress, and processing of immunogenic agents. These proteins are expressed constitutively and can be induced by stresses such as heat, oxidative stress and many more. The induction of HSP in aging could potentially maintain protein homeostasis and longevity by refolding the damaged proteins which accumulate during aging and are toxic to cells. HSP are shown to increase life span in model organisms such as C. elegans and decrease aging related proteotoxicity. Thus, decrease in HSP in aging is associated with disruption of cellular homeostasis which causes diseases such as cancer, cell senescence and neurodegeneration. HSP levels are decreased with aging in most organs including neurons. Aging also causes attenuation or alteration of many signaling pathways as well as the expression of transcription factors such as heat shock factor (HSF). The alteration in regulation and synthesis of Forkhead box O3a (FOXO3a) family of transcription factors as well as major antioxidant enzymes [manganese superoxide dismutase (MnSOD), catalase] are also seen in aging. Among many signaling mechanisms involved in altering longevity and aging, the insulin/IGF1 pathway and the Sir2 deacetylase are highly significant. This review inquires into the role of some of these pathways in longevity/aging along with HSP.
Aging can be thought of as the collision between destructive processes that act on cells and organs over the lifetime and the responses that promote homeostasis, vitality and longevity. However, the precise mechanisms that determine the rates of aging in organisms are not known.
Macromolecules such as proteins are continuously exposed to potential damaging agents that can cause loss of molecular function and depletion of cell populations over the lifetime of essential organs. One of the key homeostatic responses involved in maintaining longevity is the induction of heat shock proteins (HSPs), a conserved reaction to damaged intracellular proteins. We aim to discuss how the interplay between protein damage and its repair or removal from the cell may influence longevity and aging.
We have reviewed experiments carried out in mammalian and non-mammalian organisms on molecular chaperones and the transcription factor (heat shock factor 1, HSF1) responsible for their expression. We have discussed mechanisms through which these molecules are regulated in cells, respond to stimuli that enhance longevity and become impaired during aging.
The transcription factor HSF1 initiates the prolific induction of HSP when cells are exposed to protein damage. HSPs are molecular chaperones that protect the proteome by folding denatured polypeptides and promoting the degradation of severely damaged proteins. Activation of HSF1 is coupled functionally to fundamental pathways of longevity and orchestrates the evasion of aging through HSP induction and antagonism of protein aggregation. In addition to mediating protein quality control, some HSPs such as Hsp27 and Hsp70 directly protect cells against damage-induced entry into death pathways. However, the heat shock response declines in potency over the lifetime, and enfeeblement of the response contributes to aging by permitting the emergence of protein aggregation diseases, reduction in cellular vigor and decreased longevity.
Molecular chaperones play an important role in the deterrence of protein damage during aging and their expression is required for longevity. Chemical stimulation of HSP synthesis might therefore be a significant strategy in future design of antiaging pharmaceuticals.
Heat shock protein; Aging; Aggregation; Molecular chaperone; CHIP; Ubiquitin; Proteasome; Heat shock factor 1
HSP70 isolated from tumor-dendritic cell fusions (HSP70.PC-F) induces potent anti-tumor immunity and prevents growth of such tumors. In the present study, we have examined mechanisms underlying such anti-tumor activity of the HSP70.PC-F vaccine. The degree of anti-tumor immunity induced by HSP70.PC-F depended on intact TLR signaling in immunized animals, and mice in which the tlr2 and tlr4 genes were both inactivated did not respond to the vaccine. The reduced responses to HSP70.PC-F vaccine in such tlr knockout mice were restored by immunization of animals with HSP70.PC-F pulsed wild-type DC, indicating a key role for this cell type in HSP70.PC-F mediated immunity. Our studies also indicate a role for the HSP70 receptor SREC-1 in anti-tumor immunity induced by HSP70.PC-F. These two receptor types appeared functionally interdependent as indicated by the finding that tlr2 and tlr4 knockout decreases HSP70 binding in double knockout DC and reduces SREC-1 expression. In addition, TLR-dependent, tumor cell killing was suppressed by SREC-1 knockdown in DC, suggesting a significant role for this receptor in HSP70.PC-F-mediated tumor immunity.
Cdc37 is a molecular chaperone that physically stabilizes the catalytic domains found in protein kinases and is therefore a wide-spectrum regulator of protein phosphorylation. It is also an overexpressed oncogene that mediates carcinogenesis by stabilizing the compromised structures of mutant and/or overexpressed oncogenic kinases. Recent work shows that such dependency of malignant cells on elevated Cdc37 expression is a vulnerability that can be targeted in cancer by agents that deplete or inhibit Cdc37. Cdc37 is thus a candidate for broad-spectrum molecular cancer therapy.
Aging can be thought of as the collision between destructive processes that act on cells and organs over the lifetime and the responses that promote homeostasis, vitality and longevity. However the precise mechanism that determine the rates of aging in organisms are not known.
Macromolecules such as proteins are continuously exposed to potential damaging agents that can cause loss of molecular function and depletion of cell populations over the lifetime of essential organs. One of the key homeostatic responses involved in maintaining longevity is the induction of heat shock proteins (HSP), a conserved reaction to damaged intracellular proteins. We aim to discuss how the interplay between protein damage and its repair or removal from the cell may influence longevity and aging.
We have reviewed experiments carried out in mammalian and non-mammalian organisms on molecular chaperones and the transcription factor HSF1 responsible for their expression. We have discussed mechanisms through which these molecules are regulated in cells, respond to stimuli that enhance longevity and become impaired during aging.
The transcription factor HSF1 initiates the prolific induction of heat shock proteins when cells are exposed to protein damage. HSP are molecular chaperones that protect the proteome by folding denatured polypeptides and promoting the degradation of severely damaged proteins. Activation of HSF1 is coupled functionally to fundamental pathways of longevity and orchestrates the evasion of aging through HSP induction and antagonism of protein aggregation. In addition to mediating protein quality control, Hsp27 and Hsp70 directly protect cells against damage-induced entry into death pathways. However, the heat shock response declines in potency over the lifetime, and enfeeblement of the response contributes to aging by permitting the emergence of protein aggregation diseases, reduction in cellular vigor and decreased longevity.
Molecular chaperones play an important role in the deterrence of protein damage during aging and their expression is required for longevity. Chemical stimulation of HSP synthesis might therefore be a significant strategy in future design of anti-aging pharmaceuticals.
heat shock protein; aging; aggregation; molecular chaperone; CHIP; ubiquitin; proteasome; heat shock factor 1
Hsp70; heat shock; lysosome; ABC transporter