Targeting heat shock proteins in cancer

Heat shock proteins (HSPs) HSP27, HSP70 and HSP90 are powerful chaperones. Their expression is induced in response to a wide variety of physiological and environmental insults including anti-cancer chemotherapy, thus allowing the cell to survive to lethal conditions. Different functions of HSPs have been described to account for their cytoprotective function, including their role as molecular chaperones as they play a central role in the correct folding of misfolded proteins, but also their anti-apoptotic properties. HSPs are often overexpressed in cancer cells and this constitutive expression is necessary for cancer cells’ survival. HSPs may have oncogene-like functions and likewise mediate “non-oncogene addiction” of stressed tumor cells that must adapt to a hostile microenvironment, thereby becoming dependent for their survival on HSPs. HSP-targeting drugs have therefore emerged as potential anti-cancer agents. This review describes the different molecules and approaches being used or proposed in cancer therapy based on the in inhibition of HSP90, HSP70 and HSP27.     

Heat shock proteins in hepatocellular carcinoma: Molecular mechanism and therapeutic potential

Heat shock proteins (HSPs) are highly conserved proteins, which are expressed at low levels under normal conditions, but significantly induced in response to cellular stresses. As molecular chaperones, HSPs play crucial roles in protein homeostasis, apoptosis, invasion and cellular signaling transduction. The induction of HSPs is an important part of heat shock response, which could help cancer cells to adapt to stress conditions. Because of the constant stress condition in tumor microenvironment, HSPs overexpression is widely reported in many human cancers. In light of the significance of HSPs for cancer cells to survive and obtain invasive phenotype under stress condition, HSPs are often associated with poor prognosis and treatment resistance in many types of human cancers. It has been described that upregulation of HSPs may serve as diagnostic and prognostic markers in hepatocellular carcinoma (HCC). Targeting HSPs with specific inhibitor alone or in combination with chemotherapy regimens holds promise for the improvement of outcomes for HCC patients. In this review, we summarize the expression profiles, functions and molecular mechanisms of HSPs (HSP27, HSP70 and HSP90) as well as a HSP‐like protein (clusterin) in HCC. In addition, we address progression and challenges in targeting these HSPs as novel therapeutic strategies in HCC.     

Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

Introduction  

Heat shock proteins (HSPs) are normally induced under environmental stress to serve as chaperones for maintenance of correct protein folding but they are often overexpressed in many cancers, including breast cancer. The expression of Hsp27, an ATP-independent small HSP, is associated with cell migration and drug resistance of breast cancer cells. Breast cancer stem cells (BCSCs) have been identified as a subpopulation of breast cancer cells with markers of CD24-CD44+ or high intracellular aldehyde dehydrogenase activity (ALDH+) and proved to be associated with radiation resistance and metastasis. However, the involvement of Hsp27 in the maintenance of BCSC is largely unknown.     

Elevated levels of 70,000 dalton heat shock protein in transiently thermotolerant Chinese hamster fibroblasts and in their stable heat resistant variants

The function of one or more shock proteins (HSPs) may be to confer protection of cells against thermal damage. The quantitative relationship between heat sensitivity and concentration of several HSPs was examined in thermotolerant Chinese hamster HA-1 cells and in their heat-resistant variants. Low molecular weight HSPs (22-27 kd) showed no correlation with cell survival. The best correlation was found between concentration of 70 kd HSP and the logarithm of cell survival. There was no difference between the HSP 70 induced by heat shock and that present in a constitutive form. The 70 kd HSP may actually confer heat resistance on cells, but in any case HSP 70 appeared to be the best predictor of heat response.     

Heat Shock Protein Association with Clinico-Pathological Characteristics of Gastric Cancer in Jordan HSP70 is Predictive of Poor Prognosis

Gastric cancer (GC) is a major health problem worldwide and is one of the ten most commonly diagnosed cancers in Jordan. GC is usually diagnosed at late aggressive stages in which treatment options are limited. Recently, heat shock proteins (HSPs) were found to be overexpressed in a wide range of malignancies have been considered as promising candidate biomarkers for GC. The aim of this study was to investigate pathogenic roles of a panel of cytosolic HSPs including HSP90, HSP70, HSP60 and HSP27 in GC. Immunohistochemistry was used to assess the level of expression of these proteins in archived tumor samples (N87) representing various pathological characteristics of GC. HSP90, HSP60 and HSP27 were expressed abundantly in gastric tumors. On the other hand, HSP70 was reduced signi cantly and also found to be associated with Helicobacter pylori infection in tissues collected from GC patients. Furthermore, HSP27 was found to be associated with the level of differentiation. Our ndings indicate a role of HSP70 as a potential prognostic biomarker, patients harboring positive HSP70 expression displaying worse disease free survival than those with negative HSP70 expression. Differential expression of HSPs may play crucial roles in the initiation and progression of GC, and could be exploited as future therapeutic targets.     

Measurement and mathematical modeling of thermally induced injury and heat shock protein expression kinetics in normal and cancerous prostate cells

Purpose: Hyperthermia can induce heat shock protein (HSP) expression in tumours, which will cause enhanced tumour viability and increased resistance to additional thermal, chemotherapy, and radiation treatments. The study objective was to determine the relationship of hyperthermia protocols with HSP expression kinetics and cell death and develop corresponding computational predictive models of normal and cancerous prostate cell response.

Methods: HSP expression kinetics and cell viability were measured in PC3 prostate cancer and RWPE-1 normal prostate cells subjected to hyperthermia protocols of 44° to 60°C for 1 to 30 min. Hsp27, Hsp60, and Hsp70 expression kinetics were determined by western blotting and visualised with immunofluorescence and confocal microscopy. Based on measured HSP expression data, a mathematical model was developed for predicting thermally induced HSP expression. Cell viability was measured with propidium iodide staining and flow cytometry to quantify the injury parameters necessary for predicting cell death following hyperthermia.

Results: Significant Hsp27 and Hsp70 levels were induced in both cell types with maximum HSP expression occurring at 16 h post-heating, and diminishing substantially after 72 h. PC3 cells were slightly more sensitive to thermal stress than RWPE-1 cells. Arrhenius analysis of injury data suggested a transition between injury mechanisms at 54°C. HSP expression and injury models were effective at predicting cellular response to hyperthermia.

Conclusion: Measurement of thermally induced HSP expression kinetics and cell viability associated with hyperthermia enabled development of thermal dosimetry guidelines and predictive models for HSP expression and cell injury as a function of thermal stress to investigate and design more effective hyperthermia therapies.     

Heat shock proteins HSP27, HSP60, HSP70, and HSP90: expression in bladder carcinoma

BACKGROUND: Heat shock proteins (HSPs) are synthesized by cells in response to various stress conditions, including carcinogenesis. The expression of HSPs in neoplasia has been implicated in the regulation of apoptosis, and HSPs also can act by increasing immunity. In the current study, the authors attempted to clarify the significance of HSPs in bladder carcinoma and their effect on tumor behavior. METHODS: Expression levels of the 27-kilodalton HSP (HSP27), HSP60, HSP70, and HSP90 were studied using immunohistochemistry on tissue sections from 42 transitional cell carcinomas of the bladder (14 Grade 1 tumors; 13 Grade 2 tumors; 15 Grade 3 tumors, including 3 tumors associated with carcinoma in situ; 30 Stage Ta tumors; 7 Stage T1 tumors; and 5 Stage T2 tumors). Bladder specimens from 10 healthy patients were used as controls in the study. The selected patients had a mean follow-up of 52 months (range, 24-78 months). Among the 37 patients with superficial bladder carcinoma, 17 patients did not have any recurrence after undergoing primary resection, and 20 patients developed recurrent disease, including 4 recurrences with muscle invasion. HSP expression was evaluated according to the percentage of positively stained cells, and loss of expression was defined as < 80% of stained cells. RESULTS: In normal bladder specimens, all four HSPs (HSP27, HSP60, HSP70, and HSP90) were expressed strongly in the cytoplasm and membrane from the basal cell layer to the superficial cell layer. Loss of expression was detected in tumors: respectively, 45.2%, 38.1%, 69.0% and 23.8% of tumors showed a loss of immunostaining for HSP27, HSP60, HSP70, and HSP90. No correlation between HSP expression and grade was found. Low expression levels of HSP27 and HSP60 were correlated with higher tumor stage (87% vs. 6% [P < 0.001] and 78% vs. 9% [P < 0.01], respectively). HSP60 and HSP90 expression levels were correlated with final outcome for patients with superficial bladder carcinoma: loss of expression was associated with the risk of developing an infiltrating recurrence (97% vs. 6.0% [P < 0.001] and 88.2% vs. 52.5% [P = 0.02] for HSP60 and HSP90, respectively). CONCLUSIONS: HSPs were expressed in normal urothelium, and the current results indicated that loss of HSP60 and HSP90 expression may have prognostic relevance in patients with bladder carcinoma. The authors believe that HSP60 may be a very useful marker for patients with superficial bladder carcinoma and may be used for predicting disease progression. If these data are confirmed, low HSP60 expression levels may be usable as a prognostic marker to identify patients for whom local treatment would be insufficient.     

Significant correlation between expression of heat shock proteins 27, 70 and lymphocyte infiltration in esophageal squamous cell carcinoma

The objective of this study was to clarify the clinicopathologic and prognostic significance of heat shock proteins (HSP) 27 and 70 expression in esophageal squamous cell carcinoma (SCC). Immunohistochemical staining for HSPs 27 and 70 was performed on surgical specimens obtained from 62 patients with esophageal SCC. The expression of both HSPs 27 and 70 correlated inversely with depth of invasion (P<0.05) and pathologic stage (P<0.05), and correlated positively with lymphocyte infiltration (P<0.05). Reduction of HSP 70 expression was significantly correlated with poor prognosis (P<0.05). Patients with HSP 27-negative tumors tended to have a poor prognosis compared with patients with HSP 27-positive tumors. The present findings suggest that HSPs 27 and 70 are significant prognostic factors for esophageal SCC.     

Heat shock protein expression in human tumours grown in severe combined immunodeficient mice

The constitutional expression of heat shock proteins (HSP) 27, 70 and 90 in human breast, colon and ovarian cancer cells transplanted into severe combined immunodeficient (SCID) mice was evaluated. In addition their induced expression under chemotherapeutic stress was analyzed. The oestrogen receptor positive breast cancer cell lines (MCF-7, T47D) demonstrated an increased level of HSP 27 and 70 expression compared with oestrogen receptor negative cell lines (BT20, HBL100). After 5-fluorouracil application for 4 days, HSP 27 and 70 expression was increased in HT29 colon tumours. Hence, the human/SCID mouse model is well suited to evaluate the constitutional and induced expression of human HSPs under various experimental conditions.     

Over-expression of heat shock proteins in carcinogenic endometrium

We have previously shown that the subcellular localization of beta-catenin changes according to the cell proliferation status of the human endometrium, suggesting a role of intercellular transduction in cell growth control in human endometrium not only in the physiological but also in the carcinogenic condition. To further study the possible role of heat shock proteins (HSPs) in growth control, we immunohistochemically analyzed 92 endometrial samples, 30 of normal endometrium, 20 of endometrial hyperplasia and 42 of endometrial cancer, for expression of HSP27, HSP70, HSP90, estrogen receptor (ER) and progesterone receptor. HSP27 and HSP90 were detected in endometrial epithelium strongly in the proliferative phase and weakly in the secretory phase during the menstrual cycle according to the serum estradiol level. However, they were over-expressed in endometrial hyperplasia, especially HSP27. In endometrial cancer, HSP27 expression was heterogenic among the glands and lower than that in the proliferative phase and endometrial hyperplasia. HSP27 over-expression was also observed in samples including endometrial cancer and associated hyperplasia. Results of Western blotting followed those of immunohistochemistry. HSP70 was not changed during the menstrual cycle, as HSP27 and HSP90 were, and was rather stably expressed in endometrial hyperplasia and cancer. Our results suggest that HSP27 and HSP90 contribute to cell proliferation in endometrial epithelium and that over-expression of HSP27 in endometrial hyperplasia occurs as a result of the activated condition of ER, though in cancer it decreases according to the loss of function of ER.     

Prognostic significance of heat shock proteins 27 and 70 in patients with squamous cell carcinoma of the esophagus

BACKGROUND: Heat shock proteins (HSPs) first were defined as proteins induced by heat shock and other environmental and pathophysiologic stresses and are implicated in protein-protein interactions such as folding, translocation, and prevention of inappropriate protein aggregation. Many of their functions suggest that they play important roles in cancer. METHODS: Immunohistochemical study for HSP 27 and HSP 70 was performed on buffered formalin fixed, paraffin embedded sections of 102 esophageal squamous cell carcinoma specimens using monoclonal anti-HSP 27 antibody and anti-HSP 70 antibody. RESULTS: Normal squamous cells expressed both HSP 27 and HSP 70 with the exception of the basal layer. In cancerous tissue, expression of HSP 27 was evaluated as positive (+) (39 cases; 38%), reduced (+/-) (53 cases; 52%), or negative (-) (10 cases; 10%) and expression of HSP 70 was evaluated as (+) (14 cases; 14%), (+/-) (57 cases; 56%), or (-) (31 cases; 30%). There was a strong correlation between the expression of HSP 27 and HSP 70 (P < 0.0001). When compared with clinicopathologic features, expression of both HSP 27 and HSP 70 correlated negatively with lymph node metastases (P < 0.05), but not with depth of invasion or histologic grade. The reduction of the HSPs was associated significantly with poor postoperative survival (P < 0.0001). In addition, multivariate analysis revealed that HSP 27 (-) was the strongest prognostic factor among the clinicopathologic features. CONCLUSIONS: This study suggests that the expression of HSP 27 and HSP 70 frequently is reduced in patients with esophageal squamous cell carcinoma and therefore should be considered an independent prognostic factor of this disease.     

Heat shock proteins in breast cancer progression–A suitable case for treatment?

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 tumour 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 favour cell birth over cell death. In addition, Hsp90 plays a role in facilitating transformation by stabilising the mutated and over-expressed oncoproteins found in breast tumours, 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.     

主要人热休克蛋白在癌组织及癌旁正常组织中表达水平的比较研究

目的对２５例喉癌及癌旁正常喉组织中几种主要人热休克蛋白的ｍＲＮＡ表达水平进行检测和比较。方法采用ｍＲＮＡ狭缝杂交的方法。ＨＳＰ２７、ＨＳＣ７０和ＨＳＰ９０β探针用ＰＣＲ方法进行制备。结果ＨＳＰ９０α和ＨＳＰ７０在喉癌组织中的表达量显著高于正常喉粘膜,平均表达量均为正常喉粘膜表达量的５倍。而ＨＳＰ２７、ＨＳＣ７０和ＨＳＰ９０β的表达量,在两种组织中变化不大。结论与其他热休克蛋白相比,ＨＳＰ７０和ＨＳＰ９０α可能在喉癌的发生过程中有更为重要的作用。进一步深入研究它们在肿瘤和正常组织中不同表达的机理,将为热休克蛋白用于喉癌的生物治疗提供理论依据。     

Heat-shock protein expression in cisplatin-sensitive and -resistant human tumor cells

It has been suggested that the expression of certain heat-shock proteins (HSPs) may be prognostic markers in several tumor types. Since HSPs may be involved in determining cellular sensitivity to chemotherapeutic drugs, the possible relation between HSP expression and cisplatin (cDDP) sensitivity was studied. Three human germ-cell tumor cell lines, 1 human small-cell lung carcinoma (SCLC) cell line and 3 human colon carcinoma cell lines were used as a model for differences in intrinsic cDDP sensitivity. The constitutive expression of a panel of HSPs was studied by immunoblotting. No correlation was found between expression of HSP90, HSP73, HSP72, HSP60 and HSP27 and the extent of intrinsic cDDP sensitivity when all cell lines studied were considered. However, for the 3 cell lines derived from germ-cell carcinomas, HSP27 expression was inversely related to cDDP sensitivity, i.e. decreased HSP27 levels were associated with decreased sensitivity. Constitutive HSP expression was also studied in 2 sets of human cell lines with in vitro acquired cDDP resistance. In both resistant cell lines, decreased expression of HSP27 (as determined by Western blotting) was found as compared to the sensitive parent cell lines. Thus, acquired resistance to cDDP was also accompanied by decreased HSP27 expression. Interestingly, when basal HSP27 mRNA levels were measured in the SCLC cell line (GLC₄) and its subline with acquired resistance (GLC₄-cDDP), no significant differences were detected. Continuous cDDP incubation increased HSP27 levels and induced HSP27 phosphorylation in GLC₄ cells, but not in the resistant subline. Thus, although no general relationships between HSP expression and cDDP sensitivity are apparent, high HSP27 expression in vitro relates to high sensitivity to cDDP treatment in some tumor types. This is in accordance with reported clinical data on high HSP27 levels in tumors correlating with good prognosis. © 1996 Wiley-Liss, Inc.     

Human tumor cells killed by anthracyclines induce a tumor-specific immune response

Immunogenic cell death is characterized by the early surface exposure of chaperones including calreticulin and HSPs, which affect dendritic cell (DC) maturation and the uptake and presentation of tumor antigens. It has also been shown that it is characterized by the late release of high mobility group box 1 (HMGB1), which acts through Toll-like receptor 4 (TLR4) and augments the presentation of antigens from dying tumor cells to DCs. Most of the data on immunogenic tumor cell death were obtained using mouse models. In this study, we investigated the capacity of clinically used chemotherapeutics to induce immunogenic cell death in human tumor cell lines and primary tumor cells. We found that only anthracyclines induced a rapid translocation of calreticulin, HSP70, and HSP90 to the cell surface and the release of HMGB1 12 hours after the treatment. The interaction of immature DCs with immunogenic tumor cells led to an increased tumor cell uptake and induces moderate phenotypic maturation of DCs. Killed tumor cell-loaded DCs efficiently stimulated tumor-specific IFN-γ-producing T cells. DCs pulsed with killed immunogenic tumor cells also induced significantly lower numbers of regulatory T cells than those pulsed with nonimmunogenic tumor cells. These data indicate that human prostate cancer, ovarian cancer, and acute lymphoblastic leukemia cells share the key features of immunogenic cell death with mice tumor cells. These data also identify anthracyclines as anticancer drugs capable of inducing immunogenic cell death in sensitive human tumor cells.     

The heat shock proteins as targets for radiosensitization and chemosensitization in cancer

The heat shock proteins (HSPs) represent a class of proteins which are induced under physiologic stress to promote cell survival in the face of endogenous or exogenous injury. HSPs function predominantly as molecular chaperones, maintaining their "client" proteins in the correct conformational state in order to withstand a biologic stressor. Elevated HSP expression is also found in a range of pathologic conditions, notably malignancy. Cancer cells exploit the pro-survival phenotype endowed by HSPs to bolster their proliferative potential. Consequently, developing means of abrogating HSP expression may provide a way to render cancer cells more susceptible to radiation or chemotherapy. Here, we review the members of the HSP class and their roles in malignancy. We focus on attempts to target these proteins, particularly the small HSPs, in developing potent radiation and chemotherapy sensitizers, as well as proposed mechanisms for this sensitization effect.     

Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy

Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (>10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (>66%) of Hsp27 had shorter DFS.<0B> <0R>The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies. Int. J. Cancer (Pred. Oncol.) 79:468–475, 1998.© 1998 Wiley-Liss, Inc.     

Antibodies to HSP70 and HSP90 in serum in non-small cell lung cancer patients

Heat shock proteins (HSPs) are components of a physiologic stress response that are also over-expressed in various cancers including non-small cell lung cancer (NSCLC). During NSCLC serum-antibody screening of a NSCLC cDNA T7 phage library for immunogenic proteins we isolated HSP70 and HSP90 proteins. Isolation of these proteins suggested that corresponding antibodies could be elevated in NSCLC patient sera, a novel finding that could pilot their use as markers of NSCLC. We showed histochemically that patient sera were more reactive with each phage-expressed protein than normal sera. Antibody affinity for each phage-expressed protein was confirmed by limiting the dilution of individual sera assayed by Ab enzyme-linked immunosorbent assay (ELISA). Sera from 49 NSCLC patients assayed by Ab ELISA and normalized to 40 controls showed that HSP70 antibodies were significantly greater in patient sera than in normals (P=0.0002), while HSP90 antibodies were not significantly different (P=0.11). Analysis of the results with logistic regression and receiver operating characteristics (ROC) curves showed that HSP70 antibodies were modest markers of NSCLC (sensitivity 0.74 and specificity 0.73; area under the curve or AUC=0.731), while HSP90 antibodies appeared to be poor in both criteria with an AUC of 0.602. Further evaluation of HSP70 antibodies as potential markers of disease may be rational.