High Expression Of HSP90 Research Articles

Heat shock protein 90 relieves heat stress damage of myocardial cells by regulating Akt and PKM2 signaling invivo.

Heat shock protein 90 (Hsp90) is associated with resisting heat-stress injury to the heart, particularly in myocardial mitochondria. However, the mechanism underlying this effect remains unclear. The present study was based on the high expression of Hsp90 during heat stress (HS) and involved inducing higher expression of Hsp90 using aspirin in mouse hearts. Higher Hsp90 levels inhibited HS-induced myocardial damage and apoptosis, and mitochondrial dysfunction, by stimulating Akt (protein kinase B) activation and PKM2 (pyruvate kinase M2) signaling, and subsequently increasing mitochondrial Bcl-2 (B-cell lymphoma 2) levels and its phosphorylation. Functional inhibition of Hsp90 using geldanamycin verified that reducing the association of Hsp90 with Akt and PKM2 caused the functional decline of phosphorylated (p)-Akt and PKM2 that initiate Bcl-2 to move into mitochondria, where it is phosphorylated. Protection by Hsp90 was weakened by blocking Akt activation using Triciribine, which could not be recovered by normal initiation of the PKM2 pathway. Furthermore, increased Hsp70 levels induced by Akt activation in myocardial cells may flow into the blood to resist heat stress. The results provided in vivo mechanistic evidence that in myocardial cells, Hsp90 resists heat stress via separate activation of the Akt-Bcl-2 and PKM2-Bcl-2 signaling pathways, which contribute toward preserving cardiac function and mitochondrial homeostasis.

MicroRNA-361-Mediated Inhibition of HSP90 Expression and EMT in Cervical Cancer Is Counteracted by Oncogenic lncRNA NEAT1.

Epithelial-mesenchymal transition (EMT) is a key process contributing to cervical cancer (CC) metastasis, and microRNAs (miRNAs) modulate the expression of genes implicated in EMT. However, the accurate role of miR-361 in CC-associated EMT and the mechanisms underlying its function in CC remains largely unknown. The functional roles of miR-361 in CC cells were explored by a series of cell functional assays. Luciferase reporter assays were used to demonstrate the potential interaction between miR-361, HSP90, and long non-coding RNA (lncRNA) NEAT1. We detected a reduction of miR-361 expression in CC tissues compared with normal tissues, and miR-361 overexpression inhibited invasion and EMT phenotypes of CC cells by directly targeting a key EMT activator HSP90. Additionally, we detected significantly higher levels of HSP90 in CC tissues compared with normal tissues, and high expression of HSP90 predicted a poorer prognosis. We further identified NEAT1 as a significantly upregulated lncRNA in CC tissues and high expression of NEAT1 was associated with worse survival in CC patients. NEAT1 directly repressed miR-361 expression and played an oncogenic role in CC cell invasion and sphere formation. Conclusions: These results demonstrated that miR-361 directly targets HSP90 to inhibit the invasion and EMT features, and NEAT1 functions as an oncogenic lncRNA that suppresses miR-361 expression and induces EMT and sphere formation in CC cells, thus providing critical insights into the molecular pathways operating in this malignancy.

Multiple Progressive Thermopreconditioning Improves Cardiac Ischemia/Reperfusion-induced Left Ventricular Contractile Dysfunction and Structural Abnormality in Rat.

Triple progressive thermopreconditioning (3PTP) may induce high Hsp-70 expression to maintain cardiac function. We suggest that 3PTP may reduce myocardial ischemia/reperfusion (I/R) injury during organ transplantation through Bag3/Hsp-70 mediated defense mechanisms. Male Wistar rats were divided into sham control group and 72 h after 3PTP in a 42°C water bath (3PTP) group. Rats underwent 60 min of ischemia by occlusion of the left anterior descending coronary artery followed by 240 min reperfusion. Hemodynamic parameters, including the electrocardiogram, microcirculation, heart rate, left ventricular end-diastolic pressure, maximal rate of rise (+dp/dt), and fall (-dp/dt) in the left ventricular pressure for index of contraction and relaxation were determined. Myocardial infarct size was evaluated by the Evans blue-2,3,5-triphenyltetrazolium chloride method. 3PTP-induced protective mechanisms were determined by Western blot and immunohistochemistry. Cardiac I/R depressed cardiac microcirculation, induced S-T segment elevation, and R-R and P-R interval elongation increased infarct size associated with erythrocyte extravasation, leukocytes and macrophage/monocyte infiltration, granulocyte colony-stimulating factor, poly(ADP-ribose) polymerase 1 stain, and transferase-mediated dUTP-biotin nick end labeling positive cells. However, 3PTP evoked significant cardioprotection against I/R injury, characterized by the increased +dp/dt value and the decreased elevated left ventricular end-diastolic pressure, erythrocyte extravasation, leukocyte and macrophage/monocyte infiltration, granulocyte colony-stimulating factor expression, poly(ADP-ribose) polymerase 1 expression, transferase-mediated dUTP-biotin nick end labeling positive cells, and fragmentation and infarct area. In addition, 3PTP increased Hsp-70 and Bag3 expression and decreased Bax/Bcl-2 ratio, but did not affect the Beclin-1 and LC3-II/LC3-I ratio in the heart with I/R injury. 3PTP therapies may through Bag3 upregulation alleviate I/R injury-induced left ventricular structural deterioration and dysfunction.

Immunohistochemical expression of interlukin10 (IL10) and heat shock protein-90 (HSP-90) in prostatic carcinoma.

Specific cytokines are related to pathologically changed prostate, propose that the balance in cytokine differs in normal and pathological prostate. Of these cytokines the interleukins 10, due to its "pleiotropic" actions in inflammation and angiogenesis, and HSP-90 due to its expression in tumor cells at high levels, suggesting that it has an important role for growth and/or survival of tumor cells. Evaluation of HSP-90 and IL10 immunoreactivity in benign prostatic hyperplasia (BPH) and prostatic carcinoma and to correlate this expression with clinicopathological parameters. A retrospective study in which 83 Paraffin-embedded tissue specimens including (43) BPH, (40) prostatic carcinoma and (20) normal prostate as control were included between the period of January 2015 and January 2017. All the cases were evaluated histopathologically and stained immunohistochemically for IL10 and HSP-90. Only cytoplasmic staining was considered as positive. Immunoreactivity scoring for both markers expression was calculated based on both staining intensity and percentage. Was done using SPSS Version 21 statistical analysis software. P value of <0.05 was considered statistically significant. Statistical analysis of HSP-90 and IL10 expression revealed a highly significant correlation of expression of these two markers in advanced Gleason grading and tumor, node, and metastasis (TNM) staging cases of prostatic carcinoma. High expression of IL10 and HSP-90 is associated with high grade and stage of prostatic carcinoma. This provides a base for further studies and researches on the role of these investigated proteins as prognostic markers immunotherapy targets for carcinoma of the prostate.

High expression of HSP90 is associated with poor prognosis in patients with colorectal cancer.

BackgroundHeat shock protein 90 (HSP90) is a highly conserved chaperone with an approximate molecular weight of 90-kDa. It plays a critical role in maintaining stability and homeostasis of oncoproteins, helping cancer cells living in the unsuitable environmental conditions. The current study aims to inquire the difference of HSP90 expression in tumor tissues and normal tissues, analyze the correlation between HSP90 expression and the prognoses of patients with colorectal cancer (CRC), and investigate its role in CRC preliminarily.MethodsOnline analysis of HSP90 mRNA levels in different cancers was firstly done in Gene Expression Profiling Interactive Analysis. Then HSP90 expression was determined by immunohistochemistry between 99 CRC tissues and 81 normal tissues. Chi-square test or Fisher’s exact test was used to analyze the relationship between HSP90 and histopathologic characteristics. Kaplan–Meier analysis and Cox’s proportional hazards model were also done for further analysis of the prognostic values of HSP90. Pearson’s correlation coefficients between HSP90 expression values and other mRNA expression values were calculated based on The Cancer Genome Atlas dataset and bioinformatic analysis was done about these screened genes.ResultsColorectal cancer tissues showed significantly higher expression of HSP90 than normal tissues (55.6% vs. 3.7%, P < 0.0001). Kaplan–Meier curves showed high HSP90 expression was associated with poor prognosis (P = 0.039) in CRC patients, and multivariate Cox proportional hazards regression model analysis also indicated that HSP90 expression (HR = 1.930, 95% CI [1.113–3.349], P = 0.019) linked to poor prognosis. Moreover, 85 genes were correlated with HSP90, which were involved in metabolic process and enriched in pathways of Proteasome and Base excision repair.ConclusionsOur results suggested that HSP90 expression is inversely associated with survival outcomes and could be an independent prognostic factor for CRC patients. It mainly involved in metabolic process and exerted binding and catalytic activities.

Flower-like Mn-Doped Magnetic Nanoparticles Functionalized with αvβ3-Integrin-Ligand to Efficiently Induce Intracellular Heat after Alternating Magnetic Field Exposition, Triggering Glioma Cell Death.

Despite the potential of magnetic nanoparticles (NPs) to mediate intracellular hyperthermia when exposed to an alternating magnetic field (AMF), several studies indicate that the intracellular heating capacity of magnetic NPs depends on factors such as cytoplasm viscosity, nanoparticle aggregation within subcellular compartments, and dipolar interactions. In this work, we report the design and synthesis of monodispersed flowerlike superparamagnetic manganese iron oxide NPs with maximized SAR (specific absorption rate) and evaluate their efficacy as intracellular heaters in the human tumor-derived glioblastoma cell line U87MG. Three main strategies to tune the particle anisotropy of the core and the surface to reach the maximum heating efficiency were adopted: (1) varying the crystalline anisotropy by inserting a low amount of Mn2+ in the inverse spinel structure, (2) varying the NP shape to add an additional anisotropy source while keeping the superparamagnetic behavior, and (3) maximizing NP-cell affinity through conjugation with a biological targeting molecule to reach the NP concentration required to increase the temperature within the cell. We investigate possible effects produced by these improved NPs under the AMF (f = 96 kHz, H = 47 kA/m) exposure in the glioblastoma cell line U87MG by monitoring the expression of hsp70 gene and reactive oxygen species (ROS) production, as both effects have been described to be induced by increasing the intracellular temperature. The induced cell responses include cellular membrane permeabilization and rupture with concomitant high ROS appearance and hsp70 expression, followed by cell death. The responses were largely limited to cells that contained the NPs exposed to the AMF. Our results indicate that the developed strategies to optimize particle anisotropy in this work are a promising guidance to improve the heating efficiency of magnetic NPs in the human glioma cell line.

Abstract 2245: Improving the effectiveness of immunotherapy in breast cancer by targeting the tumor microenvironment

Abstract Large cohorts of recent clinical studies have firmly established that increased levels of tumor-infiltrating lymphocytes (TILs) in TNBC and HER2+ subtypes predicted better clinical outcome compared to the luminal subtype. These observations led to the hypothesis that women with TNBC or HER2+ subtypes may respond to a checkpoint blockade. However, early results from these trials using check point inhibitors alone or in combination with chemotherapy have shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, it has suggested that the combinations of check point blockade with therapeutics that target immunosuppression may potentiate its efficacy. TNFα exhibits paradoxical roles; it may fuel tumor cell growth, invasion and metastasis in some tumor types, while in others it induces cytotoxic cell death. We recently demonstrated that TNFα distinctly induces A20 in TNBC subtype and protects these cells from TNFα-induced cytotoxic cell death by upregulating HSP70 protein and maintaining EMT/CSC phenotype. In contrast, luminal MCF7 or ZR75-1 cells display approximately 70% apoptosis when treated with TNFα. Overexpression of A20 in luminal cells not only protected them from TNFα-induced cytotoxicity by upregulating HSP70 and EMT/CSC phenotype, but also exhibited aggressive metastatic properties in mouse xenograft models. Furthermore, we show that A20/HSP70 pathway attracts tumor-infiltrating lymphocytes (TILs) while inducing the accumulation of immunosuppressive MDSCs in syngeneic mouse models. Interestingly, pulmonary DTCs as well as the immune infiltrates from 4T1 tumor-bearing mice exhibited significantly higher HSP70 expression. Therefore, we proposed that targeting HSP70 may potentiate the efficacy of immunotherapy in preclinical models of breast cancer. As previously reported, murine 4T1 tumors do respond to check point inhibitors. We reasoned that this may be an appropriate model to test the efficacy of HSP70 inhibitor, JG-231. Expectedly, there was no difference in tumor growth and metastasis between control and anti-PDL1 treated animals, however, combination of anti-PDL1 antibody ed with JG-231 and chemotherapy (cyclophosphamide-CTX) significantly reduced primary tumor growth (&amp;gt;10 fold) and eliminated metastasis. Collectively, our pilot experiments provide a strong rationale for testing our hypothesis and may lead to a rapid translation into the clinical utility. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Maria Ouzounova, Ahmet Korkaya, Jason Gestwicki, Max S. Wicha, Esteban Celis. Improving the effectiveness of immunotherapy in breast cancer by targeting the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2245.

Elevated HSP90 associates with expression of HIF-1α and p-AKT and is predictive of poor prognosis in nasopharyngeal carcinoma.

HSP90, as a molecular chaperone, has numerous substrate proteins, including HIF-1α and p-AKT, but the relationships among HSP90, HIF-1α and p-AKT have not been investigated in NPC. We examined and analysed the correlation between expression of HSP90, HIF-1α and p-AKT and clinicopathological features of NPC. We collected 445 cases of NPC and 54 cases of non-cancerous nasopharyngeal epithelia tissues, detected expression of HSP90, HIF-1α and p-AKT proteins in these tissues by immunohistochemistry. The results indicated that overexpression of HSP90, HIF-1α and p-AKT in NPC was significantly higher than that in non-cancerous nasopharyngeal epithelia (P<0.05). The overexpression of HIF-1α in primary NPC was significantly lower than that in matched lymph node metastatic NPC (P=0.024) or recurrent NPC (P=0.039). The overexpression of HSP90 (P<0.001) and HIF-1α (P=0.031) was evidently higher in late stage NPC. NPC patients with lymph node metastasis (LNM) had a higher overexpression rate of HSP90 (P<0.001) than those without LNM. Increased HSP90 expression was positively associated with HIF-1α expression (r=0.367, P<0.001) and p-AKT (r=0.142, P=0.003) expression in NPC. Furthermore, HIF-1α was also related to p-AKT expression (r=0.114, P=0.017). The overall survival rate for NPC patients with up-regulated HSP90 was significantly lower than those with down-regulated HSP90 (P<0.001), as was found with raised HIF-1α (P=0.036) and increased p-AKT (P=0.044). Multivariate Cox regression analysis further identified that HSP90 and HIF-1α were independent poor prognostic factors for NPC. Taken together, elevated HSP90 was associated with expression of HIF-1α and p-AKT in NPC. Furthermore, high expression of HSP90 and HIF-1α could be used as a novel independent poor prognostic biomarker for patients with NPC.

Overexpression of HSP27 and HSP70 is associated with decreased survival among patients with esophageal adenocarcinoma.

BACKGROUNDOverexpression of heat shock proteins (HSPs) is associated with several malignancies and contributes to the development, progression, and metastasis of cancer, in addition to the inhibition of cellular death. In recent years, there has been active research into using HSP inhibitors in several malignancies. Due to the poor prognosis of esophageal adenocarcinoma (EAC), it would be valuable to find new biomarkers for the development of cancer treatments.AIMTo evaluate the expressions of HSP27 and HSP70 and their effect on survival in EAC.METHODSImmunohistochemical analyses and evaluations of HSP27 and HSP70 expression were performed on all available samples from 93 patients diagnosed with EAC between 1990 and 2007 at two university hospitals. Fifteen cases with Barrett’s metaplasia and 5 control cases from the same patient population were included in the analysis. HSP expression was quantitatively assessed and classified as high or low. Kaplan-Meier analyses and Cox regression models adjusting for age and sex as well as tumor site, stage, and grade were used to evaluate the effect on survival.RESULTSTumor stage and surgical treatment were the main prognostic factors. High HSP27 expression in cancer cases was a strong negative predictive factor, with a mean survival of 23 mo compared to the 49 mo in cases with a low expression (P = 0.018). The results were similar for HSP70, with a poorer survival of 17 mo in cases with high HSP70 expression, in contrast to 40 mo (P = 0.006) in cases with a low expression. A Cox regression survival analysis was performed, adjusting for possible confounding factors, and higher HSP27 and HSP70 expressions remained an independent negative prognostic factor. The HSPs’ correlation with survival was not affected by cancer treatments. When the analysis was adjusted for all factors, the odds ratios for HSP27 and HSP70 were 3.3 (CI: 1.6–6.6, P = 0.001) and 2.2 (CI: 1.2–3.9, P = 0.02), respectively.CONCLUSIONHSP27 and HSP70 overexpression is associated with poor survival in EAC, which is, to the best of our knowledge, reported for the first time.

Imbalance of the redox system and quality of tilapia fillets subjected to pre-slaughter stress.

The objective of this study was to evaluate the effect of oxidative stress on the instrumental and sensory quality of Nile tilapia fillets. The experiment was conducted in a 2x2 factorial arrangement, evaluating densities (60 and 300 kg m-3) and depuration times (1 and 24 hours) in a total of four treatments. The serum levels of cortisol and gene expression levels of catalase (CAT), glutathione peroxidase (GPx) and 70 kDa heat shock protein (HSP70) as well as the pH, color, tenderness, water-holding capacity and sensory analysis of the fillets were evaluated. High density (300 kg m-3) resulted in higher mean cortisol levels, lower expression of CAT and GPx enzymes as well as higher expression of HSP70. Fish under this treatment also exhibited fillets with greater tenderness, higher lightness, lower redness and lower sensory acceptance. The longer depuration time (24 hours) resulted in lower expression of the CAT and GPx enzymes and fillets with higher lightness. The water-holding capacity was not affected by the different treatments. Therefore, low density and longer depuration times are recommended for decreased stress and improved quality of fillets.

Muscular apoptosis but not oxidative stress increases with old age in a long-lived diver, the Weddell seal.

Seals experience repeated bouts of ischemia-reperfusion while diving, potentially exposing their tissues to increased oxidant generation and thus oxidative damage and accelerated aging. We contrasted markers of oxidative damage with antioxidant profiles across age and sex for propulsive (longissismus dorsi) and maneuvering (pectoralis) muscles of Weddell seals to determine whether previously observed morphological senescence is associated with oxidative stress. In longissismus dorsi, old (age 17-26 years) seals exhibited a nearly 2-fold increase in apoptosis over young (age 9-16 years) seals. There was no evidence of age-associated changes in lipid peroxidation or enzymatic antioxidant profiles. In pectoralis, 4-hydroxynonenal-Lys (4-HNE-Lys) levels increased 1.5-fold in old versus young seals, but lipid hydroperoxide levels and apoptotic index did not vary with age. Glutathione peroxidase activity was 1.5-fold higher in pectoralis of old versus young animals, but no other antioxidants changed with age in this muscle. With respect to sex, no differences in lipid hydroperoxides or apoptosis were observed in either muscle. Males had higher HSP70 expression (1.4-fold) and glutathione peroxidase activity (1.3-fold) than females in longissismus dorsi, although glutathione reductase activity was 1.4-fold higher in females. No antioxidants varied with sex in pectoralis. These results show that apoptosis is not associated with oxidative stress in aged Weddell seal muscles. Additionally, the data suggest that adult seals utilize sex-specific antioxidant strategies in longissismus dorsi but not pectoralis to protect skeletal muscles from oxidative damage.

Preclinical Evaluation of the Hsp70 Peptide Tracer TPP-PEG24-DFO[89Zr] for Tumor-Specific PET/CT Imaging.

High precision in vivo PET/CT imaging of solid tumors improves diagnostic credibility and clinical outcome of patients. An epitope of the oligomerization domain of Hsp70 is exclusively exposed on the membrane of a large variety of tumor types, but not on normal cells, and thus provides a universal tumor-specific target. Here we developed a novel PET tracer TPP-PEG24-DFO[89Zr] based on the tumor cell-penetrating peptide probe TPP, which specifically recognizes membrane Hsp70 (mHsp70) on tumor cells. The implemented PEG24 moiety supported tracer stability and improved biodistribution characteristics in vivo The K d of the tracer ranged in the low nanomolar range (18.9 ± 11.3 nmol/L). Fluorescein isothiocyanate (FITC)-labeled derivatives TPP-[FITC] and TPP-PEG24-[FITC] revealed comparable and specific binding to mHsp70-positive 4T1, 4T1+, a derivative of the 4T1 cell line sorted for high Hsp70 expression, and CT26 tumor cells, but not to mHsp70-negative normal fibroblasts. The rapid internalization kinetics of mHsp70 into the cytosol and the favorable biodistribution of the peptide-based tracer TPP-PEG24-DFO[89Zr] in vivo enabled a tumor-specific accumulation with a high tumor-to-background contrast and renal body clearance. The tumor-specific enrichment of the tracer in 4T1+ (6.2 ± 1.1%ID/g), 4T1 (4.3 ± 0.7%ID/g), and CT26 (2.6 ± 0.6%ID/g) mouse tumors with very high, high, and intermediate mHsp70 densities, respectively, reflected mHsp70 expression profiles of the different tumor types, whereas benign mHsp70-negative fibroblastic hyperplasia showed no tracer accumulation (0.2 ± 0.03%ID/g). The ability of our chemically optimized peptide-based tracer TPP-PEG24-DFO[89Zr] to detect mHsp70 in vivo suggests its broad applicability in targeting and imaging with high specificity for any tumor type that exhibits surface expression of Hsp70.Significance: A novel peptide-based PET tracer against the oligomerization domain of Hsp70 has potential for universal tumor-specific imaging in vivo across many tumor type. Cancer Res; 78(21); 6268-81. ©2018 AACR.

Impact of different seasons on the milk somatic and differential cell counts, milk cortisol and neutrophils functionality of three Indian native breeds of cattle

The present study was undertaken to compare the effect of different seasons on the mammary immunity of three Indian native breeds of cows (Tharparkar, Gir and Sahiwal) well adapted to the tropical region. For this milk samples were collected from cows in winter (THI=57, comfortable zone), hot-dry (HD; THI=76, heat stressful zone) and hot-humid (HH; THI = 82, severe heat stress) and estimated for milk somatic cell counts (SCC), phagocytic activity (PA) of milk neutrophils, milk cortisol and heat shock proteins and function associated genes in milk neutrophils. Milk SCC was evaluated using a cell counter and differential cell counts measured microscopically. Cortisol was quantified in skimmed milk by competitive ELISA. Milk PA was estimated using nitro blue tetrazolium assay, and for gene expression studies, milk neutrophils were isolated and studied for heat shock proteins (HSP40, HSP70, HSP90α) and cell adhesion molecules (CD11b, CD25, CD44) using real-time polymerase chain reaction. All the studied parameters increased in HD and HH seasons with highest values observed in Sahiwal cows. However, PA of neutrophil was highest in Tharparkar cows in winter and decreased gradually at higher THI values during hot seasons. Milk cortisol was positively correlated with expression of various CD molecules and HSPs (p < 0.05) in milk neutrophils but negatively correlated (p < 0.05) with PA during HH season in all breeds. The study revealed that Indian native cows were at considerable risk in HH season and Sahiwal cows were more heat stressed followed by Gir and Tharparkar cows, respectively, and thus may require managemental interventions. Also, the higher expression of HSP70 and CD25 with increasing THI levels in hot seasons makes them suitable biological markers for quantifying heat stress.

Abstract 1672: Preclinical evaluation of WXFL1024H251, a potent second-generation HSP90 inhibitor

Abstract Objective: Heat Shock Protein 90 (Hsp90) is a molecular chaperone that mediates the post-translational stability of its protein substrates, many of which are onco-proteins. Hsp90 is emerging as an important target for cancer therapy because its inactivation may abrogate multiple signaling pathways simultaneously, irrespective of the mutational status of proteins involved. We disclose here our clinical candidate WXFL1024H251, a novel second-generation small-molecule Hsp90 inhibitor with a chemical structure unrelated to the first generation, the ansamycin family of Hsp90 inhibitors. Method: The anti-proliferative activity of WXFL1024H251 was evaluated in a variety of cancer cell lines in a 96-hour viability assay. QW, Q2W or TIW intravenous administration of at doses of 5~50 mg/kg was used to evaluate the in vivo anti-tumor activity of WXFL1024H251 in BT474 cell line-derived xenograft (CDX) breast cancer model. Effects of WXFL1024H251 on HSP90 function were assessed by western blot analysis of PD markers HSP70, Her2 and p-Her2 in the BT474 tumors. Result: A panel of 20 cancer cell lines were screened for sensitivity to WXFL1024H251. Proliferation of 2 cancer cell lines A549 and BT474 were potently inhibited by WXFL1024H251, with IC50 of 0.007 μM and 0.009 μM, respectively. A549 cell line has KRAS mutation and medium to high EGFR expression. BT474 is an ER+ and ERBB2+ breast cancer cell line. In the BT474 tumor model, 3 week treatment with WXFL1024H251 showed good antitumor efficacy with T/C values of 36% at 7.5 mg/kg Q2W and 45% at 10 mg/kg QW; toxicity were acceptable with both dosing regiments. Conclusion: These preclinical studies showed good antitumor efficacy of WXFL1024H251, which was associated with client protein maturation and HSP70 up-regulation in the tumor tissue. WXFL1024H251 may represent a novel promising clinical antitumor agent for treating breast cancer and other cancer types with high expression of HSP90. Citation Format: Xiaobing yan, Charles Z. Ding, Lihong Hu, Wei Huang, Shuhui Chen, Dongdong Wu, Jiahu Wu, Guoqiang Ma, Qiyao Zhang, Rui Gao, Yusong Zhu, Tiantian Dong, Yizhong Zhu, Caixia Yan, Yan Yan. Preclinical evaluation of WXFL1024H251, a potent second-generation HSP90 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1672.

The Prognostic Significance of Hsp70/Hsp90 Expression in Breast Cancer: A Systematic Review and Meta-analysis

Studies have focused on heat shock protein (Hsp) inhibitors as potential treatment agents in breast cancer, with controversial results. Adopting a pathophysiological perspective, this systematic review aims to synthesize the evidence examining the association between Hsp70/Hsp90 expression and breast cancer prognosis, as well as prognosis-related clinicopathological indices. Secondarily, changes in Hsp70/Hsp90 expression in the continuum of breast neoplasia were assessed. Hsp70/Hsp90 expression was approached globally, quantified by means of immunohistochemistry, western blot or PCR. This study was performed in accordance with the PRISMA guidelines. Relevant studies were sought in PubMed, up to December 31, 2015. A total of 23 eligible studies were identified (7,288 breast cancer cases). High Hsp90 expre s sion was associated with worse overall survival (pooled RR=1.48, 95%CI=1.21-1.82) and marginally with worse disease-free survival. High Hsp70 expression also correlated with worse disease-free survival (pooled RR=1.77, 95%CI=1.71-2.82). Hsp70 intense expression correlated with ER positivity (pooled OR=3.51, 95%CI=1.31-9.40) and PR positivity (pooled OR=2.48, 95%CI=1.39-4.44). No significant associations were noted between Hsp70/Hsp90 expression and clinicopathological variables including histological grade, tumor size, nodal metastasis or patient age at diagnosis. No clear pattern emerged for Hsp70/Hsp90 expression along the breast neoplasia continuum. This systematic review and meta-analysis highlights the prognostic role of Hsp90 and Hsp70 expression in breast cancer. Further high-quality studies, with detailed reporting are needed to provide epidemiological evidence complementing the findings of ongoing clinical trials on Hsp inhibitors.

Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis.

BackgroundGlioblastoma is one of the most aggressive and common malignancies of the central nervous system in humans. Owing to the correlation of high Hsp90 expression with prognosis and clinical pathology features of diverse types of cancer, targeting Hsp90 with small-molecule inhibitors has become a promising anticancer strategy.PurposeIn this study, we aimed to explore the possibility of anticancer effect of NMS-E973 in giloblastoma and elucidate the mechanism.MethodsCell based MTT assay and colony formation assay were used to detect cell viability. Apoptosis was analyzed by nuclear staining with Hoechst 33258 and Annexin V/propidium iodide staining followed by flow cytometry. Western-blot and RT-PCR were used to detect gene expression. Xenograft assay was used to explore the anticancer effect of NMS-E973 in vivo.ResultsWe found that NMS-E973 induces apoptosis and inhibits cell growth in glioblastoma cells in cell culture and xenograft models. As a proapoptotic Bcl-2 member, PUMA was induced by NMS-E973 in a p53-dependent manner in glioblastoma in cell culture, thereby inducing apoptosis in glioblastoma cells. Furthermore, PUMA was induced by NMS-E973 treatment in xenograft tumors, and deficiency in PUMA significantly suppressed the antitumor effects of NMS-E973.ConclusionOur study suggests that PUMA-mediated apoptosis is important for the therapeutic responses of NMS-E973. Induction of PUMA might be a potential biomarker for predicting NMS-E973 responses.

BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC), and acquired chemotherapy resistance is a primary obstacle for the treatment of these tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6) of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549rDOX20) and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-468r5-FU2000) cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549rDOX20 and MDA-MB-468r5-FU2000 cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer.

Heat shock protein 70 and tumor-infiltrating NK cells as prognostic indicators for patients with squamous cell carcinoma of the head and neck after radiochemotherapy: A multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).

Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre‐activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor‐infiltrating CD56+ NK cells in formalin‐fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin‐based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3–4) showed significantly decreased overall survival (OS; p = 0.008), local progression‐free survival (LPFS; p = 0.034) and distant metastases‐free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0–2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16‐ (p = 0.001), p53‐ (p = 0.0003) and HPV16 DNA‐negative (p = 0.001) tumors. The absence or low numbers of tumor‐infiltrating CD56+ NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor‐infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor‐infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers.

Overexpression of cytosolic, plasma membrane bound and extracellular heat shock protein 70 (Hsp70) in primary glioblastomas.

A unique feature in several non-CNS-tumors is the overexpression of heat shock protein 70 (Hsp70, HSPA1A) in the cytosol, but also its unusual plasma membrane expression and release. Although in gliomas, cytosolic Hsp70 levels are not associated with histological grading, the role of membrane bound and released Hsp70 is still completely unknown. Membrane bound as well as cytosolic Hsp70 can be detected in viable tumor cells with the monoclonal antibody (mAb) cmHsp70.1. Herein, we analysed membrane bound Hsp70 levels in primary and secondary gliomas of different grades and on isolated glioma subpopulations (endothelial cells, CD133-positive cells, primary cultures) by immunohistochemistry and flow cytometry using cmHsp70.1 mAb. Extracellular Hsp70 was determined by a commercial Hsp70 sandwich ELISA (R&D) in plasma samples of glioblastoma patients and healthy volunteers. We found an overexpression of Hsp70 in primary glioblastomas compared to low-grade, anaplastic, or secondary gliomas as determined by immunohistochemistry. Especially in flow cytometry, a strong plasma membrane Hsp70 expression was only observed in primary but not secondary glioblastomas. Within the heterogeneous tumor mass, CD133-positive tumor-initiating and primary glioblastoma cells showed a high membrane Hsp70 expression density, whereas endothelial cells, isolated from glioblastoma tissues only showed a weak staining pattern. Also in plasma samples, secreted Hsp70 protein was significantly increased in patients harbouring primary glioblastomas compared to those with secondary and low grade glioblastomas. Taken together, we show for the first time that cytosolic, membrane bound and extracellular Hsp70 is uniquely overexpressed in primary glioblastomas.

High expression of heat shock proteins and heat shock factor-1 distinguishes an aggressive subset of clear cell renal cell carcinoma.

Heat shock proteins (HSPs) are a group of molecules induced by a variety of environmental and pathophysiological stresses, including cancer. HSPs are implicated in the regulation of apoptosis and immunity in neoplasm. Transcription factor heat shock factor 1 (HSF1) acts as the master regulator to control HSP expression, and is therefore involved in tumorigenesis. The purpose of this study was to evaluate the expression and clinicopathological relevance of HSPs and HSF1 in clear cell renal cell carcinoma (ccRCC). The expression of HSP27, HSP60, HSP70, HSP90 and HSF1 was assessed in 428 cases of ccRCC using immunohistochemistry. High expression of HSP60 and HSP70 was correlated positively with grade and stage. High expression of HSF1 was correlated positively with stage. Univariate and multivariate analyses demonstrated that 216 patients (52%) with tumour expressing three or four markers in a panel of HSP60, HSP70, HSP90 and HSF1 had a significantly heightened risk for cancer-specific mortality than tumours expressing fewer than three markers (P<0.0001; concordance index, 0.81). Immunohistochemical examination of HSPs and HSF1 provides useful prognostic information that may contribute to the design of therapeutic strategies for patients with ccRCC.