Heat Shock Protein 90 Inhibitor Research Articles

Heat shock protein 90 modulates cutaneous vasodilation during an exercise-heat stress, but not during passive whole-body heating in young women.

Heat shock protein 90 (HSP90) modulates exercise‐induced cutaneous vasodilation in young men via nitric oxide synthase (NOS), but only when core temperature is elevated ~1.0°C. While less is known about modulation of this heat loss response in women during exercise, sex differences may exist. Further, the mechanisms regulating cutaneous vasodilation can differ between exercise‐ and passive‐heat stress. Therefore, in 11 young women (23 ± 3 years), we evaluated whether HSP90 contributes to NOS‐dependent cutaneous vasodilation during exercise (Protocol 1) and passive heating (Protocol 2) and directly compared responses between end‐exercise and a matched core temperature elevation during passive heating. Cutaneous vascular conductance (CVC%max) was measured at four forearm skin sites continuously treated with (a) lactated Ringers solution (control), (b) 178 μM Geldanamycin (HSP90 inhibitor), (c) 10 mM L‐NAME (NOS inhibitor), or (d) combined 178 μM Geldanamycin and 10 mM L‐NAME. Participants completed both protocols during the early follicular (low hormone) phase of the menstrual cycle (0–7 days). Protocol 1: participants rested in the heat (35°C) for 70 min and then performed 50 min of moderate‐intensity cycling (~55% VO2peak) followed by 30 min of recovery. Protocol 2: participants were passively heated to increase rectal temperature by 1.0°C, comparable to end‐exercise. HSP90 inhibition attenuated CVC%max relative to control at end‐exercise (p < .05), but not during passive heating. While NOS inhibition and combined HSP90 + NOS inhibition attenuated CVC%max relative to control for both protocols (all p < .05), they did not differ from each other. We show that HSP90 modulates cutaneous vasodilation NOS‐dependently during exercise in young women, with no effect during passive heating, despite a similar NOS contribution.

HSP90 Inhibition and Modulation of the Proteome: Therapeutical Implications for Idiopathic Pulmonary Fibrosis (IPF).

Idiopathic Pulmonary fibrosis (IPF) is a catastrophic disease with poor outcomes and limited pharmacological approaches. Heat shock protein 90 (HSP90) has been recently involved in the wound-healing pathological response that leads to collagen deposition in patients with IPF and its inhibition represents an exciting drug target against the development of pulmonary fibrosis. Under physiological conditions, HSP90 guarantees proteostasis through the refolding of damaged proteins and the degradation of irreversibly damaged ones. Additionally, its inhibition, by specific HSP90 inhibitors (e.g., 17 AAG, 17 DAG, and AUY-922) has proven beneficial in different preclinical models of human disease. HSP90 inhibition modulates a complex subset of kinases and interferes with intracellular signaling pathways and proteome regulation. In this review, we evaluated the current evidence and rationale for the use of HSP90 inhibitors in the treatment of pulmonary fibrosis, discussed the intracellular pathways involved, described the limitations of the current understanding and provided insights for future research.

Fluoropyrimidin-2,4-dihydroxy-5-isopropylbenzamides as antitumor agents against CRC and NSCLC cancer cells

A major cause of failure of therapy in patients with non-small cell lung cancer (NSCLC) is development of acquired drug resistance leading to tumor recurrence and disease progression. In addition to the development of new generations of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), different molecular targets may provide opportunities to improve the therapeutic outcomes. In this study, we utilized the core structure 5-fluorouracil (5-FU) or tegafur, a 5-FU prodrug combined through different linkers with resorcinol to generate a series of fluoropyrimidin-2,4-dihydroxy-5-isopropylbenzamides which inhibit potent Heat Shock Protein 90 (HSP90). These compounds were found to show significant antiproliferative activity in colorectal cancer (CRC) HCT116 and NSCLC A549, H460, and H1975 (EGFR L858R/T790 M double mutation) cells. Compound 12c, developed by molecular docking analysis and enzymatic assays exhibits promising inhibitory activity of HSP90. This compound, 12c shows the most potent HSP90 inhibitory activity with an IC50 value of 27.8 ± 4.4 nM, superior to that of reference compounds AUY-922 (Luminespib) and BIIB021 whose IC50 values are 43.0 ± 0.9 nM and 56.8 ± 4.0 nM respectively. This strong HSP90 inhibitory activity of 12c leads to rapid degradation of client proteins EGFR and Akt in NSCLC cells. In addition, 12c induces significant accumulation of a sub-G1 phase population in parallel with apoptosis by showing activated caspase-3, -8 and -9 and PARP induction. These results provide a new strategy for development of novel HSP90 inhibitors for cancer treatment.

HSP90 Inhibitor Improves Lung Protection in Porcine Model of Donation After Circulatory Arrest

Ischemia-reperfusion associated with prolonged warm ischemia during donation after circulatory death (DCD) induces acute lung injury. The objective of this study was to combine exvivo lung perfusion (EVLP) and a heat shock protein-90 inhibitor (HSP90i) to recondition DCD organs and prevent primary graft dysfunction. Pigs (55 to 65 kg) were anesthetized, ventilated, and hemodynamically monitored. Cardiac arrest was induced with potassium chloride, and animals were left nonventilated for 2 hours. Lungs were procured and perfused in an EVLP platform for 4 hours by using a cellular perfusate. In the study group, the perfusate contained HSP90i and its transport vehicle (n= 4). In the control group, the perfusate contained only the transport vehicle (n= 4). Gas exchange, airway pressures, and compliance were measured. Pulmonary edema was assessed by bronchoscopy and weight measurement. Lung biopsy samples were obtained for histologic analyses and protein expression measurements. The use of HSP90i reduced lung weight gain to 8.4 ± 3.4% vs 26.6 ± 6.2% in the control group (P < .05). There was reduced edema formation. The ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen at the end of EVLP was 423 ± 65 in the study group vs 339 ± 25 mm Hg in the control group, but this difference was not statistically significant. Lactate metabolism, pulmonary vascular resistance, and pulmonary arterial pressure improved during EVLP with the use of the HSP90i. The use of HSP90i with EVLP improves the lung reconditioning process. Further research is required to confirm whether these findings translate to benefit once transplanted and observed invivo. Successful pharmacologic inhibitors may expand the donor pool in the context of DCD donors.

The HSP90 Inhibitor, AUY-922, Ameliorates the Development of Nitrogen Mustard-Induced Pulmonary Fibrosis and Lung Dysfunction in Mice

Increased levels of heat shock protein 90 (HSP90) have been recently implicated in the pathogenesis of pulmonary fibrosis and the use of HSP90 inhibitors constitutes a potential therapeutic approach. Similarly, acute exposure to nitrogen mustard (NM) is related to the development of chronic lung injury driven by TNF-α, TGF-β, ERK and HSP90. Thus, we developed a murine model of NM-induced pulmonary fibrosis by instilling C57BI/6J mice with 0.625 mg/kg mechlorethamine hydrochloride. After 24 h, mice began receiving AUY-922, a second generation HSP90 inhibitor, at 1 mg/kg 2 times per week or 2 mg/kg 3 times per week, for either 10 or 30 days. AUY-922 suppressed the NM-induced sustained inflammation, as reflected in the reduction of leukocyte and protein concentrations in bronchoalveolar lavage fluid (BALF), and inhibited the activation of pro-fibrotic biomarkers, ERK and HSP90. Furthermore, AUY-922 maintained normal lung function, decreased the overexpression and accumulation of extracellular matrix proteins, and dramatically reduced histologic evidence of fibrosis in the lungs of mice exposed to NM. The HSP90 inhibitor, AUY-922, successfully blocked the adverse effects associated with acute exposures to NM, representing a promising approach against NM-induced pulmonary fibrosis.

Discovery of novel anti-breast cancer agents derived from deguelin as inhibitors of heat shock protein 90 (HSP90)

A series of O-substituted analogues of the B,C-ring truncated scaffold of deguelin were designed as C-terminal inhibitors of heat shock protein 90 (HSP90) and investigated as novel antiproliferative agents against HER2-positive breast cancer. Among the synthesized compounds, compound 80 exhibited significant inhibition in both trastuzumab-sensitive and trastuzumab-resistant breast cancer cells, whereas compound 80 did not show any cytotoxicity in normal cells. Compound 80 markedly downregulated the expression of the major client proteins of HSP90 in both cell types, indicating that the cytotoxicity of 80 in breast cancer cells is attributed to the destabilization and inactivation of HSP90 client proteins and that HSP90 inhibition represents a promising strategy to overcome trastuzumab resistance. A molecular docking study of 80 with the homology model of a HSP90 homodimer showed that 80 fit nicely in the C-terminal domain with a higher electrostatic complementary score than that of ATP.

HSP90 inhibitors strengthen extracellular ATP-stimulated synthesis of interleukin-6 in osteoblasts: Amplification of p38 MAP kinase.

Heat shock protein 90 (HSP90) is expressed ubiquitously in a variety of cell types including osteoblasts. HSP90 acts as a key driver of proteostasis under pathophysiological conditions. Here, we investigated the involvement of HSP90 in extracellular ATP-stimulated interleukin (IL)-6 synthesis and HSP90 downstream signalling in osteoblast-like MC3T3-E1 cells. In osteoblasts, extracellular ATP stimulates the synthesis of IL-6, a bone-remodelling agent. Geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and onalespib, three different HSP90 inhibitors, amplified the ATP-stimulated IL-6 release. Geldanamycin increased IL-6 mRNA expression elicited by ATP. ATP enhanced the triiodothyronine-induced osteocalcin release, but HSP90 inhibitors suppressed the release. Extracellular ATP induced the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), p70 S6 kinase, Akt, and myosin phosphatase-targeting subunit (MYPT), a Rho-kinase substrate. SB203580, an inhibitor of p38 MAPK, suppressed ATP-stimulated IL-6 release. Inhibitors of MEK1/2 (PD98059), JNK (SP600125), upstream kinase of p70 S6 kinase (rapamycin) and Akt (deguelin), all increased IL-6 release. Y27632, a Rho-kinase inhibitor, failed to affect the IL-6 release stimulated by ATP. Geldanamycin and 17-AAG both amplified ATP-induced p38 MAPK phosphorylation, although geldanamycin inhibited the phosphorylation of Akt induced by ATP. In addition, SB203580 significantly reduced the amplification by geldanamycin of the IL-6 release. Taken together, our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 MAPK activation in osteoblasts. SIGNIFICANCE OF THE STUDY: Heat shock protein 90 (HSP90) acts as a key driver of proteostasis under pathophysiological conditions in a variety of cell types. We have previously shown that HSP90 is expressed at high levels in osteoblast-like MC3T3-E1 cells, even in their quiescent state, consistent with HSP90 performing an important physiological function in osteoblasts. In the present study, we investigated whether HSP90 is implicated in extracellular ATP-induced interleukin (IL)-6 synthesis in osteoblast-like MC3T3-E1 cells. Our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 mitogen-activated protein kinase activation in osteoblasts.

17-Aminogeldanamycin Inhibits Constitutive Nuclear Factor-Kappa B (NF-κB) Activity in Patient-Derived Melanoma Cell Lines.

Melanoma remains incurable skin cancer, and targeting heat shock protein 90 (HSP90) is a promising therapeutic approach. In this study, we investigate the effect of 17-aminogeldanamycin, a potent HSP90 inhibitor, on nuclear factor-kappa B (NF-κB) activity in BRAFV600E and NRASQ61R patient-derived melanoma cell lines. We performed time-lapse microscopy and flow cytometry to monitor changes in cell confluence and viability. The NF-κB activity was determined by immunodetection of phospho-p65 and assessment of expression of NF-κB-dependent genes by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Constitutive activity of p65/NF-κB was evident in all melanoma cell lines. Differences in its level might be associated with genetic alterations in CHUK, IL1B, MAP3K14, NFKBIE, RIPK1, and TLR4, while differences in transcript levels of NF-κB-inducible genes revealed by PCR array might result from the contribution of other regulatory mechanisms. 17-Aminogeldanamycin markedly diminished the level of phospho-p65, but the total p65 protein level was unaltered, indicating that 17-aminogeldanamycin inhibited activation of p65/NF-κB. This conclusion was supported by significantly reduced expression of selected NF-κB-dependent genes: cyclin D1 (CCND1), C-X-C motif chemokine ligand 8 (CXCL8), and vascular endothelial growth factor (VEGF), as shown at transcript and protein levels, as well as secretion of IL-8 and VEGF. Our study indicates that 17-aminogeldanamycin can be used for efficient inhibition of NF-κB activity and the simultaneous diminution of IL-8 and VEGF levels in the extracellular milieu of melanoma.

The Role of Heat Shock Protein -90 (HSP-90) in Inflammatory Disorders

In human cells, Heat Shock Protein-90 (HSP-90) is present in the cytosol, nucleoplasm, endoplasmic reticulum, and mitochondria. The eukaryotic HSP-90 is multifunctionary and essential for cell viability, signal transduction, cell-cycle control as well as transcriptional regulation. The intracellular environment does not restrict HSP-90. It has a vital role in all types of inflammatory disorders, including cancer, autoimmune diseases, infectious inflammatory conditions. Hence, pharmacological inhibition of HSP-90 is currently a choice of therapeutic target for the treatment of autoimmune diseases, cancer, and infectious diseases. Based on the biology of HSP-90, several COOH-terminal ATPase sites of HSP-90, NH2-terminal ATPase sites of HSP-90, and Histone deacetylase inhibitors are evaluated and classified under various groups. For the treatment of different inflammatory disorders, HSP-90 identified as a promising therapeutic target. The present review may guide researchers for evaluating the HSP-90 targeted pathway as a useful therapeutic target for inflammatory diseases, including cancers.

Heat shock protein 90 inhibition and multi-target approach to maximize cardioprotection in ischaemic injury.

Despite several advances in medicine, ischaemic heart disease remains a major cause of morbidity and mortality. The unravelling of molecular mechanisms underlying disease pathophysiology has revealed targets for pharmacological interventions. However, transfer of these pharmcological possibilities to clinical use has been disappointing. Considering the complexity of ischaemic disease at the cellular and molecular levels, an equally multifaceted treatment approach may be envisioned. The pharmacological principle of 'one target, one key' may fall short in such contexts, and optimal treatment may involve one or many agents directed against complementary targets. Here, we introduce a 'multi-target approach to cardioprotection' and propose heat shock protein 90 (HSP90) as a target of interest. We report on a member of a distinct class of HSP90 inhibitor possessing pleiotropic activity, which we found to exhibit potent infarct-sparing effects.

Mild microwave ablation combined with HSP90 and TGF‑β1 inhibitors enhances the therapeutic effect on osteosarcoma.

Osteosarcoma is the most common malignant bone tumour and the second leading cause of cancer-related death in children and adolescents. Microwave ablation has an excellent therapeutic effect on bone tumours by instantaneously increasing the temperature in the tumour; however, there is a risk of damaging the surrounding healthy tissues by exposure to a high temperature when the treatment power is too large. In the present study, two anti-tumour reagents, a heat shock protein 90 (HSP90) inhibitor (PF-04929113) and a transforming growth factor-β1 (TGF-β1) inhibitor (SB-525334) were employed to enhance the therapeutic effect of mild-power microwave ablation. It was revealed that microwaving to 48°C combined with HSP90 and TGF-β1 inhibitors significantly increased the apoptotic rate of VX2 cells. The same results were observed during in vivo experiments using New Zealand rabbits to model osteosarcoma. In addition, the results indicated that the expression of cytochrome c, caspase-3 and caspase-9 were upregulated in response to the treatment, which indicated that the mitochondrial apoptotic signalling pathway had been activated. These findings may provide a novel strategy for the development of microwave ablation in osteosarcoma treatment, which could effectively kill tumour cells without damaging the surrounding normal tissues.

CTEP 9557: A dose-escalation trial of combination dabrafenib, trametinib, and AT13387 in patients with BRAF mutant solid tumors.

3609 Background: Combination BRAF and MEK inhibitor therapy is associated with response in patients (pts) with BRAF mutant (mut) solid tumors; however critical limitations for the durable activity of these agents remains. Preclinically, the addition of heat shock protein 90 (HSP90) inhibitors improves the efficacy of BRAF inhibitor (BRAFi) therapy in both BRAFi -sensitive and resistant mutant cell lines. Methods: CTEP study 9557 (NCT02097225) is a phase I study designed to determine the safety and efficacy of the small molecule HSP90inhibitor, AT13387, in combination with dabrafenib (dab) and trametinib (tram) in patients with BRAF V600E/K mut solid tumors. Prior chemotherapy, immunotherapy, BRAF and/or MEK exposure was permitted. The primary objective was to determine the maximum tolerated dose (MTD). Results: From July 2015 to June 2018, 22 patients with previously treated, metastatic BRAF V600E/K mut solid tumors were enrolled using a 3 + 3 design at four dose levels (DL) (Table). Pts were predominantly female (59%) with a median age of 57.5yrs (37 -75). The most common tumor type was BRAF V600Emut colon cancer (N=12). Dose limiting toxicities (DLTs) occurred in one patient in DL3 and one in DL4, specifically grade 3 myelosuppression and fatigue, respectively. The MTD was Dab 150mg [BID/PO], Tram 2mg [QD/PO] and AT1187 260mg/m2 [D1,8,15/IV]. Twenty-one of 22 pts were eligible for efficacy assessment. Best response, per RECIST 1.1, was partial response (PR) in 2 pts – one with colon ca (TKI-naïve), one with melanoma (TKI-resistant) - stable disease (SD) in 8 pts, and disease progression (PD) in 11 with a disease control rate (PR + SD) of 47.6% (90% CI: 29% - 67%). Median time to progression was significantly longer in DL3 (3.9 mths; 1.8-9.2) compared to DL1 (1.6mths; 0.9-1.7) or DL2 (1.5; 0.6-3.6). Median PFS and OS were 1.8mths (90% CI: 1.6 – 3.7mths) and 5.1 mths (90% CI: 2.5 -10.6mths), respectively. Median OS was not reached in DL3/4. Correlative data on the expression of the key signaling proteins relating to response will be presented at the meeting. Conclusions: HSP90 inhibition combined with BRAF/MEK inhibition was determined to be safe with evidence of disease control in a heavily pre-treated population of pts with BRAF V600E/K mut solid tumors. Clinical trial information: NCT02097225 . [Table: see text]

Post-treatment with a heat shock protein 90 inhibitor prevents chronic lung injury and pulmonary fibrosis, following acute exposure of mice to HCl

Aim/Purpose: Exposure to high levels of hydrochloric acid (HCl) is associated with severe lung injury including both acute inflammation and chronic lung disease, which leads to the development of pulmonary fibrosis. Currently, there are no specific therapeutic agents for HCl-induced lung injury. Heat shock protein 90 (HSP90) has been implicated in the pathogenesis of pulmonary fibrosis. Thus, we have used a murine model of intra-tracheal acid instillation to investigate the antidotal effects of AUY-922, a small molecule HSP90 inhibitor, already in clinical trials for various types of cancer, against HCl-induced chronic lung injury and pulmonary fibrosis.Methods: HCl (0.1 N, 2 μl/g body weight) was instilled into male C57Bl/6J mice at day 0. After 24 h, mice began receiving 1 mg/kg AUY-922, 2x/week for 15 or 30 days.Results: AUY-922 suppressed the HCl-induced sustained inflammation, as reflected in the reduction of leukocyte and protein concentrations in bronchoalveolar lavage fluid, and inhibited the activation of pro-fibrotic biomarkers, ERK and HSP90. Furthermore, AUY-922 improved lung function, decreased the overexpression and accumulation of extracellular matrix proteins and dramatically reduced histologic evidence of fibrosis in the lungs of mice exposed to HCl.Conclusions: We conclude that AUY-922, and possibly other HSP90 inhibitors, successfully block the adverse effects associated with acute exposures to HCl and may represent an effective antidote against HCl-induced chronic lung injury and fibrosis.

Studies on the pIC50 of 4,5-Diarylisoxazole as HSP90 Inhibitors

Background: Heat Shock Protein 90(HSP90) inhibitors are involved in multiple anticancer pathways, which indicate many important novel molecular targets for cancer therapy. However, the characteristics of poor water solubility, liver toxicity and finite bioavailability of the present inhibitors limit clinical application. Hence, it is crucial to evaluate the characteristics of compounds and develop new drugs with hypotoxicity and high-bioactivity. Methods: Quantitative Structure-Activity Relationship (QSAR) has been an effective method for screening novel structures and predicting various properties of the synthesized compounds. Heuristic Method (HM) and Gene Expression Programming (GEP) algorithm were used to establish linear and nonlinear models severally. Results: The results showed that HM has good correlation coefficients of R2 and lower S2 as 0.79 and 0.29 for the training set and GEP has better values of 0.89 and 0.05, respectively. Conclusion: Both models have the capability of prediction but the nonlinear model developed by GEP has a more excellent predictive ability and indicates further optimization of the HSP90 inhibitors.

Combination of HSP90 and autophagy inhibitors promotes hepatocellular carcinoma apoptosis following incomplete thermal ablation.

The present study evaluated the effect of combining inhibitors (17-AAG) of heat shock protein 90 (HSP90) and autophagy (3-MA) on apoptosis using an incomplete thermal ablation animal model. A total of 28 orthotopic mice with hepatocellular carcinoma were randomly divided into 4 groups to receive different drug interventions. Following palliative laser ablation, changes in autophagy, apoptosis and Akt/mTOR expression levels were assessed in tumors. Compared with the controls, the 17-AAG-treated mice exhibited significantly decreased expression levels of phosphorylated (p)-Akt and p-mTOR with enhanced autophagy and apoptosis; no marked increases in the expression levels of p-Akt and p-mTOR were observed in the 3-MA-treated mice, with no significant changes in autophagy; however, apoptosis was enhanced. No significant decreases in p-Akt and p-mTOR or any increase in autophagy were observed in the mice receiving a combination of 17-AAG and 3-MA, but they did exhibit a marked increase in apoptosis. Compared with 17-AAG alone, the combination of 17-AAG and 3-MA resulted in a marked increase in apoptosis without enhanced autophagy. In the incomplete ablation model, the effects of autophagy and apoptosis are antagonistic. The combined use of 17-AAG and 3-MA can significantly promote apoptosis and is worthy of further study.

The HSP90 inhibitor Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy: an in vitro and in vivo approach

Oncogenic client-proteins of the chaperone Heat shock protein 90 (HSP90) insure unlimited tumor growth and are involved in resistance to chemo- and radiotherapy. The HSP90 inhibitor Onalespib initiates the degradation of oncoproteins, and might also act as a radiosensitizer. The aim of this study was therefore to evaluate the efficacy of Onalespib in combination with external beam radiotherapy in an in vitro and in vivo approach. Onalespib downregulated client proteins, lead to increased apoptosis and caused DNA-double-strands. Monotherapy and combination with radiotherapy reduced colony formation, proliferation and migration assessed in radiosensitive HCT116 and radioresistant A431 cells. In vivo, a minimal treatment regimen for 3 consecutive days of Onalespib (3 × 10 mg/kg) doubled survival, whereas Onalespib with radiotherapy (3 × 2 Gy) caused a substantial delay in tumor growth and prolonged the survival by a factor of 3 compared to the HCT116 xenografted control group. Our results demonstrate that Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy, most prominent in the radiosensitive cell models. We speculate that the depletion and downregulation of client proteins involved in signalling, migration and DNA repair mechanisms is the cause. Thus, individually, or in combination with radiotherapy Onalespib inhibits tumor growth and has the potential to improve radiotherapy outcomes, prolonging the overall survival of cancer patients.

&lt;p&gt;HSP90 Inhibitor Ganetespib (STA-9090) Inhibits Tumor Growth in c-Myc-Dependent Esophageal Squamous Cell Carcinoma&lt;/p&gt;

PurposeCurrently, the paucity of classical effective pharmacological drugs to treat esophageal squamous cell carcinoma (ESCC) is a major problem. The c-Myc (MYC) protein is a promising target as it is overexpressed in ESCC. MYC is a sensitive client protein of the heat shock protein 90 (HSP90) and, therefore, targeting the HSP90-MYC axis by inhibition of HSP90 is a potential therapeutic strategy for ESCC. Here, we evaluated the clinical application value of the HSP90 inhibitor (Ganetespib, STA-9090) as an anti-cancer agent for MYC-positive ESCC.Materials and MethodsWe first analyzed ESCC tissue microarrays and clinical tissue samples to determine MYC expression. The relationship between MYC and HSP90 was analyzed by co-immunoprecipitation assays and immunofluorescence. In in vitro cell models, cell growth was analyzed using the CCK-8 kit, and MYC protein expression was analyzed by Western blot. The in vivo antitumor activity of STA-9090 was assessed in two xenograft animal models.ResultsWe demonstrated that MYC-overexpressing ESCC cells were highly sensitive to STA-9090 treatment through suppressing ESCC cell proliferation, cell cycle progression and survival. Moreover, STA-9090 treatment decreased MYC expression, reducing the half-life of the MYC protein. We further established two xenograft mouse models using ESCC cells and clinical ESCC samples to validate the effectiveness of STA-9090 in vivo. In both xenograft models, STA-9090 substantially inhibited the growth of MYC-positive ESCC tumors in vivo. In contrast, STA-9090 treatment demonstrated no beneficial effects in mice with low-MYC expressing ESCC tumors.ConclusionIn conclusion, our data support that the HSP90 inhibitor, STA-9090, suppresses the expression of the MYC protein and interferes with HSP90-MYC protein–protein interaction. This, in turn, leads to inhibition of ESCC cell proliferation and promotion of apoptosis in ESCC cells in vitro and reduction of ESCC tumors in vivo. We propose, based on our findings, that STA-9090 is a potential novel therapeutic target for MYC-positive ESCC.

Inhibition of heat shock protein 90 attenuates post‑angioplasty intimal hyperplasia.

Intimal hyperplasia (IH) is a pathologic process that leads to restenosis after treatment for peripheral arterial disease. Heat shock protein 90 (HSP90) is a molecular chaperone that regulates protein maturation. Activation of HSP90 results in increased cell migration and proliferation. 17‑N‑allylamino‑17‑demethoxygeldanamycin (17‑AAG) and 17‑dimethylaminoethylamino‑17‑demethoxygeldanamycin (17‑DMAG) are low toxicity Food and Drug Association approved HSP90 inhibitors. The current study hypothesized that HSP90 inhibition was predicted to reduce vascular smooth muscle cell (VSMC) migration and proliferation. In addition, localized HSP90 inhibition may inhibit post‑angioplasty IH formation. For proliferation, VSMCs were treated with serum‑free media (SFM), 17‑DMAG or 17‑AAG. The selected proliferative agents were SFM, platelet derived growth factor (PDGF) or fibronectin. After three days, proliferation was measured. For migration, VSMCs were treated with SFM, 17‑AAG or 17‑DMAG with SFM, PDGF or fibronectin as chemoattractants. Balloon injury to the carotid artery was performed in rats. The groups included in the present study were the control, saline control, 17‑DMAG in 20% pluronic gel delivered topically to the adventitia or intraluminal delivery of 17‑DMAG. After 14days, arteries were fixed and sectioned for morphometric analysis. Data was analyzed using ANOVA or a student's t‑test. P<0.05 was considered to indicate a statistically significant difference. The results revealed that 17‑AAG and 17‑DMAG had no effect on cell viability. PDGF and fibronectin also increased VSMC proliferation and migration. Furthermore, both 17‑AAG and 17‑DMAG decreased cell migration and proliferation in all agonists. Topical adventitial treatment with 17‑DMAG after balloon arterial injury reduced IH. HSP90 inhibitors suppressed VSMC proliferation and migration without affecting cell viability. Topical treatment with a HSP90 inhibitor (DMAG) decreased IH formation after arterial injury. It was concluded that 17‑DMAG may be utilized as an effective therapy to prevent restenosis after revascularization.

Abstract P3-01-18: Kinase D interacting substrate 220 (Kidins220) and disease progression of breast cancer, the role of heat shock protein90 (HSP90)

Abstract Background: Kidins220 (Kinase D Interacting Substrate 220) is a transmembrane scaffold protein and was initially found as the substrate of Protein Kinase D. It has several binding partners and plays an important role in neuronal activities and attribute to the occurrence of neuronal disease. Moreover, emerging evidence has revealed the involvement of Kidins220 in regulating cancer development including melanoma, neuroblastoma, and prostate cancer. Our current study investigated the role of Kidins220 in breast cancer and the biological implications on breast cancer cells. Method: A panel of human breast cancer cell lines were used in the biological studies. Kidin220 Knockdown cell model was established using lentivirus anti-Kidins220 shRNA and the knockdown of Kidins220 was verified in gene and protein levels. Cell matrix attachment and cell migration assay were determined using an automated cell analyser (ECIS). The growth of breast cancer cells was evaluated using colorimetric method. Human breast tumours (n=106) together with adjacent background tissues were collected immediately after surgery. Transcription level of Kidins220 and HSPs in the fresh tissues was determined using quantitative transcript analyses. Results: In the clinical cohort of breast cancer, an increase in expression of Kidins220 was found in tumour tissues in comparison with adjacent normal tissues. Patients with poor prognosis (NPI&amp;gt;5.4) had the lowest expression of Kidins220 compared with patients with good and moderate prognosis. Likewise, high grade tumours had markedly reduced level of Kidins220. Furthermore, patients remain disease free had the highest Kidins220 level when compared with those who had breast cancer-related incidence. Kaplan-Meier plot showed high level of Kidins220 was linked to longer survival. Our database also revealed that the levels of expression of Kidins200 were significantly correlated with that of HSP90A (Heat Shock Protein-90A) (r=0.37, p&amp;lt;0.001), but not HSP90B. Moreover, breast cancer cells (MDA MB-231 and MCF-7) after losing Kidins220 by way of knockdown showed an increase in cellular migration and cell matrix adhesion, with little changes in cell growth compared with control cells. In these cell based models, HSP90 inhibitor was found to affect Kidins220 mediated changes in matrix adhesion. It was also noteworthy that cells with Kidins220 knockdown had dramatically reduced expression of HSP90, particularly HSP90A. Conclusion: Kidins220 may act as a tumour suppressor in regulating the disease progression of breast cancer and has a correlation with the survival of patients with breast cancer. Kidins220 regulates the aggressive property including migration and cell matrix attachment of breast cancer with the involvement of HSP90A. Citation Format: Zhe Zhao, Tracey A. Martin, Johannes Benedikt, Shuo Cai, Lin Ye, Eleri Davies, Robert E Mansel, Wen G. Jiang. Kinase D interacting substrate 220 (Kidins220) and disease progression of breast cancer, the role of heat shock protein90 (HSP90) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-18.

HSP90 Interacts with the Fibronectin N-terminal Domains and Increases Matrix Formation.

Heat shock protein 90 (HSP90) is an evolutionarily conserved chaperone protein that controls the function and stability of a wide range of cellular client proteins. Fibronectin (FN) is an extracellular client protein of HSP90, and exogenous HSP90 or inhibitors of HSP90 alter the morphology of the extracellular matrix. Here, we further characterized the HSP90 and FN interaction. FN bound to the M domain of HSP90 and interacted with both the open and closed HSP90 conformations; and the interaction was reduced in the presence of sodium molybdate. HSP90 interacted with the N-terminal regions of FN, which are known to be important for matrix assembly. The highest affinity interaction was with the 30-kDa (heparin-binding) FN fragment, which also showed the greatest colocalization in cells and accommodated both HSP90 and heparin in the complex. The strength of interaction with HSP90 was influenced by the inherent stability of the FN fragments, together with the type of motif, where HSP90 preferentially bound the type-I FN repeat over the type-II repeat. Exogenous extracellular HSP90 led to increased incorporation of both full-length and 70-kDa fragments of FN into fibrils. Together, our data suggested that HSP90 may regulate FN matrix assembly through its interaction with N-terminal FN fragments.