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Evaluation of the Heat Shock Protein 90 Inhibitor Ganetespib as a Sensitizer to Hyperthermia-Based Cancer Treatments.

ABSTRACT: Hyperthermia is being used as a radio- and chemotherapy sensitizer for a growing range of tumor subtypes in the clinic. Its potential is limited, however, by the ability of cancer cells to activate a protective mechanism known as the heat stress response (HSR). The HSR is marked by the rapid overexpression of molecular chaperones, and recent advances in drug development make their inhibition an attractive option to improve the efficacy of hyperthermia-based therapies. Our previous in vitro work showed that a single, short co-treatment with a HSR (HSP90) inhibitor ganetespib prolongs and potentiates the effects of hyperthermia on DNA repair, enhances hyperthermic sensitization to radio- and chemotherapeutic agents, and reduces thermotolerance. In the current study, we first validated these results using an extended panel of cell lines and more robust methodology. Next, we examined the effects of hyperthermia and ganetespib on global proteome changes. Finally, we evaluated the potential of ganetespib to boost the efficacy of thermo-chemotherapy and thermo-radiotherapy in a xenograft murine model of cervix cancer. Our results revealed new insights into the effects of HSR inhibition on cellular responses to heat and show that ganetespib could be employed to increase the efficacy of hyperthermia when combined with radiation.

SUBMITTER: Scutigliani EM

PROVIDER: S-EPMC9654690 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Evaluation of the Heat Shock Protein 90 Inhibitor Ganetespib as a Sensitizer to Hyperthermia-Based Cancer Treatments.

Scutigliani Enzo M EM Liang Yongxin Y IJff Marloes M Rodermond Hans H Mei Xionge X Korver Miriam P MP Orie Vaneesha S VS Hoebe Ron A RA Picavet Daisy I DI Oei Arlene A Kanaar Roland R Krawczyk Przemek M PM

Cancers 20221026 21

Hyperthermia is being used as a radio- and chemotherapy sensitizer for a growing range of tumor subtypes in the clinic. Its potential is limited, however, by the ability of cancer cells to activate a protective mechanism known as the heat stress response (HSR). The HSR is marked by the rapid overexpression of molecular chaperones, and recent advances in drug development make their inhibition an attractive option to improve the efficacy of hyperthermia-based therapies. Our previous in vitro work ...[more]

PMID: 36358669

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Project description:Gastric cancer (GC) remains the fifth most common cancer worldwide. Heat-shock protein 90 (HSP90) has become an attractive therapeutic target in treating cancers, because of its abnormally high expression in cancers. Several successful cases of HSP90 inhibitors capable of inhibiting GC inspired us to try ganetespib, a clinically promising and actively investigated second-generation HSP90 inhibitor in GC treatment. In our study, we show that ganetespib markedly reduced the growth of MGC-803 and also significantly inhibited the growth of SGC-7901 and MKN-28 in a dose-dependent manner. It induced G2/M cell-cycle arrest and apoptosis in all three cell lines, together with the related markers affected significantly. Mechanistically, ganetespib caused pronounced decrease of expression of classic HSP90 client proteins. Specifically, it greatly affected epidermal growth factor receptor (EGFR) signaling cascades by markedly decreasing the levels of total EGFR and EGFR on cell membranes. EGFR knockdown also induced cell-cycle arrest and apoptosis accompanied with a decrease of several EGFR downstream proteins. These results strongly support that EGFR signaling greatly contributes to the ganetespib inhibitory effects. Besides, we found that the responses of GC cell lines to ganetespib correlated well with their EGFR expression levels: MGC-803, as well as AGS and BGC-803, with higher EGFR expression responded to ganetespib better, whereas SGC-7901 and MKN-28 with lower EGFR levels were much less sensitive to ganetespib. Although SGC-7901 and MKN-28 were not very sensitive to ganetespib, ganetespib worked synergistically with radiation and cisplatin in killing them. Importantly, ganetespib significantly inhibited the growth of xenograft tumors in vivo as a single agent or in combination with cisplatin. Results of hematoxylin/eosin staining, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assays, and immunohistochemistry staining of phosphorylated cyclin-dependent kinase 1 (pCDK1), EGFR and Ki-67 revealed significant differences in ganetespib-treated tumors. Collectively, our data suggest that ganetespib, as a new potent treatment option, can be used for the molecularly targeted therapy of GC patients according to their expression profiles of EGFR.

Project description:HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC50) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC50>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.

Project description:IntroductionChronic inflammatory skin diseases may have a profound negative impact on the quality of life. Current treatment options may be inadequate, offering an unsatisfactory response or side effects. Therefore, ongoing efforts exist to identify novel effective and safe treatments. Heat shock protein (HSP) 90 is a chaperone that promotes the activity of a wide range of client proteins including key proinflammatory molecules involved in aberrant inflammation. Recently, a proof-of-concept clinical trial of 13 patients suggested that RGRN-305 (an HSP90 inhibitor) may be an oral treatment for psoriasis. However, HSP90 inhibition may be a novel therapeutic approach extending beyond psoriasis to include multiple immune-mediated inflammatory skin diseases.MethodsThis study aimed to investigate (i) the anti-inflammatory effects and mechanisms of HSP90 inhibition and (ii) the feasibility of topical RGRN-305 administration (new route of administration) in models of inflammation elicited by 12-O-tetradecanoylphorbol-13-acetate (TPA) in primary human keratinocytes and mice (irritative dermatitis murine model).Results/discussionIn primary human keratinocytes stimulated with TPA, a Nanostring® nCounter gene expression assay demonstrated that HSP90 inhibition with RGRN-305 suppressed many proinflammatory genes. Furthermore, when measured by quantitative real-time polymerase chain reaction (RT-qPCR), RGRN-305 significantly reduced the gene expression of TNF, IL1B, IL6 and CXCL8. We next demonstrated that topical RGRN-305 application significantly ameliorated TPA-induced skin inflammation in mice. The increase in ear thickness (a marker of inflammation) was significantly reduced (up to 89% inhibition). In accordance, RT-qPCR of the ear tissue demonstrated that RGRN-305 robustly reduced the gene expression of proinflammatory markers (Tnf, Il1b, Il6, Il17A and Defb4). Moreover, RNA sequencing revealed that RGRN-305 mitigated TPA-induced alterations in gene expression and suppressed genes implicated in inflammation. Lastly, we discovered that the anti-inflammatory effects were mediated, at least partly, by suppressing the activity of NF-κB, ERK1/2, p38 MAPK and c-Jun signaling pathways, which are consistent with previous findings in other experimental models beyond skin inflammation. In summary, HSP90 inhibition robustly suppressed TPA-induced inflammation by targeting key proinflammatory cytokines and signaling pathways. Our findings suggest that HSP90 inhibition may be a novel mechanism of action for treating immune-mediated skin disease beyond psoriasis, and it may be a topical treatment option.

Project description:Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81 ± 0.17. Six Hsp90 inhibitory compounds identified by this screening with IC50 values between 1.0 and 6 μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI50 values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9-Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy.

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Evaluation of the Heat Shock Protein 90 Inhibitor Ganetespib as a Sensitizer to Hyperthermia-Based Cancer Treatments.

Publications

Evaluation of the Heat Shock Protein 90 Inhibitor Ganetespib as a Sensitizer to Hyperthermia-Based Cancer Treatments.

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