DCZ3112, a novel Hsp90 inhibitor, exerts potent antitumor activity against HER2-positive breast cancer through disruption of Hsp90-Cdc37 interaction

doi:10.1016/j.canlet.2018.07.012

Cancer Letters

Volume 434, 10 October 2018, Pages 70-80

https://doi.org/10.1016/j.canlet.2018.07.012 Get rights and content

Highlights

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DCZ3112 is a novel Hsp90 inhibitor that disrupts Hsp90-Cdc37 interaction.
•
DCZ3112 overcomes resistance to traditional Hsp90 inhibitors.
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DCZ3112 predominantly acts in HER2-positive breast cancer.
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The combination of DCZ3112 and anti-HER2 antibodies exerts synergistic effects in breast cancer.
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DCZ3112 alone or with anti-HER2 antibodies overcomes trastuzumab resistance.

Abstract

Hsp90 regulates the stability of oncoproteins important in tumor development and progression, and represents a potential therapeutic target. However, all Hsp90 inhibitors currently in clinical trials target Hsp90 ATPase activity and exhibit low selectivity and high toxicity. In this study, we discovered a new Hsp90 inhibitor, DCZ3112, with a novel mechanism of action. DCZ3112 directly bound to the N-terminal domain of Hsp90 and inhibited Hsp90-Cdc37 interaction without inhibiting ATPase activity. DCZ3112 inhibited the proliferation predominantly in HER2-positive breast cancer cells, including those resistant to the classical Hsp90 inhibitor geldanamycin, which mainly targets ATPase. DCZ3112 produced synergistic in vitro activity in inhibiting cell proliferation, inducing G₁-phase arrest and apoptosis, and reducing AKT and ERK phosphorylation. Consistent with this, DCZ3112 alone inhibited the growth of HER2-positive BT-474 xenografts, and exhibited enhanced antitumor activity when combined with the anti-HER2 antibody trastuzumab. Importantly, DCZ3112 also significantly inhibited the growth of trastuzumab-resistant BT-474 cells, and combined treatment retained synergistic antitumor activity. Thus, our findings show that disrupting Hsp90-Cdc37 interaction may represent a promising strategy against HER2-positive breast cancer, especially those with acquired resistance to trastuzumab.

Introduction

Human epidermal growth factor receptor 2 (HER2/EGFR2/ErbB2) belongs to a family of receptor tyrosine kinases involved in regulating cell growth, differentiation, and survival [1]. HER2 is overexpressed in 25–30% of human breast cancer and is associated with high rate of recurrence and poor overall prognosis [2,3]. Thus, inhibition of the HER2 signaling pathway is a clinically established strategy for treating breast cancer. Several HER2-targeting agents, including the anti-HER2 antibodies trastuzumab [4] and pertuzumab [5,6], the EGFR/HER2 inhibitor lapatinib [7], as well as combinations of HER2-targeting agents, are currently available for treating patients with HER2-positive breast cancer [8,9]. Although these single and combinatorial regimens produce robust positive responses, the clinical benefit is usually transient, with most patients relapsing and their disease ultimately progressing after long-term treatment. Thus, there has been continuing interest in finding new HER2-targeting agents or rational drug combinations for optimal HER2 blockade to treat HER2-positive breast cancer.

Heat shock protein 90 (Hsp90) is an ATP-dependent protein chaperone involved in the regulation of cellular protein homeostasis. Hsp90 regulates the stability of client proteins, including HER2, AKT, RAF-1, CDK4 and Bcr-Abl, most of which are essential for cancer cell survival and proliferation. In fact, HER2 is one of the most well-defined client proteins in breast cancer; moreover, breast cancer is a prime target indication for Hsp90 inhibitors [[10], [11], [12], [13]]. It has been reported that Hsp90 is expressed at 2–10-fold higher levels in cancer cells than in normal cells [10]. Therefore, inhibition of HER2-mediated signaling pathways by targeting Hsp90 is a promising strategy in the treatment of HER2-positive breast cancer.

Hsp90 consists of an N-terminal domain, a middle domain and a C-terminal domain [11]. Current Hsp90 inhibitors in clinical trials all bind to the ATP pocket of the N-terminal domain and employ the same mechanism of ATP blockade to inactivate this chaperone [[12], [13], [14]]. However, none of these inhibitors has successfully reached the market owing to high toxicity and low selectivity. Thus, there is an urgent need to develop a new type of Hsp90 inhibitor distinct from the traditional strategy of inhibiting Hsp90 ATPase activity.

Hsp90 exerts its chaperone function through a process that involves transient formation of multiprotein complexes with cochaperones [15]. Disrupting interactions of Hsp90 with its cochaperones, and thereby blocking the chaperoning cycle at various stages, is also likely to achieve Hsp90 inhibition in cancer cells [16,17]. Cdc37, one of these essential cochaperones, acts through association with Hsp90 to recognize important client proteins in cancer [17]. Targeting Hsp90-Cdc37 interaction might represent a powerful strategy for treating certain kinase-dependent cancer with lower toxicity and higher selectivity compared with traditional inhibitors of Hsp90 ATPase activity. Previous studies have shown that some natural products, such as celastrol [18], withaferin A [19] and FW-04-086 [20], exhibit antitumor activity by inhibiting Hsp90 function through disruption of Hsp90-Cdc37 interaction. To date, however, no potent synthetic inhibitors of the Hsp90-Cdc37 interaction have been reported.

Our previous study showed that the designed triazine compounds targeted to N-terminal of Hsp90 exert antitumor activity by inhibiting Hsp90 function [21]. In the current study, we found that DCZ3112, a novel derivative of triazine, disrupted Hsp90-Cdc37 interaction, thereby leading to the degradation of Hsp90 client proteins. DCZ3112 showed antitumor activity predominantly in HER2-positive breast cancer and exhibited synergistic effects when combined with anti-HER2 antibodies. Importantly, DCZ3112 overcame acquired drug resistance to anti-HER2 antibodies in BT474 breast cancer cells, and also exhibited synergistic combinational effects. Thus, DCZ3112 alone or combined with anti-HER2 antibodies may constitute a targeted approach against HER2-positive breast cancer, especially for those with acquired resistance to anti-HER2 antibodies. These findings will also aid in the rational design of novel Hsp90-targeted drugs.

Section snippets

Materials

DCZ3112 was synthesized by Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). Geldanamycin (GM) was purchased from Sangon Biotech (Shanghai, China). Trastuzumab and pertuzumab was purchased from Roche (Basel, Switzerland).

Appropriate antibodies specific for Cdc37, HER2, AKT, CDK4, CDK6, PARP, Caspase-3, Cleaved caspase-3, Caspase-8, Caspase-9, Rb, P-Rb, Cyclin D, P-AKT^S473, P-ERK1/2, and EGFR were purchased from Cell Signaling Technology (Beverly, MA, USA).

DCZ3112 binds to the N-terminal domain of Hsp90 and disrupts Hsp90-Cdc37 interaction

Screening serial triazine derivatives using SPR assays led to the identification of DCZ3112 as a novel Hsp90 inhibitor (Fig. 1A). Like the Hsp90 inhibitor GM, DCZ3112 directly bound to recombinant human full-length Hsp90α in a concentration-dependent manner, exhibiting a dissociation constant (KD) of 4.98 μM (Fig. 1B). We next employed a fluorescence polarization assay, a classic method for screening Hsp90 inhibitor binding to the ATP pocket. Unexpectedly, DCZ3112 failed to compete with FITC-GM

Discussion

HER2 is one of the most sensitive client proteins of Hsp90, and Hsp90 expression is significantly higher in breast cancer cells than in normal cells [27,28]. Accordingly, Hsp90 inhibitors have been considered potential therapeutic agents for HER2-positive breast cancer, and there is some evidence that these agents exert antitumor activity [29,30]. However, the low selectivity and high toxicity of traditional Hsp90 inhibitors and the “protective” heat shock response to them limit their clinical

Conflicts of interest

None.

Acknowledgements

We thank Professor Chrisostomos Prodromou and Eileen Hickey for their generous gifts of pRSETA-yHsp90 and pUC-Hsp90α plasmids, respectively.

This research was supported by grants from the National Natural Science Foundation of China (№ 81273546) and the Shanghai Science and Technology Committee (№ 18DZ2293200).

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Citation Excerpt :
Furthermore, DCZ3112 was shown to overcome resistance to either antibody. Molecular docking studies suggest the compound operates via competitive binding to the Hsp90 NTD to displace Cdc3763. In 2019, Wang and colleagues64 used molecular dynamics simulations and mutagenesis studies to discover a novel binding interaction between Glu-47 and Gln-133 on Hsp90 and Arg-167 on Cdc37.
The 90-kiloDalton (kD) heat shock protein (Hsp90) is a ubiquitous, ATP-dependent molecular chaperone whose primary function is to ensure the proper folding of several hundred client protein substrates. Because many of these clients are overexpressed or become mutated during cancer progression, Hsp90 inhibition has been pursued as a potential strategy for cancer as one can target multiple oncoproteins and signaling pathways simultaneously. The first discovered Hsp90 inhibitors, geldanamycin and radicicol, function by competitively binding to Hsp90's N-terminal binding site and inhibiting its ATPase activity. However, most of these N-terminal inhibitors exhibited detrimental activities during clinical evaluation due to induction of the pro-survival heat shock response as well as poor selectivity amongst the four isoforms. Consequently, alternative approaches to Hsp90 inhibition have been pursued and include C-terminal inhibition, isoform-selective inhibition, and the disruption of Hsp90 protein−protein interactions. Since the Hsp90 protein folding cycle requires the assembly of Hsp90 into a large heteroprotein complex, along with various co-chaperones and immunophilins, the development of small molecules that prevent assembly of the complex offers an alternative method of Hsp90 inhibition.

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¹: These authors have contributed equally to the work.

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Original ArticlesDCZ3112, a novel Hsp90 inhibitor, exerts potent antitumor activity against HER2-positive breast cancer through disruption of Hsp90-Cdc37 interaction

Highlights

Abstract

Introduction

Section snippets

Materials

DCZ3112 binds to the N-terminal domain of Hsp90 and disrupts Hsp90-Cdc37 interaction

Discussion

Conflicts of interest

Acknowledgements

Mol. Cell. Proteomics : MCP

Canc. Cell

Canc. Cell

Curr. Opin. Pharmacol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Cell

Biochem. Pharmacol.

Canc. Cell

J. Biol. Chem.

Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene

Science

Prognostic impact of human epidermal growth factor-like receptor 2 and hormone receptor status in inflammatory breast cancer (IBC): analysis of 2,014 IBC patient cases from the California Cancer Registry

Breast Cancer Research : BCR

Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer

J. Clin. Oncol. : Official Journal of the American Society of Clinical Oncology

Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects

Canc. Res.

Lapatinib for breast cancer: a review of the current literature

Expet Opin. Drug Saf.

Lapatinib: current status and future directions in breast cancer

Oncol

Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group

J. Clin. Oncol. : Official Journal of the American Society of Clinical Oncology

Advances in the discovery and development of heat-shock protein 90 inhibitors for cancer treatment

Expet Opin. Drug Discov.

Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions

Expet Opin. Invest. Drugs

Original Articles
DCZ3112, a novel Hsp90 inhibitor, exerts potent antitumor activity against HER2-positive breast cancer through disruption of Hsp90-Cdc37 interaction