Targeting heat shock protein 90 for anti-cancer drug development.

CHAPTER FOUR Targeting heat shock protein 90 for anti-cancer drug development

Anthony Aswad and Tuoen Liu* Department of Biomedical Sciences, West Virginia School of Osteopathic Medicine, Lewisburg,

WV, United States *Corresponding author: e-mail address: tliu@osteo.wvsom.edu

Contents 1. Introduction 180 2. HSP 90 and cancer 181

3. Heat shock protein 90 as an anti-cancer drug target

184 4. Conclusions 195 References 196 Abstract Introduction: Heat shock proteins (HSPs) constitute a large family of proteins involved in protein folding and maturation. HSP expression is induced by heat shock or other stressors including cellular damage and hypoxia. The major groups, which are classified based on their molecular weight, include HSP27, HSP40, HSP60, HSP70, HSP90, and large

HSP (HSP110 and glucose-regulated protein 170). HSPs play a significant role in cellular proliferation, differentiation, survival, apoptosis, and carcinogenesis. The human HSP90 family consists of five members and has a strong association with cancer.

Objectives: The primary objective is to review the important functions of heat shock protein 90 in cancer, especially as an anti-cancer drug target.

Results: The HSP90 proteins not only play important roles in cancer development, progression, and metastasis, but also have potential clinical use as biomarkers for cancer diagnosis or assessing disease progression, and as therapeutic targets for cancer ther- apy. In this chapter, we discuss the roles of HSP90 in cancer biology and pharmacology, focusing on HSP90 as an anti-cancer drug target. An understanding of the functions and molecular mechanisms of HSP90 is critical for enhancing the accuracy of cancer diag- nosis as well as for developing more effective and less toxic chemotherapeutic agents.

Conclusion: We have provided an overview of the complex relationship between cancer and HSP90. HSP90 proteins play an important role in tumorigenesis and may be used as potential clinical biomarkers for the diagnosis and predicting prognostic outcome of patients with cancer. HSP90 proteins may be used as therapeutic targets for cancer therapy, prompting discovery and development of novel chemotherapeutic agents.

Advances in Cancer Research, Volume 152 Copyright # 2021 Elsevier Inc.

ISSN 0065-230X All rights reserved. https://doi.org/10.1016/bs.acr.2021.03.006

179 Abbreviations 17-AAG 17-N-Allylamino-17-demethoxygeldanamycintanespimy cin, tanespimycin

17-DMAG 17-dimethylaminoethylamino-17-demethoxygeldanamycin, alvespimycin

ADP adenosine diphosphate AIF apoptosis-inducing factor

AKT protein kinase B ALL acute lymphoid leukemia ATP adenosine triphosphate

ATPase adenosine triphosphatase 9 AX aminoxyrone B-RAF v-Raf murine sarcoma viral oncogene homolog B

BID BH3-interacting domain death agonist CDK cyclin-dependent kinase

CML chronic myeloid leukemia EGFR epidermal growth factor receptor

ER endoplasmic reticulum ERK Ras/extracellular signal-regulated kinase

GA geldanamycin GRP glucose-regulated protein HAD histone deacetylase

HER2 human epidermal growth factor receptor 2 HIF hypoxia inducible factors

HSC protein heat-shock cognate protein HSE heat shock element

HSF heat shock factor HSP heat shock protein HSR heat shock response

IL interleukin JAK Janus kinase NPC nasopharyngeal carcinoma

PI3K phosphatidylinositol-4,5-bisphosphate 3-kinase

STAT signal transducer and activator of transcription

1. Introduction Heat shock proteins (HSPs) are a group of proteins that function to reverse or inhibit denaturation or unfolding of cellular proteins in response to stress or high temperature. Traditionally, HSPs have also been known as molecular chaperones because of their physiological and protective roles in cells. They facilitate protein folding and maintenance of natural struc- ture and function when cells are exposed to homeostatic challenges such as extreme temperature, anoxia, hypoxia, heavy metals, drugs, or other chemical

180 Anthony Aswad and Tuoen Liu agents that may induce stress or protein denaturation (Liu, Daniels, & Cao,

2012; Macario & Conway de Macario, 2007). HSP are generally classified based on their molecular weight with the majority of them belonging to the groups of HSP27(HSPB1), HSP40, HSP60, HSP70, HSP90, and large

HSP [HSP110 and glucose-regulated protein 170 (GRP170)] (Ciocca &

Calderwood, 2005). Heat shock factors (HSFs) act as inducible trans- criptional regulators of HSP and are required for the expression of a major- ity of the HSPs. Heat shock elements (HSEs) are cis-acting sequences located upstream of HSP genes where HSFs bind to and induce HSP gene expression (A˚ kerfelt, Morimoto, & Sistonen, 2010). Except for the small

HSP group, other HSP family members, including HSP90 proteins, are

ATP-dependent proteins with adenosine triphosphatase (ATPase) activity (Bepperling et al., 2012). In other words, they are ATP binding chaperones with intrinsic ATPase functions which hydrolyze ATP into ADP.

Hydrolysis of ATP initiates the conformational change of HSPs and further causes substrate binding to them ( Jakob, Scheibel, Bose, Reinstein, &

Buchner, 1996; Sullivan & Pipas, 2002).

2. HSP 90 and cancer The nomenclature of human HSP is based on the system assigned by the Human Genome Organization (HUGO) Gene Nomenclature

Committee which uses the Entrez Gene database from the National

Center of Biotechnology Information. The HSP90 family is composed of five members encoded by the HSPC genes (HSPC1 to HSPC5) (Table 1)

Table 1 Human HSP90 (HSPC) family members.

HSPC Gene name Protein name Old/other common name(s)

Human gene ID 1 HSPC1 HSPC1 HSP90AA1; HSPN; LAP2; HSP86;

HSPC1; HSPCA; HSP89; HSP90; HSP90A; HSP90N; HSPCAL1;

HSPCAL4; FLJ31884 3320 2 HSPC2 HSPC2 HSP90AA2; HSPCA; HSPCAL3;

HSP90ALPHA 3324 3 HSPC3 HSPC3 HSP90AB1; HSPC2; HSPCB; D6S182;

HSP90B; FLJ26984; HSP90-beta 3326 4 HSPC4 HSPC4 HSP90B1; ECGP; GP96; TRA1; GRP94;

Endoplasmin 7184 5 HSPC5 HSPC5 TRAP1; HSP75; HSP90L

10,131 181 HSP90 as a drug target (Kampinga et al., 2009). The HSP90 family has received much attention due to their important roles in cancer biology and as the potential targets for chemotherapeutic agents. HSP90 is probably the most well studied member of the family. A large volume of review papers have described the structure, function of HSP90 proteins, and its role in health and disease (e.g., cancer, neurodegenerative, psychiatric, and cardiovascular diseases) (Bohush, Bieganowski, & Filipek, 2019; Condelli et al., 2019; Criado- Marrero et al., 2018; Ranek, Stachowski, Kirk, & Willis, 2018).

HSP90 is primarily located in the cytoplasm (Pearl & Prodromou, 2006) and forms flexible homodimers and its basic structure comprises three parts: the amino-terminal (N-terminal) domain (
25kDa), the middle domain (
40kDa), and the carboxyl-terminal (C-terminal) domain (
12kDa).

The N-terminal domain shows homology not only among the HSP90 family members but also to members of the ATPase/kinase superfamily including DNA gyrase, histidine kinase, and DNA mismatch repair enzyme MutL (Prodromou & Pearl, 2003). This domain is the binding site for nucleotides and drugs such as geldanamycin (GA) and tanespimycin (17-allylamino, 17-demethoxygeldanamycin, 17-AAG). The middle domain, which contains the catalytic loop that consists of three regions (a 3-layer α-β-α sandwich, a 3-turn α-helix and irregular loops, and a 6-turn α-helix), serves as the binding site for the γ-phosphate of ATP (also known as the Bergerat pocket) and HSP90 client proteins. The C-terminal domain functions in facilitation of HSP90 dimerization and co-chaperones binding. It possesses an alternative ATP-binding site when the Bergerat pocket is occupied, and is also the binding site for small molecules such as nucleotides, novobiocin and cisplatin (S€oti, Ra´cz, & Csermely, 2002; Whitesell & Lindquist,

2005). The structure of HSP90 dimers and the functions of each domain are shown in Fig. 1 (Whitesell & Lindquist, 2005).

As mentioned above, HSP90 family members are ATP-dependent molecules with intrinsic ATPase activity. The ATPase activity of HSP90 is crucial for their mechanism of action that assists in protein folding, client protein maturation and trafficking. More specifically, binding of ATP in the ATP binding cleft of the N-terminal domain of HSP90 induces the following conformational changes: part of the N-domain (ATP-lid) trans- locates over the ATP binding pocket and attaches to the corresponding

N-domain of the other homodimer, resulting in a twisted, compacted dimer. As a result, the N- and middle domains get closer together and form the “split ATPase” site. After ATP hydrolysis, the N-domains of the HSP90 homo-dimers dissociate with the release of ADP and phosphate, while the

HSP90 returns to its original open conformation. The ATPase cycle of

HSP90 is modeled in Fig. 2.

182 Anthony Aswad and Tuoen Liu N-Terminus: ATP binding domain.

ATPase acƟvity.

Middle Domain:

Client Protein and Co-chaperone Binding Region C- Terminus:

Region of HSP90 DimerizaƟon Example N-terminus inhibitors: geldanamycin, 17- AAG, 17-DMAG

Example Middle domain inhibitors:

MG132, Platycodin D Example C-terminus domain inhibitors:

NCT-50, Aminoxyrone Fig. 1 Basic Structure of HSP90 and common target sites of HSP inhibitors. Functional

HSP90 forms homodimers with three different domains joined together by flexible linkers. The N-terminal domain contains the ATP binding site and is the target of

HSP90 inhibitors including geldanamycin and its derivatives, such as 17-AAG and

17-DMAG (Mellatyar et al., 2018). The middle domain is the site of client protein and co-chaperon binding. Some HSP90 inhibitors such as platycodin D inhibit the binding of HSP90 to its co-chaperon or client proteins (Li et al., 2017). Other inhibitors, like

MG132, cleave HSP90 at the middle domain (Park, Park, Yoo, Park, & Lee, 2017). The

C-terminal domain of HSP90 is responsible for HSP90 dimerization. Some inhibitors such as Aminoxyrone target this domain and prevent HSP90 dimerization (Bhatia et al., 2018).

ATP ATP Misfolded Client Protein (i.e. Raf, etc.) Properly Refolded

Client Protein (i.e.

Raf, etc.) N-Terminus: ATP binding domain Middle Domain:

Client Protein Binding Region C-Terminus:

Region of DimerizaƟon Misfolded Client Protein (i.e. Raf, etc.)

ATP ATP ADP ADP Open ConformaƟon Closed ConformaƟon

Fig. 2 The model of ATPase cycle of HSP90. Binding of ATP in the ATP binding cleft of the N-terminal domain of HSP90 induces the conformational changes. After ATP hydro- lysis, the N-domain of the HSP90 homo-dimers dissociate with the release of ADP and phosphate, while the HSP90 returns to its original open conformation.

HSP90 client proteins include those who are involved in crucial signal transduction pathways such as AKT (PI3K/AKT pathway), IL-6 receptor (JAK/STAT pathway), Bcr-Abl (RAS/ERK pathway), cyclin-dependent kinases (CDKs, cell cycling), and IκB kinases (NF-κB pathway) (Suzuki et al., 2015). The cancer relevant HSP90 client proteins include EGFR,

IGF-1R, Cdk4, AKT, ErbB2, c-Met, Bcr-Abl, RET, androgen receptors,

Fms like tyrosine kinase 3 (FLT3), B-Raf, NF-kB, Raf-1, HER2/Neu,

NPM-ALK, p53, neuronal nitric oxide synthase (nNOS) and HIF-1α (Kamal, Boehm, & Burrows, 2004; Suzuki et al., 2015). Some HSP90 inhib- itors, such as GA and radicicol, bind specifically to HSP90 in a way similar to ATP, thus inhibiting HSP90 ATPase activity (Hoter, El-Sabban, &

Naim, 2018).

HSP90 has involvement in carcinogenesis through the regulation of tumor growth, adhesion, invasion, metastasis, angiogenesis and apoptosis.

Overexpression of HSP90 has been associated with poor cancer prognosis.

Multiple studies reveal that HSP90 is overexpressed in various cancer types including pancreatic, ovarian, breast, lung, endometrial, oropharyngeal squamous cell carcinoma, bladder cancer, leukemia, and multiple myeloma (Burrows, Zhang, & Kamal, 2004; Huang, Chen, et al., 2014; Kolosenko,

Grander, & Tamm, 2014; McCarthy et al., 2008; Patel et al., 2014; Shi et al.,

2014; Tian et al., 2014; Zˇ a´ckova´ et al., 2013).

3. Heat shock protein 90 as an anti-cancer drug target

Due to these crucial roles, HSP90 has been considered a potential therapeutic target to inhibit tumor development and progression, with var- ious HSP90 inhibitors tested in clinical trials (Wu et al., 2017). Some molec- ular mechanisms of how inhibition of HSP90 regulates cancer cell functions have been revealed. For example, focal-adhesion kinase (FAK) and integrin linked kinase (ILK) are two key players promoting cell-adhesion. Inhibition of HSP90 stimulates the protease-mediated degradation of FAK and ILK in a variety of cancer cells (Aoyagi, Fujita, & Tsuruo, 2005; Ochel, Schulte,

Nguyen, Trepel, & Neckers, 1999). Hepatocyte growth factor (HGF) stimulates cell motility and angiogenesis via the activation of a downstream tyrosine kinase signaling cascade. Inhibition of HSP90 decreases cell motility and angiogenesis via disruption of HIF functions in human T24 bladder can- cer cells (Koga, Tsutsumi, & Neckers, 2007). HSP90 inhibitors also decrease angiogenesis via inhibition of vascular endothelial growth factor (VEGF) receptor expression and signaling in colorectal cancer cells (Wang et al.,

2016). Inhibition of HSP90 down-regulates both HIF-1α and NF-κB levels

184 Anthony Aswad and Tuoen Liu leading to inhibition of epithelial-mesenchymal transition (EMT), motility, and invasiveness in colorectal cancer cells (Nagaraju et al., 2015). Inositol hexakisphosphate kinase-2 (IP6K2) facilitates mammalian cell death which is required for p53-mediated apoptosis (Koldobskiy et al., 2010). HSP90 physiologically binds to IP6K2 and inhibits its catalytic activity, further preventing apoptosis. Thus, inhibition of HSP90-IP6K2 interaction leads to cell death in cancer cells (Chakraborty et al., 2008).

There are several advantages for the development of HSP90 inhibitors as anti-cancer drugs: (1) HSP90 inhibitors can simultaneously target multiple signaling pathways because many of the signaling proteins are HSP90 client proteins. Therefore, tumor cells are much less likely to escape a single-target therapy. (2) HSP90 inhibitors can maximize target-specific damage in tumor tissues with minimal toxicities in normal tissues. (3) The combination of

HSP90 inhibitors and proteasome inhibitors leads to the accumulation of unfolded proteins, which are insoluble and toxic to cancer cells. Here, we discuss the important HSP90 inhibitors which have been reported.

Geldanamycin (GA), a benzoquinone ansamycin antibiotic, was reported as the first HSP90 inhibitor to be evaluated as an antitumor agent.

It showed potent antitumor activity but with significant hepatotoxicity in animal models (Supko, Hickman, Grever, & Malspeis, 1995). GA binds to HSP90 and inhibits cell migration associated with downregulation of

HIF-1α and phosphorylation of FAK in glioma cells. It also induces apopto- sis in a caspase-dependent manner through activation of caspase-3 together with release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria in glioma cells (Nomura et al., 2004). 17-allylamino- 17-demethoxy-geldanamycin (17-AAG), also known as tanespimycin, is a derivative of geldanamycin that binds to the ATP binding, N-terminus of

HSP90 (Talaei et al., 2019). 17-AAG has been studied in various cancers in both preclinical and clinical trials. Pre-clinical trials have demonstrated

17-AAG’s potential pharmacological use in various cancers such as prostate and colon, but also illustrated its poor water solubility and consequential hepatotoxicity (Talaei et al., 2019). However, 17-AAG still reached clinical trials and was tested in an array of blood cancers (e.g., chronic lymphocytic leukemia), and solid tumors (e.g., metastatic thyroid cancer). These trials, both phase I and phase II, indicated that 17-AAG alone did not show significant clinical effects (Talaei et al., 2019). However,17-AAG may be used in combinational therapies to improve the efficacy of current cancer therapies such as radiation and with other pharmacologic interventions.

For instance, 17-AAG enhanced the effects of Raf-kinase inhibitors in patients with renal cancer (Talaei et al., 2019). 17-AAG entered phase III

185 HSP90 as a drug target clinical trials for patients with multiple myeloma but its development was halted due to lapsed patent protection (Georgakis, Li, & Younes, 2006;

The Myeloma Beacon Staff, 2010).Currently, the major utility for

17-AAG lies in its potential for combinational therapies, as its adverse side effects limit its efficacy as a single agent.

Another chemical, 17-Dimethylaminoethylamino-17-demethoxygel- danamycin (17-DMAG) is also a derivative of geldanamycin and was devel- oped to address the limitations of 17-AAG (Talaei et al., 2019). Like geldanamycin, 17-DMAG binds to the ATP-binding region of HSP90 and inhibits its chaperone ability, thus preventing client protein stabilization, leading to subsequent client protein degradation. In pre-clinical trials,

17-DMAG demonstrated tumor suppression and induced apoptosis in various cancers such as neuroblastomas and colon (Mellatyar et al., 2018).

In phase I and II trials, 17-DMAG demonstrated therapeutic potential with less of the toxic side effects of 17-AAG, although 17-DMAG did pose risks of ocular and cardiac toxicities (Mellatyar et al., 2018). Even with such clinical phase trials, 17-DMAG has not been approved by the FDA for can- cer therapy. Retaspimycin hydrochloride (IPI-504) is a potent HSP90 inhib- itor and also a derivative of GA. It has shown antitumor activity in phase I/II trials in patients with gastrointestinal stromal tumors and soft-tissue sarcomas.

However, the development of IPI-504 was discontinued in 2013 due to safety concerns of hepatotoxicity(Wagner et al., 2013).

BIIB021 is a novel HSP90 inhibitor which sensitizes esophageal squamous cell carcinoma cells to radiation treatment. Phase II studies of

BIIB021 showed positive results in patients with gastrointestinal stromal tumors (Dickson et al., 2013; Wang, Bao, et al., 2014). A phase I study of BIIB028, a selective HSP90 inhibitor, showed positive results in patients with refractory metastatic or locally advanced solid tumors (Hong et al.,

2013). A phase I trial of PF-04929113 (SNX-5422, an orally bioavailable selective HSP90 inhibitor) by oral administration showed good tolerance and encouraging antitumor activity in patients with refractory solid tumors and hematologic malignancies (Rajan et al., 2011; Reddy et al., 2013).

HSP90 inhibitor NVP-AUY922 induces cell death in adrenocortical, lung, colorectal, thyroid, neuroendocrine carcinoid, multiple myeloma, adult

T-cell leukemia-lymphoma, and chronic myeloid leukemia (CML) cancer cells and is currently being tested in clinical trials in patients with lung cancer, non-Hodgkin lymphoma and relapsed or refractory multiple mye- loma( Johnson et al., 2015; Lecia, 2016; Lee, Sung, Bartlett, Kwon, & Lee,

2015; Oki et al., 2015; Seggewiss-Bernhardt et al., 2015). SST0116CL1 (4-Amino substituted resorcino-isoxazole) is a potent second-generation

186 Anthony Aswad and Tuoen Liu selective HSP90 inhibitor which has better solubility and less or absent hepa- totoxicity that showed antitumor effects in murine models of leukemia , gastric and ovarian carcinomas (Vesci, Milazzo, Carolo, Pace, & Gianini, 2014).

It inhibits tumor growth via binding to the ATP binding pocket of

HSP90 and interfering with HSP90 chaperone function (Vesci et al., 2014).

AT13387 (onalespib) is another potent second-generation selective

HSP90 inhibitor which inhibits cell growth in C666-1EBV-positive naso- pharyngeal carcinoma (NPC) cells and induced cellular senescence by downregulation of multiple HSP90 client oncoproteins including EGFR,

AKT and Cdk4 in vitro and decreased both the number and size of

C666-1 tumors in xenograft mouse models of NPC in vivo (Chan et al.,

2013). AT13387 was tested in clinical phase I trials for patients with advanced solid tumors and has demonstrated preliminary antitumor activity alone or in combination with imatinib (Do et al., 2015; Shapiro et al., 2015;

Wagner et al., 2016). Gamitrinibs is a class of small molecules selectively targeting HSP90 in human tumor mitochondria (Kang et al., 2009).

Gamitrinibs can accumulate in the mitochondria of human tumor cells to inhibit HSP90 activity by acting as an ATPase antagonist with

“mitochondriotoxic” activity, inducing cancer cell death and inhibiting the growth of human tumor xenografts without toxicity to normal tissues in mice (Kang et al., 2009). In addition, gamitrinibs exhibited preclinical activity and favorable safety in a xenograft mouse model of advanced pros- tate cancer in vivo (Kang et al., 2010).

Aminoxyrone (AX) is a peptidomimetic HSP90 inhibitor that targets the

C-terminal domain of HSP90 (Bhatia et al., 2018) and was first tested in tyrosine kinase inhibitor resistant CML cells (ponatinib resistant cell line

BAIF3 and imatinib resistant cell lines K562, SUP-B15, and KCL 22) and patient derived samples. AX was found to mimic the 28-helical con- formation required to specifically bind to the C-terminal domain and prevent HSP90 dimerization. Subsequent destabilization and degradation of BCR-ABL1 was observed in the absence of the heat shock response (Bhatia et al., 2018). Patient derived samples were used to assess AX’s ability to suppress leukemia stem cells and it was shown that AX significantly inhibited proliferation and induced apoptosis in leukemic fractions. Using cord blood samples also demonstrated that AX had significantly less cyto- toxic effects on healthy cells than on the previously mentioned leukemic fractions, demonstrating AX’s potential as a therapeutic pharmacologic

HSP90 inhibitor in CML (Bhatia et al., 2018).

NCT-50 [5-methoxy-N-(3-methoxy-4-(2-(pyridin-4-yl) ethoxy) phenyl)-2,2-dimethyl-2H-chromene-6-carboxamide] is an analog of

187 HSP90 as a drug target novobiocin and deguelin that has HSP90 inhibitory effects in non-small cell lung cancer (NSCLC) (Hyun et al., 2018). Both the antibiotic novobiocin and rotenoid deguelin have HSP90 inhibitory and anticancer capabilities.

Similar to AX, NCT-50 binds to the C-terminus of HSP90 and prevents

HSP90 interaction with its client proteins, disrupting the HSP90 chaperone function and eventually leading to cell death. It was noted that the anticancer potential was observed with minimal cytotoxic effects in healthy cells (Hyun et al., 2018).

AT533 is a HSP90 inhibitor that has demonstrated potential anti-tumor capabilities in breast cancer (Zhang et al., 2020). AT533 targets the ATPase pocket of HSP90 and disrupts its chaperone function, subsequentially dis- rupting HIF 1 alpha/VEGF signaling in MCF-7 and MDA-MB-231 breast cancer cell lines. By disrupting this process, AT533 was shown to reduce breast cancer viability in both in vitro cell and in vivo MDA-MB-231 cell xenograft models (Zhang et al., 2020).

Histone deacetylases (HDACs) can regulate the function of some non-histone structures including HSP90. The HDAC6 has demonstrated an important role in HSP90 chaperone capability (Hsieh, Tu, Pan, Liou,

& Yang, 2019). MPT0G211 is an HDAC inhibitor that selectively inhibits the activity of HDAC6. MPT0G211 was shown to inhibit HDAC6 func- tion which resulted in a subsequent hyper-acetylation of HSP90 in breast cancer cell and mouse models (Hsieh et al., 2019). In the hyper-acetylated state, HSP90 function is diminished leading to the degradation of oncogenic client proteins and consequentially reduced viability of breast cancer cells.

Thus, MPT0G2011 demonstrates the potential for HSP90 inhibitors to molecularly act on proteins upstream of HSP90 for cancer therapy. (Hsieh et al., 2019).

Another mechanism of HSP90 inhibition is to deactivate the protein via direct cleavage. Carbobenzoxy, -Leu, -Leu, -leucinal, or MG132, is a proteasome inhibitor that has been shown to induce HSP90 cleavage at the middle domain in K562 CML cells (Park et al., 2017). Specifically,

MG132 increases the generation of reactive oxygen species (ROS) by upregulating the antioxidant inhibitor VDUP-1 and downregulating the antioxidant glutathione. Generation of ROS induces caspase 10 activity which cleaves HSP90. MG132 induced cleavage was only observed in the K562 cell line although it was tested in other cell lines, suggesting that

MG132 use may be limited to only certain cancer types (Park et al., 2017).

NW457, a radicicol derivative, has demonstrated HSP90 inhibitory cap- ability in the HCT116 human colorectal cancer cell line (Kinzel et al., 2016).

188 Anthony Aswad and Tuoen Liu Administration of NW457 in vitro induced a significant decrease in HSP90 cancer client proteins EGFR, EPHAL and BRAF. The degradation of these client proteins was not associated with hepatotoxicity like in

17-AAG. NW457’s reduced toxicity is attributed to its greater bio- availability and water solubility when compared to the original HSP90 inhibitors (Kinzel et al., 2016). NW457 alone only shortly delayed tumor growth, but when combined with radiation, tumor growth was signifi- cantly reduced in vivo. This synergistic effect of NW457 with radiation illustrates the potential for HSP90 inhibitors to enhance current cancer therapies (Kinzel et al., 2016).

KW-2478 is a nonansamycin HSP90 inhibitor that was first found to inhibit the proliferation of multiple myeloma cells through the degradation of novel HSP90 client proteins like FGFR3 (Nakashima et al., 2010). In phase I studies for the use of KW-2478 in patients with multiple myeloma or non-Hodgkin lymphoma, intravenous administration of KW-2478 was well-tolerated and contributed to stabilization of disease progression (Yong et al., 2016). Phase I and phase II studies revealed the utility of KW-2478 in combination therapy with bortezomib, a proteasome inhibitor, in patients with relapsing or refractory multiple myeloma. A significant inhibition in tumor regression was not observed, but the tolerability of KW-2478 indicates its potential in future pharmacological interventions for multiple myeloma treatment (Cavenagh et al., 2017).

Platycodin D, a bio-active isolate from roots of Platycodon grandifloras, shows anticancer activity through downregulating EGTR and HFR-2 proteins in breast cancer models. It is also an HSP90 inhibitor which binds to the pocket of HSP90’s necessary co-chaperone, Cdc37, and disrupts the chaperone capability of HSP90. (Li et al., 2017). This mechanism is different from disruption of the ATPase activity of HSP90, which is a common molecular mechanism for many of the HSP90 inhibitors.

HCP1-HCP6 are coumarin pyrazoline derivatives that target the

N-terminus of HSP90 and have demonstrated the ability to induce apoptosis in the A549 human lung cancer cell line. These compounds also blocked the autophagic reflex, which has become a promising target in cancer therapy (Wei et al., 2018).

Many other HSP90 inhibitors are in developmental stages and show anti- tumor properties against various cancers (Costa, Raghavendra, & Penido,

2020; Koren 3rd & Blagg, 2020; Wu et al., 2017). HSP90 inhibitors with their applications and molecular mechanisms in cancer are summarized in alphabetic order in Table 2. Since the study of the earliest HSP90 inhibitor

189 HSP90 as a drug target Table 2 Development of HSP90 inhibitors for cancer therapy.

Drug name Tested cancer types Molecular mechanism References

2-Amino-7-[4-fluoro-2- (3-pyridyl)phenyl]-4- methyl-7,8-dihydro-6H- quinazolin-5-one oxime

HCT-116 colorectal xenograft model Targets N-terminal ATPase site

Amici et al. (2014) 3,4-diarylpyrazole (CCT018159)

HCT116 colon cancer cells Inhibits ATPase activity

Cheung et al. (2005) 17-AAG Multiple cancer cell lines

Geldanamycin derivative. Binds to the ATP binding region, preventing client protein stabilization and inducing degradation

Amici et al. (2014), Talaei et al. (2019) 17-DMAG Multiple cancer cell lines

Geldanamycin derivative. Binds to the ATP binding region, preventing client protein stabilization and inducing degradation

Mellatyar et al. (2018) 17-DMCHAG Prostate cancer Geldanamycin derivative. Binds to the

ATP binding region, preventing client protein stabilization and inducing degradation

Wang et al. (2015) Aminoxyrone (AX) Multiple CML cell lines (including ponatinib and imatinib resistant cell lines) and patient derived CML cell lines

Binds to C-terminus and prevents HSP90 dimerization

Bhatia et al. (2018) AT13387 Nasopharyngeal, GI Induces senescence

Chan et al. (2013) AT 533 Human breast cancer cell lines (MDA-MB-231 and MCF-7) and human umbilical vein endothelial cells

Binds to the ATP binding region preventing client protein stabilization and inducing degradation. Prevents angiogenesis

Zhang et al. (2020) BIIB021 Various trails in both solid tumors and hematological cancers

Binds to the ATP binding region, preventing client protein stabilization and inducing degradation

He and Hu (2018), Yan, Zhang, Zhang, Xuan, and Wang (2017)

BJ-B11 CML K562 cells Induces apoptosis Ju et al. (2011)

Celastrol CML cells Depletes Bcr-Abl and induces apoptosis

Lu, Jin, Qiu, Lai, and Pan (2010) CH5164840 Gastric, breast

Induces oncogenic client protein degradation and apoptosis

Ono et al. (2012) CUDC-305 Multiple cancer cell lines, xenograft model of U87MG glioblastoma, animal models of

MDA-MB-468 breast cancer and MV4-11 acute myelogenous leukemia

Inhibits multiple signaling pathways (PI3K/AKT and RAF/MEK/ERK), induces apoptosis

Bao et al. (2009) DMAG-N-oxide B16 melanoma cells Inhibits migration and integrin/ extracellular matrix-dependent cytoskeletal organization

Tsutsumi et al. (2008) ()-epigallocatechin gallate (EGCG)

Prostate Binds to HSP90 C-terminus Moses, Henry, Ricke, and Gasiewicz (2015)

FK228 K562 leukemia cells Hyperacetylation of HSP70

Wang et al. (2007) FW-04-806 Breast Inhibits apoptosis by binding to

N-terminal of HSP90 and inhibits HSP90/Cdc37 interaction

Huang, Ye, et al. (2014) Gedunin MCF-7 and SkBr3 breast cancer cells

Not clear Brandt, Schmidt, Prisinzano, and Blagg (2008)

Continued Table 2 Development of HSP90 inhibitors for cancer therapy.—cont’d

Drug name Tested cancer types Molecular mechanism References

Ganetespib Gastric, breast, colorectal Induces G2/M cell cycle arrest and apoptosis

He et al. (2014), Proia et al. (2014) Geldanamycin (GA), original HSP90 inhibitor

Tested in many various cancer models Binds to the N-terminus and blocks

ATPase activity Sanchez, Carter, Cohen, and Blagg (2020)

HCP1-6 Lung cancer (cell line: A549) Binds to the N-terminus of HSP90.

Also blocks the autophagic reflex Wei et al. (2018)

Herbimycin A (HMA) Thyroid Inhibits growth and reverse EMT via activation of E-cadherin, p21 and p27 and inactivates p53 and PI3K/AKT pathway

Kim et al. (2014) HSP990 Glioblastoma Enhances PI3k inhibition

Wachsberger et al. (2014) IPI-493 Human GI stroma tumor xenografts

Induces apoptosis and necrosis, inhibits receptor kinase signaling

Floris et al. (2011) IPI-504 Lung cancer, mantle cell lymphoma

Down-regulates ER chaperone GRP78 Roue et al. (2011),

Sequist et al. (2010) KU135 Human leukemia cells Binds to HSP90 and causes degradation of HSP90 client proteins, induces cell cycle arrest and apoptosis

Shelton et al. (2009) KW-2478 Clinical trials multiple myeloma (Non-Hodgkins lymphoma)

Downregulates client protein Cavenagh et al. (2017), Nakashima et al. (2010), Yong et al. (2016)

MG132 CML (K562 cell line) Cleaves the middle domain

Park et al. (2017) MPT0G211 Human breast cancer cell lines (MDA-MB-231 and MCF-7)

HDAC6 inhibitor. Hyper-acetylation of HSP90 Hsieh et al. (2019)

Mycoepoxydiene Cervical cancer Modulates kinase signaling

Lin et al. (2015) N-(4-hydroxy-3-(2- hydroxynaphthalene- 1-yl)phenyl)- arylsulfonamides

SKBr3 breast cancer cells Inhibits ATPase activity with weak activity

Ganesh et al. (2008) NCT-50 NSCLC cell lines Interacts with the C-terminal domain and impairs client protein function

Hyun et al. (2018) Novobiocin HSP90 in vitro constructs

Interacts with C-terminus Marcu, Chadli, Bouhouche, Catelli, and Neckers (2000)

NVP-AUY 922 NCSLC, breast cancer and gastric cancer, non-Hodgkin lymphoma and relapsed or refractory multiple myeloma, colorectal, thyroid, neuroendocrine carcinoid, adult

T-cell leukemia-lymphoma, and chronic myeloid leukemia

Prevents stabilization of client proteins and reduces their expression

Johnson et al. (2015), Lecia (2016), Lee et al. (2015), Oki et al. (2015),

Seggewiss-Bernhardt et al. (2015) NVP-BEP800 Multiple myeloma

Inhibits survival pathway and causes client protein depletion

St€uhmer et al. (2009) NW457 Colorectal cancer Prevents protein client stabilization

Kinzel et al. (2016) NXD30001 Glioblastoma multiforme mouse model

Inhibits HSP90 target proteins Zhu et al. (2010) Onalespib

Prostate cancer cells Blocks mRNA splicing of androgen receptor

Ferraldeschi et al. (2016) Platycodin D Lung cancer

Prevents interaction between HSP90 and its co-chaperon, Cdc37

Li et al. (2017) Continued Table 2 Development of HSP90 inhibitors for cancer therapy.—cont’d

Drug name Tested cancer types Molecular mechanism References

PU-H71 Triple negative breast cancer Inhibits RAS/RAF/MAPK pathway, induces apoptosis via degradation of

AKT, BCL-XL Caldas-Lopes et al. (2009) TAS-116 Lung cancer mouse model

Depletes HSP90 client proteins Ohkubo et al. (2015)

SNX-2112 Liver, multiple myeloma Abrogates signaling via Akt and ERK

Okawa et al. (2009) SNX-25a multiple cancer cell lines

Arrests cell cycle, induce apoptosis Wang, Wang, et al. (2014)

SNX-7081 CLL Dysregulates proteins involved in DNA repair, replication and cell cycle

Che et al. (2013) SST0116CL1 (4-Amino substituted resorcinol- isoxazole)

Various cancer cell lines including NSLC, breast carcinoma and fibrosarcoma

Binds to ATP binding pocket of HSP90 and causes client protein degradation

Vesci et al. (2014) STA-1474 Osteosarcoma cell line

Activates caspase-3 activation and downregulates p-Met/Met and p-Akt/Akt

McCleese et al. (2009) Sti1 HSP90 in vitro constructs

Inhibitor of HSP90 ATPase. Binding to HSP90 prevents the N-terminal dimerization during the ATPase cycle

Richter, Muschler, Hainzl, Reinstein, and Buchner (2003)

Withaferin A Pancreatic Induces apoptosis and degradation of

HSP90 client proteins Yu et al. (2010) WK88-1 Lung

Decreases expression of HSP90 client proteins including EGFR, ErbB2,

ErbB3, MET and AKT Jang et al. (2014) XL888 NCI-N87 gastric cancer mouse xenograft model

Reduces HSP90 client proteins Bussenius et al. (2012)

GA in 1990s, much progress has been made toward clinical development of HSP90 inhibitors, which has been driven by optimization of their phar- maceutical properties including pharmacodynamics, pharmacokinetics and toxicology.

However, some of earlier clinical trials of HSP90 inhibitors failed due to their side effects or lack of anticancer activities due to the interaction of HSPs with each other such as HSP27 and HSP72, when one HSP is inhibited, other HSPs may be stimulated for overexpression to perform the same func- tions of inhibited HSP (Sidera & Patsavoudi, 2014).The clinical indications for the use of HSP90 inhibitors are still not fully understood and until now, no specific HSP90 inhibitor has been approved by the US Food and Drug

Administration (FDA) for cancer treatment. One possible reason may be due to the HSP90 inhibitor causing organ-specific toxicities (liver or ocular) or lack of convincing anticancer efficacy due to the crucial functions of HSPs in cells. Therefore, in order to optimally develop novel HSP90 inhibitor for cancer therapy there is a need to better understand the varying roles of

HSPs in cancer and normal cells and further investigate their mechanisms of action.

4. Conclusions Here we have provided an overview of the complex relationship between cancer and HSP90. HSP90 proteins play an important role in tumorigenesis and may be used as potential clinical biomarkers for the diag- nosis and predicting prognostic outcome of patients with cancer. HSP90 proteins are molecular chaperones and their expression and functions are activated during stress stimulation. Most HSP90 proteins have similar func- tions in carcinogenesis, prevention of apoptosis and conferring of drug resis- tance. In addition, HSP90 proteins may be used as therapeutic targets for cancer therapy, prompting discovery and development of novel chemother- apeutic agents. A variety of anti-cancer drugs that target HSP’s have been approved by the USA FDA for cancer treatment. For example, sorafenib (Nexavar®), a kinase inhibitor that reduces the expression of GRP78 in cancer cells, was approved by the USA FDA for treatment of renal cell car- cinoma (2005), hepatocellular carcinoma (2007) and locally recurrent or metastatic, progressive, differentiated thyroid carcinoma (2013) (Roberts et al., 2015). Ruxolitinib (Jakafi®) is a JAK inhibitor that also decreases the expression of HSP70 and HSP90 in cancer cells and animal models. It was approved by the USA FDA for the treatment of intermediate or

195 HSP90 as a drug target high-risk myelofibrosis (2011) and polycythemia vera (2014) (Tavallai,

Booth, Roberts, Poklepovic, & Dent, 2016). However, none of the specific

HSP90 inhibitors has been approved by the USA FDA for the treatment of patients with cancer. We propose three major possible reasons for this: (1) HSP are critical for the survival of the cells. Once the activities of

HSP are inhibited, the basic functions of cancer cells as well as normal cells will be severely affected. We still do not fully understand the different requirements in the amount of HSP between cancer cells and normal cells, which may provide a necessary therapeutic window for discovery and devel- opment of more efficacious and less toxic novel anticancer HSP inhibitors. (2) HSP inhibitors can cause severe organ-specific toxicities (liver or ocular) which are not easily managed. The functions of HSP are essential for both normal and tumor cells. It is difficult to identify the functions of HSP that are only present in cancer cells but not in normal cells. Identifying functions of cancer specific HSP may overcome HSP inhibitor induced organ-specific toxicities. (3) Some HSP inhibitors may lack convincing anticancer activity.

Different HSP family members interact and collaborate with each other in a signaling network to regulate cellular pathways. Inhibition of one HSP can stimulate the overexpression of other HSP members, compensating inhib- itory effects caused by a single HSP inhibitor. Therefore, combination of different HSP inhibitors may be a good strategy to enhance their anticancer efficacy in cancer therapy. In conclusion, HSP90 proteins have a significant role in cancer development and progression. Understanding the functions and molecular mechanisms of HSP90 proteins is critical for enhancing the accuracy of cancer diagnosis and for the development of more effective chemotherapeutic agents.

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