Hsp90 inhibitors as novel cancer chemotherapeutic agents

Hsp90 inhibitors as novel cancer chemotherapeutic agents

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002 A TRENDS Guide to Cancer Therapeutics | Review Hsp90 inhibitors as novel cancer chemother...

292KB Sizes 0 Downloads 59 Views

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002

A TRENDS Guide to Cancer Therapeutics

|

Review

Hsp90 inhibitors as novel cancer chemotherapeutic agents Len Neckers Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for the stability and function of multiple mutated, chimeric and over-expressed signaling proteins that promote the growth and/or survival of cancer cells. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, Akt, ErbB2 and hypoxia-inducible factor 1α (HIF-1α). Hsp90 inhibitors, by interacting specifically with a single molecular target, cause the destabilization and eventual degradation of Hsp90 client proteins, and they have shown promising antitumor activity in preclinical model systems. One Hsp90 inhibitor, 17-allylaminogeldanamycin (17AAG), is currently in phase I clinical trial. Because of the chemoprotective activity of several proteins that are Hsp90 clients, the combination of an Hsp90 inhibitor with a standard chemotherapeutic agent could dramatically increase the in vivo efficacy of the therapeutic agent. In the postgenomic era, identification of novel molecular targets for cancer therapeutics offers the promise of great specificity coupled with reduced systemic toxicity, but simultaneously faces the potential peril of being unable to deal successfully with diseases that are frequently caused by multiple genetic abnormalities (e.g. cancer). Indeed, a pessimistic viewpoint might hold that the more finely tuned our molecular therapies become, the less they will be able to confront the inherent genetic instability of cancer cells effectively. Partly because of their genetic plasticity, cancer cells are very efficient at adapting to a noxious environment, whether that be hypoxia, deprivation of certain hormones or growth factors, or exposure to chemotherapy or irradiation. Thus, hormone-dependent tumor cells eventually become hormone-independent, either by receptor mutation or by other means. Likewise, tumor cells exposed to hypoxia induce a multi-faceted transcriptional response geared to survive such an environment. Similarly, cancer cells exposed to levels of chemotherapy that are initially fatal eventually activate multiple and overlapping signaling pathways to protect themselves from further harm. One approach to this dilemma might be to target the very machinery that allows cancer cells to adapt so successfully to environmental stress. Cells respond to environmental stress by increasing synthesis of several molecular chaperones (also known as heat shock proteins, or Hsps, because they were first observed in cells exposed to elevated temperature). These housekeeping proteins, as their name implies, assist general protein folding and prevent nonfunctional side reactions such as the nonspecific aggregation of misfolded or unfolded proteins. One of the most abundant molecular chaperones in eukaryotes is Hsp90. Although drugs targeting other chaperone proteins remain to be identified, naturally http://www.trends.com

occurring specific inhibitors of Hsp90 have not only been identified, but have also been amply documented to have antitumor activity in various preclinical models. One such compound, the benzoquinone ansamycin 17-allylaminogeldanamycin (17AAG), is now in phase I clinical trial at five centers worldwide. Using geldanamycin to validate Hsp90 as a viable target for cancer therapeutics By the early 1990s, several groups reported the observation that Hsps in general, and Hsp90 in particular, were over-expressed in a wide variety of cancer cells and in virally transformed cells [1]. Hsp90 had been found in complex with the tyrosine kinase v-Src [2] and the serine/threonine kinase Raf-1 [3], but the contribution of the chaperone to the function of either enzyme remained obscure. Srivastava [4] had demonstrated that Hsps derived from tumor cells were excellent immunogens capable of inducing a robust host immune response, probably because tumor-cell-derived Hsps served as chaperones for various tumor antigens. In 1992, Ciocca et al. [5] related Hsp over-expression in breast cancer cells to acquired resistance to some forms of chemotherapy. With hindsight, one can see that these studies, in sum, suggested that Hsps might chaperone signaling proteins on which cancer cells were highly dependent. Also in 1992, Whitesell et al. reported that the antibiotic benzoquinone ansamycins exerted potent tumoricidal activity both in vitro and in vivo that was mechanistically and temporally distinct from their reported ability to inhibit kinases [6]. In fact, further observation ruled out direct kinase inhibition as a mechanism of action of these drugs, even though v-Src kinase activity was eventually lost from cells treated with these agents. Unexpectedly, the two 1992 papers published by Ciocca and Whitesell

1471-4914/02/$ – see front matter ©2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4914(02)02316-X

Len Neckers Cell and Cancer Biology Branch, National Cancer Institute, National Institutes of Health, 9610 Medical Center Drive, Suite 300, Rockville, MD 20850, USA. e-mail: [email protected]

S55

Review

|

A TRENDS Guide to Cancer Therapeutics

Figure 1. Inhibition of Hsp90 interferes with multiple signal transduction pathways Shown is a partial list of heat shock protein 90 (Hsp90) client proteins that are destabilized by geldanamycin (GA) and 17-allylaminogeldanamycin (17AAG). These include: the soluble kinases, v-Src, Akt, Raf-1 and Bcr-Abl; the transmembrane kinase ErbB2; and the transcription factors mutant p53 and hypoxiainducible factor 1α (HIF-1α). This list indicates why targeting a molecular chaperone is an attractive strategy for cancer therapy because inhibiting a single molecular target results in the interference in multiple signaling pathways that mediate cancer cell growth and survival.

Figure 2. Opposing activities of Hsp90 Heat shock protein 90 (Hsp90) participates in two multichaperone complexes with opposing activities. Hsp90 modifies its conformation, depending on the nucleotide occupancy of its amino-terminal nucleotide-binding pocket, to attract a unique group of co-chaperone proteins. When ATP is bound, the assembled ‘super-chaperone machine’, including the co-chaperone proteins p23 and p50Cdc37, binds to and stabilizes Hsp90 client proteins. When ADP is bound, the assembled superchaperone machine, including Hsp70 and p60Hop, promotes client protein ubiquitination, probably by recruitment of a chaperone-binding E3 protein, resulting in client protein targeting to, and degradation by, the proteasome. When geldanamycin (GA) or 17-allylaminogeldanamycin (17AAG) binds to the aminoterminal nucleotide pocket of Hsp90, it forces the chaperone to adopt the conformation that favors assembly of the destabilizing and proteasometargeting chaperone machine, while preventing assembly of the stabilizing chaperone complex.

S56

v-Src Soluble Akt kinases Raf-1 Bcr-Abl

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002

Mutant p53

Hsp90

Transcription factors

HIF-1α ErbB2

Transmembrane kinases TRENDS in Molecular Medicine

three months apart would have much in common, for both were addressing the importance of molecular chaperones to the growth and survival of cancer cells. When Whitesell et al. reported in 1994 that benzoquinone ansamycins, and geldanamycin (GA) in particular, bound specifically to Hsp90, inhibited the association of the chaperone with v-Src protein, and led to the eventual destabilization of the protein [7], researchers finally had the means to interfere pharmacologically with Hsp90 and to study its function in cancer cells. As a result of these studies, it rapidly became clear that Hsp90 was a novel and very exciting target for cancer therapy. Hsp90 is constitutively expressed at 2–10-fold higher levels in tumor cells compared with their normal counterparts, suggesting that it could be crucially important for the growth and/or survival of tumor cells [8]. Because GA binding to Hsp90 uniformly results in client protein destabilization, usually mediated by the proteasome, GA has proven to be an invaluable tool in expanding the list of Hsp90-dependent signaling proteins (Fig. 1).The number of these proteins has grown dramatically during the past six years, and the list now includes several mutated and/or over-expressed signaling proteins implicated in cancer, such as mutated p53 [9], Akt kinase [10,11], Raf-1 kinase [12], Bcr-Abl kinase [13,14], ErbB2 transmembrane kinase [15–17], cyclin-dependent kinases Cdk4 and Cdk6 [18], the cell-cycle-associated kinase Wee1 [19], certain basic helix–loop–helix transcription factors Hsp70 p50Cdc37 p60Hop p23

ATP

Hsp90

Targets client protein to proteasome

ADP GA 17-AAG

Hsp90

Stabilizes and protects client protein TRENDS in Molecular Medicine

including hypoxia-inducible factor 1α (HIF-1α) [20], and steroid receptors including estrogen and androgen receptors [21–24]. An Hsp90-dependent chaperoning process is even crucially important in the enhancement in telomerase expression associated with cancer progression [25]. While we and others were using GA to identify Hsp90-dependent signaling proteins, our own and two other laboratories identified the drug-binding site on Hsp90 to be a hydrophobic ATP/ADP-binding pocket in the amino terminus of the chaperone [26–28]. Nucleotide binding to this pocket alters Hsp90 conformation sufficiently to define distinct, non-overlapping subsets of co-chaperone proteins with which Hsp90 interacts, thus forming a ‘super-chaperone machine’ with very different characteristics [29]. While one Hsp90 conformation determines assembly of a machine that protects and stabilizes client proteins, a second conformation dictates assembly of a machine with a diametrically opposed function – namely to oversee the degradation of Hsp90 client proteins (Fig. 2) [30]. In this way, nucleotide binding to Hsp90 determines the half-life of an Hsp90 client. Incredibly, GA replaces nucleotide in the Hsp90-binding pocket with an affinity much greater than that of either ATP or ADP, and directs assembly of the super-chaperone machine that favors client protein degradation (Fig. 3) [30].Thus, in the presence of GA, the halflifes of Hsp90 clients are uniformly reduced, so that in some cases their steady-state level becomes undetectable. Very recently, several groups have identified E3 ubiquitin ligases containing chaperone-interacting motifs. At least one of these proteins, CHIP (for ‘carboxyl terminus of Hsc70-interacting protein’), has been shown to promote the ubiquitination and degradation of some Hsp90 clients [31,32]. Whether a single chaperone-binding E3 or a family of such proteins mediates GA-induced Hsp90 client protein degradation remains to be discovered, but it might soon be possible to modulate client protein stability by pharmacological manipulation at this level, as well as at the level of Hsp90 itself. Finally, Rosen and colleagues have demonstrated the feasibility of using GA to recruit select non-Hsp90 client proteins to the GA-bound Hsp90 super-chaperone machine [33]. Building on the demonstration some years ago that the 17 position of GA was amenable to derivatization without loss of Hsp90 binding, these investigators coupled the selective phosphoinositide 3-kinase (PI3-kinase) inhibitor LY294002 to GA by a carbon linker and demonstrated Hsp90-dependent PI3-kinase inhibition in vitro. In fact, one of the hybrid molecules displayed more-potent inhibition of PI3-kinase than did native LY294002. Screening of these hybrid inhibitors in cell culture systems is underway. http://www.trends.com

A TRENDS Guide to Cancer Therapeutics

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002

Use of Hsp90 inhibitors as single agents – cytostatic or cytotoxic? Although GA itself proved to be too hepatotoxic for clinical use, a better tolerated derivative (17AAG) that also binds Hsp90 has shown promising antitumor activity, as well as predicted biological activity, in preclinical models [34,35], and is now in phase I trial as a single agent. Preliminary data obtained from these trials demonstrate predicted biological activity achieved at drug concentrations below the maximally tolerated dose [36]. In addition, the findings demonstrate that dose-limiting hepatotoxicity occurs after several days of daily drug administration, while once weekly administration of a much higher drug dose is significantly better tolerated. Thus, these phase I trials are providing valuable information about how best to schedule 17AAG administration to achieve maximal benefit with the least toxicity. Exciting laboratory studies that have emerged during the past few years suggest defined clinical settings in which 17AAG, or another Hsp90 inhibitor, will have the most clinical efficacy (see Box 1). As Hostein et al. have recently pointed out [11], GA and 17AAG can either produce cytostasis or cause apoptosis in vitro and, given the plethora of Hsp90 client proteins, it is difficult to ascertain those factors that determine the outcome of drug exposure. Such understanding is crucial for optimal clinical use of the drug. If cytostasis were its major mechanism of action in vivo, the drug would have to be administered chronically, although in vivo cytotoxic activity would favor an intermittent dosing regimen. In examining four coloncarcinoma-derived cell lines, Hostein and colleagues demonstrated that 17AAG was able to downregulate several Hsp90 client proteins and to cause cytostasis, but the drug only led to apoptosis in three of the four lines. By contrast, apoptosis was readily induced in the resistant line by a topoisomerase II inhibitor. The lack of Bax expression and the over-expression of Bag-1 protein in the cell line resistant to 17AAG-induced apoptosis suggests that one or both of these proteins might help determine the apoptogenic response to 17AAG. Intriguingly, Bax is a proapoptotic Bcl-2 family member and Bag-1 has been shown to interact with Hsp70 and to exhibit antiapoptotic properties. Rosen and colleagues have suggested that the retinoblastoma (Rb) status of the cell plays a role in GAand 17AAG-induced apoptosis [37]. In Rb-positive cell lines, these investigators have observed a G1 cell-cycle arrest in response to 17AAG and other Hsp90 inhibitors, while cells lacking Rb expression arrest in G2/M phase of the cell cycle and are much more prone to undergo eventual apoptosis.Although possibly a contributing factor, Rb status might not be the only determinant of 17AAG apoptogenic http://www.trends.com

Dimerization domain Negativelycharged domain

Review

Figure 3. Schematic diagram of an Hsp90 monomer

ATP/ADP GA,17AAG, radicicol

NH2

|

COOH Novobiocin ATP? TRENDS in Molecular Medicine

potential because the one colon carcinoma cell line in the study by Hostein et al. that was resistant to 17AAGinduced apoptosis nevertheless accumulated in the G2/M phase of the cell cycle in response to drug addition [11]. Thus, the factors determining whether the response of a cell to Hsp90 inhibition will be growth arrest or apoptosis remain to be fully understood. Until that time arrives, the use of 17AAG as a single agent in a clinical setting will be subject to some difficulty. For example, on the basis of data obtained from the current phase I trials, daily administration of the drug is more toxic than once weekly administration. If cytostasis were the primary result of cumulative drug effects in vivo, chronic administration at an effective dose will prove difficult to maintain for an extended period of time. However, if 17AAG as a single agent can cause apoptosis in vivo, administration of quite high levels on a once weekly or perhaps twice weekly basis should be possible. Eventually, a patient’s tumor might have to be molecularly profiled in order to determine how 17AAG should best be administered.

The amino-terminal ATP/ADPbinding pocket is also the binding site for the Hsp90 inhibitors, geldanamycin (GA), 17-allylaminogeldanamycin (17AAG) and radicicol. The negatively charged domain, of unknown function, links the amino-terminal region to the carboxyl-terminal domain. The carboxy-terminal domain contains the dimerization domain and binding site for a third Hsp90 inhibitor, novobiocin. The novobiocin site could represent a previously unrecognized ATP-binding domain.

Box 1. Use of Hsp90 inhibitors in cancer therapy Heat shock protein 90 (Hsp90) inhibitors as single agents in situations in which the Hsp90 client protein is necessary for cancer development or progression • Bcr-Abl-positive leukemias • Other leukemias/lymphomas dependent on a chimeric protein; for example, anaplastic large cell lymphoma (NPM/ALK?) and acute promyelocytic leukemia (PML/RAR?) • Androgen- or estrogen-receptor-dependent cancers (prostate and breast) • Hormone-independent breast and prostate cancers in which receptor mutation can be documented Hsp90 inhibitors used to modify the response to standard chemotherapeutic agents • Used with taxol or doxorubicin in ErbB2 or Akt over-expressing tumors (breast, ovarian, prostate and lung cancer) • Used with GleevecTM in Bcr-Abl-positive leukemias

S57

Review

|

A TRENDS Guide to Cancer Therapeutics

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002

In certain special cases wherein an Hsp90 client protein is directly implicated in the pathogenesis of a particular cancer, single agent administration of an Hsp90 inhibitor, such as 17AAG, might be quite effective. For example, distinct fusion products of the Bcr and Abl genes, p210Bcr-Abl or p185Bcr-Abl, have been directly implicated in the development of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL), respectively. We recently reported that the Bcr-Abl protein is dependent on association with Hsp90 for its stability and that treatment of cells with GA or 17AAG leads to rapid destruction of Bcr-Abl [13]. Bhalla and colleagues have since reported that high (5 µM) concentrations of either GA or 17AAG induced apoptosis in CML-derived cell lines (K562), as well as in cells transfected with the Bcr-Abl gene [38]. Shiotsu et al. had previously shown that a second type of Hsp90 inhibitor, radicicol, had a similar pro-apoptotic effect on Bcr-Abl-expressing CML cells in vitro, and also significantly prolonged the survival of severe combined immunodeficient (SCID) mice inoculated with K562 cells [14]. Similar results have been obtained with STI-571 (GleevecTM), a moderately specific Bcr-Abl kinase inhibitor, confirming that inhibition (or degradation) of Bcr-Abl is sufficient to induce apoptosis. Steroid receptors were among the first Hsp90 client proteins to be identified. Thus, the androgen receptor requires association with Hsp90 to attain a high-affinity ligand-binding conformation. In the absence of Hsp90, or in the presence of GA or radicicol, androgenic stimulation of an androgen-responsive reporter gene was dosedependently inhibited [21,24]. Furthermore, a recent study reported that 17AAG induced the degradation of androgen receptors in vivo in prostate cancer xenografts, correlating with growth inhibition of the tumor [39]. Thus, androgen-dependent prostate cancer can be included among those tumors that are responsive to 17AAG as a single agent. As androgen-independent prostate cancer could depend on a constitutively active, mutated androgen receptor, 17AAG might be effective in a broader group of prostate cancer patients, and the use of 17AAG in patients who have failed hormonal therapy is also suggested. The estrogen receptor is a well-validated therapeutic target in breast cancer. Therapies aimed at blocking the estrogen receptor, although initially effective, eventually lose their activity. Acquired resistance to anti-estrogens involves multiple mechanisms, including mutations in the estrogen receptor. Whitesell and colleagues have recently shown that Hsp90 inhibitors, including 17AAG and radicicol derivatives, effectively destabilize the estrogen receptor in vitro and in vivo, and delay the growth of estrogen-responsive human tumor xenografts [23]. These data strongly suggest that an Hsp90 inhibitor such as

S58

17AAG might be effective in patients whose breast cancer has lost responsiveness to anti-estrogens. Rationale for use of Hsp90 inhibitors to modify cellular response to chemotherapy The clinical benefit of 17AAG or other Hsp90 inhibitors as single agents appears promising in certain defined settings. At the same time, exciting preclinical studies point to the probable wide-ranging use of such compounds when used in combination with standard agents. Even in the case of Bcr-Abl-positive leukemias, low-dose GA effectively sensitized cells to previously ineffective levels of doxorubicin. Thus, at concentrations that themselves were not apoptogenic (2% of the concentration used by Bhalla et al.), GA promoted caspase activation in both K562 cells and Bcr-Abl-expressing HL60 cells when combined with low-dose doxorubicin [40]. The relatively specific inhibitor of Bcr-Abl tyrosine kinase activity, GleevecTM (STI-571), produces a high rate of hematological remission in CML; however, in CML patients in blast crisis and in Bcr-Abl-expressing ALL patients, remissions have not been durable, despite continuous drug administration.These drug failures appear to be a result of either amplification and over-expression of Bcr-Abl, or point mutation of the protein at the site where GleevecTM binds (the ATP-binding site of the kinase) [41]. Because 17AAG destabilizes Bcr-Abl protein by altering its association with Hsp90, its activity should not be affected by either point mutation or over-expression of the kinase. Studies are now underway to test whether a combination of 17AAG with GleevecTM will reduce the rate of GleevecTM failure, as well as to determine the efficacy of 17AAG alone following GleevecTM failure. One of the most Hsp90-dependent client proteins is ErbB2 (HER-2/neu), whose over-expression in breast cancer antagonizes taxol cytotoxicity. Rosen and colleagues have shown that a combination of 17AAG and taxol was more cytotoxic than either agent alone [42,43]. However, the Rb protein status of the cells was important. In cells that were Rb-negative, the order of drug addition was not important; however, in cells that were Rb-positive, exposure to 17AAG before taxol enabled the cells to arrest in G1 phase of the cell cycle and become resistant to taxolinduced toxicity. This antagonism probably occurred because of the previously demonstrated ability of 17AAG to induce an Rb-dependent G1 arrest in a variety of tumor types. In contrast to taxol, the cytotoxicity produced by doxorubicin was also synergistically increased by 17AAG but, in this case, order of drug addition was not important, as doxorubicin toxicity is not cell-cycle dependent. A similar enhancement of taxol-mediated cytotoxicity by concomitant administration of 17AAG has been http://www.trends.com

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002

demonstrated in non-small-cell lung cancer models, both in vitro and in vivo, by Nguyen et al. [44,45]. Although a combination of the two agents produced additive toxicity in cells expressing low levels of ErbB2, synergy was observed in those cell lines in which ErbB2 was highly expressed (up to 20-fold reduction in taxol IC50, the concentration of inhibitor required for 50% inhibition of activity, was achieved). Again, order of addition was important. Because the cell lines examined were Rb-positive, pre-treatment with 17AAG produced G1 arrest and rendered cells less sensitive to taxol. In mice bearing xenografts of ErbB2 over-expressing tumor cells, the combination of 17AAG and taxol profoundly suppressed tumor growth and significantly prolonged survival. Immunohistochemical analysis of tumors from treated animals revealed loss of ErbB2 expression, marked reduction in vascular endothelial growth factor expression and marked apoptosis. Finally, as most small-cell lung cancer tumors do not express functional Rb proteins, they should be particularly sensitive to drug combinations that include 17AAG [46,47]. ErbB2 is over-expressed in 30–40% of breast, ovarian and non-small-cell lung cancer, and its over-expression correlates with enhanced resistance to multiple chemotherapeutic agents besides taxol, including cisplatin, etoposide and doxorubicin. Thus, it is reasonable to test the efficacy of 17AAG in combination with any of these agents in vivo. Other proteins that promote cell survival, including Akt and HIF-1α, are also Hsp90 client proteins and thus are destabilized by GA and 17AAG. Inappropriate Akt activation has been detected in a large number of ovarian carcinomas, and HIF-1α expression is frequently found in many solid tumors [48,49]. Although not yet directly shown, it is highly likely that these proteins also subvert the effectiveness of standard chemotherapeutics, suggesting that a combination of 17AAG with standard agents could prove beneficial in many instances. Hsp90 inhibition as an anticancer modality: the future Although a benzoquinone ansamycin is the first Hsp90 inhibitor to have reached the clinic, other natural products of different chemical structure have also been shown to inhibit Hsp90 both in vitro and in vivo. Thus, radicicol and its derivatives also bind in the nucleotide pocket of Hsp90, with nearly identical biological effects as 17AAG [50–52]. Likewise, novobiocin and other coumarin antibiotics bind to the carboxyl terminus of Hsp90 and also disrupt its ability to chaperone client proteins [53,54]. Whether either of these classes of natural products will produce a better alternative to 17AAG remains to be seen, but the possibility remains that, if the dose-limiting toxicity http://www.trends.com

A TRENDS Guide to Cancer Therapeutics

|

Review

of 17AAG is quinone dependent [35], this could be reduced with either a radicicol-based or novobiocin-based compound, as neither contains a quinone moiety. Several groups are now creating synthetic Hsp90 inhibitors, based either on GA or radicicol, with the purpose of removing those chemical constituents in the natural molecules that might be most responsible for generating non-target-based toxicity in vivo. Although these attempts are still in the early stages, micromolar binding affinities have already been achieved [55]. The ultimate toxicity produced as a result of inhibiting Hsp90 in normal tissues remains to be determined. In the current phase I clinical trials of 17AAG, dose-limiting Hsp90-dependent toxicity has not yet been definitively observed. Although Hsp90 inhibition will probably be immunosuppressive, other cancer chemotherapeutics have similar characteristics and have nevertheless proven to be quite useful. Hsp90 inhibition might also be of value in several non-cancer settings, especially in the prevention of restenosis and in protection from ischemia [56,57]. Finally, although the focus to date has been on the identification of small-molecule Hsp90 inhibitors like GA, the chaperone is most certainly regulated by posttranslational modification, thus identifying another avenue for pharmacological manipulation. For example, two studies have demonstrated that Hsp90 phosphorylation is coupled to the release of the chaperone from its client protein [58,59]. GA inhibits Hsp90 phosphorylation, suggesting that this post-translational modification is conformation dependent, and the phosphatase inhibitor okadaic acid leads to dramatic hyperphosphorylation of Hsp90, suggesting that cycles of phosphorylation and dephosphorylation of the chaperone are continuously occurring in a regulated manner. Conclusions Clearly, much has been learned in the past few years about the function of Hsp90 and its importance for the survival of cancer cells. However, much remains to be learned. For proteins that are neither mutated nor chimeric, the determinants of Hsp90 dependence remain essentially unknown. What domain structures are shared among certain kinases, transcription factors and steroid receptors, not to mention other proteins such as telomerase, that dictate a requirement for Hsp90 association? Is Hsp90 buffering the many hundreds to thousands of silent mutations found in cancer cells, much as it is thought to buffer the much more infrequent mutations that normally accumulate during an organism’s lifespan [60]? The further development and application of Hsp90 inhibitors should help answer these questions. Within the next ten years, the

S59

Review

|

A TRENDS Guide to Cancer Therapeutics

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002

continued use of Hsp90 inhibitors in the clinic, guided by additional experimental and preclinical observations, should determine whether targeted inhibition of this chaperone is a viable approach to cancer treatment.

References 1 Yufu,Y. et al. (1992) High constitutive expression of heat shock protein 90 α in human acute leukemia cells. Leuk. Res. 16, 597–605 2 Xu,Y. and Lindquist, S. (1993) Heat-shock protein hsp90 governs the activity of pp60v-src kinase. Proc. Natl.Acad. Sci. U. S.A. 90, 7074–7078 3 Stancato, L.F. et al. (1997) The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase. J. Biol. Chem. 272, 4013–4020 4 Srivastava, P.K. and Maki, R.G. (1991) Stress-induced proteins in immune response to cancer. Curr.Top. Microbiol. Immunol. 167, 109–123 5 Ciocca, D.R. et al. (1992) Response of human breast cancer cells to heat shock and chemotherapeutic drugs. Cancer Res. 52, 3648–3654 6 Whitesell, L. et al. (1992) Benzoquinonoid ansamycins possess selective tumoricidal activity unrelated to src kinase inhibition. Cancer Res. 52, 1721–1728 7 Whitesell, L. et al. (1994) Inhibition of heat shock protein HSP90pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc. Natl.Acad. Sci. U. S.A. 91, 8324–8328 8 Ferrarini, M. et al. (1992) Unusual expression and localization of heat-shock proteins in human tumor cells. Int. J. Cancer 51, 613–619 9 Blagosklonny, M.V. et al. (1996) Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90. Proc. Natl.Acad. Sci. U. S.A. 93, 8379–8383 10 Sato, S. et al. (2000) Modulation of Akt kinase activity by binding to Hsp90. Proc. Natl.Acad. Sci. U. S.A. 97, 10832–10837 11 Hostein, I. et al. (2001) Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res. 61, 4003–4009 12 Schulte,T.W. et al. (1996) Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1–MEK–mitogen-activated protein kinase signalling pathway. Mol. Cell. Biol. 16, 5839–5845 13 An, W.G. et al. (2000) The heat shock protein 90 antagonist geldanamycin alters chaperone association with p210bcr-abl and vsrc proteins before their degradation by the proteasome. Cell Growth Differ. 11, 355–360 14 Shiotsu,Y. et al. (2000) Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G(1) phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex. Blood 96, 2284–2291 15 Miller, P. et al. (1994) Binding of benzoquinoid ansamycins to p100 correlates with their ability to deplete the erbB2 gene product p185. Biochem. Biophys. Res. Commun. 201, 1313–1319 16 Miller, P. et al. (1994) Depletion of the erbB-2 gene product p185 by benzoquinoid ansamycins. Cancer Res. 54, 2724–2730 17 Chavany, C. et al. (1996) p185erbB2 binds to GRP94 in vivo. Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinone ansamycins precedes depletion of p185erbB2. J. Biol. Chem. 271, 4974–4977 18 Stepanova, L. et al. (1996) Mammalian p50Cdc37 is a protein kinasetargeting subunit of Hsp90 that binds and stabilizes Cdk4. Genes Dev. 10, 1491–1502 19 Aligue, R. et al. (1994) A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90. EMBO J. 13, 6099–6106 20 Minet, E. et al. (1999) Hypoxia-induced activation of HIF-1: role of HIF-1α–Hsp90 interaction. FEBS Lett. 460, 251–256 21 Fang,Y. et al. (1996) Hsp90 regulates androgen receptor hormone binding affinity in vivo. J. Biol. Chem. 271, 28697–28702 22 Segnitz, B. and Gehring, U. (1997) The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin. J. Biol. Chem. 272, 18694–18701 23 Bagatell, R. et al. (2001) Destabilization of steroid receptors by heat shock protein 90-binding drugs: a ligand-independent approach to hormonal therapy of breast cancer. Clin. Cancer Res. 7, 2076–2084 24 Haendler, B. et al. (2001) Androgen receptor signalling: comparative

S60

25 26

27

28

29 30

31 32 33

34

35

36

37

38

39

40

41 42

43

44

45

46

47 48

49

analysis of androgen response elements and implication of heat-shock protein 90 and 14-3-3eta. Mol. Cell. Endocrinol. 173, 63–73 Holt, S.E. et al. (1999) Functional requirement of p23 and Hsp90 in telomerase complexes. Genes Dev. 13, 817–826 Grenert, J.P. et al. (1997) The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation. J. Biol. Chem. 272, 23843–23850 Stebbins, C.E. et al. (1997) Crystal structure of an Hsp90–geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89, 239–250 Prodromou, C. et al. (1997) Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90, 65–75 Scheibel,T. and Buchner, J. (1998) The Hsp90 complex – a superchaperone machine as a novel drug target. Biochem.Pharmacol. 56, 675–682 Schneider, C. et al. (1996) Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc. Natl.Acad. Sci. U. S.A. 93, 14536–14541 Connell, P. et al. (2001) The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat. Cell Biol. 3, 93–96 Meacham, G.C. et al. (2001) The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nat. Cell Biol. 3, 100–105 Chiosis, G. et al. (2001) LY294002-geldanamycin heterodimers as selective inhibitors of the PI3K and PI3K-related family. Bioorg. Med. Chem. Lett. 11, 909–913 Schulte,T.W. and Neckers, L.M. (1998) The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin. Cancer Chemother. Pharmacol. 42, 273–279 Kelland, L.R. et al. (1999) DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90. J. Natl. Cancer Inst. 91, 1940–1949 Agnew, E.B. et al. (2001) Measurement of the novel antitumor agent 17-(allylamino)-17-demethoxygeldanamycin in human plasma by high-performance liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl. 755, 237–243 Srethapakdi, M. et al. (2000) Inhibition of Hsp90 function by ansamycins causes retinoblastoma gene product-dependent G1 arrest. Cancer Res. 60, 3940–3946 Nimmanapalli, R. et al. (2001) Geldanamycin and its analogue 17-allylamino-17-demethoxygeldanamycin lowers Bcr-Abl levels and induces apoptosis and differentiation of Bcr-Abl-positive human leukemic blasts. Cancer Res. 61, 1799–1804 Solit, D. et al. 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin. Cancer Res. (in press) Blagosklonny, M.V. et al. (2001) The Hsp90 inhibitor geldanamycin selectively sensitizes Bcr-Abl-expressing leukemia cells to cytotoxic chemotherapy. Leukemia 15, 1537–1543 Gorre, M.E. et al. (2001) Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 Munster, P.N. et al. (2001) Modulation of Hsp90 function by ansamycins sensitizes breast cancer cells to chemotherapy-induced apoptosis in an RB- and schedule-dependent manner. Clin. Cancer Res. 7, 2155–2158 Sausville, E.A. (2001) Combining cytotoxics and 17-allylamino, 17-demethoxygeldanamycin: sequence and tumor biology matters. Clin Cancer Res. 7, 2228–2236 Nguyen, D.M. et al. (2001) Enhancement of paclitaxel-mediated cytotoxicity in lung cancer cells by 17-allylamino geldanamycin: in vitro and in vivo analysis. Ann.Thorac. Surg. 72, 371–378, discussion 378–379 Nguyen, D.M. et al. (1999) Sequence-dependent enhancement of paclitaxel toxicity in non-small cell lung cancer by 17-allylamino 17-demethoxygeldanamycin. J.Thorac. Cardiovasc. Surg. 118, 908–915 Horowitz, J.M. et al. (1990) Frequent inactivation of the retinoblastoma anti-oncogene is restricted to a subset of human tumor cells. Proc. Natl.Acad. Sci. U. S.A. 87, 2775–2779 Lai, S.L. et al. (1995) Molecular genetic characterization of neuroendocrine lung cancer cell lines. Anticancer Res. 15, 225–232 Zhong, H. et al. (1999) Overexpression of hypoxia-inducible factor 1α in common human cancers and their metastases. Cancer Res. 59, 5830–5835 Kurose, K. et al. (2001) Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. Am. J. Pathol. 158, 2097–2106

http://www.trends.com

Trends in Molecular Medicine Vol. 8 No. 4 (Suppl.), 2002

50 Sharma, S.V. et al. (1998) Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol. Oncogene 16, 2639–2645 51 Schulte,T.W. et al. (1998) Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin. Cell Stress Chaperones 3, 100–108 52 Roe, S.M. et al. (1999) Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J. Med. Chem. 42, 260–266 53 Marcu, M.G. et al. (2000) The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone. J. Biol. Chem. 275, 37181–37186 54 Marcu, M.G. et al. (2000) Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins. J. Natl. Cancer Inst. 92, 242–248 55 Chiosis, G. et al. (2001) A small molecule designed to bind to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and the

56

57

58

59

60

A TRENDS Guide to Cancer Therapeutics

|

Review

growth arrest and differentiation of breast cancer cells. Chem. Biol. 8, 289–299 Slepian, M.J. et al. (1996) Pre-conditioning of smooth muscle cells via induction of the heat shock response limits proliferation following mechanical injury. Biochem. Biophys. Res. Commun. 225, 600–607 Conde, A.G. et al. (1997) Induction of heat shock proteins by tyrosine kinase inhibitors in rat cardiomyocytes and myogenic cells confers protection against simulated ischemia. J. Mol. Cell. Cardiol. 29, 1927–1938 Mimnaugh, E.G. et al. (1995) Possible role for serine/threonine phosphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and the pp60v-src tyrosine kinase. J. Biol. Chem. 270, 28654–28659 Zhao,Y.G. et al. (2001) Hsp90 phosphorylation is linked to its chaperoning function. Assembly of the reovirus cell attachment protein. J. Biol. Chem. 276, 32822–32827 Rutherford, S.L. and Lindquist, S. (1998) Hsp90 as a capacitor for morphological evolution. Nature 396, 336–342

The TRENDS Guides: quality content brought to you in association with the TRENDS journals The TRENDS Guides focus on areas of growing interest and provide informative and timely reviews to address these emerging information needs. The TRENDS Guides are published and indexed as supplements to our established and highly regarded TRENDS journal titles. Look out for these TRENDS Guides titles coming in 2002: A TRENDS Guide to Imaging Technologies Guest Editor: Aaron Fenster (The John P. Robarts Research Institute, Canada) A TRENDS Guide to Infectious Diseases Guest Editors: Steven Projan (Wyeth-Ayerst, USA), Paul Hagan (The University of Glasgow, UK) and John Rosamond (AstraZeneca, USA) A TRENDS Guide to Proteomics (III) Guest Editors: Ruedi Aebersold (Institute for Systems Biology, USA) and Ben Cravatt (The Scripps Research Institute, USA)

TRENDS Guides on BioMedNet Published TRENDS Guides are currently available online free-of-charge through BioMedNet. Please visit http://journals.bmn.com/supp for free full-text access to articles published so far in the TRENDS Guide series: A TRENDS Guide to Cancer Biology A TRENDS Guide to Neurodegenerative Diseases and Repair A TRENDS Guide to Proteomics (I and II) A TRENDS Guide to New Technologies for Life Sciences

http://www.trends.com

S61